Research ArticleNaja naja karachiensis Envenomation BiochemicalParameters for Cardiac Liver and Renal Damage alongwith Their Neutralization by Medicinal Plants
Muhammad Hassham Hassan Bin Asad1 Ghulam Murtaza1
Muhammad Ubaid1 Durr-e-Sabih2 Ashif Sajjad3 Rubada Mehmood2
Qaisar Mahmood4 Muhammad Muzzmil Ansari5 Sabiha Karim6
Zahid Mehmood3 and Izhar Hussain1
1 Department of Pharmacy COMSATS Institute of Information Technology Abbottabad 22060 Pakistan2Multan Institute of Nuclear Medicine and Radiotherapy (MINAR) 377 Nishtar Hospital Multan 60000 Pakistan3 Institute of Biochemistry University of Balochistan Quetta 87300 Pakistan4Department of Environmental Sciences COMSATS Institute of Information Technology Abbottabad 22060 Pakistan5 Roba-al-Safwa Pharmacy Alsafwa Hospital 67 Makkah Saudi Arabia6University College of Pharmacy University of the Punjab Lahore 54000 Pakistan
Correspondence should be addressed to Ghulam Murtaza gmdogar356gmailcom and Izhar Hussain izharhussainciitnetpk
Received 17 January 2014 Accepted 27 March 2014 Published 27 April 2014
Academic Editor Andrei Surguchov
Copyright copy 2014 Muhammad Hassham Hassan Bin Asad et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited
Naja naja karachiensis envenomation was found to hit more drastically heart liver and kidneys 400 120583gkg of venom-raisedmoderate serum levels of ALT (72 plusmn 470UL 01 gt 119875 gt 005) AST (157 plusmn 2424UL 01 gt 119875 gt 005) urea (42 plusmn 308mgdL005 gt 119875 gt 002) creatinine (174 plusmn 003mgdL 001 gt 119875 gt 0001) CK-MB (21 plusmn 15UL 005 gt 119875 gt 002) and LDH(2064 plusmn 1598UL 119875 lt 0001) were injected in experimental rabbits However lethality was enhanced with 800120583gkg of venom interms of significant release of ALT (86plusmn50UL 005 gt 119875 gt 002) AST (251plusmn182UL 001 gt 119875 gt 0001) urea (576plusmn384mgdL002 gt 119875 gt 001) creatinine (21 plusmn 010mgdL 002 gt 119875 gt 001) CK-MB (77 plusmn 1122UL 005 gt 119875 gt 002) and LDH(2562 plusmn 2514UL 119875 ≪ 0001) Among twenty-eight tested medicinal plant extracts only Stenolobium stans (L) Seem was foundthe best antivenom (119875 gt 05) compared to the efficacy of standard antidote (ALT = 525 plusmn 351UL AST = 695 plusmn 1855UL urea= 315 plusmn 050mgdL creatinine = 108 plusmn 002mgdL CK-MB = 09 plusmn 085UL and LDH = 763 plusmn 601UL) Other plant extractswere proved less beneficial and partly neutralized the toxicities posed by cobra venom However it is essential in future to isolateand characterize bioactive compound(s) from Stenolobium stans (L) Seem extract to overcome the complications of snake bite
1 Introduction
One of the animals which represent Pakistan and Indiathroughout the world is cobra in particular it is rearing out ofa wicker basket and dancing to the sound of turban-wearingsnake charmer music In fact they are deadly venomousamong all species of the snakes due to high rate of mortalitywhich strongly reasoned for their notoriety [1] At the endof the nineteenth century all cobra populations have beenmerged into single species naja (formerly known as naia)
however subsequently ten subspecies of Naja naja wereidentified [1] Later on Deraniyagala classified Naja naja(India) into several subspecies and considered Naja najakarachiensis one of them widely distributed in SouthernPunjab province of Pakistan [2] They are extremely toxicdue to their severe side effects in the victims Major signsand symptoms of cobra envenomation are edema necrosispain respiratory paralysis vomiting headache hypotensioncardiac arrest coagulopathies (elevated PT aPTT and TT)bleeding wounds hematuria mucus discharge proteinuria
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 970540 13 pageshttpdxdoiorg1011552014970540
2 BioMed Research International
and increased creatinine and urea levels along with alteredconsciousness [3 4]
Indeed snake venom is a complex mixture of varioussizes of polypeptides [5 6] (lt15 kDa 5 to 10 kDa and10 to 150 kDa) carbohydrates lipids metal ions organicmolecules purines and amines [7 8] Snake bite enveno-mation has been attributed to pose drastic changes in thephysiology of the victims that could be accessed clinicallyby measuring various biochemical parameters Among toxiccomponents phospholipases A
2(cell destroying enzymes
cause edema lipolytic or myolytic) [9 10] phosphodi-esterases (nucleic acid degrading enzyme causes hypoten-sionshock) [11 12] 51015840 nucleotidases (digest the sugar moitiescausing the delay in coagulation) [13] alkaline phosphatases(hydrolyze phosphate esters nonspecifically cause multipletoxicities via adenosine generation) [14] hyaluronidases(spreading factor causes digestion necrosis and leakage ofblood vessels) [15] L-amino oxidases (deamination leads tocell damageapoptosis) [16] and proteases (hydrolyze variousproteins) [17] are included
Laboratory animals particularly rabbits have beenselected previously to monitor various biochemical changesrelated to liver heart and renal toxicities [18 19] Aspartateaminotransferase (GOTAST) and alanine aminotransferase(GPTALT) are surrogate markers for liver toxicities whilecreatine kinase isoenzyme (CK-MB) is used exclusively toaccess cardiac tissue damage [20 21] Elevated creatinine andurea levels are indicators of 75 renal damage [20] whereashigh level of lactate dehydrogenase (LDH) implies generaltoxicity related to the liver heart kidneys and skeletonmuscles [22]
Diverse immunological properties of different cobrashave fascinated protein chemists and other researchers tocarry out efforts for the development of new antidotes Dueto this reason for the first time venom from Naja najakarachiensis is selected to evaluate its toxicity in vivo andto search out effective as well as cheap treatment fromfolk herbal remedies Pakistan is a hub of medicinal plantswhere mostly rural communities rely on natural herbs totreat their problems of snake bite [6 23] It is thereforenecessary to evaluate scientifically their folklore claims asanti-snake venom in traditional system of medicine Dueto this reason various medicinal plants (Albizia lebbeck (L)Benth Allium cepa L Allium sativum L Althaea officinalisL Bauhinia variegata L Brassica nigra (L) W D J KochCalotropis procera (Aiton)WTAitonCedrus deodara (Roxbex D Don) G Don Citrullus colocynthis (L) Schrad Citruslimon (L) Burm f Cuminum cyminum L Enicostemahyssopifolium (Willd) I Verd Fagonia cretica L Leucascapitata DesfMatthiola incana (L) W T AitonMomordicacharantiaLNerium indicumMillOcimum sanctumL Pinusroxburghii Sarg Pistacia integerrima J L Stewart Psoraleacorylifolia L Rhazya stricta Decne Rubia cordifolia LSapindus mukorossi Gaertn Solanum xanthocarpum Schardand Wendle Stenolobium stans (L) Seem Terminalia arjuna(Roxb exDC)Wight andArnTrichodesma indicum (L) Smand Zingiber officinale Roscoe) were collected to test theirpotentials against toxicities induced byNaja naja karachiensisenvenomation (Table 1)
2 Material and Methods
21 Collection and Milking of Snake Venom Black Pakistanicobra snakes (Naja naja karachiensis) were collected fromCholistan desert located in southern Punjab province ofPakistan After collection they were properly identified by azoologist Venom from Naja naja karachiensis was collectedby pressing their glands below their eyes in low light environ-ment After collection it was lyophilized and stored in lightresistant container at 2∘C to 8∘C For further experimentsvenom was used in terms of its dry weight [6]
22 Collection and Preparation of Plant Extracts Medicinalplants were collected from different areas in Pakistan Aftercollection they were duly identified by expert botanist(Professor Dr Altaf Ahmad Dasti) and voucher specimenswere deposited in the herbarium of the Institute of Pure andApplied Biology Bahauddin Zakariya University MultanPakistan Complete description about evaluated medicinalplants is summarized in Table 1 One kilogram choppedplant material was soaked in 5 L of methanol as solvent inextraction bottlesThe homogenates were kept for a period ofa month at optimum temperature After filtration filtrate wasevaporated by using water bath and extracts were weighedand stored for further experimentation [24]
23 Experimental Animals Seventy-one healthy growingmale rabbits (1 plusmn 05 kg) were selected for this study aftergetting permission from local ethical committee Animalswere acclimatized for a week in the laboratory bymaintainingtheir standards for chow water and light Subsequently theywere divided into various groups in different cages suchthat experimental rabbits in group I were used for baselinemeasurements of various biochemical parameters Animalsbelonging to groups II and III were evaluated for differentdoses of cobra venom while group IV was served as controlGroup V was redivided into twenty-nine groups such thateach subgroup (V1ndashV29) was assigned to a single medicinalplant extract [25]
24 In Vivo Antivenom Activity of Medicinal Plant ExtractsVenom from Naja naja karachiensis (400 120583gkg and800 120583gkg) was injected subcutaneously to evaluate itstoxicity to the heart liver and kidneys Before injectionof venom experimental animals were anaesthetizedby administration of ketamine (50mgkg) To determineantivenomactivity of various plantsrsquo extract they (100mgkg)were incubated (at 37∘C for 30 minutes) with fixed amountof venom (800120583gkg) before injection while saline was usedas control [26ndash28]
25 Biochemical Assays For serum analysis of various bio-chemical parameters blood was collected from marginal earartery by the use of hypodermic syringe needle after 3 hoursof envenomation [25] Separated serumwas tested for alanineaminotransferase (GPTALT) aspartate aminotransferase(GOTAST) urea creatinine creatine kinase isoenzyme MB(CK-MB) and lactate dehydrogenase (LDH) by the use of
BioMed Research International 3
Table1Detaileddescrip
tionfore
valuated
medicinalplantshaving
folklore
evidencesa
santivenom
Srnum
ber
Botanicaln
ameo
fmedicinalplants
Family
Partused
Phytochemicalsreported
Reference
1Albizialeb
beck
(L)Be
nth
Fabaceae
Seed
Carboh
ydratesproteinsalkaloidsflavanoidstanninsechinocystic
acidand
different
aminoacids
[6]
2Alliu
mcepa
LAmaryllid
aceae
Bulb
11gcarboh
ydrates12
gproteins06gfib
ersandH
2Ocontent(868g)10
0gof
material
[6]
3Alliu
msativ
umL
Amaryllid
aceae
Bulb
Quercetinscordinines
AandBallicinthiosulfin
ates2
mercapto-L-cyste
ines
anthocyaninsalliinasepo
lysaccharid
essativin
Iand
sativ
inIIand
glycosides
ofkaem
pferol
[6]
4Althaeaoffi
cinalisL
Malvaceae
Root
37sta
rch11mucilagefattyoilpectinflavon
oidspheno
licacidsasparagines
phytosterolsucroseandbu
tyric
acid
[6]
5Ba
uhiniavarie
gata
LFabaceae
Root
Tann
insfatty
oillupeolkaempferol-3-glucosid
egums120573-sito
sterol57-dehydroxy
and57-dimetho
xy-flavanon
e-4-0-a-L-rham
nopyrano
syl-120573
-Dglucop
yranosides
[6]
6Brassicanigra(L)WD
JK
och
Brassic
aceae
Seed
Essentialoilsin
igrin
andglucoside
[6]
7Ca
lotro
pisp
rocera
(Aito
n)WTA
iton
Apocyn
aceae
Exud
ates
andflo
wer
Resin
stann
inscalotro
pin
steroluscharincalotropagenin
calotoxin
andcalactin
[6]
8Ce
drus
deodara(Roxbex
DD
on)G
Don
Pinaceae
Bark
Taxifolin
dew
ardioldew
arenegumascorbica
cidhimadarolcantdarol
cholesterin
allo
himachalolcedrinosidehimachaloldewarolcedrin
dihydrod
ehydrodiconiferylalcoh
olessentia
loiliso
centdaroland
dihydrom
yricetin
[6]
9Citru
slim
on(L)Bu
rmf
Rutaceae
Fruit
4-120573-glucopyrano
sided-x-pinene
camph
ened-lim
onenelin
aloo
lichangin
4-120573-glucopyrano
sideno
milinica
cidandessentialoil
[6]
10Citru
lluscolocynthis(L)Schrad
Cucurbita
ceae
Fruit
Manyalkaloidsglycosidestanninscitrulluincitrulluicaciddihydric
alcoho
lph
ydroxybenzylcitrulluenehentria
contaneelaterincitrullolmethyletherbitter
oilcitbittoland
sapo
nins
[6]
11Cu
minum
cyminum
LAp
iaceae
Seed
Essentialoilandcumin
oilcon
tain
sminaldehyde13-p-m
enthadien-7-aland
14-p-m
enthadien-7-al
[6]
12En
icoste
mahysso
pifoliu
m(W
illd)IV
erd
Gentia
naceae
Who
leplant
Sapo
narin
isovetexin
sylysw
ertin
betulinswertia
marinapigenin
genk
wanin
erythrocentaurinesw
ertio
sideiso
swertisinenicoflavineand
swertisin
[6]
13Fagoniacretica
LZy
goph
yllaceae
Leaves
Ursolicacidpinito
lnahagenin
sapo
ninglycosidessapon
in-Iandsapo
nin-II
docosyld
ocosanoatefro
mn-hexane
extractanddifferent
proteins
from
aqueou
sextract
[6]
14Leucas
capitataDesf
Lamiaceae
Who
leplant
Alkaloidas
wellase
ssentia
loil
[6]
15Matthiolaincana
(L)WTA
iton
Brassic
aceae
Seeds
Oilric
hin
chloroph
yllsabNPK
and
Na120574-lino
lenica
cidandcaroteno
ids
Sulfo
raph
eneisa
nim
portantcom
ponent
[6]
16Mom
ordica
charantia
LCu
curbita
ceae
Fruit
Caroteno
idsmom
orcharasides
AandB
glucosidesstig
mastadien-3-beta-ol
squalenem
ycosessteroidalglucosidemom
ordicinesI
andIIstig
masterolvicine
cucurbitane
triterpenoidsm
omordicosid
escycloeucaleno
ltaraxerol
spinasterollo
phenoldiosgeninthiocyano
gen
24-m
ethylencycloarteno
land
phenylprop
anoids
[6]
17Ne
rium
indicum
Mill
Apocyn
aceae
Root
andleaves
Nerioderin
neriodo
rinkarabinand
odorin
[6]
18Ocim
umsanctum
LLamiaceae
Who
leplant
Essentialoils
arefou
ndric
hin
32
carvacrol713eugeno
l17
caryop
hyllene
204methyleugenoleugenolmethyletherlinaloo
landmethylchavicolcineole
[6]
4 BioMed Research International
Table1Con
tinued
Srnum
ber
Botanicaln
ameo
fmedicinalplants
Family
Partused
Phytochemicalsreported
Reference
19Pinu
sroxburghiiSarg
Pinaceae
Oleoresin
Careen120572
-pinene120573-pinene120573-carene120573-lo
ngifo
lenelon
gifoleneand
long
icyclene
[6]
20Pista
ciaintegerrim
aJLStewart
Anacardiaceae
Galls
13essentialoilric
hin
camph
enecineoleA-
terpineolA-
pinenearomadendrene
caprylicacidand
d-lim
onenea
bund
antingalls
[6]
21Psoralea
corylifoliaL
Fabaceae
Seeds
Limon
enelin
aloo
lpsoraleneangelicinn
eobavachalconebakuchiol4-te
rpineol
corylid
inn
eobavaiso
flavonebavachinin
isobavachin120572
-elemenegeranylacetate
stigmasterolbaku
chioltraincon
taneisopsoralidinisopsoralenbakuchalcon
eiso
neob
avachalcon
epsoralenpsoralid
inraffi
nosecorylifo
linincorylifo
leanand
corylifolin
[6]
22Rh
azya
strictaDecne
Apocyn
aceae
Leaves
Enzymes
(NADPH
depend
ent)
glycosides
(rob
lnin3-7-rhamno
sidea
ndiso
rham
netin
-3-7-rhamno
side)alkaloid(sew
arine)and
triterpenes
(ursolicacid
Mgqu
inateand120573-sito
sterol)alon
gwith
flavono
idsrhazianosides
AandB
[6]
23Ru
biacordifolia
LRu
biaceae
Stem
Pseudo
purpurinxanthop
urpu
rinpurpu
rinm
unjistin
alizarinand
mun
jistin
are
foun
dwith
theirg
lycosid
es
[6]
24Sapind
usmukorossiGaertn
Sapind
aceae
Fruit120573-Sito
sterolsapind
osideA
sapindo
sideB
kaempferolquercetinsapon
inand
emarginatosid
e[6]
25Stenolob
ium
stans
(L)Seem
Bign
oniaceae
Root120573-C
arotene120573-sito
sterol120572-amarinezeaxanthinind
olem
etabolizingenzymes
phenolicacidsoleano
licacidtriterpeno
idsursolic
acidind
ole-oxygenaseand
luteinzeaxanthin
[6]
26Term
inaliaarjuna
(Roxbex
DC)W
ight
andArn
Com
bretaceae
Bark120573-Sito
sterolarjuno
licacidtom
entosic
acidellagica
cidessentialoilarjunetin
arjuninecalcium
saltsaluminium
andmagnesiu
msaltscoloringagents
redu
cing
sugarstannin(pyrocatecho
l)andalactone
[6]
27Trich
odesmaindicum
(L)Sm
Boraginaceae
Who
leplantSeedso
ilisric
hin
linoleicoleicste
aricpalmiticandlin
olenicacidsAp
arto
fit
hexacosaneethylhexacosano
ateand2124-hexacos-adieno
icacid
ethyleste
rsare
foun
d[6]
28Zingiberoffi
cinaleR
oscoe
Zing
iberaceae
Rhizom
eD-C
urcumenecineolpotassiu
moxalatecamph
enecitralbo
rneolsin
giberin
eshogaol120572-curcumene120572-bergamotene120573-gam
ma-bisabo
lenegingeroland
120573-phelland
rene
23
BioMed Research International 5
Table2Biochemicalparametersb
eforea
ndaft
ersnakev
enom
administratio
nin
different
experim
entalgroup
sofrabbits
Toxicitydeterm
ined
Markersof
snake
veno
mtoxicity
Group
Ibaseline
measurements
(meanplusmnSE
M)
Group
IIandIIIv
enom
injection
Group
IVnegativ
econ
trol
(meanplusmnSE
M)
Normalreference
values
repo
rted
References
04m
gkg
(meanplusmnSE
M)
08m
gkg
(meanplusmnSE
M)
Liver
GPT
ALT
(52plusmn346
)UL
(72plusmn470)U
L01gt119875gt005
(86plusmn50)
UL
005gt119875gt002
(52plusmn348)U
L(48ndash80)U
L[19
]
GOTAST
(65plusmn657)U
L(157plusmn2424)
UL
01gt119875gt005
(251plusmn182)U
L001gt119875gt0001
(67plusmn321)U
L(14
ndash113)U
L[19
]
Kidn
eys
Urea
(28plusmn17
3)mgdL
(42plusmn308)m
gdL
005gt119875gt002
(576plusmn384)m
gdL
002gt119875gt001
(28plusmn033)m
gdL
(10ndash
28)m
gdL
[26]
Creatin
ine
(10plusmn0313)
mgdL
(174plusmn003)m
gdL
001gt119875gt0001
(21plusmn010)m
gdL
002gt119875gt001
(1plusmn006)m
gdL
(05ndash25)m
gdL
[26]
Heart
CK-M
B(132plusmn20)
UL
(21plusmn
15)U
L005gt119875gt002
(77plusmn1122)
UL
005gt119875gt002
(13plusmn056)U
L(lt25)U
Llowast
[2021]
LDH
(714plusmn318)U
L(206
4plusmn1598)
UL
119875lt0001
(2562plusmn2514
)UL
119875≪0001
(720plusmn470)U
L(559ndash2077)
UL
[46]
lowastCK
-MBactiv
ityislessthan
2in
healthyrabbitshow
everitisu
sually10ndash30
oftotalC
Kactiv
ity
6 BioMed Research International
0
50
100
150
200
250
300Se
rum
leve
l of G
PTG
OT
(UL
)
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
LRh
azya
stric
ta D
ecne
Rubi
a co
rdifo
lia L
Sa
pind
us m
ukor
ossi
Gae
rtn
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C) W
ight
and
Arn
Trich
odes
ma
indi
cum
(L)
SmZi
ngib
er o
fficin
ale R
osco
eRe
fere
nce s
tand
ard
anti
dote
GPT levelGOT level
Various antidotes used (100mgkg)
Figure 1 Comparison of various medicinal plant extracts with reference standard antidote in neutralization of increased GPT and GOTserum level posed by Naja naja karachiensis venom in rabbits
kits (manufactured by Merck) according to the DGKC andIFCCmethod on the Selectra Junior (Vital Scientific BVTheNetherlands) [26]
26 Statistical Analysis All numerical values were expressedas mean (3 replicates) plusmn standard error of mean (SEM) Theywere calculated by the use of Microsoft Excel 2007 and Stu-dentrsquos t-test was applied to compare the efficacy of evaluatedsamples with standard antidote (reference standard)
3 Results
Venom from Naja naja karachiensis was proved to causesevere complications in dose-dependent manner Venom atthe dose of 400 120583gkg led to release of moderate serum levelsof ALT (72plusmn470UL 01 gt 119875 gt 005) AST (157plusmn2424UL01 gt 119875 gt 005) urea (42 plusmn 308mgdL 005 gt 119875 gt 002)creatinine (174 plusmn 003mgdL 001 gt 119875 gt 0001) CK-MB(21 plusmn 15UL 005 gt 119875 gt 002) and LDH (2064 plusmn 1598UL119875 lt 0001) and thus indicated toxicities to the heart liver andkidneys At 800 120583gkg of cobra venom severe tissue damagewas observed in terms of significant release of ALT (86 plusmn50UL 005 gt 119875 gt 002) AST (251 plusmn 182UL 001 gt 119875 gt0001) urea (576 plusmn 384mgdL 002 gt 119875 gt 001) creatinine
(21 plusmn 010mgdL 002 gt 119875 gt 001) CK-MB (77plusmn 1122UL005 gt 119875 gt 002) and LDH (2562 plusmn 2514UL 119875 ≪ 0001)Complete detail about various biochemical parameters forbaseline measurements different doses of cobra venom andsaline (as negative control) is summarized in Table 2
To neutralize in vivo 800 120583gkg of cobra venom twenty-eight medicinal plant extracts were evaluated It was noticedthat extract of Stenolobium stans (L) Seem was the bestantidote (119875 gt 05) compared to reference standard Alliumsativum L Althaea officinalis L Citrullus colocynthis (L)Schrad Leucas capitataDesf Pinus roxburghii Sarg Psoraleacorylifolia L Rubia cordifolia L and Sapindus mukorossiGaertn were found to be valuable plants to protect liverdamage (119875 gt 05) as standard antidote (ALT = 525 plusmn351UL and AST = 695 plusmn 1855UL) however requiredALT and AST levels were not observed with remainingplant extracts (05 gt 119875 gt 005) as shown in Table 3 andcomparison is shown in Figure 1 Extracts of Leucas capitataDesf and Althaea officinalis L were observed to be helpful(119875 gt 05) as reference standard (urea = 315 plusmn 050mgdLand creatinine = 108 plusmn 002mgdL) nevertheless remainingplants were found less valuable (05 gt 119875 gt 001) tominimize urea and creatinine levels to maintain kidney func-tions (Table 4 and Figure 2) When compared with standardantidote (CK-MB = 90 plusmn 085UL) eight medicinal plants
BioMed Research International 7
Table 3 Hepatoprotective activity of various medicinal plant extracts on ALT and AST levels to neutralize snake bite envenomation inexperimental rabbits
Sr number Group V(subgroup) Name of evaluated sample GPTALT GOTAST
UL(mean plusmn SEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 62 plusmn 702 05 gt 119875 gt 01 99 plusmn 1855 05 gt 119875 gt 01
2 (V2) Allium cepa L 68 plusmn 1203 05 gt 119875 gt 01 1665 plusmn 050 05 gt 119875 gt 01
3 (V3) Allium sativum L 49 plusmn 501 119875 gt 05 825 plusmn 1855 119875 gt 05
4 (V4) Althaea officinalisL 70 plusmn 2005 119875 gt 05 745 plusmn 1454 119875 gt 05
5 (V5) Bauhinia variegata L 78 plusmn 404 05 gt 119875 gt 01 1495 plusmn 199 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 45 plusmn 000 05 gt 119875 gt 01 55 plusmn 100 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 315 plusmn 05 05 gt 119875 gt 01 141 plusmn 101 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 625 plusmn 252 05 gt 119875 gt 01 194 plusmn 1212 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 633 plusmn 414 05 gt 119875 gt 01 72 plusmn 202 119875 gt 05
10 (V9) Citrus limon (L) Burm f 305 plusmn 1363 05 gt 119875 gt 01 49 plusmn 2424 119875 gt 05
11 (V10) Citrullus colocynthis (L) Schrad 475 plusmn 555 119875 gt 05 74 plusmn 909 119875 gt 05
12 (V11) Cuminum cyminum L 67 plusmn 1616 119875 gt 05 157 plusmn 1414 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 8525 plusmn 126 01 gt 119875 gt
0051685 plusmn 757 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 80 plusmn 782 05 gt 119875 gt 01 170 plusmn 1313 05 gt 119875 gt 01
15 (V14) Leucas capitata Desf 455 plusmn 752 119875 gt 05 535 plusmn 050 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 62 plusmn 808 05 gt 119875 gt 01 121 plusmn 505 05 gt 119875 gt 01
17 (V16) Momordica charantia L 625 plusmn 752 05 gt 119875 gt 01 247 plusmn 4613 05 gt 119875 gt 01
18 (V17) Nerium indicumMill 88 plusmn 1955 05 gt 119875 gt 01 235 plusmn 1805 01 gt 119875 gt
005
19 (V18) Ocimum sanctum L 615 plusmn 752 05 gt 119875 gt 01 158 plusmn 1002 05 gt 119875 gt 01
20 (V19) Pinus roxburghii Sarg 53 plusmn 902 119875 gt 05 57 plusmn 1303 119875 gt 05
21 (V20) Pistacia integerrima J L Stewart 415 plusmn 15 05 gt 119875 gt 01 545 plusmn 050 119875 gt 05
22 (V21) Psoralea corylifolia L 54 plusmn 100 119875 gt 05 665 plusmn 1153 119875 gt 05
23 (V22) Rhazya stricta Decne 66 plusmn 2424 119875 gt 05 1325 plusmn 1262 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 655 plusmn 1767 119875 gt 05 129 plusmn 6641 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 56 plusmn 601 119875 gt 05 785 plusmn 1354 119875 gt 05
26 (V25) Stenolobium stans (L) Seem 465 plusmn 656 119875 gt 05 705 plusmn 353 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 475 plusmn 151 05 gt 119875 gt 01 78 plusmn 606 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 52 plusmn 000 119875 gt 05 125 plusmn 401 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 665 plusmn 3259 119875 gt 05 1175 plusmn 952 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
525 plusmn 351 Select tocompare 695 plusmn 1855 Select to
compare
(Allium cepa L Althaea officinalis L Bauhinia variegataL Cedrus deodara (Roxb ex D Don) G Don Fagoniacretica L Leucas capitataDesf Momordica charantia L andOcimum sanctum L) were found equally capable (119875 gt 05) ofnormalizing high values of CK-MB however rest of all wereproved less beneficial (05 gt 119875 gt 01) To combat highlyraised values of LDH Althaea officinalis L Leucas capitataDesf and Terminalia arjuna (Roxb ex DC) Wight and Arnwere proved useful to some extent (05 gt 119875 gt 01) although
remaining plant extracts could not be shortlisted (01 gt 119875 gt0001) as standard antisera (LDH = 763 plusmn 601UL) Overalldetail about LDH is discussed in Table 5 and comparison isshown in Figure 3
4 Discussion
Snake bite has been responsible for tens of thousands ofdeaths worldwide and numerous physical handicaps [29]
8 BioMed Research International
Table 4 Nephroprotective activity of different medicinal plant extracts on urea and creatinine levels to neutralize snake bite envenomationin experimental rabbits
Sr number Group V(subgroup) Name of evaluated sample Urea Creatinine
mgdL(mean plusmn SEM)
119875 valuecomment
mgdL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 57 plusmn 00 002 gt 119875 gt 001 16 plusmn 020 05 gt 119875 gt 01
2 (V2) Allium cepa L 51 plusmn 1103 05 gt 119875 gt 01 150 plusmn 017 05 gt 119875 gt 01
3 (V3) Allium sativum L 50 plusmn 401 05 gt 119875 gt 01 130 plusmn 006 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 26 plusmn 505 119875 gt 05 118 plusmn 024 119875 gt 05
5 (V5) Bauhinia variegata L 50 plusmn 126 05 gt 119875 gt 01 136 plusmn 024 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 225 plusmn 451 05 gt 119875 gt 01 107 plusmn 011 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 425 plusmn 252 05 gt 119875 gt 01 130 plusmn 001 01 gt 119875 gt 005
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 39 plusmn 202 05 gt 119875 gt 01 125 plusmn 003 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 44 plusmn 404 05 gt 119875 gt 01 144 plusmn 007 05 gt 119875 gt 01
10 (V9) Citrus limon (L) Burm f 395 plusmn 252 05 gt 119875 gt 01 140 plusmn 003 01 gt 119875 gt 005
11 (V10) Citrullus colocynthis (L) Schrad 495 plusmn 353 05 gt 119875 gt 01 152 plusmn 015 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 54 plusmn 101 005 gt 119875 gt 002 148 plusmn 001 005 gt 119875 gt 002
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 395 plusmn 454 05 gt 119875 gt 01 135 plusmn 005 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 43 plusmn 227 05 gt 119875 gt 01 123 plusmn 013 119875 lt 05
15 (V14) Leucas capitata Desf 315 plusmn 050 119875 gt 05 107 plusmn 005 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 435 plusmn 353 05 gt 119875 gt 01 144 plusmn 014 05 gt 119875 gt 01
17 (V16) Momordica charantia L 57 plusmn 501 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
18 (V17) Nerium indicumMill 47 plusmn 000 119875 lt 005 124 plusmn 010 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 495 plusmn 752 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
20 (V19) Pinus roxburghii Sarg 45 plusmn 100 119875 gt 005 14 plusmn 005 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 665 plusmn 050 002 gt 119875 gt 001 168 plusmn 010 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 425 plusmn 250 05 gt 119875 gt 01 127 plusmn 015 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 535 plusmn 353 05 gt 119875 gt 01 167 plusmn 0005 005 gt 119875 gt 002
24 (V23) Rubia cordifolia L 36 plusmn 101 05 gt 119875 gt 01 093 plusmn 048 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 44 plusmn 100 01 gt 119875 gt 005 143 plusmn 012 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 255 plusmn 651 119875 gt 05 11 plusmn 006 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 385 plusmn 151 05 gt 119875 gt 01 127 plusmn 023 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 37 plusmn 100 05 gt 119875 gt 01 046 plusmn 001 005 gt 119875 gt 002
29 (V28) Zingiber officinale Roscoe 47 plusmn 100 005 gt 119875 gt 002 130 plusmn 001 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
315 plusmn 050 Select tocompare 108 plusmn 002 Select to
compare
Generally snake venom is an intricate mixture of variousproteins (gt90) and most of them are enzymes particularly(40) phospholipases A
2[30] Among different complica-
tions hepatic injury is one of the deadly venomous effectsproduced by cobra bite [31 32] Naja naja karachiensisvenom caused significant increase in ALT and AST levels(dose dependently) that are surrogate markers for livertoxicity either by direct action or immunological (hyper-sensitivity) reaction The allergic reactions reactions are notdose dependent which clarify lethal effects to the cytoplasm
and mitochondrial membranes of the hepatic cells [25 33]Phospholipases enzymes abundant in Naja naja karachien-sis venom are responsible for breakdown of membranousphospholipids and therefore resulted in cellular injury alongwith inflammation [24] PLA
2is attributed to decrease in
Na+K+ ATPase activities and led to greater influx of sodiumions and water molecules into the cell Subsequently plasmamembrane lipid bilayer disorganized and eventually resultedin hepatic cells death [33 34] In addition PLA
2is also found
to cause anticoagulation in victims [6] All pharmacological
BioMed Research International 9
Table 5 Cardioprotective activity of medicinal plant extracts on LDH and CK-MB levels to neutralize snake bite envenomation inexperimental rabbits
Sr numberGroup V(subgroup) Name of evaluated sample LDH CK-MB
UL (mean plusmnSEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 1357 plusmn 100 001 gt 119875 gt 0001 41 plusmn 085 05 gt 119875 gt 01
2 (V2) Allium cepa L 934 plusmn 1303 01 gt 119875 gt 005 148 plusmn 165 119875 gt 05
3 (V3) Allium sativum L 1177 plusmn 2056 005 gt 119875 gt 002 66 plusmn 330 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 9755 plusmn 3360 05 gt 119875 gt 01 148 plusmn 330 119875 gt 05
5 (V5) Bauhinia variegata L 1972 plusmn 300 119875 gt 0001 83 plusmn 676 119875 gt 05
6 (V6) Brassica nigra (L) W D J Koch 8555 plusmn 050 005 gt 119875 gt 002 90 plusmn 085 05 gt 119875 gt 01
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 1022 plusmn 501 002 gt 119875 gt 001 66 plusmn 330 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 1114 plusmn 100 002 gt 119875 gt 001 618 plusmn 109 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 1230 plusmn 2357 005 gt 119875 gt 002 412 plusmn 3166 119875 gt 05
10 (V9) Citrus limon (L) Burm f 1831 plusmn 6569 005 gt 119875 gt 002 73 plusmn 141 05 gt 119875 gt 01
11 (V10) Citrullus colocynthis (L) Schrad 827 plusmn 651 01 gt 119875 gt 005 05 plusmn 166 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 1589 plusmn 2256 002 gt 119875 gt 001 58 plusmn 080 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 1615 plusmn 151 119875 gt 0001 985 plusmn 166 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 1418 plusmn 1303 002 gt 119875 gt 001 11 plusmn 252 119875 gt 05
15 (V14) Leucas capitata Desf 783 plusmn 1002 05 gt 119875 gt 01 14 plusmn 080 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 1428 plusmn 651 119875 gt 0001 08 plusmn 166 05 gt 119875 gt 01
17 (V16) Momordica charantia L 14755 plusmn 351 119875 gt 0001 156 plusmn 245 119875 gt 05
18 (V17) Nerium indicumMill 1268 plusmn 1203 002 gt 119875 gt 001 05 plusmn 165 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 1335 plusmn 1203 002 gt 119875 gt 001 123 plusmn 245 119875 gt 05
20 (V19) Pinus roxburghii Sarg 1050 plusmn 100 002 gt 119875 gt 001 08 plusmn 165 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 11355 plusmn 05 002 gt 119875 gt 001 131 plusmn 165 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 11535 plusmn 05 119875 gt 0001 173 plusmn 250 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 1538 plusmn 2005 002 gt 119875 gt 001 41 plusmn 085 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 1078 plusmn 1604 005 gt 119875 gt 002 66 plusmn 000 05 gt 119875 gt 01
25 (V24) Sapindus mukorossi Gaertn 14605 plusmn 551 001 gt 119875 gt 0001 41 plusmn 080 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 787 plusmn 2808 119875 gt 05 13 plusmn 176 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 7985 plusmn 1454 05 gt 119875 gt 01 66 plusmn 000 05 gt 119875 gt 01
28 (V27) Trichodesma indicum (L) Sm 9785 plusmn 451 005 gt 119875 gt 002 66 plusmn 300 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 888 plusmn 200 005 gt 119875 gt 002 173 plusmn 080 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
763 plusmn 601 Select to compare 09 plusmn 085 Select to compare
effects are due to phospholipid hydrolysis or phospholipidcompeting bidingmechanismwith other coagulation factorsparticularly FXa or by both ways however it is very hardto pinpoint exact one [35] Anticoagulant response posed bycobra PLA
2(anticoagulant enzyme) may aggravate hepatic
injury as many anticoagulant agents cause liver damage thatare idiosyncratic in nature [36]
Cardiac injury particularly systolic heart arrest is one ofthewell-known toxicities related toNaja naja subspecies [30]In present study Pakistani cobra venom was found to release
two cystolic enzymes (LDH and CK-MB) that are sensitiveindicators of myocyte injury [37] It is due to the presenceof myotoxic PLA
2and other cardiotoxin(s) that are salient
features of cobra venom that are responsible for cellularnecrosis and cytotoxicity [38 39] Cytotoxic effect of Najanaja karachiensis venom may not be overlooked even whenantisera administration is late cause of toxic components tothe microvasculature and thrombus produced which poseshindrance in access of immunoglobulins to the site of snakebite [26 29 39]
10 BioMed Research International
0
10
20
30
40
50
60
70Se
rum
leve
l of u
rea a
nd cr
eatin
ine (
mg
dL)
UreaCreatinine
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
L
Rhaz
ya st
ricta
Dec
neRu
bia
cord
ifolia
L
Sapi
ndus
muk
oros
si G
aert
nSt
enol
obiu
m st
ans (
L) S
eem
Term
inal
ia a
rjuna
(Rox
b ex
DC
) Wig
ht an
d A
rnTr
ichod
esm
a in
dicu
m (L
) Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antidotes tested (100mgkg)
Figure 2 Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea andcreatinine level posed by Naja naja karachiensis venom in rabbits
Snake venom has been responsible for detrimental effectsto the renal tissues [40] Likewise Naja naja krachiensisvenomwas found to cause severe renal damage by significantrise in serum urea and creatinine levels Indeed phospho-lipases enzymes are responsible for the increase in vascularpermeability with hemorrhagic effects to the vital tissues inthe victims Subsequently numerous lesions are producedrelated to the glomerular membrane and renal tubules eitherinterstitial or vascular [29 41 42] Presence of lymphocytes(white blood cells) and oedema in the cortical as well asmedullary regions of renal tubules further confirmed theidea of renal damage (data has not shown) as reportedpreviously with different snake venoms like Hemiscorpiuslepturus [26 43] Apart of it pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope99mTc confirmed that kidneys and urinary bladder are themost saturated organs (gt70) after intravenous injection inexperimental rabbits (unpublished data by our group)
Medicinal plants of Pakistan are used to inhibit snakevenom (PLA
2) enzymes [6] Due to this effect present
study was designed to select twenty-eight medicinal plantsof Pakistan to test their potentials as an antidote againsttoxicities produced by Naja naja karachiensis venom It
was fascinating that extract of Stenolobium stans (L) Seemshowed significant neutralization compared to that of refer-ence standard (antisera) as reported previously by Asad etal for inhibition of phospholipases A
2anticoagulant activity
[6 44] Present study indicated that extract of Stenolobiumstans (L) Seem possesses an endogenous inhibitor(s) tonullify venom (PLA
2) induced toxicities Extracts of these
plants material are routinely used in Pakistan by simpleapplication in the form of paste to the affected area [45]This practice provides effective first aid treatment as tinymolecules of an antidote diffuse favorably at the site of snakebite before hospitalization and abrogated the spreading oftoxins [39] It is owing to the various secondary metaboliteslike phenols flavonoids terpenoids xanthenes quinonoidsand so forth as reported earlier to mask various enzymaticactions of cobra venom [6 23] Indeed secondarymetabolitespose hindrance in binding of different snake venom enzymesto their potential targets therefore antidotal effect evokedOther plants extract were not proved significantly effectiveto neutralize cobra venom therefore cannot be declareduseful plants in venom therapy It is the need of the time toisolate lethal component(s) of cobra venom attributed to itsmajor toxicities Furthermore characterization of antivenom
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
2 BioMed Research International
and increased creatinine and urea levels along with alteredconsciousness [3 4]
Indeed snake venom is a complex mixture of varioussizes of polypeptides [5 6] (lt15 kDa 5 to 10 kDa and10 to 150 kDa) carbohydrates lipids metal ions organicmolecules purines and amines [7 8] Snake bite enveno-mation has been attributed to pose drastic changes in thephysiology of the victims that could be accessed clinicallyby measuring various biochemical parameters Among toxiccomponents phospholipases A
2(cell destroying enzymes
cause edema lipolytic or myolytic) [9 10] phosphodi-esterases (nucleic acid degrading enzyme causes hypoten-sionshock) [11 12] 51015840 nucleotidases (digest the sugar moitiescausing the delay in coagulation) [13] alkaline phosphatases(hydrolyze phosphate esters nonspecifically cause multipletoxicities via adenosine generation) [14] hyaluronidases(spreading factor causes digestion necrosis and leakage ofblood vessels) [15] L-amino oxidases (deamination leads tocell damageapoptosis) [16] and proteases (hydrolyze variousproteins) [17] are included
Laboratory animals particularly rabbits have beenselected previously to monitor various biochemical changesrelated to liver heart and renal toxicities [18 19] Aspartateaminotransferase (GOTAST) and alanine aminotransferase(GPTALT) are surrogate markers for liver toxicities whilecreatine kinase isoenzyme (CK-MB) is used exclusively toaccess cardiac tissue damage [20 21] Elevated creatinine andurea levels are indicators of 75 renal damage [20] whereashigh level of lactate dehydrogenase (LDH) implies generaltoxicity related to the liver heart kidneys and skeletonmuscles [22]
Diverse immunological properties of different cobrashave fascinated protein chemists and other researchers tocarry out efforts for the development of new antidotes Dueto this reason for the first time venom from Naja najakarachiensis is selected to evaluate its toxicity in vivo andto search out effective as well as cheap treatment fromfolk herbal remedies Pakistan is a hub of medicinal plantswhere mostly rural communities rely on natural herbs totreat their problems of snake bite [6 23] It is thereforenecessary to evaluate scientifically their folklore claims asanti-snake venom in traditional system of medicine Dueto this reason various medicinal plants (Albizia lebbeck (L)Benth Allium cepa L Allium sativum L Althaea officinalisL Bauhinia variegata L Brassica nigra (L) W D J KochCalotropis procera (Aiton)WTAitonCedrus deodara (Roxbex D Don) G Don Citrullus colocynthis (L) Schrad Citruslimon (L) Burm f Cuminum cyminum L Enicostemahyssopifolium (Willd) I Verd Fagonia cretica L Leucascapitata DesfMatthiola incana (L) W T AitonMomordicacharantiaLNerium indicumMillOcimum sanctumL Pinusroxburghii Sarg Pistacia integerrima J L Stewart Psoraleacorylifolia L Rhazya stricta Decne Rubia cordifolia LSapindus mukorossi Gaertn Solanum xanthocarpum Schardand Wendle Stenolobium stans (L) Seem Terminalia arjuna(Roxb exDC)Wight andArnTrichodesma indicum (L) Smand Zingiber officinale Roscoe) were collected to test theirpotentials against toxicities induced byNaja naja karachiensisenvenomation (Table 1)
2 Material and Methods
21 Collection and Milking of Snake Venom Black Pakistanicobra snakes (Naja naja karachiensis) were collected fromCholistan desert located in southern Punjab province ofPakistan After collection they were properly identified by azoologist Venom from Naja naja karachiensis was collectedby pressing their glands below their eyes in low light environ-ment After collection it was lyophilized and stored in lightresistant container at 2∘C to 8∘C For further experimentsvenom was used in terms of its dry weight [6]
22 Collection and Preparation of Plant Extracts Medicinalplants were collected from different areas in Pakistan Aftercollection they were duly identified by expert botanist(Professor Dr Altaf Ahmad Dasti) and voucher specimenswere deposited in the herbarium of the Institute of Pure andApplied Biology Bahauddin Zakariya University MultanPakistan Complete description about evaluated medicinalplants is summarized in Table 1 One kilogram choppedplant material was soaked in 5 L of methanol as solvent inextraction bottlesThe homogenates were kept for a period ofa month at optimum temperature After filtration filtrate wasevaporated by using water bath and extracts were weighedand stored for further experimentation [24]
23 Experimental Animals Seventy-one healthy growingmale rabbits (1 plusmn 05 kg) were selected for this study aftergetting permission from local ethical committee Animalswere acclimatized for a week in the laboratory bymaintainingtheir standards for chow water and light Subsequently theywere divided into various groups in different cages suchthat experimental rabbits in group I were used for baselinemeasurements of various biochemical parameters Animalsbelonging to groups II and III were evaluated for differentdoses of cobra venom while group IV was served as controlGroup V was redivided into twenty-nine groups such thateach subgroup (V1ndashV29) was assigned to a single medicinalplant extract [25]
24 In Vivo Antivenom Activity of Medicinal Plant ExtractsVenom from Naja naja karachiensis (400 120583gkg and800 120583gkg) was injected subcutaneously to evaluate itstoxicity to the heart liver and kidneys Before injectionof venom experimental animals were anaesthetizedby administration of ketamine (50mgkg) To determineantivenomactivity of various plantsrsquo extract they (100mgkg)were incubated (at 37∘C for 30 minutes) with fixed amountof venom (800120583gkg) before injection while saline was usedas control [26ndash28]
25 Biochemical Assays For serum analysis of various bio-chemical parameters blood was collected from marginal earartery by the use of hypodermic syringe needle after 3 hoursof envenomation [25] Separated serumwas tested for alanineaminotransferase (GPTALT) aspartate aminotransferase(GOTAST) urea creatinine creatine kinase isoenzyme MB(CK-MB) and lactate dehydrogenase (LDH) by the use of
BioMed Research International 3
Table1Detaileddescrip
tionfore
valuated
medicinalplantshaving
folklore
evidencesa
santivenom
Srnum
ber
Botanicaln
ameo
fmedicinalplants
Family
Partused
Phytochemicalsreported
Reference
1Albizialeb
beck
(L)Be
nth
Fabaceae
Seed
Carboh
ydratesproteinsalkaloidsflavanoidstanninsechinocystic
acidand
different
aminoacids
[6]
2Alliu
mcepa
LAmaryllid
aceae
Bulb
11gcarboh
ydrates12
gproteins06gfib
ersandH
2Ocontent(868g)10
0gof
material
[6]
3Alliu
msativ
umL
Amaryllid
aceae
Bulb
Quercetinscordinines
AandBallicinthiosulfin
ates2
mercapto-L-cyste
ines
anthocyaninsalliinasepo
lysaccharid
essativin
Iand
sativ
inIIand
glycosides
ofkaem
pferol
[6]
4Althaeaoffi
cinalisL
Malvaceae
Root
37sta
rch11mucilagefattyoilpectinflavon
oidspheno
licacidsasparagines
phytosterolsucroseandbu
tyric
acid
[6]
5Ba
uhiniavarie
gata
LFabaceae
Root
Tann
insfatty
oillupeolkaempferol-3-glucosid
egums120573-sito
sterol57-dehydroxy
and57-dimetho
xy-flavanon
e-4-0-a-L-rham
nopyrano
syl-120573
-Dglucop
yranosides
[6]
6Brassicanigra(L)WD
JK
och
Brassic
aceae
Seed
Essentialoilsin
igrin
andglucoside
[6]
7Ca
lotro
pisp
rocera
(Aito
n)WTA
iton
Apocyn
aceae
Exud
ates
andflo
wer
Resin
stann
inscalotro
pin
steroluscharincalotropagenin
calotoxin
andcalactin
[6]
8Ce
drus
deodara(Roxbex
DD
on)G
Don
Pinaceae
Bark
Taxifolin
dew
ardioldew
arenegumascorbica
cidhimadarolcantdarol
cholesterin
allo
himachalolcedrinosidehimachaloldewarolcedrin
dihydrod
ehydrodiconiferylalcoh
olessentia
loiliso
centdaroland
dihydrom
yricetin
[6]
9Citru
slim
on(L)Bu
rmf
Rutaceae
Fruit
4-120573-glucopyrano
sided-x-pinene
camph
ened-lim
onenelin
aloo
lichangin
4-120573-glucopyrano
sideno
milinica
cidandessentialoil
[6]
10Citru
lluscolocynthis(L)Schrad
Cucurbita
ceae
Fruit
Manyalkaloidsglycosidestanninscitrulluincitrulluicaciddihydric
alcoho
lph
ydroxybenzylcitrulluenehentria
contaneelaterincitrullolmethyletherbitter
oilcitbittoland
sapo
nins
[6]
11Cu
minum
cyminum
LAp
iaceae
Seed
Essentialoilandcumin
oilcon
tain
sminaldehyde13-p-m
enthadien-7-aland
14-p-m
enthadien-7-al
[6]
12En
icoste
mahysso
pifoliu
m(W
illd)IV
erd
Gentia
naceae
Who
leplant
Sapo
narin
isovetexin
sylysw
ertin
betulinswertia
marinapigenin
genk
wanin
erythrocentaurinesw
ertio
sideiso
swertisinenicoflavineand
swertisin
[6]
13Fagoniacretica
LZy
goph
yllaceae
Leaves
Ursolicacidpinito
lnahagenin
sapo
ninglycosidessapon
in-Iandsapo
nin-II
docosyld
ocosanoatefro
mn-hexane
extractanddifferent
proteins
from
aqueou
sextract
[6]
14Leucas
capitataDesf
Lamiaceae
Who
leplant
Alkaloidas
wellase
ssentia
loil
[6]
15Matthiolaincana
(L)WTA
iton
Brassic
aceae
Seeds
Oilric
hin
chloroph
yllsabNPK
and
Na120574-lino
lenica
cidandcaroteno
ids
Sulfo
raph
eneisa
nim
portantcom
ponent
[6]
16Mom
ordica
charantia
LCu
curbita
ceae
Fruit
Caroteno
idsmom
orcharasides
AandB
glucosidesstig
mastadien-3-beta-ol
squalenem
ycosessteroidalglucosidemom
ordicinesI
andIIstig
masterolvicine
cucurbitane
triterpenoidsm
omordicosid
escycloeucaleno
ltaraxerol
spinasterollo
phenoldiosgeninthiocyano
gen
24-m
ethylencycloarteno
land
phenylprop
anoids
[6]
17Ne
rium
indicum
Mill
Apocyn
aceae
Root
andleaves
Nerioderin
neriodo
rinkarabinand
odorin
[6]
18Ocim
umsanctum
LLamiaceae
Who
leplant
Essentialoils
arefou
ndric
hin
32
carvacrol713eugeno
l17
caryop
hyllene
204methyleugenoleugenolmethyletherlinaloo
landmethylchavicolcineole
[6]
4 BioMed Research International
Table1Con
tinued
Srnum
ber
Botanicaln
ameo
fmedicinalplants
Family
Partused
Phytochemicalsreported
Reference
19Pinu
sroxburghiiSarg
Pinaceae
Oleoresin
Careen120572
-pinene120573-pinene120573-carene120573-lo
ngifo
lenelon
gifoleneand
long
icyclene
[6]
20Pista
ciaintegerrim
aJLStewart
Anacardiaceae
Galls
13essentialoilric
hin
camph
enecineoleA-
terpineolA-
pinenearomadendrene
caprylicacidand
d-lim
onenea
bund
antingalls
[6]
21Psoralea
corylifoliaL
Fabaceae
Seeds
Limon
enelin
aloo
lpsoraleneangelicinn
eobavachalconebakuchiol4-te
rpineol
corylid
inn
eobavaiso
flavonebavachinin
isobavachin120572
-elemenegeranylacetate
stigmasterolbaku
chioltraincon
taneisopsoralidinisopsoralenbakuchalcon
eiso
neob
avachalcon
epsoralenpsoralid
inraffi
nosecorylifo
linincorylifo
leanand
corylifolin
[6]
22Rh
azya
strictaDecne
Apocyn
aceae
Leaves
Enzymes
(NADPH
depend
ent)
glycosides
(rob
lnin3-7-rhamno
sidea
ndiso
rham
netin
-3-7-rhamno
side)alkaloid(sew
arine)and
triterpenes
(ursolicacid
Mgqu
inateand120573-sito
sterol)alon
gwith
flavono
idsrhazianosides
AandB
[6]
23Ru
biacordifolia
LRu
biaceae
Stem
Pseudo
purpurinxanthop
urpu
rinpurpu
rinm
unjistin
alizarinand
mun
jistin
are
foun
dwith
theirg
lycosid
es
[6]
24Sapind
usmukorossiGaertn
Sapind
aceae
Fruit120573-Sito
sterolsapind
osideA
sapindo
sideB
kaempferolquercetinsapon
inand
emarginatosid
e[6]
25Stenolob
ium
stans
(L)Seem
Bign
oniaceae
Root120573-C
arotene120573-sito
sterol120572-amarinezeaxanthinind
olem
etabolizingenzymes
phenolicacidsoleano
licacidtriterpeno
idsursolic
acidind
ole-oxygenaseand
luteinzeaxanthin
[6]
26Term
inaliaarjuna
(Roxbex
DC)W
ight
andArn
Com
bretaceae
Bark120573-Sito
sterolarjuno
licacidtom
entosic
acidellagica
cidessentialoilarjunetin
arjuninecalcium
saltsaluminium
andmagnesiu
msaltscoloringagents
redu
cing
sugarstannin(pyrocatecho
l)andalactone
[6]
27Trich
odesmaindicum
(L)Sm
Boraginaceae
Who
leplantSeedso
ilisric
hin
linoleicoleicste
aricpalmiticandlin
olenicacidsAp
arto
fit
hexacosaneethylhexacosano
ateand2124-hexacos-adieno
icacid
ethyleste
rsare
foun
d[6]
28Zingiberoffi
cinaleR
oscoe
Zing
iberaceae
Rhizom
eD-C
urcumenecineolpotassiu
moxalatecamph
enecitralbo
rneolsin
giberin
eshogaol120572-curcumene120572-bergamotene120573-gam
ma-bisabo
lenegingeroland
120573-phelland
rene
23
BioMed Research International 5
Table2Biochemicalparametersb
eforea
ndaft
ersnakev
enom
administratio
nin
different
experim
entalgroup
sofrabbits
Toxicitydeterm
ined
Markersof
snake
veno
mtoxicity
Group
Ibaseline
measurements
(meanplusmnSE
M)
Group
IIandIIIv
enom
injection
Group
IVnegativ
econ
trol
(meanplusmnSE
M)
Normalreference
values
repo
rted
References
04m
gkg
(meanplusmnSE
M)
08m
gkg
(meanplusmnSE
M)
Liver
GPT
ALT
(52plusmn346
)UL
(72plusmn470)U
L01gt119875gt005
(86plusmn50)
UL
005gt119875gt002
(52plusmn348)U
L(48ndash80)U
L[19
]
GOTAST
(65plusmn657)U
L(157plusmn2424)
UL
01gt119875gt005
(251plusmn182)U
L001gt119875gt0001
(67plusmn321)U
L(14
ndash113)U
L[19
]
Kidn
eys
Urea
(28plusmn17
3)mgdL
(42plusmn308)m
gdL
005gt119875gt002
(576plusmn384)m
gdL
002gt119875gt001
(28plusmn033)m
gdL
(10ndash
28)m
gdL
[26]
Creatin
ine
(10plusmn0313)
mgdL
(174plusmn003)m
gdL
001gt119875gt0001
(21plusmn010)m
gdL
002gt119875gt001
(1plusmn006)m
gdL
(05ndash25)m
gdL
[26]
Heart
CK-M
B(132plusmn20)
UL
(21plusmn
15)U
L005gt119875gt002
(77plusmn1122)
UL
005gt119875gt002
(13plusmn056)U
L(lt25)U
Llowast
[2021]
LDH
(714plusmn318)U
L(206
4plusmn1598)
UL
119875lt0001
(2562plusmn2514
)UL
119875≪0001
(720plusmn470)U
L(559ndash2077)
UL
[46]
lowastCK
-MBactiv
ityislessthan
2in
healthyrabbitshow
everitisu
sually10ndash30
oftotalC
Kactiv
ity
6 BioMed Research International
0
50
100
150
200
250
300Se
rum
leve
l of G
PTG
OT
(UL
)
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
LRh
azya
stric
ta D
ecne
Rubi
a co
rdifo
lia L
Sa
pind
us m
ukor
ossi
Gae
rtn
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C) W
ight
and
Arn
Trich
odes
ma
indi
cum
(L)
SmZi
ngib
er o
fficin
ale R
osco
eRe
fere
nce s
tand
ard
anti
dote
GPT levelGOT level
Various antidotes used (100mgkg)
Figure 1 Comparison of various medicinal plant extracts with reference standard antidote in neutralization of increased GPT and GOTserum level posed by Naja naja karachiensis venom in rabbits
kits (manufactured by Merck) according to the DGKC andIFCCmethod on the Selectra Junior (Vital Scientific BVTheNetherlands) [26]
26 Statistical Analysis All numerical values were expressedas mean (3 replicates) plusmn standard error of mean (SEM) Theywere calculated by the use of Microsoft Excel 2007 and Stu-dentrsquos t-test was applied to compare the efficacy of evaluatedsamples with standard antidote (reference standard)
3 Results
Venom from Naja naja karachiensis was proved to causesevere complications in dose-dependent manner Venom atthe dose of 400 120583gkg led to release of moderate serum levelsof ALT (72plusmn470UL 01 gt 119875 gt 005) AST (157plusmn2424UL01 gt 119875 gt 005) urea (42 plusmn 308mgdL 005 gt 119875 gt 002)creatinine (174 plusmn 003mgdL 001 gt 119875 gt 0001) CK-MB(21 plusmn 15UL 005 gt 119875 gt 002) and LDH (2064 plusmn 1598UL119875 lt 0001) and thus indicated toxicities to the heart liver andkidneys At 800 120583gkg of cobra venom severe tissue damagewas observed in terms of significant release of ALT (86 plusmn50UL 005 gt 119875 gt 002) AST (251 plusmn 182UL 001 gt 119875 gt0001) urea (576 plusmn 384mgdL 002 gt 119875 gt 001) creatinine
(21 plusmn 010mgdL 002 gt 119875 gt 001) CK-MB (77plusmn 1122UL005 gt 119875 gt 002) and LDH (2562 plusmn 2514UL 119875 ≪ 0001)Complete detail about various biochemical parameters forbaseline measurements different doses of cobra venom andsaline (as negative control) is summarized in Table 2
To neutralize in vivo 800 120583gkg of cobra venom twenty-eight medicinal plant extracts were evaluated It was noticedthat extract of Stenolobium stans (L) Seem was the bestantidote (119875 gt 05) compared to reference standard Alliumsativum L Althaea officinalis L Citrullus colocynthis (L)Schrad Leucas capitataDesf Pinus roxburghii Sarg Psoraleacorylifolia L Rubia cordifolia L and Sapindus mukorossiGaertn were found to be valuable plants to protect liverdamage (119875 gt 05) as standard antidote (ALT = 525 plusmn351UL and AST = 695 plusmn 1855UL) however requiredALT and AST levels were not observed with remainingplant extracts (05 gt 119875 gt 005) as shown in Table 3 andcomparison is shown in Figure 1 Extracts of Leucas capitataDesf and Althaea officinalis L were observed to be helpful(119875 gt 05) as reference standard (urea = 315 plusmn 050mgdLand creatinine = 108 plusmn 002mgdL) nevertheless remainingplants were found less valuable (05 gt 119875 gt 001) tominimize urea and creatinine levels to maintain kidney func-tions (Table 4 and Figure 2) When compared with standardantidote (CK-MB = 90 plusmn 085UL) eight medicinal plants
BioMed Research International 7
Table 3 Hepatoprotective activity of various medicinal plant extracts on ALT and AST levels to neutralize snake bite envenomation inexperimental rabbits
Sr number Group V(subgroup) Name of evaluated sample GPTALT GOTAST
UL(mean plusmn SEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 62 plusmn 702 05 gt 119875 gt 01 99 plusmn 1855 05 gt 119875 gt 01
2 (V2) Allium cepa L 68 plusmn 1203 05 gt 119875 gt 01 1665 plusmn 050 05 gt 119875 gt 01
3 (V3) Allium sativum L 49 plusmn 501 119875 gt 05 825 plusmn 1855 119875 gt 05
4 (V4) Althaea officinalisL 70 plusmn 2005 119875 gt 05 745 plusmn 1454 119875 gt 05
5 (V5) Bauhinia variegata L 78 plusmn 404 05 gt 119875 gt 01 1495 plusmn 199 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 45 plusmn 000 05 gt 119875 gt 01 55 plusmn 100 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 315 plusmn 05 05 gt 119875 gt 01 141 plusmn 101 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 625 plusmn 252 05 gt 119875 gt 01 194 plusmn 1212 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 633 plusmn 414 05 gt 119875 gt 01 72 plusmn 202 119875 gt 05
10 (V9) Citrus limon (L) Burm f 305 plusmn 1363 05 gt 119875 gt 01 49 plusmn 2424 119875 gt 05
11 (V10) Citrullus colocynthis (L) Schrad 475 plusmn 555 119875 gt 05 74 plusmn 909 119875 gt 05
12 (V11) Cuminum cyminum L 67 plusmn 1616 119875 gt 05 157 plusmn 1414 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 8525 plusmn 126 01 gt 119875 gt
0051685 plusmn 757 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 80 plusmn 782 05 gt 119875 gt 01 170 plusmn 1313 05 gt 119875 gt 01
15 (V14) Leucas capitata Desf 455 plusmn 752 119875 gt 05 535 plusmn 050 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 62 plusmn 808 05 gt 119875 gt 01 121 plusmn 505 05 gt 119875 gt 01
17 (V16) Momordica charantia L 625 plusmn 752 05 gt 119875 gt 01 247 plusmn 4613 05 gt 119875 gt 01
18 (V17) Nerium indicumMill 88 plusmn 1955 05 gt 119875 gt 01 235 plusmn 1805 01 gt 119875 gt
005
19 (V18) Ocimum sanctum L 615 plusmn 752 05 gt 119875 gt 01 158 plusmn 1002 05 gt 119875 gt 01
20 (V19) Pinus roxburghii Sarg 53 plusmn 902 119875 gt 05 57 plusmn 1303 119875 gt 05
21 (V20) Pistacia integerrima J L Stewart 415 plusmn 15 05 gt 119875 gt 01 545 plusmn 050 119875 gt 05
22 (V21) Psoralea corylifolia L 54 plusmn 100 119875 gt 05 665 plusmn 1153 119875 gt 05
23 (V22) Rhazya stricta Decne 66 plusmn 2424 119875 gt 05 1325 plusmn 1262 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 655 plusmn 1767 119875 gt 05 129 plusmn 6641 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 56 plusmn 601 119875 gt 05 785 plusmn 1354 119875 gt 05
26 (V25) Stenolobium stans (L) Seem 465 plusmn 656 119875 gt 05 705 plusmn 353 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 475 plusmn 151 05 gt 119875 gt 01 78 plusmn 606 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 52 plusmn 000 119875 gt 05 125 plusmn 401 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 665 plusmn 3259 119875 gt 05 1175 plusmn 952 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
525 plusmn 351 Select tocompare 695 plusmn 1855 Select to
compare
(Allium cepa L Althaea officinalis L Bauhinia variegataL Cedrus deodara (Roxb ex D Don) G Don Fagoniacretica L Leucas capitataDesf Momordica charantia L andOcimum sanctum L) were found equally capable (119875 gt 05) ofnormalizing high values of CK-MB however rest of all wereproved less beneficial (05 gt 119875 gt 01) To combat highlyraised values of LDH Althaea officinalis L Leucas capitataDesf and Terminalia arjuna (Roxb ex DC) Wight and Arnwere proved useful to some extent (05 gt 119875 gt 01) although
remaining plant extracts could not be shortlisted (01 gt 119875 gt0001) as standard antisera (LDH = 763 plusmn 601UL) Overalldetail about LDH is discussed in Table 5 and comparison isshown in Figure 3
4 Discussion
Snake bite has been responsible for tens of thousands ofdeaths worldwide and numerous physical handicaps [29]
8 BioMed Research International
Table 4 Nephroprotective activity of different medicinal plant extracts on urea and creatinine levels to neutralize snake bite envenomationin experimental rabbits
Sr number Group V(subgroup) Name of evaluated sample Urea Creatinine
mgdL(mean plusmn SEM)
119875 valuecomment
mgdL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 57 plusmn 00 002 gt 119875 gt 001 16 plusmn 020 05 gt 119875 gt 01
2 (V2) Allium cepa L 51 plusmn 1103 05 gt 119875 gt 01 150 plusmn 017 05 gt 119875 gt 01
3 (V3) Allium sativum L 50 plusmn 401 05 gt 119875 gt 01 130 plusmn 006 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 26 plusmn 505 119875 gt 05 118 plusmn 024 119875 gt 05
5 (V5) Bauhinia variegata L 50 plusmn 126 05 gt 119875 gt 01 136 plusmn 024 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 225 plusmn 451 05 gt 119875 gt 01 107 plusmn 011 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 425 plusmn 252 05 gt 119875 gt 01 130 plusmn 001 01 gt 119875 gt 005
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 39 plusmn 202 05 gt 119875 gt 01 125 plusmn 003 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 44 plusmn 404 05 gt 119875 gt 01 144 plusmn 007 05 gt 119875 gt 01
10 (V9) Citrus limon (L) Burm f 395 plusmn 252 05 gt 119875 gt 01 140 plusmn 003 01 gt 119875 gt 005
11 (V10) Citrullus colocynthis (L) Schrad 495 plusmn 353 05 gt 119875 gt 01 152 plusmn 015 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 54 plusmn 101 005 gt 119875 gt 002 148 plusmn 001 005 gt 119875 gt 002
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 395 plusmn 454 05 gt 119875 gt 01 135 plusmn 005 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 43 plusmn 227 05 gt 119875 gt 01 123 plusmn 013 119875 lt 05
15 (V14) Leucas capitata Desf 315 plusmn 050 119875 gt 05 107 plusmn 005 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 435 plusmn 353 05 gt 119875 gt 01 144 plusmn 014 05 gt 119875 gt 01
17 (V16) Momordica charantia L 57 plusmn 501 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
18 (V17) Nerium indicumMill 47 plusmn 000 119875 lt 005 124 plusmn 010 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 495 plusmn 752 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
20 (V19) Pinus roxburghii Sarg 45 plusmn 100 119875 gt 005 14 plusmn 005 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 665 plusmn 050 002 gt 119875 gt 001 168 plusmn 010 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 425 plusmn 250 05 gt 119875 gt 01 127 plusmn 015 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 535 plusmn 353 05 gt 119875 gt 01 167 plusmn 0005 005 gt 119875 gt 002
24 (V23) Rubia cordifolia L 36 plusmn 101 05 gt 119875 gt 01 093 plusmn 048 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 44 plusmn 100 01 gt 119875 gt 005 143 plusmn 012 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 255 plusmn 651 119875 gt 05 11 plusmn 006 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 385 plusmn 151 05 gt 119875 gt 01 127 plusmn 023 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 37 plusmn 100 05 gt 119875 gt 01 046 plusmn 001 005 gt 119875 gt 002
29 (V28) Zingiber officinale Roscoe 47 plusmn 100 005 gt 119875 gt 002 130 plusmn 001 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
315 plusmn 050 Select tocompare 108 plusmn 002 Select to
compare
Generally snake venom is an intricate mixture of variousproteins (gt90) and most of them are enzymes particularly(40) phospholipases A
2[30] Among different complica-
tions hepatic injury is one of the deadly venomous effectsproduced by cobra bite [31 32] Naja naja karachiensisvenom caused significant increase in ALT and AST levels(dose dependently) that are surrogate markers for livertoxicity either by direct action or immunological (hyper-sensitivity) reaction The allergic reactions reactions are notdose dependent which clarify lethal effects to the cytoplasm
and mitochondrial membranes of the hepatic cells [25 33]Phospholipases enzymes abundant in Naja naja karachien-sis venom are responsible for breakdown of membranousphospholipids and therefore resulted in cellular injury alongwith inflammation [24] PLA
2is attributed to decrease in
Na+K+ ATPase activities and led to greater influx of sodiumions and water molecules into the cell Subsequently plasmamembrane lipid bilayer disorganized and eventually resultedin hepatic cells death [33 34] In addition PLA
2is also found
to cause anticoagulation in victims [6] All pharmacological
BioMed Research International 9
Table 5 Cardioprotective activity of medicinal plant extracts on LDH and CK-MB levels to neutralize snake bite envenomation inexperimental rabbits
Sr numberGroup V(subgroup) Name of evaluated sample LDH CK-MB
UL (mean plusmnSEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 1357 plusmn 100 001 gt 119875 gt 0001 41 plusmn 085 05 gt 119875 gt 01
2 (V2) Allium cepa L 934 plusmn 1303 01 gt 119875 gt 005 148 plusmn 165 119875 gt 05
3 (V3) Allium sativum L 1177 plusmn 2056 005 gt 119875 gt 002 66 plusmn 330 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 9755 plusmn 3360 05 gt 119875 gt 01 148 plusmn 330 119875 gt 05
5 (V5) Bauhinia variegata L 1972 plusmn 300 119875 gt 0001 83 plusmn 676 119875 gt 05
6 (V6) Brassica nigra (L) W D J Koch 8555 plusmn 050 005 gt 119875 gt 002 90 plusmn 085 05 gt 119875 gt 01
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 1022 plusmn 501 002 gt 119875 gt 001 66 plusmn 330 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 1114 plusmn 100 002 gt 119875 gt 001 618 plusmn 109 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 1230 plusmn 2357 005 gt 119875 gt 002 412 plusmn 3166 119875 gt 05
10 (V9) Citrus limon (L) Burm f 1831 plusmn 6569 005 gt 119875 gt 002 73 plusmn 141 05 gt 119875 gt 01
11 (V10) Citrullus colocynthis (L) Schrad 827 plusmn 651 01 gt 119875 gt 005 05 plusmn 166 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 1589 plusmn 2256 002 gt 119875 gt 001 58 plusmn 080 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 1615 plusmn 151 119875 gt 0001 985 plusmn 166 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 1418 plusmn 1303 002 gt 119875 gt 001 11 plusmn 252 119875 gt 05
15 (V14) Leucas capitata Desf 783 plusmn 1002 05 gt 119875 gt 01 14 plusmn 080 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 1428 plusmn 651 119875 gt 0001 08 plusmn 166 05 gt 119875 gt 01
17 (V16) Momordica charantia L 14755 plusmn 351 119875 gt 0001 156 plusmn 245 119875 gt 05
18 (V17) Nerium indicumMill 1268 plusmn 1203 002 gt 119875 gt 001 05 plusmn 165 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 1335 plusmn 1203 002 gt 119875 gt 001 123 plusmn 245 119875 gt 05
20 (V19) Pinus roxburghii Sarg 1050 plusmn 100 002 gt 119875 gt 001 08 plusmn 165 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 11355 plusmn 05 002 gt 119875 gt 001 131 plusmn 165 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 11535 plusmn 05 119875 gt 0001 173 plusmn 250 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 1538 plusmn 2005 002 gt 119875 gt 001 41 plusmn 085 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 1078 plusmn 1604 005 gt 119875 gt 002 66 plusmn 000 05 gt 119875 gt 01
25 (V24) Sapindus mukorossi Gaertn 14605 plusmn 551 001 gt 119875 gt 0001 41 plusmn 080 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 787 plusmn 2808 119875 gt 05 13 plusmn 176 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 7985 plusmn 1454 05 gt 119875 gt 01 66 plusmn 000 05 gt 119875 gt 01
28 (V27) Trichodesma indicum (L) Sm 9785 plusmn 451 005 gt 119875 gt 002 66 plusmn 300 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 888 plusmn 200 005 gt 119875 gt 002 173 plusmn 080 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
763 plusmn 601 Select to compare 09 plusmn 085 Select to compare
effects are due to phospholipid hydrolysis or phospholipidcompeting bidingmechanismwith other coagulation factorsparticularly FXa or by both ways however it is very hardto pinpoint exact one [35] Anticoagulant response posed bycobra PLA
2(anticoagulant enzyme) may aggravate hepatic
injury as many anticoagulant agents cause liver damage thatare idiosyncratic in nature [36]
Cardiac injury particularly systolic heart arrest is one ofthewell-known toxicities related toNaja naja subspecies [30]In present study Pakistani cobra venom was found to release
two cystolic enzymes (LDH and CK-MB) that are sensitiveindicators of myocyte injury [37] It is due to the presenceof myotoxic PLA
2and other cardiotoxin(s) that are salient
features of cobra venom that are responsible for cellularnecrosis and cytotoxicity [38 39] Cytotoxic effect of Najanaja karachiensis venom may not be overlooked even whenantisera administration is late cause of toxic components tothe microvasculature and thrombus produced which poseshindrance in access of immunoglobulins to the site of snakebite [26 29 39]
10 BioMed Research International
0
10
20
30
40
50
60
70Se
rum
leve
l of u
rea a
nd cr
eatin
ine (
mg
dL)
UreaCreatinine
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
L
Rhaz
ya st
ricta
Dec
neRu
bia
cord
ifolia
L
Sapi
ndus
muk
oros
si G
aert
nSt
enol
obiu
m st
ans (
L) S
eem
Term
inal
ia a
rjuna
(Rox
b ex
DC
) Wig
ht an
d A
rnTr
ichod
esm
a in
dicu
m (L
) Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antidotes tested (100mgkg)
Figure 2 Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea andcreatinine level posed by Naja naja karachiensis venom in rabbits
Snake venom has been responsible for detrimental effectsto the renal tissues [40] Likewise Naja naja krachiensisvenomwas found to cause severe renal damage by significantrise in serum urea and creatinine levels Indeed phospho-lipases enzymes are responsible for the increase in vascularpermeability with hemorrhagic effects to the vital tissues inthe victims Subsequently numerous lesions are producedrelated to the glomerular membrane and renal tubules eitherinterstitial or vascular [29 41 42] Presence of lymphocytes(white blood cells) and oedema in the cortical as well asmedullary regions of renal tubules further confirmed theidea of renal damage (data has not shown) as reportedpreviously with different snake venoms like Hemiscorpiuslepturus [26 43] Apart of it pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope99mTc confirmed that kidneys and urinary bladder are themost saturated organs (gt70) after intravenous injection inexperimental rabbits (unpublished data by our group)
Medicinal plants of Pakistan are used to inhibit snakevenom (PLA
2) enzymes [6] Due to this effect present
study was designed to select twenty-eight medicinal plantsof Pakistan to test their potentials as an antidote againsttoxicities produced by Naja naja karachiensis venom It
was fascinating that extract of Stenolobium stans (L) Seemshowed significant neutralization compared to that of refer-ence standard (antisera) as reported previously by Asad etal for inhibition of phospholipases A
2anticoagulant activity
[6 44] Present study indicated that extract of Stenolobiumstans (L) Seem possesses an endogenous inhibitor(s) tonullify venom (PLA
2) induced toxicities Extracts of these
plants material are routinely used in Pakistan by simpleapplication in the form of paste to the affected area [45]This practice provides effective first aid treatment as tinymolecules of an antidote diffuse favorably at the site of snakebite before hospitalization and abrogated the spreading oftoxins [39] It is owing to the various secondary metaboliteslike phenols flavonoids terpenoids xanthenes quinonoidsand so forth as reported earlier to mask various enzymaticactions of cobra venom [6 23] Indeed secondarymetabolitespose hindrance in binding of different snake venom enzymesto their potential targets therefore antidotal effect evokedOther plants extract were not proved significantly effectiveto neutralize cobra venom therefore cannot be declareduseful plants in venom therapy It is the need of the time toisolate lethal component(s) of cobra venom attributed to itsmajor toxicities Furthermore characterization of antivenom
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 3
Table1Detaileddescrip
tionfore
valuated
medicinalplantshaving
folklore
evidencesa
santivenom
Srnum
ber
Botanicaln
ameo
fmedicinalplants
Family
Partused
Phytochemicalsreported
Reference
1Albizialeb
beck
(L)Be
nth
Fabaceae
Seed
Carboh
ydratesproteinsalkaloidsflavanoidstanninsechinocystic
acidand
different
aminoacids
[6]
2Alliu
mcepa
LAmaryllid
aceae
Bulb
11gcarboh
ydrates12
gproteins06gfib
ersandH
2Ocontent(868g)10
0gof
material
[6]
3Alliu
msativ
umL
Amaryllid
aceae
Bulb
Quercetinscordinines
AandBallicinthiosulfin
ates2
mercapto-L-cyste
ines
anthocyaninsalliinasepo
lysaccharid
essativin
Iand
sativ
inIIand
glycosides
ofkaem
pferol
[6]
4Althaeaoffi
cinalisL
Malvaceae
Root
37sta
rch11mucilagefattyoilpectinflavon
oidspheno
licacidsasparagines
phytosterolsucroseandbu
tyric
acid
[6]
5Ba
uhiniavarie
gata
LFabaceae
Root
Tann
insfatty
oillupeolkaempferol-3-glucosid
egums120573-sito
sterol57-dehydroxy
and57-dimetho
xy-flavanon
e-4-0-a-L-rham
nopyrano
syl-120573
-Dglucop
yranosides
[6]
6Brassicanigra(L)WD
JK
och
Brassic
aceae
Seed
Essentialoilsin
igrin
andglucoside
[6]
7Ca
lotro
pisp
rocera
(Aito
n)WTA
iton
Apocyn
aceae
Exud
ates
andflo
wer
Resin
stann
inscalotro
pin
steroluscharincalotropagenin
calotoxin
andcalactin
[6]
8Ce
drus
deodara(Roxbex
DD
on)G
Don
Pinaceae
Bark
Taxifolin
dew
ardioldew
arenegumascorbica
cidhimadarolcantdarol
cholesterin
allo
himachalolcedrinosidehimachaloldewarolcedrin
dihydrod
ehydrodiconiferylalcoh
olessentia
loiliso
centdaroland
dihydrom
yricetin
[6]
9Citru
slim
on(L)Bu
rmf
Rutaceae
Fruit
4-120573-glucopyrano
sided-x-pinene
camph
ened-lim
onenelin
aloo
lichangin
4-120573-glucopyrano
sideno
milinica
cidandessentialoil
[6]
10Citru
lluscolocynthis(L)Schrad
Cucurbita
ceae
Fruit
Manyalkaloidsglycosidestanninscitrulluincitrulluicaciddihydric
alcoho
lph
ydroxybenzylcitrulluenehentria
contaneelaterincitrullolmethyletherbitter
oilcitbittoland
sapo
nins
[6]
11Cu
minum
cyminum
LAp
iaceae
Seed
Essentialoilandcumin
oilcon
tain
sminaldehyde13-p-m
enthadien-7-aland
14-p-m
enthadien-7-al
[6]
12En
icoste
mahysso
pifoliu
m(W
illd)IV
erd
Gentia
naceae
Who
leplant
Sapo
narin
isovetexin
sylysw
ertin
betulinswertia
marinapigenin
genk
wanin
erythrocentaurinesw
ertio
sideiso
swertisinenicoflavineand
swertisin
[6]
13Fagoniacretica
LZy
goph
yllaceae
Leaves
Ursolicacidpinito
lnahagenin
sapo
ninglycosidessapon
in-Iandsapo
nin-II
docosyld
ocosanoatefro
mn-hexane
extractanddifferent
proteins
from
aqueou
sextract
[6]
14Leucas
capitataDesf
Lamiaceae
Who
leplant
Alkaloidas
wellase
ssentia
loil
[6]
15Matthiolaincana
(L)WTA
iton
Brassic
aceae
Seeds
Oilric
hin
chloroph
yllsabNPK
and
Na120574-lino
lenica
cidandcaroteno
ids
Sulfo
raph
eneisa
nim
portantcom
ponent
[6]
16Mom
ordica
charantia
LCu
curbita
ceae
Fruit
Caroteno
idsmom
orcharasides
AandB
glucosidesstig
mastadien-3-beta-ol
squalenem
ycosessteroidalglucosidemom
ordicinesI
andIIstig
masterolvicine
cucurbitane
triterpenoidsm
omordicosid
escycloeucaleno
ltaraxerol
spinasterollo
phenoldiosgeninthiocyano
gen
24-m
ethylencycloarteno
land
phenylprop
anoids
[6]
17Ne
rium
indicum
Mill
Apocyn
aceae
Root
andleaves
Nerioderin
neriodo
rinkarabinand
odorin
[6]
18Ocim
umsanctum
LLamiaceae
Who
leplant
Essentialoils
arefou
ndric
hin
32
carvacrol713eugeno
l17
caryop
hyllene
204methyleugenoleugenolmethyletherlinaloo
landmethylchavicolcineole
[6]
4 BioMed Research International
Table1Con
tinued
Srnum
ber
Botanicaln
ameo
fmedicinalplants
Family
Partused
Phytochemicalsreported
Reference
19Pinu
sroxburghiiSarg
Pinaceae
Oleoresin
Careen120572
-pinene120573-pinene120573-carene120573-lo
ngifo
lenelon
gifoleneand
long
icyclene
[6]
20Pista
ciaintegerrim
aJLStewart
Anacardiaceae
Galls
13essentialoilric
hin
camph
enecineoleA-
terpineolA-
pinenearomadendrene
caprylicacidand
d-lim
onenea
bund
antingalls
[6]
21Psoralea
corylifoliaL
Fabaceae
Seeds
Limon
enelin
aloo
lpsoraleneangelicinn
eobavachalconebakuchiol4-te
rpineol
corylid
inn
eobavaiso
flavonebavachinin
isobavachin120572
-elemenegeranylacetate
stigmasterolbaku
chioltraincon
taneisopsoralidinisopsoralenbakuchalcon
eiso
neob
avachalcon
epsoralenpsoralid
inraffi
nosecorylifo
linincorylifo
leanand
corylifolin
[6]
22Rh
azya
strictaDecne
Apocyn
aceae
Leaves
Enzymes
(NADPH
depend
ent)
glycosides
(rob
lnin3-7-rhamno
sidea
ndiso
rham
netin
-3-7-rhamno
side)alkaloid(sew
arine)and
triterpenes
(ursolicacid
Mgqu
inateand120573-sito
sterol)alon
gwith
flavono
idsrhazianosides
AandB
[6]
23Ru
biacordifolia
LRu
biaceae
Stem
Pseudo
purpurinxanthop
urpu
rinpurpu
rinm
unjistin
alizarinand
mun
jistin
are
foun
dwith
theirg
lycosid
es
[6]
24Sapind
usmukorossiGaertn
Sapind
aceae
Fruit120573-Sito
sterolsapind
osideA
sapindo
sideB
kaempferolquercetinsapon
inand
emarginatosid
e[6]
25Stenolob
ium
stans
(L)Seem
Bign
oniaceae
Root120573-C
arotene120573-sito
sterol120572-amarinezeaxanthinind
olem
etabolizingenzymes
phenolicacidsoleano
licacidtriterpeno
idsursolic
acidind
ole-oxygenaseand
luteinzeaxanthin
[6]
26Term
inaliaarjuna
(Roxbex
DC)W
ight
andArn
Com
bretaceae
Bark120573-Sito
sterolarjuno
licacidtom
entosic
acidellagica
cidessentialoilarjunetin
arjuninecalcium
saltsaluminium
andmagnesiu
msaltscoloringagents
redu
cing
sugarstannin(pyrocatecho
l)andalactone
[6]
27Trich
odesmaindicum
(L)Sm
Boraginaceae
Who
leplantSeedso
ilisric
hin
linoleicoleicste
aricpalmiticandlin
olenicacidsAp
arto
fit
hexacosaneethylhexacosano
ateand2124-hexacos-adieno
icacid
ethyleste
rsare
foun
d[6]
28Zingiberoffi
cinaleR
oscoe
Zing
iberaceae
Rhizom
eD-C
urcumenecineolpotassiu
moxalatecamph
enecitralbo
rneolsin
giberin
eshogaol120572-curcumene120572-bergamotene120573-gam
ma-bisabo
lenegingeroland
120573-phelland
rene
23
BioMed Research International 5
Table2Biochemicalparametersb
eforea
ndaft
ersnakev
enom
administratio
nin
different
experim
entalgroup
sofrabbits
Toxicitydeterm
ined
Markersof
snake
veno
mtoxicity
Group
Ibaseline
measurements
(meanplusmnSE
M)
Group
IIandIIIv
enom
injection
Group
IVnegativ
econ
trol
(meanplusmnSE
M)
Normalreference
values
repo
rted
References
04m
gkg
(meanplusmnSE
M)
08m
gkg
(meanplusmnSE
M)
Liver
GPT
ALT
(52plusmn346
)UL
(72plusmn470)U
L01gt119875gt005
(86plusmn50)
UL
005gt119875gt002
(52plusmn348)U
L(48ndash80)U
L[19
]
GOTAST
(65plusmn657)U
L(157plusmn2424)
UL
01gt119875gt005
(251plusmn182)U
L001gt119875gt0001
(67plusmn321)U
L(14
ndash113)U
L[19
]
Kidn
eys
Urea
(28plusmn17
3)mgdL
(42plusmn308)m
gdL
005gt119875gt002
(576plusmn384)m
gdL
002gt119875gt001
(28plusmn033)m
gdL
(10ndash
28)m
gdL
[26]
Creatin
ine
(10plusmn0313)
mgdL
(174plusmn003)m
gdL
001gt119875gt0001
(21plusmn010)m
gdL
002gt119875gt001
(1plusmn006)m
gdL
(05ndash25)m
gdL
[26]
Heart
CK-M
B(132plusmn20)
UL
(21plusmn
15)U
L005gt119875gt002
(77plusmn1122)
UL
005gt119875gt002
(13plusmn056)U
L(lt25)U
Llowast
[2021]
LDH
(714plusmn318)U
L(206
4plusmn1598)
UL
119875lt0001
(2562plusmn2514
)UL
119875≪0001
(720plusmn470)U
L(559ndash2077)
UL
[46]
lowastCK
-MBactiv
ityislessthan
2in
healthyrabbitshow
everitisu
sually10ndash30
oftotalC
Kactiv
ity
6 BioMed Research International
0
50
100
150
200
250
300Se
rum
leve
l of G
PTG
OT
(UL
)
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
LRh
azya
stric
ta D
ecne
Rubi
a co
rdifo
lia L
Sa
pind
us m
ukor
ossi
Gae
rtn
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C) W
ight
and
Arn
Trich
odes
ma
indi
cum
(L)
SmZi
ngib
er o
fficin
ale R
osco
eRe
fere
nce s
tand
ard
anti
dote
GPT levelGOT level
Various antidotes used (100mgkg)
Figure 1 Comparison of various medicinal plant extracts with reference standard antidote in neutralization of increased GPT and GOTserum level posed by Naja naja karachiensis venom in rabbits
kits (manufactured by Merck) according to the DGKC andIFCCmethod on the Selectra Junior (Vital Scientific BVTheNetherlands) [26]
26 Statistical Analysis All numerical values were expressedas mean (3 replicates) plusmn standard error of mean (SEM) Theywere calculated by the use of Microsoft Excel 2007 and Stu-dentrsquos t-test was applied to compare the efficacy of evaluatedsamples with standard antidote (reference standard)
3 Results
Venom from Naja naja karachiensis was proved to causesevere complications in dose-dependent manner Venom atthe dose of 400 120583gkg led to release of moderate serum levelsof ALT (72plusmn470UL 01 gt 119875 gt 005) AST (157plusmn2424UL01 gt 119875 gt 005) urea (42 plusmn 308mgdL 005 gt 119875 gt 002)creatinine (174 plusmn 003mgdL 001 gt 119875 gt 0001) CK-MB(21 plusmn 15UL 005 gt 119875 gt 002) and LDH (2064 plusmn 1598UL119875 lt 0001) and thus indicated toxicities to the heart liver andkidneys At 800 120583gkg of cobra venom severe tissue damagewas observed in terms of significant release of ALT (86 plusmn50UL 005 gt 119875 gt 002) AST (251 plusmn 182UL 001 gt 119875 gt0001) urea (576 plusmn 384mgdL 002 gt 119875 gt 001) creatinine
(21 plusmn 010mgdL 002 gt 119875 gt 001) CK-MB (77plusmn 1122UL005 gt 119875 gt 002) and LDH (2562 plusmn 2514UL 119875 ≪ 0001)Complete detail about various biochemical parameters forbaseline measurements different doses of cobra venom andsaline (as negative control) is summarized in Table 2
To neutralize in vivo 800 120583gkg of cobra venom twenty-eight medicinal plant extracts were evaluated It was noticedthat extract of Stenolobium stans (L) Seem was the bestantidote (119875 gt 05) compared to reference standard Alliumsativum L Althaea officinalis L Citrullus colocynthis (L)Schrad Leucas capitataDesf Pinus roxburghii Sarg Psoraleacorylifolia L Rubia cordifolia L and Sapindus mukorossiGaertn were found to be valuable plants to protect liverdamage (119875 gt 05) as standard antidote (ALT = 525 plusmn351UL and AST = 695 plusmn 1855UL) however requiredALT and AST levels were not observed with remainingplant extracts (05 gt 119875 gt 005) as shown in Table 3 andcomparison is shown in Figure 1 Extracts of Leucas capitataDesf and Althaea officinalis L were observed to be helpful(119875 gt 05) as reference standard (urea = 315 plusmn 050mgdLand creatinine = 108 plusmn 002mgdL) nevertheless remainingplants were found less valuable (05 gt 119875 gt 001) tominimize urea and creatinine levels to maintain kidney func-tions (Table 4 and Figure 2) When compared with standardantidote (CK-MB = 90 plusmn 085UL) eight medicinal plants
BioMed Research International 7
Table 3 Hepatoprotective activity of various medicinal plant extracts on ALT and AST levels to neutralize snake bite envenomation inexperimental rabbits
Sr number Group V(subgroup) Name of evaluated sample GPTALT GOTAST
UL(mean plusmn SEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 62 plusmn 702 05 gt 119875 gt 01 99 plusmn 1855 05 gt 119875 gt 01
2 (V2) Allium cepa L 68 plusmn 1203 05 gt 119875 gt 01 1665 plusmn 050 05 gt 119875 gt 01
3 (V3) Allium sativum L 49 plusmn 501 119875 gt 05 825 plusmn 1855 119875 gt 05
4 (V4) Althaea officinalisL 70 plusmn 2005 119875 gt 05 745 plusmn 1454 119875 gt 05
5 (V5) Bauhinia variegata L 78 plusmn 404 05 gt 119875 gt 01 1495 plusmn 199 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 45 plusmn 000 05 gt 119875 gt 01 55 plusmn 100 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 315 plusmn 05 05 gt 119875 gt 01 141 plusmn 101 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 625 plusmn 252 05 gt 119875 gt 01 194 plusmn 1212 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 633 plusmn 414 05 gt 119875 gt 01 72 plusmn 202 119875 gt 05
10 (V9) Citrus limon (L) Burm f 305 plusmn 1363 05 gt 119875 gt 01 49 plusmn 2424 119875 gt 05
11 (V10) Citrullus colocynthis (L) Schrad 475 plusmn 555 119875 gt 05 74 plusmn 909 119875 gt 05
12 (V11) Cuminum cyminum L 67 plusmn 1616 119875 gt 05 157 plusmn 1414 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 8525 plusmn 126 01 gt 119875 gt
0051685 plusmn 757 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 80 plusmn 782 05 gt 119875 gt 01 170 plusmn 1313 05 gt 119875 gt 01
15 (V14) Leucas capitata Desf 455 plusmn 752 119875 gt 05 535 plusmn 050 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 62 plusmn 808 05 gt 119875 gt 01 121 plusmn 505 05 gt 119875 gt 01
17 (V16) Momordica charantia L 625 plusmn 752 05 gt 119875 gt 01 247 plusmn 4613 05 gt 119875 gt 01
18 (V17) Nerium indicumMill 88 plusmn 1955 05 gt 119875 gt 01 235 plusmn 1805 01 gt 119875 gt
005
19 (V18) Ocimum sanctum L 615 plusmn 752 05 gt 119875 gt 01 158 plusmn 1002 05 gt 119875 gt 01
20 (V19) Pinus roxburghii Sarg 53 plusmn 902 119875 gt 05 57 plusmn 1303 119875 gt 05
21 (V20) Pistacia integerrima J L Stewart 415 plusmn 15 05 gt 119875 gt 01 545 plusmn 050 119875 gt 05
22 (V21) Psoralea corylifolia L 54 plusmn 100 119875 gt 05 665 plusmn 1153 119875 gt 05
23 (V22) Rhazya stricta Decne 66 plusmn 2424 119875 gt 05 1325 plusmn 1262 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 655 plusmn 1767 119875 gt 05 129 plusmn 6641 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 56 plusmn 601 119875 gt 05 785 plusmn 1354 119875 gt 05
26 (V25) Stenolobium stans (L) Seem 465 plusmn 656 119875 gt 05 705 plusmn 353 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 475 plusmn 151 05 gt 119875 gt 01 78 plusmn 606 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 52 plusmn 000 119875 gt 05 125 plusmn 401 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 665 plusmn 3259 119875 gt 05 1175 plusmn 952 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
525 plusmn 351 Select tocompare 695 plusmn 1855 Select to
compare
(Allium cepa L Althaea officinalis L Bauhinia variegataL Cedrus deodara (Roxb ex D Don) G Don Fagoniacretica L Leucas capitataDesf Momordica charantia L andOcimum sanctum L) were found equally capable (119875 gt 05) ofnormalizing high values of CK-MB however rest of all wereproved less beneficial (05 gt 119875 gt 01) To combat highlyraised values of LDH Althaea officinalis L Leucas capitataDesf and Terminalia arjuna (Roxb ex DC) Wight and Arnwere proved useful to some extent (05 gt 119875 gt 01) although
remaining plant extracts could not be shortlisted (01 gt 119875 gt0001) as standard antisera (LDH = 763 plusmn 601UL) Overalldetail about LDH is discussed in Table 5 and comparison isshown in Figure 3
4 Discussion
Snake bite has been responsible for tens of thousands ofdeaths worldwide and numerous physical handicaps [29]
8 BioMed Research International
Table 4 Nephroprotective activity of different medicinal plant extracts on urea and creatinine levels to neutralize snake bite envenomationin experimental rabbits
Sr number Group V(subgroup) Name of evaluated sample Urea Creatinine
mgdL(mean plusmn SEM)
119875 valuecomment
mgdL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 57 plusmn 00 002 gt 119875 gt 001 16 plusmn 020 05 gt 119875 gt 01
2 (V2) Allium cepa L 51 plusmn 1103 05 gt 119875 gt 01 150 plusmn 017 05 gt 119875 gt 01
3 (V3) Allium sativum L 50 plusmn 401 05 gt 119875 gt 01 130 plusmn 006 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 26 plusmn 505 119875 gt 05 118 plusmn 024 119875 gt 05
5 (V5) Bauhinia variegata L 50 plusmn 126 05 gt 119875 gt 01 136 plusmn 024 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 225 plusmn 451 05 gt 119875 gt 01 107 plusmn 011 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 425 plusmn 252 05 gt 119875 gt 01 130 plusmn 001 01 gt 119875 gt 005
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 39 plusmn 202 05 gt 119875 gt 01 125 plusmn 003 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 44 plusmn 404 05 gt 119875 gt 01 144 plusmn 007 05 gt 119875 gt 01
10 (V9) Citrus limon (L) Burm f 395 plusmn 252 05 gt 119875 gt 01 140 plusmn 003 01 gt 119875 gt 005
11 (V10) Citrullus colocynthis (L) Schrad 495 plusmn 353 05 gt 119875 gt 01 152 plusmn 015 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 54 plusmn 101 005 gt 119875 gt 002 148 plusmn 001 005 gt 119875 gt 002
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 395 plusmn 454 05 gt 119875 gt 01 135 plusmn 005 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 43 plusmn 227 05 gt 119875 gt 01 123 plusmn 013 119875 lt 05
15 (V14) Leucas capitata Desf 315 plusmn 050 119875 gt 05 107 plusmn 005 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 435 plusmn 353 05 gt 119875 gt 01 144 plusmn 014 05 gt 119875 gt 01
17 (V16) Momordica charantia L 57 plusmn 501 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
18 (V17) Nerium indicumMill 47 plusmn 000 119875 lt 005 124 plusmn 010 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 495 plusmn 752 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
20 (V19) Pinus roxburghii Sarg 45 plusmn 100 119875 gt 005 14 plusmn 005 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 665 plusmn 050 002 gt 119875 gt 001 168 plusmn 010 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 425 plusmn 250 05 gt 119875 gt 01 127 plusmn 015 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 535 plusmn 353 05 gt 119875 gt 01 167 plusmn 0005 005 gt 119875 gt 002
24 (V23) Rubia cordifolia L 36 plusmn 101 05 gt 119875 gt 01 093 plusmn 048 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 44 plusmn 100 01 gt 119875 gt 005 143 plusmn 012 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 255 plusmn 651 119875 gt 05 11 plusmn 006 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 385 plusmn 151 05 gt 119875 gt 01 127 plusmn 023 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 37 plusmn 100 05 gt 119875 gt 01 046 plusmn 001 005 gt 119875 gt 002
29 (V28) Zingiber officinale Roscoe 47 plusmn 100 005 gt 119875 gt 002 130 plusmn 001 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
315 plusmn 050 Select tocompare 108 plusmn 002 Select to
compare
Generally snake venom is an intricate mixture of variousproteins (gt90) and most of them are enzymes particularly(40) phospholipases A
2[30] Among different complica-
tions hepatic injury is one of the deadly venomous effectsproduced by cobra bite [31 32] Naja naja karachiensisvenom caused significant increase in ALT and AST levels(dose dependently) that are surrogate markers for livertoxicity either by direct action or immunological (hyper-sensitivity) reaction The allergic reactions reactions are notdose dependent which clarify lethal effects to the cytoplasm
and mitochondrial membranes of the hepatic cells [25 33]Phospholipases enzymes abundant in Naja naja karachien-sis venom are responsible for breakdown of membranousphospholipids and therefore resulted in cellular injury alongwith inflammation [24] PLA
2is attributed to decrease in
Na+K+ ATPase activities and led to greater influx of sodiumions and water molecules into the cell Subsequently plasmamembrane lipid bilayer disorganized and eventually resultedin hepatic cells death [33 34] In addition PLA
2is also found
to cause anticoagulation in victims [6] All pharmacological
BioMed Research International 9
Table 5 Cardioprotective activity of medicinal plant extracts on LDH and CK-MB levels to neutralize snake bite envenomation inexperimental rabbits
Sr numberGroup V(subgroup) Name of evaluated sample LDH CK-MB
UL (mean plusmnSEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 1357 plusmn 100 001 gt 119875 gt 0001 41 plusmn 085 05 gt 119875 gt 01
2 (V2) Allium cepa L 934 plusmn 1303 01 gt 119875 gt 005 148 plusmn 165 119875 gt 05
3 (V3) Allium sativum L 1177 plusmn 2056 005 gt 119875 gt 002 66 plusmn 330 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 9755 plusmn 3360 05 gt 119875 gt 01 148 plusmn 330 119875 gt 05
5 (V5) Bauhinia variegata L 1972 plusmn 300 119875 gt 0001 83 plusmn 676 119875 gt 05
6 (V6) Brassica nigra (L) W D J Koch 8555 plusmn 050 005 gt 119875 gt 002 90 plusmn 085 05 gt 119875 gt 01
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 1022 plusmn 501 002 gt 119875 gt 001 66 plusmn 330 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 1114 plusmn 100 002 gt 119875 gt 001 618 plusmn 109 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 1230 plusmn 2357 005 gt 119875 gt 002 412 plusmn 3166 119875 gt 05
10 (V9) Citrus limon (L) Burm f 1831 plusmn 6569 005 gt 119875 gt 002 73 plusmn 141 05 gt 119875 gt 01
11 (V10) Citrullus colocynthis (L) Schrad 827 plusmn 651 01 gt 119875 gt 005 05 plusmn 166 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 1589 plusmn 2256 002 gt 119875 gt 001 58 plusmn 080 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 1615 plusmn 151 119875 gt 0001 985 plusmn 166 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 1418 plusmn 1303 002 gt 119875 gt 001 11 plusmn 252 119875 gt 05
15 (V14) Leucas capitata Desf 783 plusmn 1002 05 gt 119875 gt 01 14 plusmn 080 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 1428 plusmn 651 119875 gt 0001 08 plusmn 166 05 gt 119875 gt 01
17 (V16) Momordica charantia L 14755 plusmn 351 119875 gt 0001 156 plusmn 245 119875 gt 05
18 (V17) Nerium indicumMill 1268 plusmn 1203 002 gt 119875 gt 001 05 plusmn 165 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 1335 plusmn 1203 002 gt 119875 gt 001 123 plusmn 245 119875 gt 05
20 (V19) Pinus roxburghii Sarg 1050 plusmn 100 002 gt 119875 gt 001 08 plusmn 165 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 11355 plusmn 05 002 gt 119875 gt 001 131 plusmn 165 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 11535 plusmn 05 119875 gt 0001 173 plusmn 250 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 1538 plusmn 2005 002 gt 119875 gt 001 41 plusmn 085 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 1078 plusmn 1604 005 gt 119875 gt 002 66 plusmn 000 05 gt 119875 gt 01
25 (V24) Sapindus mukorossi Gaertn 14605 plusmn 551 001 gt 119875 gt 0001 41 plusmn 080 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 787 plusmn 2808 119875 gt 05 13 plusmn 176 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 7985 plusmn 1454 05 gt 119875 gt 01 66 plusmn 000 05 gt 119875 gt 01
28 (V27) Trichodesma indicum (L) Sm 9785 plusmn 451 005 gt 119875 gt 002 66 plusmn 300 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 888 plusmn 200 005 gt 119875 gt 002 173 plusmn 080 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
763 plusmn 601 Select to compare 09 plusmn 085 Select to compare
effects are due to phospholipid hydrolysis or phospholipidcompeting bidingmechanismwith other coagulation factorsparticularly FXa or by both ways however it is very hardto pinpoint exact one [35] Anticoagulant response posed bycobra PLA
2(anticoagulant enzyme) may aggravate hepatic
injury as many anticoagulant agents cause liver damage thatare idiosyncratic in nature [36]
Cardiac injury particularly systolic heart arrest is one ofthewell-known toxicities related toNaja naja subspecies [30]In present study Pakistani cobra venom was found to release
two cystolic enzymes (LDH and CK-MB) that are sensitiveindicators of myocyte injury [37] It is due to the presenceof myotoxic PLA
2and other cardiotoxin(s) that are salient
features of cobra venom that are responsible for cellularnecrosis and cytotoxicity [38 39] Cytotoxic effect of Najanaja karachiensis venom may not be overlooked even whenantisera administration is late cause of toxic components tothe microvasculature and thrombus produced which poseshindrance in access of immunoglobulins to the site of snakebite [26 29 39]
10 BioMed Research International
0
10
20
30
40
50
60
70Se
rum
leve
l of u
rea a
nd cr
eatin
ine (
mg
dL)
UreaCreatinine
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
L
Rhaz
ya st
ricta
Dec
neRu
bia
cord
ifolia
L
Sapi
ndus
muk
oros
si G
aert
nSt
enol
obiu
m st
ans (
L) S
eem
Term
inal
ia a
rjuna
(Rox
b ex
DC
) Wig
ht an
d A
rnTr
ichod
esm
a in
dicu
m (L
) Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antidotes tested (100mgkg)
Figure 2 Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea andcreatinine level posed by Naja naja karachiensis venom in rabbits
Snake venom has been responsible for detrimental effectsto the renal tissues [40] Likewise Naja naja krachiensisvenomwas found to cause severe renal damage by significantrise in serum urea and creatinine levels Indeed phospho-lipases enzymes are responsible for the increase in vascularpermeability with hemorrhagic effects to the vital tissues inthe victims Subsequently numerous lesions are producedrelated to the glomerular membrane and renal tubules eitherinterstitial or vascular [29 41 42] Presence of lymphocytes(white blood cells) and oedema in the cortical as well asmedullary regions of renal tubules further confirmed theidea of renal damage (data has not shown) as reportedpreviously with different snake venoms like Hemiscorpiuslepturus [26 43] Apart of it pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope99mTc confirmed that kidneys and urinary bladder are themost saturated organs (gt70) after intravenous injection inexperimental rabbits (unpublished data by our group)
Medicinal plants of Pakistan are used to inhibit snakevenom (PLA
2) enzymes [6] Due to this effect present
study was designed to select twenty-eight medicinal plantsof Pakistan to test their potentials as an antidote againsttoxicities produced by Naja naja karachiensis venom It
was fascinating that extract of Stenolobium stans (L) Seemshowed significant neutralization compared to that of refer-ence standard (antisera) as reported previously by Asad etal for inhibition of phospholipases A
2anticoagulant activity
[6 44] Present study indicated that extract of Stenolobiumstans (L) Seem possesses an endogenous inhibitor(s) tonullify venom (PLA
2) induced toxicities Extracts of these
plants material are routinely used in Pakistan by simpleapplication in the form of paste to the affected area [45]This practice provides effective first aid treatment as tinymolecules of an antidote diffuse favorably at the site of snakebite before hospitalization and abrogated the spreading oftoxins [39] It is owing to the various secondary metaboliteslike phenols flavonoids terpenoids xanthenes quinonoidsand so forth as reported earlier to mask various enzymaticactions of cobra venom [6 23] Indeed secondarymetabolitespose hindrance in binding of different snake venom enzymesto their potential targets therefore antidotal effect evokedOther plants extract were not proved significantly effectiveto neutralize cobra venom therefore cannot be declareduseful plants in venom therapy It is the need of the time toisolate lethal component(s) of cobra venom attributed to itsmajor toxicities Furthermore characterization of antivenom
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
4 BioMed Research International
Table1Con
tinued
Srnum
ber
Botanicaln
ameo
fmedicinalplants
Family
Partused
Phytochemicalsreported
Reference
19Pinu
sroxburghiiSarg
Pinaceae
Oleoresin
Careen120572
-pinene120573-pinene120573-carene120573-lo
ngifo
lenelon
gifoleneand
long
icyclene
[6]
20Pista
ciaintegerrim
aJLStewart
Anacardiaceae
Galls
13essentialoilric
hin
camph
enecineoleA-
terpineolA-
pinenearomadendrene
caprylicacidand
d-lim
onenea
bund
antingalls
[6]
21Psoralea
corylifoliaL
Fabaceae
Seeds
Limon
enelin
aloo
lpsoraleneangelicinn
eobavachalconebakuchiol4-te
rpineol
corylid
inn
eobavaiso
flavonebavachinin
isobavachin120572
-elemenegeranylacetate
stigmasterolbaku
chioltraincon
taneisopsoralidinisopsoralenbakuchalcon
eiso
neob
avachalcon
epsoralenpsoralid
inraffi
nosecorylifo
linincorylifo
leanand
corylifolin
[6]
22Rh
azya
strictaDecne
Apocyn
aceae
Leaves
Enzymes
(NADPH
depend
ent)
glycosides
(rob
lnin3-7-rhamno
sidea
ndiso
rham
netin
-3-7-rhamno
side)alkaloid(sew
arine)and
triterpenes
(ursolicacid
Mgqu
inateand120573-sito
sterol)alon
gwith
flavono
idsrhazianosides
AandB
[6]
23Ru
biacordifolia
LRu
biaceae
Stem
Pseudo
purpurinxanthop
urpu
rinpurpu
rinm
unjistin
alizarinand
mun
jistin
are
foun
dwith
theirg
lycosid
es
[6]
24Sapind
usmukorossiGaertn
Sapind
aceae
Fruit120573-Sito
sterolsapind
osideA
sapindo
sideB
kaempferolquercetinsapon
inand
emarginatosid
e[6]
25Stenolob
ium
stans
(L)Seem
Bign
oniaceae
Root120573-C
arotene120573-sito
sterol120572-amarinezeaxanthinind
olem
etabolizingenzymes
phenolicacidsoleano
licacidtriterpeno
idsursolic
acidind
ole-oxygenaseand
luteinzeaxanthin
[6]
26Term
inaliaarjuna
(Roxbex
DC)W
ight
andArn
Com
bretaceae
Bark120573-Sito
sterolarjuno
licacidtom
entosic
acidellagica
cidessentialoilarjunetin
arjuninecalcium
saltsaluminium
andmagnesiu
msaltscoloringagents
redu
cing
sugarstannin(pyrocatecho
l)andalactone
[6]
27Trich
odesmaindicum
(L)Sm
Boraginaceae
Who
leplantSeedso
ilisric
hin
linoleicoleicste
aricpalmiticandlin
olenicacidsAp
arto
fit
hexacosaneethylhexacosano
ateand2124-hexacos-adieno
icacid
ethyleste
rsare
foun
d[6]
28Zingiberoffi
cinaleR
oscoe
Zing
iberaceae
Rhizom
eD-C
urcumenecineolpotassiu
moxalatecamph
enecitralbo
rneolsin
giberin
eshogaol120572-curcumene120572-bergamotene120573-gam
ma-bisabo
lenegingeroland
120573-phelland
rene
23
BioMed Research International 5
Table2Biochemicalparametersb
eforea
ndaft
ersnakev
enom
administratio
nin
different
experim
entalgroup
sofrabbits
Toxicitydeterm
ined
Markersof
snake
veno
mtoxicity
Group
Ibaseline
measurements
(meanplusmnSE
M)
Group
IIandIIIv
enom
injection
Group
IVnegativ
econ
trol
(meanplusmnSE
M)
Normalreference
values
repo
rted
References
04m
gkg
(meanplusmnSE
M)
08m
gkg
(meanplusmnSE
M)
Liver
GPT
ALT
(52plusmn346
)UL
(72plusmn470)U
L01gt119875gt005
(86plusmn50)
UL
005gt119875gt002
(52plusmn348)U
L(48ndash80)U
L[19
]
GOTAST
(65plusmn657)U
L(157plusmn2424)
UL
01gt119875gt005
(251plusmn182)U
L001gt119875gt0001
(67plusmn321)U
L(14
ndash113)U
L[19
]
Kidn
eys
Urea
(28plusmn17
3)mgdL
(42plusmn308)m
gdL
005gt119875gt002
(576plusmn384)m
gdL
002gt119875gt001
(28plusmn033)m
gdL
(10ndash
28)m
gdL
[26]
Creatin
ine
(10plusmn0313)
mgdL
(174plusmn003)m
gdL
001gt119875gt0001
(21plusmn010)m
gdL
002gt119875gt001
(1plusmn006)m
gdL
(05ndash25)m
gdL
[26]
Heart
CK-M
B(132plusmn20)
UL
(21plusmn
15)U
L005gt119875gt002
(77plusmn1122)
UL
005gt119875gt002
(13plusmn056)U
L(lt25)U
Llowast
[2021]
LDH
(714plusmn318)U
L(206
4plusmn1598)
UL
119875lt0001
(2562plusmn2514
)UL
119875≪0001
(720plusmn470)U
L(559ndash2077)
UL
[46]
lowastCK
-MBactiv
ityislessthan
2in
healthyrabbitshow
everitisu
sually10ndash30
oftotalC
Kactiv
ity
6 BioMed Research International
0
50
100
150
200
250
300Se
rum
leve
l of G
PTG
OT
(UL
)
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
LRh
azya
stric
ta D
ecne
Rubi
a co
rdifo
lia L
Sa
pind
us m
ukor
ossi
Gae
rtn
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C) W
ight
and
Arn
Trich
odes
ma
indi
cum
(L)
SmZi
ngib
er o
fficin
ale R
osco
eRe
fere
nce s
tand
ard
anti
dote
GPT levelGOT level
Various antidotes used (100mgkg)
Figure 1 Comparison of various medicinal plant extracts with reference standard antidote in neutralization of increased GPT and GOTserum level posed by Naja naja karachiensis venom in rabbits
kits (manufactured by Merck) according to the DGKC andIFCCmethod on the Selectra Junior (Vital Scientific BVTheNetherlands) [26]
26 Statistical Analysis All numerical values were expressedas mean (3 replicates) plusmn standard error of mean (SEM) Theywere calculated by the use of Microsoft Excel 2007 and Stu-dentrsquos t-test was applied to compare the efficacy of evaluatedsamples with standard antidote (reference standard)
3 Results
Venom from Naja naja karachiensis was proved to causesevere complications in dose-dependent manner Venom atthe dose of 400 120583gkg led to release of moderate serum levelsof ALT (72plusmn470UL 01 gt 119875 gt 005) AST (157plusmn2424UL01 gt 119875 gt 005) urea (42 plusmn 308mgdL 005 gt 119875 gt 002)creatinine (174 plusmn 003mgdL 001 gt 119875 gt 0001) CK-MB(21 plusmn 15UL 005 gt 119875 gt 002) and LDH (2064 plusmn 1598UL119875 lt 0001) and thus indicated toxicities to the heart liver andkidneys At 800 120583gkg of cobra venom severe tissue damagewas observed in terms of significant release of ALT (86 plusmn50UL 005 gt 119875 gt 002) AST (251 plusmn 182UL 001 gt 119875 gt0001) urea (576 plusmn 384mgdL 002 gt 119875 gt 001) creatinine
(21 plusmn 010mgdL 002 gt 119875 gt 001) CK-MB (77plusmn 1122UL005 gt 119875 gt 002) and LDH (2562 plusmn 2514UL 119875 ≪ 0001)Complete detail about various biochemical parameters forbaseline measurements different doses of cobra venom andsaline (as negative control) is summarized in Table 2
To neutralize in vivo 800 120583gkg of cobra venom twenty-eight medicinal plant extracts were evaluated It was noticedthat extract of Stenolobium stans (L) Seem was the bestantidote (119875 gt 05) compared to reference standard Alliumsativum L Althaea officinalis L Citrullus colocynthis (L)Schrad Leucas capitataDesf Pinus roxburghii Sarg Psoraleacorylifolia L Rubia cordifolia L and Sapindus mukorossiGaertn were found to be valuable plants to protect liverdamage (119875 gt 05) as standard antidote (ALT = 525 plusmn351UL and AST = 695 plusmn 1855UL) however requiredALT and AST levels were not observed with remainingplant extracts (05 gt 119875 gt 005) as shown in Table 3 andcomparison is shown in Figure 1 Extracts of Leucas capitataDesf and Althaea officinalis L were observed to be helpful(119875 gt 05) as reference standard (urea = 315 plusmn 050mgdLand creatinine = 108 plusmn 002mgdL) nevertheless remainingplants were found less valuable (05 gt 119875 gt 001) tominimize urea and creatinine levels to maintain kidney func-tions (Table 4 and Figure 2) When compared with standardantidote (CK-MB = 90 plusmn 085UL) eight medicinal plants
BioMed Research International 7
Table 3 Hepatoprotective activity of various medicinal plant extracts on ALT and AST levels to neutralize snake bite envenomation inexperimental rabbits
Sr number Group V(subgroup) Name of evaluated sample GPTALT GOTAST
UL(mean plusmn SEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 62 plusmn 702 05 gt 119875 gt 01 99 plusmn 1855 05 gt 119875 gt 01
2 (V2) Allium cepa L 68 plusmn 1203 05 gt 119875 gt 01 1665 plusmn 050 05 gt 119875 gt 01
3 (V3) Allium sativum L 49 plusmn 501 119875 gt 05 825 plusmn 1855 119875 gt 05
4 (V4) Althaea officinalisL 70 plusmn 2005 119875 gt 05 745 plusmn 1454 119875 gt 05
5 (V5) Bauhinia variegata L 78 plusmn 404 05 gt 119875 gt 01 1495 plusmn 199 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 45 plusmn 000 05 gt 119875 gt 01 55 plusmn 100 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 315 plusmn 05 05 gt 119875 gt 01 141 plusmn 101 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 625 plusmn 252 05 gt 119875 gt 01 194 plusmn 1212 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 633 plusmn 414 05 gt 119875 gt 01 72 plusmn 202 119875 gt 05
10 (V9) Citrus limon (L) Burm f 305 plusmn 1363 05 gt 119875 gt 01 49 plusmn 2424 119875 gt 05
11 (V10) Citrullus colocynthis (L) Schrad 475 plusmn 555 119875 gt 05 74 plusmn 909 119875 gt 05
12 (V11) Cuminum cyminum L 67 plusmn 1616 119875 gt 05 157 plusmn 1414 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 8525 plusmn 126 01 gt 119875 gt
0051685 plusmn 757 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 80 plusmn 782 05 gt 119875 gt 01 170 plusmn 1313 05 gt 119875 gt 01
15 (V14) Leucas capitata Desf 455 plusmn 752 119875 gt 05 535 plusmn 050 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 62 plusmn 808 05 gt 119875 gt 01 121 plusmn 505 05 gt 119875 gt 01
17 (V16) Momordica charantia L 625 plusmn 752 05 gt 119875 gt 01 247 plusmn 4613 05 gt 119875 gt 01
18 (V17) Nerium indicumMill 88 plusmn 1955 05 gt 119875 gt 01 235 plusmn 1805 01 gt 119875 gt
005
19 (V18) Ocimum sanctum L 615 plusmn 752 05 gt 119875 gt 01 158 plusmn 1002 05 gt 119875 gt 01
20 (V19) Pinus roxburghii Sarg 53 plusmn 902 119875 gt 05 57 plusmn 1303 119875 gt 05
21 (V20) Pistacia integerrima J L Stewart 415 plusmn 15 05 gt 119875 gt 01 545 plusmn 050 119875 gt 05
22 (V21) Psoralea corylifolia L 54 plusmn 100 119875 gt 05 665 plusmn 1153 119875 gt 05
23 (V22) Rhazya stricta Decne 66 plusmn 2424 119875 gt 05 1325 plusmn 1262 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 655 plusmn 1767 119875 gt 05 129 plusmn 6641 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 56 plusmn 601 119875 gt 05 785 plusmn 1354 119875 gt 05
26 (V25) Stenolobium stans (L) Seem 465 plusmn 656 119875 gt 05 705 plusmn 353 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 475 plusmn 151 05 gt 119875 gt 01 78 plusmn 606 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 52 plusmn 000 119875 gt 05 125 plusmn 401 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 665 plusmn 3259 119875 gt 05 1175 plusmn 952 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
525 plusmn 351 Select tocompare 695 plusmn 1855 Select to
compare
(Allium cepa L Althaea officinalis L Bauhinia variegataL Cedrus deodara (Roxb ex D Don) G Don Fagoniacretica L Leucas capitataDesf Momordica charantia L andOcimum sanctum L) were found equally capable (119875 gt 05) ofnormalizing high values of CK-MB however rest of all wereproved less beneficial (05 gt 119875 gt 01) To combat highlyraised values of LDH Althaea officinalis L Leucas capitataDesf and Terminalia arjuna (Roxb ex DC) Wight and Arnwere proved useful to some extent (05 gt 119875 gt 01) although
remaining plant extracts could not be shortlisted (01 gt 119875 gt0001) as standard antisera (LDH = 763 plusmn 601UL) Overalldetail about LDH is discussed in Table 5 and comparison isshown in Figure 3
4 Discussion
Snake bite has been responsible for tens of thousands ofdeaths worldwide and numerous physical handicaps [29]
8 BioMed Research International
Table 4 Nephroprotective activity of different medicinal plant extracts on urea and creatinine levels to neutralize snake bite envenomationin experimental rabbits
Sr number Group V(subgroup) Name of evaluated sample Urea Creatinine
mgdL(mean plusmn SEM)
119875 valuecomment
mgdL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 57 plusmn 00 002 gt 119875 gt 001 16 plusmn 020 05 gt 119875 gt 01
2 (V2) Allium cepa L 51 plusmn 1103 05 gt 119875 gt 01 150 plusmn 017 05 gt 119875 gt 01
3 (V3) Allium sativum L 50 plusmn 401 05 gt 119875 gt 01 130 plusmn 006 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 26 plusmn 505 119875 gt 05 118 plusmn 024 119875 gt 05
5 (V5) Bauhinia variegata L 50 plusmn 126 05 gt 119875 gt 01 136 plusmn 024 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 225 plusmn 451 05 gt 119875 gt 01 107 plusmn 011 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 425 plusmn 252 05 gt 119875 gt 01 130 plusmn 001 01 gt 119875 gt 005
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 39 plusmn 202 05 gt 119875 gt 01 125 plusmn 003 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 44 plusmn 404 05 gt 119875 gt 01 144 plusmn 007 05 gt 119875 gt 01
10 (V9) Citrus limon (L) Burm f 395 plusmn 252 05 gt 119875 gt 01 140 plusmn 003 01 gt 119875 gt 005
11 (V10) Citrullus colocynthis (L) Schrad 495 plusmn 353 05 gt 119875 gt 01 152 plusmn 015 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 54 plusmn 101 005 gt 119875 gt 002 148 plusmn 001 005 gt 119875 gt 002
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 395 plusmn 454 05 gt 119875 gt 01 135 plusmn 005 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 43 plusmn 227 05 gt 119875 gt 01 123 plusmn 013 119875 lt 05
15 (V14) Leucas capitata Desf 315 plusmn 050 119875 gt 05 107 plusmn 005 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 435 plusmn 353 05 gt 119875 gt 01 144 plusmn 014 05 gt 119875 gt 01
17 (V16) Momordica charantia L 57 plusmn 501 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
18 (V17) Nerium indicumMill 47 plusmn 000 119875 lt 005 124 plusmn 010 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 495 plusmn 752 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
20 (V19) Pinus roxburghii Sarg 45 plusmn 100 119875 gt 005 14 plusmn 005 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 665 plusmn 050 002 gt 119875 gt 001 168 plusmn 010 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 425 plusmn 250 05 gt 119875 gt 01 127 plusmn 015 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 535 plusmn 353 05 gt 119875 gt 01 167 plusmn 0005 005 gt 119875 gt 002
24 (V23) Rubia cordifolia L 36 plusmn 101 05 gt 119875 gt 01 093 plusmn 048 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 44 plusmn 100 01 gt 119875 gt 005 143 plusmn 012 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 255 plusmn 651 119875 gt 05 11 plusmn 006 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 385 plusmn 151 05 gt 119875 gt 01 127 plusmn 023 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 37 plusmn 100 05 gt 119875 gt 01 046 plusmn 001 005 gt 119875 gt 002
29 (V28) Zingiber officinale Roscoe 47 plusmn 100 005 gt 119875 gt 002 130 plusmn 001 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
315 plusmn 050 Select tocompare 108 plusmn 002 Select to
compare
Generally snake venom is an intricate mixture of variousproteins (gt90) and most of them are enzymes particularly(40) phospholipases A
2[30] Among different complica-
tions hepatic injury is one of the deadly venomous effectsproduced by cobra bite [31 32] Naja naja karachiensisvenom caused significant increase in ALT and AST levels(dose dependently) that are surrogate markers for livertoxicity either by direct action or immunological (hyper-sensitivity) reaction The allergic reactions reactions are notdose dependent which clarify lethal effects to the cytoplasm
and mitochondrial membranes of the hepatic cells [25 33]Phospholipases enzymes abundant in Naja naja karachien-sis venom are responsible for breakdown of membranousphospholipids and therefore resulted in cellular injury alongwith inflammation [24] PLA
2is attributed to decrease in
Na+K+ ATPase activities and led to greater influx of sodiumions and water molecules into the cell Subsequently plasmamembrane lipid bilayer disorganized and eventually resultedin hepatic cells death [33 34] In addition PLA
2is also found
to cause anticoagulation in victims [6] All pharmacological
BioMed Research International 9
Table 5 Cardioprotective activity of medicinal plant extracts on LDH and CK-MB levels to neutralize snake bite envenomation inexperimental rabbits
Sr numberGroup V(subgroup) Name of evaluated sample LDH CK-MB
UL (mean plusmnSEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 1357 plusmn 100 001 gt 119875 gt 0001 41 plusmn 085 05 gt 119875 gt 01
2 (V2) Allium cepa L 934 plusmn 1303 01 gt 119875 gt 005 148 plusmn 165 119875 gt 05
3 (V3) Allium sativum L 1177 plusmn 2056 005 gt 119875 gt 002 66 plusmn 330 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 9755 plusmn 3360 05 gt 119875 gt 01 148 plusmn 330 119875 gt 05
5 (V5) Bauhinia variegata L 1972 plusmn 300 119875 gt 0001 83 plusmn 676 119875 gt 05
6 (V6) Brassica nigra (L) W D J Koch 8555 plusmn 050 005 gt 119875 gt 002 90 plusmn 085 05 gt 119875 gt 01
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 1022 plusmn 501 002 gt 119875 gt 001 66 plusmn 330 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 1114 plusmn 100 002 gt 119875 gt 001 618 plusmn 109 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 1230 plusmn 2357 005 gt 119875 gt 002 412 plusmn 3166 119875 gt 05
10 (V9) Citrus limon (L) Burm f 1831 plusmn 6569 005 gt 119875 gt 002 73 plusmn 141 05 gt 119875 gt 01
11 (V10) Citrullus colocynthis (L) Schrad 827 plusmn 651 01 gt 119875 gt 005 05 plusmn 166 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 1589 plusmn 2256 002 gt 119875 gt 001 58 plusmn 080 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 1615 plusmn 151 119875 gt 0001 985 plusmn 166 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 1418 plusmn 1303 002 gt 119875 gt 001 11 plusmn 252 119875 gt 05
15 (V14) Leucas capitata Desf 783 plusmn 1002 05 gt 119875 gt 01 14 plusmn 080 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 1428 plusmn 651 119875 gt 0001 08 plusmn 166 05 gt 119875 gt 01
17 (V16) Momordica charantia L 14755 plusmn 351 119875 gt 0001 156 plusmn 245 119875 gt 05
18 (V17) Nerium indicumMill 1268 plusmn 1203 002 gt 119875 gt 001 05 plusmn 165 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 1335 plusmn 1203 002 gt 119875 gt 001 123 plusmn 245 119875 gt 05
20 (V19) Pinus roxburghii Sarg 1050 plusmn 100 002 gt 119875 gt 001 08 plusmn 165 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 11355 plusmn 05 002 gt 119875 gt 001 131 plusmn 165 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 11535 plusmn 05 119875 gt 0001 173 plusmn 250 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 1538 plusmn 2005 002 gt 119875 gt 001 41 plusmn 085 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 1078 plusmn 1604 005 gt 119875 gt 002 66 plusmn 000 05 gt 119875 gt 01
25 (V24) Sapindus mukorossi Gaertn 14605 plusmn 551 001 gt 119875 gt 0001 41 plusmn 080 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 787 plusmn 2808 119875 gt 05 13 plusmn 176 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 7985 plusmn 1454 05 gt 119875 gt 01 66 plusmn 000 05 gt 119875 gt 01
28 (V27) Trichodesma indicum (L) Sm 9785 plusmn 451 005 gt 119875 gt 002 66 plusmn 300 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 888 plusmn 200 005 gt 119875 gt 002 173 plusmn 080 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
763 plusmn 601 Select to compare 09 plusmn 085 Select to compare
effects are due to phospholipid hydrolysis or phospholipidcompeting bidingmechanismwith other coagulation factorsparticularly FXa or by both ways however it is very hardto pinpoint exact one [35] Anticoagulant response posed bycobra PLA
2(anticoagulant enzyme) may aggravate hepatic
injury as many anticoagulant agents cause liver damage thatare idiosyncratic in nature [36]
Cardiac injury particularly systolic heart arrest is one ofthewell-known toxicities related toNaja naja subspecies [30]In present study Pakistani cobra venom was found to release
two cystolic enzymes (LDH and CK-MB) that are sensitiveindicators of myocyte injury [37] It is due to the presenceof myotoxic PLA
2and other cardiotoxin(s) that are salient
features of cobra venom that are responsible for cellularnecrosis and cytotoxicity [38 39] Cytotoxic effect of Najanaja karachiensis venom may not be overlooked even whenantisera administration is late cause of toxic components tothe microvasculature and thrombus produced which poseshindrance in access of immunoglobulins to the site of snakebite [26 29 39]
10 BioMed Research International
0
10
20
30
40
50
60
70Se
rum
leve
l of u
rea a
nd cr
eatin
ine (
mg
dL)
UreaCreatinine
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
L
Rhaz
ya st
ricta
Dec
neRu
bia
cord
ifolia
L
Sapi
ndus
muk
oros
si G
aert
nSt
enol
obiu
m st
ans (
L) S
eem
Term
inal
ia a
rjuna
(Rox
b ex
DC
) Wig
ht an
d A
rnTr
ichod
esm
a in
dicu
m (L
) Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antidotes tested (100mgkg)
Figure 2 Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea andcreatinine level posed by Naja naja karachiensis venom in rabbits
Snake venom has been responsible for detrimental effectsto the renal tissues [40] Likewise Naja naja krachiensisvenomwas found to cause severe renal damage by significantrise in serum urea and creatinine levels Indeed phospho-lipases enzymes are responsible for the increase in vascularpermeability with hemorrhagic effects to the vital tissues inthe victims Subsequently numerous lesions are producedrelated to the glomerular membrane and renal tubules eitherinterstitial or vascular [29 41 42] Presence of lymphocytes(white blood cells) and oedema in the cortical as well asmedullary regions of renal tubules further confirmed theidea of renal damage (data has not shown) as reportedpreviously with different snake venoms like Hemiscorpiuslepturus [26 43] Apart of it pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope99mTc confirmed that kidneys and urinary bladder are themost saturated organs (gt70) after intravenous injection inexperimental rabbits (unpublished data by our group)
Medicinal plants of Pakistan are used to inhibit snakevenom (PLA
2) enzymes [6] Due to this effect present
study was designed to select twenty-eight medicinal plantsof Pakistan to test their potentials as an antidote againsttoxicities produced by Naja naja karachiensis venom It
was fascinating that extract of Stenolobium stans (L) Seemshowed significant neutralization compared to that of refer-ence standard (antisera) as reported previously by Asad etal for inhibition of phospholipases A
2anticoagulant activity
[6 44] Present study indicated that extract of Stenolobiumstans (L) Seem possesses an endogenous inhibitor(s) tonullify venom (PLA
2) induced toxicities Extracts of these
plants material are routinely used in Pakistan by simpleapplication in the form of paste to the affected area [45]This practice provides effective first aid treatment as tinymolecules of an antidote diffuse favorably at the site of snakebite before hospitalization and abrogated the spreading oftoxins [39] It is owing to the various secondary metaboliteslike phenols flavonoids terpenoids xanthenes quinonoidsand so forth as reported earlier to mask various enzymaticactions of cobra venom [6 23] Indeed secondarymetabolitespose hindrance in binding of different snake venom enzymesto their potential targets therefore antidotal effect evokedOther plants extract were not proved significantly effectiveto neutralize cobra venom therefore cannot be declareduseful plants in venom therapy It is the need of the time toisolate lethal component(s) of cobra venom attributed to itsmajor toxicities Furthermore characterization of antivenom
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Volume 2014
Zoology
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Microbiology
BioMed Research International 5
Table2Biochemicalparametersb
eforea
ndaft
ersnakev
enom
administratio
nin
different
experim
entalgroup
sofrabbits
Toxicitydeterm
ined
Markersof
snake
veno
mtoxicity
Group
Ibaseline
measurements
(meanplusmnSE
M)
Group
IIandIIIv
enom
injection
Group
IVnegativ
econ
trol
(meanplusmnSE
M)
Normalreference
values
repo
rted
References
04m
gkg
(meanplusmnSE
M)
08m
gkg
(meanplusmnSE
M)
Liver
GPT
ALT
(52plusmn346
)UL
(72plusmn470)U
L01gt119875gt005
(86plusmn50)
UL
005gt119875gt002
(52plusmn348)U
L(48ndash80)U
L[19
]
GOTAST
(65plusmn657)U
L(157plusmn2424)
UL
01gt119875gt005
(251plusmn182)U
L001gt119875gt0001
(67plusmn321)U
L(14
ndash113)U
L[19
]
Kidn
eys
Urea
(28plusmn17
3)mgdL
(42plusmn308)m
gdL
005gt119875gt002
(576plusmn384)m
gdL
002gt119875gt001
(28plusmn033)m
gdL
(10ndash
28)m
gdL
[26]
Creatin
ine
(10plusmn0313)
mgdL
(174plusmn003)m
gdL
001gt119875gt0001
(21plusmn010)m
gdL
002gt119875gt001
(1plusmn006)m
gdL
(05ndash25)m
gdL
[26]
Heart
CK-M
B(132plusmn20)
UL
(21plusmn
15)U
L005gt119875gt002
(77plusmn1122)
UL
005gt119875gt002
(13plusmn056)U
L(lt25)U
Llowast
[2021]
LDH
(714plusmn318)U
L(206
4plusmn1598)
UL
119875lt0001
(2562plusmn2514
)UL
119875≪0001
(720plusmn470)U
L(559ndash2077)
UL
[46]
lowastCK
-MBactiv
ityislessthan
2in
healthyrabbitshow
everitisu
sually10ndash30
oftotalC
Kactiv
ity
6 BioMed Research International
0
50
100
150
200
250
300Se
rum
leve
l of G
PTG
OT
(UL
)
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
LRh
azya
stric
ta D
ecne
Rubi
a co
rdifo
lia L
Sa
pind
us m
ukor
ossi
Gae
rtn
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C) W
ight
and
Arn
Trich
odes
ma
indi
cum
(L)
SmZi
ngib
er o
fficin
ale R
osco
eRe
fere
nce s
tand
ard
anti
dote
GPT levelGOT level
Various antidotes used (100mgkg)
Figure 1 Comparison of various medicinal plant extracts with reference standard antidote in neutralization of increased GPT and GOTserum level posed by Naja naja karachiensis venom in rabbits
kits (manufactured by Merck) according to the DGKC andIFCCmethod on the Selectra Junior (Vital Scientific BVTheNetherlands) [26]
26 Statistical Analysis All numerical values were expressedas mean (3 replicates) plusmn standard error of mean (SEM) Theywere calculated by the use of Microsoft Excel 2007 and Stu-dentrsquos t-test was applied to compare the efficacy of evaluatedsamples with standard antidote (reference standard)
3 Results
Venom from Naja naja karachiensis was proved to causesevere complications in dose-dependent manner Venom atthe dose of 400 120583gkg led to release of moderate serum levelsof ALT (72plusmn470UL 01 gt 119875 gt 005) AST (157plusmn2424UL01 gt 119875 gt 005) urea (42 plusmn 308mgdL 005 gt 119875 gt 002)creatinine (174 plusmn 003mgdL 001 gt 119875 gt 0001) CK-MB(21 plusmn 15UL 005 gt 119875 gt 002) and LDH (2064 plusmn 1598UL119875 lt 0001) and thus indicated toxicities to the heart liver andkidneys At 800 120583gkg of cobra venom severe tissue damagewas observed in terms of significant release of ALT (86 plusmn50UL 005 gt 119875 gt 002) AST (251 plusmn 182UL 001 gt 119875 gt0001) urea (576 plusmn 384mgdL 002 gt 119875 gt 001) creatinine
(21 plusmn 010mgdL 002 gt 119875 gt 001) CK-MB (77plusmn 1122UL005 gt 119875 gt 002) and LDH (2562 plusmn 2514UL 119875 ≪ 0001)Complete detail about various biochemical parameters forbaseline measurements different doses of cobra venom andsaline (as negative control) is summarized in Table 2
To neutralize in vivo 800 120583gkg of cobra venom twenty-eight medicinal plant extracts were evaluated It was noticedthat extract of Stenolobium stans (L) Seem was the bestantidote (119875 gt 05) compared to reference standard Alliumsativum L Althaea officinalis L Citrullus colocynthis (L)Schrad Leucas capitataDesf Pinus roxburghii Sarg Psoraleacorylifolia L Rubia cordifolia L and Sapindus mukorossiGaertn were found to be valuable plants to protect liverdamage (119875 gt 05) as standard antidote (ALT = 525 plusmn351UL and AST = 695 plusmn 1855UL) however requiredALT and AST levels were not observed with remainingplant extracts (05 gt 119875 gt 005) as shown in Table 3 andcomparison is shown in Figure 1 Extracts of Leucas capitataDesf and Althaea officinalis L were observed to be helpful(119875 gt 05) as reference standard (urea = 315 plusmn 050mgdLand creatinine = 108 plusmn 002mgdL) nevertheless remainingplants were found less valuable (05 gt 119875 gt 001) tominimize urea and creatinine levels to maintain kidney func-tions (Table 4 and Figure 2) When compared with standardantidote (CK-MB = 90 plusmn 085UL) eight medicinal plants
BioMed Research International 7
Table 3 Hepatoprotective activity of various medicinal plant extracts on ALT and AST levels to neutralize snake bite envenomation inexperimental rabbits
Sr number Group V(subgroup) Name of evaluated sample GPTALT GOTAST
UL(mean plusmn SEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 62 plusmn 702 05 gt 119875 gt 01 99 plusmn 1855 05 gt 119875 gt 01
2 (V2) Allium cepa L 68 plusmn 1203 05 gt 119875 gt 01 1665 plusmn 050 05 gt 119875 gt 01
3 (V3) Allium sativum L 49 plusmn 501 119875 gt 05 825 plusmn 1855 119875 gt 05
4 (V4) Althaea officinalisL 70 plusmn 2005 119875 gt 05 745 plusmn 1454 119875 gt 05
5 (V5) Bauhinia variegata L 78 plusmn 404 05 gt 119875 gt 01 1495 plusmn 199 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 45 plusmn 000 05 gt 119875 gt 01 55 plusmn 100 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 315 plusmn 05 05 gt 119875 gt 01 141 plusmn 101 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 625 plusmn 252 05 gt 119875 gt 01 194 plusmn 1212 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 633 plusmn 414 05 gt 119875 gt 01 72 plusmn 202 119875 gt 05
10 (V9) Citrus limon (L) Burm f 305 plusmn 1363 05 gt 119875 gt 01 49 plusmn 2424 119875 gt 05
11 (V10) Citrullus colocynthis (L) Schrad 475 plusmn 555 119875 gt 05 74 plusmn 909 119875 gt 05
12 (V11) Cuminum cyminum L 67 plusmn 1616 119875 gt 05 157 plusmn 1414 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 8525 plusmn 126 01 gt 119875 gt
0051685 plusmn 757 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 80 plusmn 782 05 gt 119875 gt 01 170 plusmn 1313 05 gt 119875 gt 01
15 (V14) Leucas capitata Desf 455 plusmn 752 119875 gt 05 535 plusmn 050 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 62 plusmn 808 05 gt 119875 gt 01 121 plusmn 505 05 gt 119875 gt 01
17 (V16) Momordica charantia L 625 plusmn 752 05 gt 119875 gt 01 247 plusmn 4613 05 gt 119875 gt 01
18 (V17) Nerium indicumMill 88 plusmn 1955 05 gt 119875 gt 01 235 plusmn 1805 01 gt 119875 gt
005
19 (V18) Ocimum sanctum L 615 plusmn 752 05 gt 119875 gt 01 158 plusmn 1002 05 gt 119875 gt 01
20 (V19) Pinus roxburghii Sarg 53 plusmn 902 119875 gt 05 57 plusmn 1303 119875 gt 05
21 (V20) Pistacia integerrima J L Stewart 415 plusmn 15 05 gt 119875 gt 01 545 plusmn 050 119875 gt 05
22 (V21) Psoralea corylifolia L 54 plusmn 100 119875 gt 05 665 plusmn 1153 119875 gt 05
23 (V22) Rhazya stricta Decne 66 plusmn 2424 119875 gt 05 1325 plusmn 1262 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 655 plusmn 1767 119875 gt 05 129 plusmn 6641 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 56 plusmn 601 119875 gt 05 785 plusmn 1354 119875 gt 05
26 (V25) Stenolobium stans (L) Seem 465 plusmn 656 119875 gt 05 705 plusmn 353 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 475 plusmn 151 05 gt 119875 gt 01 78 plusmn 606 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 52 plusmn 000 119875 gt 05 125 plusmn 401 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 665 plusmn 3259 119875 gt 05 1175 plusmn 952 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
525 plusmn 351 Select tocompare 695 plusmn 1855 Select to
compare
(Allium cepa L Althaea officinalis L Bauhinia variegataL Cedrus deodara (Roxb ex D Don) G Don Fagoniacretica L Leucas capitataDesf Momordica charantia L andOcimum sanctum L) were found equally capable (119875 gt 05) ofnormalizing high values of CK-MB however rest of all wereproved less beneficial (05 gt 119875 gt 01) To combat highlyraised values of LDH Althaea officinalis L Leucas capitataDesf and Terminalia arjuna (Roxb ex DC) Wight and Arnwere proved useful to some extent (05 gt 119875 gt 01) although
remaining plant extracts could not be shortlisted (01 gt 119875 gt0001) as standard antisera (LDH = 763 plusmn 601UL) Overalldetail about LDH is discussed in Table 5 and comparison isshown in Figure 3
4 Discussion
Snake bite has been responsible for tens of thousands ofdeaths worldwide and numerous physical handicaps [29]
8 BioMed Research International
Table 4 Nephroprotective activity of different medicinal plant extracts on urea and creatinine levels to neutralize snake bite envenomationin experimental rabbits
Sr number Group V(subgroup) Name of evaluated sample Urea Creatinine
mgdL(mean plusmn SEM)
119875 valuecomment
mgdL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 57 plusmn 00 002 gt 119875 gt 001 16 plusmn 020 05 gt 119875 gt 01
2 (V2) Allium cepa L 51 plusmn 1103 05 gt 119875 gt 01 150 plusmn 017 05 gt 119875 gt 01
3 (V3) Allium sativum L 50 plusmn 401 05 gt 119875 gt 01 130 plusmn 006 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 26 plusmn 505 119875 gt 05 118 plusmn 024 119875 gt 05
5 (V5) Bauhinia variegata L 50 plusmn 126 05 gt 119875 gt 01 136 plusmn 024 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 225 plusmn 451 05 gt 119875 gt 01 107 plusmn 011 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 425 plusmn 252 05 gt 119875 gt 01 130 plusmn 001 01 gt 119875 gt 005
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 39 plusmn 202 05 gt 119875 gt 01 125 plusmn 003 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 44 plusmn 404 05 gt 119875 gt 01 144 plusmn 007 05 gt 119875 gt 01
10 (V9) Citrus limon (L) Burm f 395 plusmn 252 05 gt 119875 gt 01 140 plusmn 003 01 gt 119875 gt 005
11 (V10) Citrullus colocynthis (L) Schrad 495 plusmn 353 05 gt 119875 gt 01 152 plusmn 015 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 54 plusmn 101 005 gt 119875 gt 002 148 plusmn 001 005 gt 119875 gt 002
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 395 plusmn 454 05 gt 119875 gt 01 135 plusmn 005 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 43 plusmn 227 05 gt 119875 gt 01 123 plusmn 013 119875 lt 05
15 (V14) Leucas capitata Desf 315 plusmn 050 119875 gt 05 107 plusmn 005 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 435 plusmn 353 05 gt 119875 gt 01 144 plusmn 014 05 gt 119875 gt 01
17 (V16) Momordica charantia L 57 plusmn 501 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
18 (V17) Nerium indicumMill 47 plusmn 000 119875 lt 005 124 plusmn 010 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 495 plusmn 752 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
20 (V19) Pinus roxburghii Sarg 45 plusmn 100 119875 gt 005 14 plusmn 005 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 665 plusmn 050 002 gt 119875 gt 001 168 plusmn 010 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 425 plusmn 250 05 gt 119875 gt 01 127 plusmn 015 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 535 plusmn 353 05 gt 119875 gt 01 167 plusmn 0005 005 gt 119875 gt 002
24 (V23) Rubia cordifolia L 36 plusmn 101 05 gt 119875 gt 01 093 plusmn 048 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 44 plusmn 100 01 gt 119875 gt 005 143 plusmn 012 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 255 plusmn 651 119875 gt 05 11 plusmn 006 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 385 plusmn 151 05 gt 119875 gt 01 127 plusmn 023 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 37 plusmn 100 05 gt 119875 gt 01 046 plusmn 001 005 gt 119875 gt 002
29 (V28) Zingiber officinale Roscoe 47 plusmn 100 005 gt 119875 gt 002 130 plusmn 001 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
315 plusmn 050 Select tocompare 108 plusmn 002 Select to
compare
Generally snake venom is an intricate mixture of variousproteins (gt90) and most of them are enzymes particularly(40) phospholipases A
2[30] Among different complica-
tions hepatic injury is one of the deadly venomous effectsproduced by cobra bite [31 32] Naja naja karachiensisvenom caused significant increase in ALT and AST levels(dose dependently) that are surrogate markers for livertoxicity either by direct action or immunological (hyper-sensitivity) reaction The allergic reactions reactions are notdose dependent which clarify lethal effects to the cytoplasm
and mitochondrial membranes of the hepatic cells [25 33]Phospholipases enzymes abundant in Naja naja karachien-sis venom are responsible for breakdown of membranousphospholipids and therefore resulted in cellular injury alongwith inflammation [24] PLA
2is attributed to decrease in
Na+K+ ATPase activities and led to greater influx of sodiumions and water molecules into the cell Subsequently plasmamembrane lipid bilayer disorganized and eventually resultedin hepatic cells death [33 34] In addition PLA
2is also found
to cause anticoagulation in victims [6] All pharmacological
BioMed Research International 9
Table 5 Cardioprotective activity of medicinal plant extracts on LDH and CK-MB levels to neutralize snake bite envenomation inexperimental rabbits
Sr numberGroup V(subgroup) Name of evaluated sample LDH CK-MB
UL (mean plusmnSEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 1357 plusmn 100 001 gt 119875 gt 0001 41 plusmn 085 05 gt 119875 gt 01
2 (V2) Allium cepa L 934 plusmn 1303 01 gt 119875 gt 005 148 plusmn 165 119875 gt 05
3 (V3) Allium sativum L 1177 plusmn 2056 005 gt 119875 gt 002 66 plusmn 330 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 9755 plusmn 3360 05 gt 119875 gt 01 148 plusmn 330 119875 gt 05
5 (V5) Bauhinia variegata L 1972 plusmn 300 119875 gt 0001 83 plusmn 676 119875 gt 05
6 (V6) Brassica nigra (L) W D J Koch 8555 plusmn 050 005 gt 119875 gt 002 90 plusmn 085 05 gt 119875 gt 01
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 1022 plusmn 501 002 gt 119875 gt 001 66 plusmn 330 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 1114 plusmn 100 002 gt 119875 gt 001 618 plusmn 109 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 1230 plusmn 2357 005 gt 119875 gt 002 412 plusmn 3166 119875 gt 05
10 (V9) Citrus limon (L) Burm f 1831 plusmn 6569 005 gt 119875 gt 002 73 plusmn 141 05 gt 119875 gt 01
11 (V10) Citrullus colocynthis (L) Schrad 827 plusmn 651 01 gt 119875 gt 005 05 plusmn 166 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 1589 plusmn 2256 002 gt 119875 gt 001 58 plusmn 080 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 1615 plusmn 151 119875 gt 0001 985 plusmn 166 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 1418 plusmn 1303 002 gt 119875 gt 001 11 plusmn 252 119875 gt 05
15 (V14) Leucas capitata Desf 783 plusmn 1002 05 gt 119875 gt 01 14 plusmn 080 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 1428 plusmn 651 119875 gt 0001 08 plusmn 166 05 gt 119875 gt 01
17 (V16) Momordica charantia L 14755 plusmn 351 119875 gt 0001 156 plusmn 245 119875 gt 05
18 (V17) Nerium indicumMill 1268 plusmn 1203 002 gt 119875 gt 001 05 plusmn 165 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 1335 plusmn 1203 002 gt 119875 gt 001 123 plusmn 245 119875 gt 05
20 (V19) Pinus roxburghii Sarg 1050 plusmn 100 002 gt 119875 gt 001 08 plusmn 165 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 11355 plusmn 05 002 gt 119875 gt 001 131 plusmn 165 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 11535 plusmn 05 119875 gt 0001 173 plusmn 250 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 1538 plusmn 2005 002 gt 119875 gt 001 41 plusmn 085 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 1078 plusmn 1604 005 gt 119875 gt 002 66 plusmn 000 05 gt 119875 gt 01
25 (V24) Sapindus mukorossi Gaertn 14605 plusmn 551 001 gt 119875 gt 0001 41 plusmn 080 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 787 plusmn 2808 119875 gt 05 13 plusmn 176 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 7985 plusmn 1454 05 gt 119875 gt 01 66 plusmn 000 05 gt 119875 gt 01
28 (V27) Trichodesma indicum (L) Sm 9785 plusmn 451 005 gt 119875 gt 002 66 plusmn 300 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 888 plusmn 200 005 gt 119875 gt 002 173 plusmn 080 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
763 plusmn 601 Select to compare 09 plusmn 085 Select to compare
effects are due to phospholipid hydrolysis or phospholipidcompeting bidingmechanismwith other coagulation factorsparticularly FXa or by both ways however it is very hardto pinpoint exact one [35] Anticoagulant response posed bycobra PLA
2(anticoagulant enzyme) may aggravate hepatic
injury as many anticoagulant agents cause liver damage thatare idiosyncratic in nature [36]
Cardiac injury particularly systolic heart arrest is one ofthewell-known toxicities related toNaja naja subspecies [30]In present study Pakistani cobra venom was found to release
two cystolic enzymes (LDH and CK-MB) that are sensitiveindicators of myocyte injury [37] It is due to the presenceof myotoxic PLA
2and other cardiotoxin(s) that are salient
features of cobra venom that are responsible for cellularnecrosis and cytotoxicity [38 39] Cytotoxic effect of Najanaja karachiensis venom may not be overlooked even whenantisera administration is late cause of toxic components tothe microvasculature and thrombus produced which poseshindrance in access of immunoglobulins to the site of snakebite [26 29 39]
10 BioMed Research International
0
10
20
30
40
50
60
70Se
rum
leve
l of u
rea a
nd cr
eatin
ine (
mg
dL)
UreaCreatinine
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
L
Rhaz
ya st
ricta
Dec
neRu
bia
cord
ifolia
L
Sapi
ndus
muk
oros
si G
aert
nSt
enol
obiu
m st
ans (
L) S
eem
Term
inal
ia a
rjuna
(Rox
b ex
DC
) Wig
ht an
d A
rnTr
ichod
esm
a in
dicu
m (L
) Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antidotes tested (100mgkg)
Figure 2 Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea andcreatinine level posed by Naja naja karachiensis venom in rabbits
Snake venom has been responsible for detrimental effectsto the renal tissues [40] Likewise Naja naja krachiensisvenomwas found to cause severe renal damage by significantrise in serum urea and creatinine levels Indeed phospho-lipases enzymes are responsible for the increase in vascularpermeability with hemorrhagic effects to the vital tissues inthe victims Subsequently numerous lesions are producedrelated to the glomerular membrane and renal tubules eitherinterstitial or vascular [29 41 42] Presence of lymphocytes(white blood cells) and oedema in the cortical as well asmedullary regions of renal tubules further confirmed theidea of renal damage (data has not shown) as reportedpreviously with different snake venoms like Hemiscorpiuslepturus [26 43] Apart of it pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope99mTc confirmed that kidneys and urinary bladder are themost saturated organs (gt70) after intravenous injection inexperimental rabbits (unpublished data by our group)
Medicinal plants of Pakistan are used to inhibit snakevenom (PLA
2) enzymes [6] Due to this effect present
study was designed to select twenty-eight medicinal plantsof Pakistan to test their potentials as an antidote againsttoxicities produced by Naja naja karachiensis venom It
was fascinating that extract of Stenolobium stans (L) Seemshowed significant neutralization compared to that of refer-ence standard (antisera) as reported previously by Asad etal for inhibition of phospholipases A
2anticoagulant activity
[6 44] Present study indicated that extract of Stenolobiumstans (L) Seem possesses an endogenous inhibitor(s) tonullify venom (PLA
2) induced toxicities Extracts of these
plants material are routinely used in Pakistan by simpleapplication in the form of paste to the affected area [45]This practice provides effective first aid treatment as tinymolecules of an antidote diffuse favorably at the site of snakebite before hospitalization and abrogated the spreading oftoxins [39] It is owing to the various secondary metaboliteslike phenols flavonoids terpenoids xanthenes quinonoidsand so forth as reported earlier to mask various enzymaticactions of cobra venom [6 23] Indeed secondarymetabolitespose hindrance in binding of different snake venom enzymesto their potential targets therefore antidotal effect evokedOther plants extract were not proved significantly effectiveto neutralize cobra venom therefore cannot be declareduseful plants in venom therapy It is the need of the time toisolate lethal component(s) of cobra venom attributed to itsmajor toxicities Furthermore characterization of antivenom
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
6 BioMed Research International
0
50
100
150
200
250
300Se
rum
leve
l of G
PTG
OT
(UL
)
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
LRh
azya
stric
ta D
ecne
Rubi
a co
rdifo
lia L
Sa
pind
us m
ukor
ossi
Gae
rtn
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C) W
ight
and
Arn
Trich
odes
ma
indi
cum
(L)
SmZi
ngib
er o
fficin
ale R
osco
eRe
fere
nce s
tand
ard
anti
dote
GPT levelGOT level
Various antidotes used (100mgkg)
Figure 1 Comparison of various medicinal plant extracts with reference standard antidote in neutralization of increased GPT and GOTserum level posed by Naja naja karachiensis venom in rabbits
kits (manufactured by Merck) according to the DGKC andIFCCmethod on the Selectra Junior (Vital Scientific BVTheNetherlands) [26]
26 Statistical Analysis All numerical values were expressedas mean (3 replicates) plusmn standard error of mean (SEM) Theywere calculated by the use of Microsoft Excel 2007 and Stu-dentrsquos t-test was applied to compare the efficacy of evaluatedsamples with standard antidote (reference standard)
3 Results
Venom from Naja naja karachiensis was proved to causesevere complications in dose-dependent manner Venom atthe dose of 400 120583gkg led to release of moderate serum levelsof ALT (72plusmn470UL 01 gt 119875 gt 005) AST (157plusmn2424UL01 gt 119875 gt 005) urea (42 plusmn 308mgdL 005 gt 119875 gt 002)creatinine (174 plusmn 003mgdL 001 gt 119875 gt 0001) CK-MB(21 plusmn 15UL 005 gt 119875 gt 002) and LDH (2064 plusmn 1598UL119875 lt 0001) and thus indicated toxicities to the heart liver andkidneys At 800 120583gkg of cobra venom severe tissue damagewas observed in terms of significant release of ALT (86 plusmn50UL 005 gt 119875 gt 002) AST (251 plusmn 182UL 001 gt 119875 gt0001) urea (576 plusmn 384mgdL 002 gt 119875 gt 001) creatinine
(21 plusmn 010mgdL 002 gt 119875 gt 001) CK-MB (77plusmn 1122UL005 gt 119875 gt 002) and LDH (2562 plusmn 2514UL 119875 ≪ 0001)Complete detail about various biochemical parameters forbaseline measurements different doses of cobra venom andsaline (as negative control) is summarized in Table 2
To neutralize in vivo 800 120583gkg of cobra venom twenty-eight medicinal plant extracts were evaluated It was noticedthat extract of Stenolobium stans (L) Seem was the bestantidote (119875 gt 05) compared to reference standard Alliumsativum L Althaea officinalis L Citrullus colocynthis (L)Schrad Leucas capitataDesf Pinus roxburghii Sarg Psoraleacorylifolia L Rubia cordifolia L and Sapindus mukorossiGaertn were found to be valuable plants to protect liverdamage (119875 gt 05) as standard antidote (ALT = 525 plusmn351UL and AST = 695 plusmn 1855UL) however requiredALT and AST levels were not observed with remainingplant extracts (05 gt 119875 gt 005) as shown in Table 3 andcomparison is shown in Figure 1 Extracts of Leucas capitataDesf and Althaea officinalis L were observed to be helpful(119875 gt 05) as reference standard (urea = 315 plusmn 050mgdLand creatinine = 108 plusmn 002mgdL) nevertheless remainingplants were found less valuable (05 gt 119875 gt 001) tominimize urea and creatinine levels to maintain kidney func-tions (Table 4 and Figure 2) When compared with standardantidote (CK-MB = 90 plusmn 085UL) eight medicinal plants
BioMed Research International 7
Table 3 Hepatoprotective activity of various medicinal plant extracts on ALT and AST levels to neutralize snake bite envenomation inexperimental rabbits
Sr number Group V(subgroup) Name of evaluated sample GPTALT GOTAST
UL(mean plusmn SEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 62 plusmn 702 05 gt 119875 gt 01 99 plusmn 1855 05 gt 119875 gt 01
2 (V2) Allium cepa L 68 plusmn 1203 05 gt 119875 gt 01 1665 plusmn 050 05 gt 119875 gt 01
3 (V3) Allium sativum L 49 plusmn 501 119875 gt 05 825 plusmn 1855 119875 gt 05
4 (V4) Althaea officinalisL 70 plusmn 2005 119875 gt 05 745 plusmn 1454 119875 gt 05
5 (V5) Bauhinia variegata L 78 plusmn 404 05 gt 119875 gt 01 1495 plusmn 199 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 45 plusmn 000 05 gt 119875 gt 01 55 plusmn 100 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 315 plusmn 05 05 gt 119875 gt 01 141 plusmn 101 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 625 plusmn 252 05 gt 119875 gt 01 194 plusmn 1212 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 633 plusmn 414 05 gt 119875 gt 01 72 plusmn 202 119875 gt 05
10 (V9) Citrus limon (L) Burm f 305 plusmn 1363 05 gt 119875 gt 01 49 plusmn 2424 119875 gt 05
11 (V10) Citrullus colocynthis (L) Schrad 475 plusmn 555 119875 gt 05 74 plusmn 909 119875 gt 05
12 (V11) Cuminum cyminum L 67 plusmn 1616 119875 gt 05 157 plusmn 1414 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 8525 plusmn 126 01 gt 119875 gt
0051685 plusmn 757 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 80 plusmn 782 05 gt 119875 gt 01 170 plusmn 1313 05 gt 119875 gt 01
15 (V14) Leucas capitata Desf 455 plusmn 752 119875 gt 05 535 plusmn 050 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 62 plusmn 808 05 gt 119875 gt 01 121 plusmn 505 05 gt 119875 gt 01
17 (V16) Momordica charantia L 625 plusmn 752 05 gt 119875 gt 01 247 plusmn 4613 05 gt 119875 gt 01
18 (V17) Nerium indicumMill 88 plusmn 1955 05 gt 119875 gt 01 235 plusmn 1805 01 gt 119875 gt
005
19 (V18) Ocimum sanctum L 615 plusmn 752 05 gt 119875 gt 01 158 plusmn 1002 05 gt 119875 gt 01
20 (V19) Pinus roxburghii Sarg 53 plusmn 902 119875 gt 05 57 plusmn 1303 119875 gt 05
21 (V20) Pistacia integerrima J L Stewart 415 plusmn 15 05 gt 119875 gt 01 545 plusmn 050 119875 gt 05
22 (V21) Psoralea corylifolia L 54 plusmn 100 119875 gt 05 665 plusmn 1153 119875 gt 05
23 (V22) Rhazya stricta Decne 66 plusmn 2424 119875 gt 05 1325 plusmn 1262 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 655 plusmn 1767 119875 gt 05 129 plusmn 6641 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 56 plusmn 601 119875 gt 05 785 plusmn 1354 119875 gt 05
26 (V25) Stenolobium stans (L) Seem 465 plusmn 656 119875 gt 05 705 plusmn 353 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 475 plusmn 151 05 gt 119875 gt 01 78 plusmn 606 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 52 plusmn 000 119875 gt 05 125 plusmn 401 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 665 plusmn 3259 119875 gt 05 1175 plusmn 952 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
525 plusmn 351 Select tocompare 695 plusmn 1855 Select to
compare
(Allium cepa L Althaea officinalis L Bauhinia variegataL Cedrus deodara (Roxb ex D Don) G Don Fagoniacretica L Leucas capitataDesf Momordica charantia L andOcimum sanctum L) were found equally capable (119875 gt 05) ofnormalizing high values of CK-MB however rest of all wereproved less beneficial (05 gt 119875 gt 01) To combat highlyraised values of LDH Althaea officinalis L Leucas capitataDesf and Terminalia arjuna (Roxb ex DC) Wight and Arnwere proved useful to some extent (05 gt 119875 gt 01) although
remaining plant extracts could not be shortlisted (01 gt 119875 gt0001) as standard antisera (LDH = 763 plusmn 601UL) Overalldetail about LDH is discussed in Table 5 and comparison isshown in Figure 3
4 Discussion
Snake bite has been responsible for tens of thousands ofdeaths worldwide and numerous physical handicaps [29]
8 BioMed Research International
Table 4 Nephroprotective activity of different medicinal plant extracts on urea and creatinine levels to neutralize snake bite envenomationin experimental rabbits
Sr number Group V(subgroup) Name of evaluated sample Urea Creatinine
mgdL(mean plusmn SEM)
119875 valuecomment
mgdL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 57 plusmn 00 002 gt 119875 gt 001 16 plusmn 020 05 gt 119875 gt 01
2 (V2) Allium cepa L 51 plusmn 1103 05 gt 119875 gt 01 150 plusmn 017 05 gt 119875 gt 01
3 (V3) Allium sativum L 50 plusmn 401 05 gt 119875 gt 01 130 plusmn 006 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 26 plusmn 505 119875 gt 05 118 plusmn 024 119875 gt 05
5 (V5) Bauhinia variegata L 50 plusmn 126 05 gt 119875 gt 01 136 plusmn 024 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 225 plusmn 451 05 gt 119875 gt 01 107 plusmn 011 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 425 plusmn 252 05 gt 119875 gt 01 130 plusmn 001 01 gt 119875 gt 005
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 39 plusmn 202 05 gt 119875 gt 01 125 plusmn 003 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 44 plusmn 404 05 gt 119875 gt 01 144 plusmn 007 05 gt 119875 gt 01
10 (V9) Citrus limon (L) Burm f 395 plusmn 252 05 gt 119875 gt 01 140 plusmn 003 01 gt 119875 gt 005
11 (V10) Citrullus colocynthis (L) Schrad 495 plusmn 353 05 gt 119875 gt 01 152 plusmn 015 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 54 plusmn 101 005 gt 119875 gt 002 148 plusmn 001 005 gt 119875 gt 002
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 395 plusmn 454 05 gt 119875 gt 01 135 plusmn 005 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 43 plusmn 227 05 gt 119875 gt 01 123 plusmn 013 119875 lt 05
15 (V14) Leucas capitata Desf 315 plusmn 050 119875 gt 05 107 plusmn 005 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 435 plusmn 353 05 gt 119875 gt 01 144 plusmn 014 05 gt 119875 gt 01
17 (V16) Momordica charantia L 57 plusmn 501 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
18 (V17) Nerium indicumMill 47 plusmn 000 119875 lt 005 124 plusmn 010 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 495 plusmn 752 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
20 (V19) Pinus roxburghii Sarg 45 plusmn 100 119875 gt 005 14 plusmn 005 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 665 plusmn 050 002 gt 119875 gt 001 168 plusmn 010 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 425 plusmn 250 05 gt 119875 gt 01 127 plusmn 015 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 535 plusmn 353 05 gt 119875 gt 01 167 plusmn 0005 005 gt 119875 gt 002
24 (V23) Rubia cordifolia L 36 plusmn 101 05 gt 119875 gt 01 093 plusmn 048 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 44 plusmn 100 01 gt 119875 gt 005 143 plusmn 012 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 255 plusmn 651 119875 gt 05 11 plusmn 006 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 385 plusmn 151 05 gt 119875 gt 01 127 plusmn 023 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 37 plusmn 100 05 gt 119875 gt 01 046 plusmn 001 005 gt 119875 gt 002
29 (V28) Zingiber officinale Roscoe 47 plusmn 100 005 gt 119875 gt 002 130 plusmn 001 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
315 plusmn 050 Select tocompare 108 plusmn 002 Select to
compare
Generally snake venom is an intricate mixture of variousproteins (gt90) and most of them are enzymes particularly(40) phospholipases A
2[30] Among different complica-
tions hepatic injury is one of the deadly venomous effectsproduced by cobra bite [31 32] Naja naja karachiensisvenom caused significant increase in ALT and AST levels(dose dependently) that are surrogate markers for livertoxicity either by direct action or immunological (hyper-sensitivity) reaction The allergic reactions reactions are notdose dependent which clarify lethal effects to the cytoplasm
and mitochondrial membranes of the hepatic cells [25 33]Phospholipases enzymes abundant in Naja naja karachien-sis venom are responsible for breakdown of membranousphospholipids and therefore resulted in cellular injury alongwith inflammation [24] PLA
2is attributed to decrease in
Na+K+ ATPase activities and led to greater influx of sodiumions and water molecules into the cell Subsequently plasmamembrane lipid bilayer disorganized and eventually resultedin hepatic cells death [33 34] In addition PLA
2is also found
to cause anticoagulation in victims [6] All pharmacological
BioMed Research International 9
Table 5 Cardioprotective activity of medicinal plant extracts on LDH and CK-MB levels to neutralize snake bite envenomation inexperimental rabbits
Sr numberGroup V(subgroup) Name of evaluated sample LDH CK-MB
UL (mean plusmnSEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 1357 plusmn 100 001 gt 119875 gt 0001 41 plusmn 085 05 gt 119875 gt 01
2 (V2) Allium cepa L 934 plusmn 1303 01 gt 119875 gt 005 148 plusmn 165 119875 gt 05
3 (V3) Allium sativum L 1177 plusmn 2056 005 gt 119875 gt 002 66 plusmn 330 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 9755 plusmn 3360 05 gt 119875 gt 01 148 plusmn 330 119875 gt 05
5 (V5) Bauhinia variegata L 1972 plusmn 300 119875 gt 0001 83 plusmn 676 119875 gt 05
6 (V6) Brassica nigra (L) W D J Koch 8555 plusmn 050 005 gt 119875 gt 002 90 plusmn 085 05 gt 119875 gt 01
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 1022 plusmn 501 002 gt 119875 gt 001 66 plusmn 330 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 1114 plusmn 100 002 gt 119875 gt 001 618 plusmn 109 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 1230 plusmn 2357 005 gt 119875 gt 002 412 plusmn 3166 119875 gt 05
10 (V9) Citrus limon (L) Burm f 1831 plusmn 6569 005 gt 119875 gt 002 73 plusmn 141 05 gt 119875 gt 01
11 (V10) Citrullus colocynthis (L) Schrad 827 plusmn 651 01 gt 119875 gt 005 05 plusmn 166 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 1589 plusmn 2256 002 gt 119875 gt 001 58 plusmn 080 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 1615 plusmn 151 119875 gt 0001 985 plusmn 166 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 1418 plusmn 1303 002 gt 119875 gt 001 11 plusmn 252 119875 gt 05
15 (V14) Leucas capitata Desf 783 plusmn 1002 05 gt 119875 gt 01 14 plusmn 080 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 1428 plusmn 651 119875 gt 0001 08 plusmn 166 05 gt 119875 gt 01
17 (V16) Momordica charantia L 14755 plusmn 351 119875 gt 0001 156 plusmn 245 119875 gt 05
18 (V17) Nerium indicumMill 1268 plusmn 1203 002 gt 119875 gt 001 05 plusmn 165 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 1335 plusmn 1203 002 gt 119875 gt 001 123 plusmn 245 119875 gt 05
20 (V19) Pinus roxburghii Sarg 1050 plusmn 100 002 gt 119875 gt 001 08 plusmn 165 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 11355 plusmn 05 002 gt 119875 gt 001 131 plusmn 165 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 11535 plusmn 05 119875 gt 0001 173 plusmn 250 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 1538 plusmn 2005 002 gt 119875 gt 001 41 plusmn 085 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 1078 plusmn 1604 005 gt 119875 gt 002 66 plusmn 000 05 gt 119875 gt 01
25 (V24) Sapindus mukorossi Gaertn 14605 plusmn 551 001 gt 119875 gt 0001 41 plusmn 080 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 787 plusmn 2808 119875 gt 05 13 plusmn 176 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 7985 plusmn 1454 05 gt 119875 gt 01 66 plusmn 000 05 gt 119875 gt 01
28 (V27) Trichodesma indicum (L) Sm 9785 plusmn 451 005 gt 119875 gt 002 66 plusmn 300 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 888 plusmn 200 005 gt 119875 gt 002 173 plusmn 080 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
763 plusmn 601 Select to compare 09 plusmn 085 Select to compare
effects are due to phospholipid hydrolysis or phospholipidcompeting bidingmechanismwith other coagulation factorsparticularly FXa or by both ways however it is very hardto pinpoint exact one [35] Anticoagulant response posed bycobra PLA
2(anticoagulant enzyme) may aggravate hepatic
injury as many anticoagulant agents cause liver damage thatare idiosyncratic in nature [36]
Cardiac injury particularly systolic heart arrest is one ofthewell-known toxicities related toNaja naja subspecies [30]In present study Pakistani cobra venom was found to release
two cystolic enzymes (LDH and CK-MB) that are sensitiveindicators of myocyte injury [37] It is due to the presenceof myotoxic PLA
2and other cardiotoxin(s) that are salient
features of cobra venom that are responsible for cellularnecrosis and cytotoxicity [38 39] Cytotoxic effect of Najanaja karachiensis venom may not be overlooked even whenantisera administration is late cause of toxic components tothe microvasculature and thrombus produced which poseshindrance in access of immunoglobulins to the site of snakebite [26 29 39]
10 BioMed Research International
0
10
20
30
40
50
60
70Se
rum
leve
l of u
rea a
nd cr
eatin
ine (
mg
dL)
UreaCreatinine
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
L
Rhaz
ya st
ricta
Dec
neRu
bia
cord
ifolia
L
Sapi
ndus
muk
oros
si G
aert
nSt
enol
obiu
m st
ans (
L) S
eem
Term
inal
ia a
rjuna
(Rox
b ex
DC
) Wig
ht an
d A
rnTr
ichod
esm
a in
dicu
m (L
) Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antidotes tested (100mgkg)
Figure 2 Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea andcreatinine level posed by Naja naja karachiensis venom in rabbits
Snake venom has been responsible for detrimental effectsto the renal tissues [40] Likewise Naja naja krachiensisvenomwas found to cause severe renal damage by significantrise in serum urea and creatinine levels Indeed phospho-lipases enzymes are responsible for the increase in vascularpermeability with hemorrhagic effects to the vital tissues inthe victims Subsequently numerous lesions are producedrelated to the glomerular membrane and renal tubules eitherinterstitial or vascular [29 41 42] Presence of lymphocytes(white blood cells) and oedema in the cortical as well asmedullary regions of renal tubules further confirmed theidea of renal damage (data has not shown) as reportedpreviously with different snake venoms like Hemiscorpiuslepturus [26 43] Apart of it pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope99mTc confirmed that kidneys and urinary bladder are themost saturated organs (gt70) after intravenous injection inexperimental rabbits (unpublished data by our group)
Medicinal plants of Pakistan are used to inhibit snakevenom (PLA
2) enzymes [6] Due to this effect present
study was designed to select twenty-eight medicinal plantsof Pakistan to test their potentials as an antidote againsttoxicities produced by Naja naja karachiensis venom It
was fascinating that extract of Stenolobium stans (L) Seemshowed significant neutralization compared to that of refer-ence standard (antisera) as reported previously by Asad etal for inhibition of phospholipases A
2anticoagulant activity
[6 44] Present study indicated that extract of Stenolobiumstans (L) Seem possesses an endogenous inhibitor(s) tonullify venom (PLA
2) induced toxicities Extracts of these
plants material are routinely used in Pakistan by simpleapplication in the form of paste to the affected area [45]This practice provides effective first aid treatment as tinymolecules of an antidote diffuse favorably at the site of snakebite before hospitalization and abrogated the spreading oftoxins [39] It is owing to the various secondary metaboliteslike phenols flavonoids terpenoids xanthenes quinonoidsand so forth as reported earlier to mask various enzymaticactions of cobra venom [6 23] Indeed secondarymetabolitespose hindrance in binding of different snake venom enzymesto their potential targets therefore antidotal effect evokedOther plants extract were not proved significantly effectiveto neutralize cobra venom therefore cannot be declareduseful plants in venom therapy It is the need of the time toisolate lethal component(s) of cobra venom attributed to itsmajor toxicities Furthermore characterization of antivenom
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
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Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 7
Table 3 Hepatoprotective activity of various medicinal plant extracts on ALT and AST levels to neutralize snake bite envenomation inexperimental rabbits
Sr number Group V(subgroup) Name of evaluated sample GPTALT GOTAST
UL(mean plusmn SEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 62 plusmn 702 05 gt 119875 gt 01 99 plusmn 1855 05 gt 119875 gt 01
2 (V2) Allium cepa L 68 plusmn 1203 05 gt 119875 gt 01 1665 plusmn 050 05 gt 119875 gt 01
3 (V3) Allium sativum L 49 plusmn 501 119875 gt 05 825 plusmn 1855 119875 gt 05
4 (V4) Althaea officinalisL 70 plusmn 2005 119875 gt 05 745 plusmn 1454 119875 gt 05
5 (V5) Bauhinia variegata L 78 plusmn 404 05 gt 119875 gt 01 1495 plusmn 199 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 45 plusmn 000 05 gt 119875 gt 01 55 plusmn 100 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 315 plusmn 05 05 gt 119875 gt 01 141 plusmn 101 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 625 plusmn 252 05 gt 119875 gt 01 194 plusmn 1212 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 633 plusmn 414 05 gt 119875 gt 01 72 plusmn 202 119875 gt 05
10 (V9) Citrus limon (L) Burm f 305 plusmn 1363 05 gt 119875 gt 01 49 plusmn 2424 119875 gt 05
11 (V10) Citrullus colocynthis (L) Schrad 475 plusmn 555 119875 gt 05 74 plusmn 909 119875 gt 05
12 (V11) Cuminum cyminum L 67 plusmn 1616 119875 gt 05 157 plusmn 1414 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 8525 plusmn 126 01 gt 119875 gt
0051685 plusmn 757 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 80 plusmn 782 05 gt 119875 gt 01 170 plusmn 1313 05 gt 119875 gt 01
15 (V14) Leucas capitata Desf 455 plusmn 752 119875 gt 05 535 plusmn 050 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 62 plusmn 808 05 gt 119875 gt 01 121 plusmn 505 05 gt 119875 gt 01
17 (V16) Momordica charantia L 625 plusmn 752 05 gt 119875 gt 01 247 plusmn 4613 05 gt 119875 gt 01
18 (V17) Nerium indicumMill 88 plusmn 1955 05 gt 119875 gt 01 235 plusmn 1805 01 gt 119875 gt
005
19 (V18) Ocimum sanctum L 615 plusmn 752 05 gt 119875 gt 01 158 plusmn 1002 05 gt 119875 gt 01
20 (V19) Pinus roxburghii Sarg 53 plusmn 902 119875 gt 05 57 plusmn 1303 119875 gt 05
21 (V20) Pistacia integerrima J L Stewart 415 plusmn 15 05 gt 119875 gt 01 545 plusmn 050 119875 gt 05
22 (V21) Psoralea corylifolia L 54 plusmn 100 119875 gt 05 665 plusmn 1153 119875 gt 05
23 (V22) Rhazya stricta Decne 66 plusmn 2424 119875 gt 05 1325 plusmn 1262 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 655 plusmn 1767 119875 gt 05 129 plusmn 6641 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 56 plusmn 601 119875 gt 05 785 plusmn 1354 119875 gt 05
26 (V25) Stenolobium stans (L) Seem 465 plusmn 656 119875 gt 05 705 plusmn 353 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 475 plusmn 151 05 gt 119875 gt 01 78 plusmn 606 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 52 plusmn 000 119875 gt 05 125 plusmn 401 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 665 plusmn 3259 119875 gt 05 1175 plusmn 952 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
525 plusmn 351 Select tocompare 695 plusmn 1855 Select to
compare
(Allium cepa L Althaea officinalis L Bauhinia variegataL Cedrus deodara (Roxb ex D Don) G Don Fagoniacretica L Leucas capitataDesf Momordica charantia L andOcimum sanctum L) were found equally capable (119875 gt 05) ofnormalizing high values of CK-MB however rest of all wereproved less beneficial (05 gt 119875 gt 01) To combat highlyraised values of LDH Althaea officinalis L Leucas capitataDesf and Terminalia arjuna (Roxb ex DC) Wight and Arnwere proved useful to some extent (05 gt 119875 gt 01) although
remaining plant extracts could not be shortlisted (01 gt 119875 gt0001) as standard antisera (LDH = 763 plusmn 601UL) Overalldetail about LDH is discussed in Table 5 and comparison isshown in Figure 3
4 Discussion
Snake bite has been responsible for tens of thousands ofdeaths worldwide and numerous physical handicaps [29]
8 BioMed Research International
Table 4 Nephroprotective activity of different medicinal plant extracts on urea and creatinine levels to neutralize snake bite envenomationin experimental rabbits
Sr number Group V(subgroup) Name of evaluated sample Urea Creatinine
mgdL(mean plusmn SEM)
119875 valuecomment
mgdL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 57 plusmn 00 002 gt 119875 gt 001 16 plusmn 020 05 gt 119875 gt 01
2 (V2) Allium cepa L 51 plusmn 1103 05 gt 119875 gt 01 150 plusmn 017 05 gt 119875 gt 01
3 (V3) Allium sativum L 50 plusmn 401 05 gt 119875 gt 01 130 plusmn 006 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 26 plusmn 505 119875 gt 05 118 plusmn 024 119875 gt 05
5 (V5) Bauhinia variegata L 50 plusmn 126 05 gt 119875 gt 01 136 plusmn 024 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 225 plusmn 451 05 gt 119875 gt 01 107 plusmn 011 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 425 plusmn 252 05 gt 119875 gt 01 130 plusmn 001 01 gt 119875 gt 005
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 39 plusmn 202 05 gt 119875 gt 01 125 plusmn 003 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 44 plusmn 404 05 gt 119875 gt 01 144 plusmn 007 05 gt 119875 gt 01
10 (V9) Citrus limon (L) Burm f 395 plusmn 252 05 gt 119875 gt 01 140 plusmn 003 01 gt 119875 gt 005
11 (V10) Citrullus colocynthis (L) Schrad 495 plusmn 353 05 gt 119875 gt 01 152 plusmn 015 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 54 plusmn 101 005 gt 119875 gt 002 148 plusmn 001 005 gt 119875 gt 002
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 395 plusmn 454 05 gt 119875 gt 01 135 plusmn 005 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 43 plusmn 227 05 gt 119875 gt 01 123 plusmn 013 119875 lt 05
15 (V14) Leucas capitata Desf 315 plusmn 050 119875 gt 05 107 plusmn 005 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 435 plusmn 353 05 gt 119875 gt 01 144 plusmn 014 05 gt 119875 gt 01
17 (V16) Momordica charantia L 57 plusmn 501 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
18 (V17) Nerium indicumMill 47 plusmn 000 119875 lt 005 124 plusmn 010 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 495 plusmn 752 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
20 (V19) Pinus roxburghii Sarg 45 plusmn 100 119875 gt 005 14 plusmn 005 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 665 plusmn 050 002 gt 119875 gt 001 168 plusmn 010 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 425 plusmn 250 05 gt 119875 gt 01 127 plusmn 015 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 535 plusmn 353 05 gt 119875 gt 01 167 plusmn 0005 005 gt 119875 gt 002
24 (V23) Rubia cordifolia L 36 plusmn 101 05 gt 119875 gt 01 093 plusmn 048 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 44 plusmn 100 01 gt 119875 gt 005 143 plusmn 012 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 255 plusmn 651 119875 gt 05 11 plusmn 006 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 385 plusmn 151 05 gt 119875 gt 01 127 plusmn 023 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 37 plusmn 100 05 gt 119875 gt 01 046 plusmn 001 005 gt 119875 gt 002
29 (V28) Zingiber officinale Roscoe 47 plusmn 100 005 gt 119875 gt 002 130 plusmn 001 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
315 plusmn 050 Select tocompare 108 plusmn 002 Select to
compare
Generally snake venom is an intricate mixture of variousproteins (gt90) and most of them are enzymes particularly(40) phospholipases A
2[30] Among different complica-
tions hepatic injury is one of the deadly venomous effectsproduced by cobra bite [31 32] Naja naja karachiensisvenom caused significant increase in ALT and AST levels(dose dependently) that are surrogate markers for livertoxicity either by direct action or immunological (hyper-sensitivity) reaction The allergic reactions reactions are notdose dependent which clarify lethal effects to the cytoplasm
and mitochondrial membranes of the hepatic cells [25 33]Phospholipases enzymes abundant in Naja naja karachien-sis venom are responsible for breakdown of membranousphospholipids and therefore resulted in cellular injury alongwith inflammation [24] PLA
2is attributed to decrease in
Na+K+ ATPase activities and led to greater influx of sodiumions and water molecules into the cell Subsequently plasmamembrane lipid bilayer disorganized and eventually resultedin hepatic cells death [33 34] In addition PLA
2is also found
to cause anticoagulation in victims [6] All pharmacological
BioMed Research International 9
Table 5 Cardioprotective activity of medicinal plant extracts on LDH and CK-MB levels to neutralize snake bite envenomation inexperimental rabbits
Sr numberGroup V(subgroup) Name of evaluated sample LDH CK-MB
UL (mean plusmnSEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 1357 plusmn 100 001 gt 119875 gt 0001 41 plusmn 085 05 gt 119875 gt 01
2 (V2) Allium cepa L 934 plusmn 1303 01 gt 119875 gt 005 148 plusmn 165 119875 gt 05
3 (V3) Allium sativum L 1177 plusmn 2056 005 gt 119875 gt 002 66 plusmn 330 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 9755 plusmn 3360 05 gt 119875 gt 01 148 plusmn 330 119875 gt 05
5 (V5) Bauhinia variegata L 1972 plusmn 300 119875 gt 0001 83 plusmn 676 119875 gt 05
6 (V6) Brassica nigra (L) W D J Koch 8555 plusmn 050 005 gt 119875 gt 002 90 plusmn 085 05 gt 119875 gt 01
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 1022 plusmn 501 002 gt 119875 gt 001 66 plusmn 330 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 1114 plusmn 100 002 gt 119875 gt 001 618 plusmn 109 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 1230 plusmn 2357 005 gt 119875 gt 002 412 plusmn 3166 119875 gt 05
10 (V9) Citrus limon (L) Burm f 1831 plusmn 6569 005 gt 119875 gt 002 73 plusmn 141 05 gt 119875 gt 01
11 (V10) Citrullus colocynthis (L) Schrad 827 plusmn 651 01 gt 119875 gt 005 05 plusmn 166 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 1589 plusmn 2256 002 gt 119875 gt 001 58 plusmn 080 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 1615 plusmn 151 119875 gt 0001 985 plusmn 166 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 1418 plusmn 1303 002 gt 119875 gt 001 11 plusmn 252 119875 gt 05
15 (V14) Leucas capitata Desf 783 plusmn 1002 05 gt 119875 gt 01 14 plusmn 080 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 1428 plusmn 651 119875 gt 0001 08 plusmn 166 05 gt 119875 gt 01
17 (V16) Momordica charantia L 14755 plusmn 351 119875 gt 0001 156 plusmn 245 119875 gt 05
18 (V17) Nerium indicumMill 1268 plusmn 1203 002 gt 119875 gt 001 05 plusmn 165 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 1335 plusmn 1203 002 gt 119875 gt 001 123 plusmn 245 119875 gt 05
20 (V19) Pinus roxburghii Sarg 1050 plusmn 100 002 gt 119875 gt 001 08 plusmn 165 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 11355 plusmn 05 002 gt 119875 gt 001 131 plusmn 165 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 11535 plusmn 05 119875 gt 0001 173 plusmn 250 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 1538 plusmn 2005 002 gt 119875 gt 001 41 plusmn 085 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 1078 plusmn 1604 005 gt 119875 gt 002 66 plusmn 000 05 gt 119875 gt 01
25 (V24) Sapindus mukorossi Gaertn 14605 plusmn 551 001 gt 119875 gt 0001 41 plusmn 080 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 787 plusmn 2808 119875 gt 05 13 plusmn 176 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 7985 plusmn 1454 05 gt 119875 gt 01 66 plusmn 000 05 gt 119875 gt 01
28 (V27) Trichodesma indicum (L) Sm 9785 plusmn 451 005 gt 119875 gt 002 66 plusmn 300 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 888 plusmn 200 005 gt 119875 gt 002 173 plusmn 080 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
763 plusmn 601 Select to compare 09 plusmn 085 Select to compare
effects are due to phospholipid hydrolysis or phospholipidcompeting bidingmechanismwith other coagulation factorsparticularly FXa or by both ways however it is very hardto pinpoint exact one [35] Anticoagulant response posed bycobra PLA
2(anticoagulant enzyme) may aggravate hepatic
injury as many anticoagulant agents cause liver damage thatare idiosyncratic in nature [36]
Cardiac injury particularly systolic heart arrest is one ofthewell-known toxicities related toNaja naja subspecies [30]In present study Pakistani cobra venom was found to release
two cystolic enzymes (LDH and CK-MB) that are sensitiveindicators of myocyte injury [37] It is due to the presenceof myotoxic PLA
2and other cardiotoxin(s) that are salient
features of cobra venom that are responsible for cellularnecrosis and cytotoxicity [38 39] Cytotoxic effect of Najanaja karachiensis venom may not be overlooked even whenantisera administration is late cause of toxic components tothe microvasculature and thrombus produced which poseshindrance in access of immunoglobulins to the site of snakebite [26 29 39]
10 BioMed Research International
0
10
20
30
40
50
60
70Se
rum
leve
l of u
rea a
nd cr
eatin
ine (
mg
dL)
UreaCreatinine
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
L
Rhaz
ya st
ricta
Dec
neRu
bia
cord
ifolia
L
Sapi
ndus
muk
oros
si G
aert
nSt
enol
obiu
m st
ans (
L) S
eem
Term
inal
ia a
rjuna
(Rox
b ex
DC
) Wig
ht an
d A
rnTr
ichod
esm
a in
dicu
m (L
) Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antidotes tested (100mgkg)
Figure 2 Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea andcreatinine level posed by Naja naja karachiensis venom in rabbits
Snake venom has been responsible for detrimental effectsto the renal tissues [40] Likewise Naja naja krachiensisvenomwas found to cause severe renal damage by significantrise in serum urea and creatinine levels Indeed phospho-lipases enzymes are responsible for the increase in vascularpermeability with hemorrhagic effects to the vital tissues inthe victims Subsequently numerous lesions are producedrelated to the glomerular membrane and renal tubules eitherinterstitial or vascular [29 41 42] Presence of lymphocytes(white blood cells) and oedema in the cortical as well asmedullary regions of renal tubules further confirmed theidea of renal damage (data has not shown) as reportedpreviously with different snake venoms like Hemiscorpiuslepturus [26 43] Apart of it pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope99mTc confirmed that kidneys and urinary bladder are themost saturated organs (gt70) after intravenous injection inexperimental rabbits (unpublished data by our group)
Medicinal plants of Pakistan are used to inhibit snakevenom (PLA
2) enzymes [6] Due to this effect present
study was designed to select twenty-eight medicinal plantsof Pakistan to test their potentials as an antidote againsttoxicities produced by Naja naja karachiensis venom It
was fascinating that extract of Stenolobium stans (L) Seemshowed significant neutralization compared to that of refer-ence standard (antisera) as reported previously by Asad etal for inhibition of phospholipases A
2anticoagulant activity
[6 44] Present study indicated that extract of Stenolobiumstans (L) Seem possesses an endogenous inhibitor(s) tonullify venom (PLA
2) induced toxicities Extracts of these
plants material are routinely used in Pakistan by simpleapplication in the form of paste to the affected area [45]This practice provides effective first aid treatment as tinymolecules of an antidote diffuse favorably at the site of snakebite before hospitalization and abrogated the spreading oftoxins [39] It is owing to the various secondary metaboliteslike phenols flavonoids terpenoids xanthenes quinonoidsand so forth as reported earlier to mask various enzymaticactions of cobra venom [6 23] Indeed secondarymetabolitespose hindrance in binding of different snake venom enzymesto their potential targets therefore antidotal effect evokedOther plants extract were not proved significantly effectiveto neutralize cobra venom therefore cannot be declareduseful plants in venom therapy It is the need of the time toisolate lethal component(s) of cobra venom attributed to itsmajor toxicities Furthermore characterization of antivenom
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
8 BioMed Research International
Table 4 Nephroprotective activity of different medicinal plant extracts on urea and creatinine levels to neutralize snake bite envenomationin experimental rabbits
Sr number Group V(subgroup) Name of evaluated sample Urea Creatinine
mgdL(mean plusmn SEM)
119875 valuecomment
mgdL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 57 plusmn 00 002 gt 119875 gt 001 16 plusmn 020 05 gt 119875 gt 01
2 (V2) Allium cepa L 51 plusmn 1103 05 gt 119875 gt 01 150 plusmn 017 05 gt 119875 gt 01
3 (V3) Allium sativum L 50 plusmn 401 05 gt 119875 gt 01 130 plusmn 006 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 26 plusmn 505 119875 gt 05 118 plusmn 024 119875 gt 05
5 (V5) Bauhinia variegata L 50 plusmn 126 05 gt 119875 gt 01 136 plusmn 024 05 gt 119875 gt 01
6 (V6) Brassica nigra (L) W D J Koch 225 plusmn 451 05 gt 119875 gt 01 107 plusmn 011 119875 gt 05
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 425 plusmn 252 05 gt 119875 gt 01 130 plusmn 001 01 gt 119875 gt 005
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 39 plusmn 202 05 gt 119875 gt 01 125 plusmn 003 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 44 plusmn 404 05 gt 119875 gt 01 144 plusmn 007 05 gt 119875 gt 01
10 (V9) Citrus limon (L) Burm f 395 plusmn 252 05 gt 119875 gt 01 140 plusmn 003 01 gt 119875 gt 005
11 (V10) Citrullus colocynthis (L) Schrad 495 plusmn 353 05 gt 119875 gt 01 152 plusmn 015 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 54 plusmn 101 005 gt 119875 gt 002 148 plusmn 001 005 gt 119875 gt 002
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 395 plusmn 454 05 gt 119875 gt 01 135 plusmn 005 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 43 plusmn 227 05 gt 119875 gt 01 123 plusmn 013 119875 lt 05
15 (V14) Leucas capitata Desf 315 plusmn 050 119875 gt 05 107 plusmn 005 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 435 plusmn 353 05 gt 119875 gt 01 144 plusmn 014 05 gt 119875 gt 01
17 (V16) Momordica charantia L 57 plusmn 501 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
18 (V17) Nerium indicumMill 47 plusmn 000 119875 lt 005 124 plusmn 010 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 495 plusmn 752 05 gt 119875 gt 01 15 plusmn 0005 005 gt 119875 gt 002
20 (V19) Pinus roxburghii Sarg 45 plusmn 100 119875 gt 005 14 plusmn 005 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 665 plusmn 050 002 gt 119875 gt 001 168 plusmn 010 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 425 plusmn 250 05 gt 119875 gt 01 127 plusmn 015 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 535 plusmn 353 05 gt 119875 gt 01 167 plusmn 0005 005 gt 119875 gt 002
24 (V23) Rubia cordifolia L 36 plusmn 101 05 gt 119875 gt 01 093 plusmn 048 119875 gt 05
25 (V24) Sapindus mukorossi Gaertn 44 plusmn 100 01 gt 119875 gt 005 143 plusmn 012 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 255 plusmn 651 119875 gt 05 11 plusmn 006 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 385 plusmn 151 05 gt 119875 gt 01 127 plusmn 023 119875 gt 05
28 (V27) Trichodesma indicum (L) Sm 37 plusmn 100 05 gt 119875 gt 01 046 plusmn 001 005 gt 119875 gt 002
29 (V28) Zingiber officinale Roscoe 47 plusmn 100 005 gt 119875 gt 002 130 plusmn 001 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
315 plusmn 050 Select tocompare 108 plusmn 002 Select to
compare
Generally snake venom is an intricate mixture of variousproteins (gt90) and most of them are enzymes particularly(40) phospholipases A
2[30] Among different complica-
tions hepatic injury is one of the deadly venomous effectsproduced by cobra bite [31 32] Naja naja karachiensisvenom caused significant increase in ALT and AST levels(dose dependently) that are surrogate markers for livertoxicity either by direct action or immunological (hyper-sensitivity) reaction The allergic reactions reactions are notdose dependent which clarify lethal effects to the cytoplasm
and mitochondrial membranes of the hepatic cells [25 33]Phospholipases enzymes abundant in Naja naja karachien-sis venom are responsible for breakdown of membranousphospholipids and therefore resulted in cellular injury alongwith inflammation [24] PLA
2is attributed to decrease in
Na+K+ ATPase activities and led to greater influx of sodiumions and water molecules into the cell Subsequently plasmamembrane lipid bilayer disorganized and eventually resultedin hepatic cells death [33 34] In addition PLA
2is also found
to cause anticoagulation in victims [6] All pharmacological
BioMed Research International 9
Table 5 Cardioprotective activity of medicinal plant extracts on LDH and CK-MB levels to neutralize snake bite envenomation inexperimental rabbits
Sr numberGroup V(subgroup) Name of evaluated sample LDH CK-MB
UL (mean plusmnSEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 1357 plusmn 100 001 gt 119875 gt 0001 41 plusmn 085 05 gt 119875 gt 01
2 (V2) Allium cepa L 934 plusmn 1303 01 gt 119875 gt 005 148 plusmn 165 119875 gt 05
3 (V3) Allium sativum L 1177 plusmn 2056 005 gt 119875 gt 002 66 plusmn 330 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 9755 plusmn 3360 05 gt 119875 gt 01 148 plusmn 330 119875 gt 05
5 (V5) Bauhinia variegata L 1972 plusmn 300 119875 gt 0001 83 plusmn 676 119875 gt 05
6 (V6) Brassica nigra (L) W D J Koch 8555 plusmn 050 005 gt 119875 gt 002 90 plusmn 085 05 gt 119875 gt 01
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 1022 plusmn 501 002 gt 119875 gt 001 66 plusmn 330 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 1114 plusmn 100 002 gt 119875 gt 001 618 plusmn 109 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 1230 plusmn 2357 005 gt 119875 gt 002 412 plusmn 3166 119875 gt 05
10 (V9) Citrus limon (L) Burm f 1831 plusmn 6569 005 gt 119875 gt 002 73 plusmn 141 05 gt 119875 gt 01
11 (V10) Citrullus colocynthis (L) Schrad 827 plusmn 651 01 gt 119875 gt 005 05 plusmn 166 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 1589 plusmn 2256 002 gt 119875 gt 001 58 plusmn 080 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 1615 plusmn 151 119875 gt 0001 985 plusmn 166 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 1418 plusmn 1303 002 gt 119875 gt 001 11 plusmn 252 119875 gt 05
15 (V14) Leucas capitata Desf 783 plusmn 1002 05 gt 119875 gt 01 14 plusmn 080 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 1428 plusmn 651 119875 gt 0001 08 plusmn 166 05 gt 119875 gt 01
17 (V16) Momordica charantia L 14755 plusmn 351 119875 gt 0001 156 plusmn 245 119875 gt 05
18 (V17) Nerium indicumMill 1268 plusmn 1203 002 gt 119875 gt 001 05 plusmn 165 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 1335 plusmn 1203 002 gt 119875 gt 001 123 plusmn 245 119875 gt 05
20 (V19) Pinus roxburghii Sarg 1050 plusmn 100 002 gt 119875 gt 001 08 plusmn 165 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 11355 plusmn 05 002 gt 119875 gt 001 131 plusmn 165 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 11535 plusmn 05 119875 gt 0001 173 plusmn 250 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 1538 plusmn 2005 002 gt 119875 gt 001 41 plusmn 085 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 1078 plusmn 1604 005 gt 119875 gt 002 66 plusmn 000 05 gt 119875 gt 01
25 (V24) Sapindus mukorossi Gaertn 14605 plusmn 551 001 gt 119875 gt 0001 41 plusmn 080 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 787 plusmn 2808 119875 gt 05 13 plusmn 176 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 7985 plusmn 1454 05 gt 119875 gt 01 66 plusmn 000 05 gt 119875 gt 01
28 (V27) Trichodesma indicum (L) Sm 9785 plusmn 451 005 gt 119875 gt 002 66 plusmn 300 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 888 plusmn 200 005 gt 119875 gt 002 173 plusmn 080 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
763 plusmn 601 Select to compare 09 plusmn 085 Select to compare
effects are due to phospholipid hydrolysis or phospholipidcompeting bidingmechanismwith other coagulation factorsparticularly FXa or by both ways however it is very hardto pinpoint exact one [35] Anticoagulant response posed bycobra PLA
2(anticoagulant enzyme) may aggravate hepatic
injury as many anticoagulant agents cause liver damage thatare idiosyncratic in nature [36]
Cardiac injury particularly systolic heart arrest is one ofthewell-known toxicities related toNaja naja subspecies [30]In present study Pakistani cobra venom was found to release
two cystolic enzymes (LDH and CK-MB) that are sensitiveindicators of myocyte injury [37] It is due to the presenceof myotoxic PLA
2and other cardiotoxin(s) that are salient
features of cobra venom that are responsible for cellularnecrosis and cytotoxicity [38 39] Cytotoxic effect of Najanaja karachiensis venom may not be overlooked even whenantisera administration is late cause of toxic components tothe microvasculature and thrombus produced which poseshindrance in access of immunoglobulins to the site of snakebite [26 29 39]
10 BioMed Research International
0
10
20
30
40
50
60
70Se
rum
leve
l of u
rea a
nd cr
eatin
ine (
mg
dL)
UreaCreatinine
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
L
Rhaz
ya st
ricta
Dec
neRu
bia
cord
ifolia
L
Sapi
ndus
muk
oros
si G
aert
nSt
enol
obiu
m st
ans (
L) S
eem
Term
inal
ia a
rjuna
(Rox
b ex
DC
) Wig
ht an
d A
rnTr
ichod
esm
a in
dicu
m (L
) Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antidotes tested (100mgkg)
Figure 2 Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea andcreatinine level posed by Naja naja karachiensis venom in rabbits
Snake venom has been responsible for detrimental effectsto the renal tissues [40] Likewise Naja naja krachiensisvenomwas found to cause severe renal damage by significantrise in serum urea and creatinine levels Indeed phospho-lipases enzymes are responsible for the increase in vascularpermeability with hemorrhagic effects to the vital tissues inthe victims Subsequently numerous lesions are producedrelated to the glomerular membrane and renal tubules eitherinterstitial or vascular [29 41 42] Presence of lymphocytes(white blood cells) and oedema in the cortical as well asmedullary regions of renal tubules further confirmed theidea of renal damage (data has not shown) as reportedpreviously with different snake venoms like Hemiscorpiuslepturus [26 43] Apart of it pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope99mTc confirmed that kidneys and urinary bladder are themost saturated organs (gt70) after intravenous injection inexperimental rabbits (unpublished data by our group)
Medicinal plants of Pakistan are used to inhibit snakevenom (PLA
2) enzymes [6] Due to this effect present
study was designed to select twenty-eight medicinal plantsof Pakistan to test their potentials as an antidote againsttoxicities produced by Naja naja karachiensis venom It
was fascinating that extract of Stenolobium stans (L) Seemshowed significant neutralization compared to that of refer-ence standard (antisera) as reported previously by Asad etal for inhibition of phospholipases A
2anticoagulant activity
[6 44] Present study indicated that extract of Stenolobiumstans (L) Seem possesses an endogenous inhibitor(s) tonullify venom (PLA
2) induced toxicities Extracts of these
plants material are routinely used in Pakistan by simpleapplication in the form of paste to the affected area [45]This practice provides effective first aid treatment as tinymolecules of an antidote diffuse favorably at the site of snakebite before hospitalization and abrogated the spreading oftoxins [39] It is owing to the various secondary metaboliteslike phenols flavonoids terpenoids xanthenes quinonoidsand so forth as reported earlier to mask various enzymaticactions of cobra venom [6 23] Indeed secondarymetabolitespose hindrance in binding of different snake venom enzymesto their potential targets therefore antidotal effect evokedOther plants extract were not proved significantly effectiveto neutralize cobra venom therefore cannot be declareduseful plants in venom therapy It is the need of the time toisolate lethal component(s) of cobra venom attributed to itsmajor toxicities Furthermore characterization of antivenom
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 9
Table 5 Cardioprotective activity of medicinal plant extracts on LDH and CK-MB levels to neutralize snake bite envenomation inexperimental rabbits
Sr numberGroup V(subgroup) Name of evaluated sample LDH CK-MB
UL (mean plusmnSEM)
119875 valuecomment
UL(mean plusmn SEM)
119875 valuecomment
1 (V1) Albizia lebbeck (L) Benth 1357 plusmn 100 001 gt 119875 gt 0001 41 plusmn 085 05 gt 119875 gt 01
2 (V2) Allium cepa L 934 plusmn 1303 01 gt 119875 gt 005 148 plusmn 165 119875 gt 05
3 (V3) Allium sativum L 1177 plusmn 2056 005 gt 119875 gt 002 66 plusmn 330 05 gt 119875 gt 01
4 (V4) Althaea officinalisL 9755 plusmn 3360 05 gt 119875 gt 01 148 plusmn 330 119875 gt 05
5 (V5) Bauhinia variegata L 1972 plusmn 300 119875 gt 0001 83 plusmn 676 119875 gt 05
6 (V6) Brassica nigra (L) W D J Koch 8555 plusmn 050 005 gt 119875 gt 002 90 plusmn 085 05 gt 119875 gt 01
7 (V7a) Calotropis procera (Aiton) W TAiton (exudates) 1022 plusmn 501 002 gt 119875 gt 001 66 plusmn 330 05 gt 119875 gt 01
8 (V7b) Calotropis procera (Aiton) W TAiton (flowers) 1114 plusmn 100 002 gt 119875 gt 001 618 plusmn 109 05 gt 119875 gt 01
9 (V8) Cedrus deodara (Roxb ex DDon) G Don 1230 plusmn 2357 005 gt 119875 gt 002 412 plusmn 3166 119875 gt 05
10 (V9) Citrus limon (L) Burm f 1831 plusmn 6569 005 gt 119875 gt 002 73 plusmn 141 05 gt 119875 gt 01
11 (V10) Citrullus colocynthis (L) Schrad 827 plusmn 651 01 gt 119875 gt 005 05 plusmn 166 05 gt 119875 gt 01
12 (V11) Cuminum cyminum L 1589 plusmn 2256 002 gt 119875 gt 001 58 plusmn 080 05 gt 119875 gt 01
13 (V12) Enicostemma hyssopifolium(Willd) I Verd 1615 plusmn 151 119875 gt 0001 985 plusmn 166 05 gt 119875 gt 01
14 (V13) Fagonia cretica L 1418 plusmn 1303 002 gt 119875 gt 001 11 plusmn 252 119875 gt 05
15 (V14) Leucas capitata Desf 783 plusmn 1002 05 gt 119875 gt 01 14 plusmn 080 119875 gt 05
16 (V15) Matthiola incana (L) W T Aiton 1428 plusmn 651 119875 gt 0001 08 plusmn 166 05 gt 119875 gt 01
17 (V16) Momordica charantia L 14755 plusmn 351 119875 gt 0001 156 plusmn 245 119875 gt 05
18 (V17) Nerium indicumMill 1268 plusmn 1203 002 gt 119875 gt 001 05 plusmn 165 05 gt 119875 gt 01
19 (V18) Ocimum sanctum L 1335 plusmn 1203 002 gt 119875 gt 001 123 plusmn 245 119875 gt 05
20 (V19) Pinus roxburghii Sarg 1050 plusmn 100 002 gt 119875 gt 001 08 plusmn 165 05 gt 119875 gt 01
21 (V20) Pistacia integerrima J L Stewart 11355 plusmn 05 002 gt 119875 gt 001 131 plusmn 165 05 gt 119875 gt 01
22 (V21) Psoralea corylifolia L 11535 plusmn 05 119875 gt 0001 173 plusmn 250 05 gt 119875 gt 01
23 (V22) Rhazya stricta Decne 1538 plusmn 2005 002 gt 119875 gt 001 41 plusmn 085 05 gt 119875 gt 01
24 (V23) Rubia cordifolia L 1078 plusmn 1604 005 gt 119875 gt 002 66 plusmn 000 05 gt 119875 gt 01
25 (V24) Sapindus mukorossi Gaertn 14605 plusmn 551 001 gt 119875 gt 0001 41 plusmn 080 05 gt 119875 gt 01
26 (V25) Stenolobium stans (L) Seem 787 plusmn 2808 119875 gt 05 13 plusmn 176 119875 gt 05
27 (V26) Terminalia arjuna (Roxb exDC) Wight and Arn 7985 plusmn 1454 05 gt 119875 gt 01 66 plusmn 000 05 gt 119875 gt 01
28 (V27) Trichodesma indicum (L) Sm 9785 plusmn 451 005 gt 119875 gt 002 66 plusmn 300 05 gt 119875 gt 01
29 (V28) Zingiber officinale Roscoe 888 plusmn 200 005 gt 119875 gt 002 173 plusmn 080 05 gt 119875 gt 01
30 (V29)Reference standard antidote(standard antiseraimmunoglobulinrsquos)
763 plusmn 601 Select to compare 09 plusmn 085 Select to compare
effects are due to phospholipid hydrolysis or phospholipidcompeting bidingmechanismwith other coagulation factorsparticularly FXa or by both ways however it is very hardto pinpoint exact one [35] Anticoagulant response posed bycobra PLA
2(anticoagulant enzyme) may aggravate hepatic
injury as many anticoagulant agents cause liver damage thatare idiosyncratic in nature [36]
Cardiac injury particularly systolic heart arrest is one ofthewell-known toxicities related toNaja naja subspecies [30]In present study Pakistani cobra venom was found to release
two cystolic enzymes (LDH and CK-MB) that are sensitiveindicators of myocyte injury [37] It is due to the presenceof myotoxic PLA
2and other cardiotoxin(s) that are salient
features of cobra venom that are responsible for cellularnecrosis and cytotoxicity [38 39] Cytotoxic effect of Najanaja karachiensis venom may not be overlooked even whenantisera administration is late cause of toxic components tothe microvasculature and thrombus produced which poseshindrance in access of immunoglobulins to the site of snakebite [26 29 39]
10 BioMed Research International
0
10
20
30
40
50
60
70Se
rum
leve
l of u
rea a
nd cr
eatin
ine (
mg
dL)
UreaCreatinine
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
L
Rhaz
ya st
ricta
Dec
neRu
bia
cord
ifolia
L
Sapi
ndus
muk
oros
si G
aert
nSt
enol
obiu
m st
ans (
L) S
eem
Term
inal
ia a
rjuna
(Rox
b ex
DC
) Wig
ht an
d A
rnTr
ichod
esm
a in
dicu
m (L
) Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antidotes tested (100mgkg)
Figure 2 Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea andcreatinine level posed by Naja naja karachiensis venom in rabbits
Snake venom has been responsible for detrimental effectsto the renal tissues [40] Likewise Naja naja krachiensisvenomwas found to cause severe renal damage by significantrise in serum urea and creatinine levels Indeed phospho-lipases enzymes are responsible for the increase in vascularpermeability with hemorrhagic effects to the vital tissues inthe victims Subsequently numerous lesions are producedrelated to the glomerular membrane and renal tubules eitherinterstitial or vascular [29 41 42] Presence of lymphocytes(white blood cells) and oedema in the cortical as well asmedullary regions of renal tubules further confirmed theidea of renal damage (data has not shown) as reportedpreviously with different snake venoms like Hemiscorpiuslepturus [26 43] Apart of it pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope99mTc confirmed that kidneys and urinary bladder are themost saturated organs (gt70) after intravenous injection inexperimental rabbits (unpublished data by our group)
Medicinal plants of Pakistan are used to inhibit snakevenom (PLA
2) enzymes [6] Due to this effect present
study was designed to select twenty-eight medicinal plantsof Pakistan to test their potentials as an antidote againsttoxicities produced by Naja naja karachiensis venom It
was fascinating that extract of Stenolobium stans (L) Seemshowed significant neutralization compared to that of refer-ence standard (antisera) as reported previously by Asad etal for inhibition of phospholipases A
2anticoagulant activity
[6 44] Present study indicated that extract of Stenolobiumstans (L) Seem possesses an endogenous inhibitor(s) tonullify venom (PLA
2) induced toxicities Extracts of these
plants material are routinely used in Pakistan by simpleapplication in the form of paste to the affected area [45]This practice provides effective first aid treatment as tinymolecules of an antidote diffuse favorably at the site of snakebite before hospitalization and abrogated the spreading oftoxins [39] It is owing to the various secondary metaboliteslike phenols flavonoids terpenoids xanthenes quinonoidsand so forth as reported earlier to mask various enzymaticactions of cobra venom [6 23] Indeed secondarymetabolitespose hindrance in binding of different snake venom enzymesto their potential targets therefore antidotal effect evokedOther plants extract were not proved significantly effectiveto neutralize cobra venom therefore cannot be declareduseful plants in venom therapy It is the need of the time toisolate lethal component(s) of cobra venom attributed to itsmajor toxicities Furthermore characterization of antivenom
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
10 BioMed Research International
0
10
20
30
40
50
60
70Se
rum
leve
l of u
rea a
nd cr
eatin
ine (
mg
dL)
UreaCreatinine
Albi
zia
lebbe
ck (L
) Be
nth
Alliu
m ce
pa L
Al
lium
sativ
um L
Al
thae
a offi
cinal
is L
Bauh
inia
varie
gata
L
Bras
sica
nigr
a (L
) W
D J
Koc
hCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCa
lotro
pis p
roce
ra (A
iton)
W T
Aito
nCe
drus
deo
dara
(Rox
b ex
D D
on) G
Don
Citru
s lim
on (L
) Bu
rm f
Ci
trullu
s col
ocyn
this
(L)
Schr
adCu
min
um cy
min
um L
En
icoste
mm
a hy
ssopi
foliu
m (W
illd
) I V
erd
Fago
nia
cret
ica L
Le
ucas
capi
tata
Des
fM
atth
iola
inca
na (L
) W
T A
iton
Mom
ordi
ca ch
aran
tia L
Ne
rium
indi
cum
Mill
Ocim
um sa
nctu
m L
Pi
nus r
oxbu
rghi
i Sar
gPi
stacia
inte
gerr
ima
J L
Ste
war
tPs
oral
ea co
rylif
olia
L
Rhaz
ya st
ricta
Dec
neRu
bia
cord
ifolia
L
Sapi
ndus
muk
oros
si G
aert
nSt
enol
obiu
m st
ans (
L) S
eem
Term
inal
ia a
rjuna
(Rox
b ex
DC
) Wig
ht an
d A
rnTr
ichod
esm
a in
dicu
m (L
) Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antidotes tested (100mgkg)
Figure 2 Comparison of various medicinal plant extracts with reference standard antivenom in neutralization of elevated serum urea andcreatinine level posed by Naja naja karachiensis venom in rabbits
Snake venom has been responsible for detrimental effectsto the renal tissues [40] Likewise Naja naja krachiensisvenomwas found to cause severe renal damage by significantrise in serum urea and creatinine levels Indeed phospho-lipases enzymes are responsible for the increase in vascularpermeability with hemorrhagic effects to the vital tissues inthe victims Subsequently numerous lesions are producedrelated to the glomerular membrane and renal tubules eitherinterstitial or vascular [29 41 42] Presence of lymphocytes(white blood cells) and oedema in the cortical as well asmedullary regions of renal tubules further confirmed theidea of renal damage (data has not shown) as reportedpreviously with different snake venoms like Hemiscorpiuslepturus [26 43] Apart of it pharmacokinetic studies ofNaja naja karachiensis venom with short lived radioisotope99mTc confirmed that kidneys and urinary bladder are themost saturated organs (gt70) after intravenous injection inexperimental rabbits (unpublished data by our group)
Medicinal plants of Pakistan are used to inhibit snakevenom (PLA
2) enzymes [6] Due to this effect present
study was designed to select twenty-eight medicinal plantsof Pakistan to test their potentials as an antidote againsttoxicities produced by Naja naja karachiensis venom It
was fascinating that extract of Stenolobium stans (L) Seemshowed significant neutralization compared to that of refer-ence standard (antisera) as reported previously by Asad etal for inhibition of phospholipases A
2anticoagulant activity
[6 44] Present study indicated that extract of Stenolobiumstans (L) Seem possesses an endogenous inhibitor(s) tonullify venom (PLA
2) induced toxicities Extracts of these
plants material are routinely used in Pakistan by simpleapplication in the form of paste to the affected area [45]This practice provides effective first aid treatment as tinymolecules of an antidote diffuse favorably at the site of snakebite before hospitalization and abrogated the spreading oftoxins [39] It is owing to the various secondary metaboliteslike phenols flavonoids terpenoids xanthenes quinonoidsand so forth as reported earlier to mask various enzymaticactions of cobra venom [6 23] Indeed secondarymetabolitespose hindrance in binding of different snake venom enzymesto their potential targets therefore antidotal effect evokedOther plants extract were not proved significantly effectiveto neutralize cobra venom therefore cannot be declareduseful plants in venom therapy It is the need of the time toisolate lethal component(s) of cobra venom attributed to itsmajor toxicities Furthermore characterization of antivenom
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 11
1000
0
2000CK
-MB
(UL
)S
erum
leve
l of L
DH
LDHCK-MB
0-Al
bizi
a leb
beck
(L)
Bent
hAl
lium
cepa
L
Alliu
m sa
tivum
L
Alth
aea
officin
alis
LBa
uhin
ia va
riega
ta L
Br
assic
a ni
gra
(L)
W D
J K
och
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Calo
tropi
s pro
cera
(Aito
n) W
T A
iton
Cedr
us d
eoda
ra (R
oxb
ex D
Don
) G D
onCi
trus l
imon
(L)
Burm
f
Citru
llus c
oloc
ynth
is (L
) Sc
hrad
Cum
inum
cym
inum
L
Enico
stem
ma
hysso
pifo
lium
(Will
d) I
Ver
dFa
goni
a cr
etica
L
Leuc
as ca
pita
ta D
esf
Mat
thio
la in
cana
(L)
W T
Aito
nM
omor
dica
char
antia
L
Neriu
m in
dicu
m M
illO
cimum
sanc
tum
L
Pinu
s rox
burg
hii S
arg
Pista
cia in
tege
rrim
a J
L S
tew
art
Psor
alea
cory
lifol
ia L
Rhaz
ya st
ricta
Dec
ne
Rubi
a co
rdifo
lia L
Sapi
ndus
muk
oros
si G
aert
n
Sten
olob
ium
stan
s (L
) See
mTe
rmin
alia
arju
na (R
oxb
ex D
C)
Trich
odes
ma
indi
cum
(L)
Sm
Zing
iber
offi
cinal
e Ros
coe
Refe
renc
e sta
ndar
d an
ti do
te
Various antivenoms tested (100mgkg)
Wig
ht an
d A
rn
Figure 3 Comparison of various medicinal plant extracts with reference standard antisera in neutralization of LDH and CK-MB levelselevated by Naja naja karachiensis venom in rabbits
compound(s) from medicinal plant extracts would be worthfull for complete and effective treatment of snake bite in thefuture
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are cordially thankful to Dr Khan MuhammadSajid (Deputy Chief Scientist MINAR) and Israr Ahmad(Radiopharmacist MINAR) for their valuable suggestionsSpecial thanks are due to Director COMSATS Institute ofInformationTechnologyAbbottabad Pakistan for the grant ofstudy leave to conduct research inMultan Institute of NuclearMedicine and Radiotherapy (MINAR) Nishtar HospitalMultan Pakistan
References
[1] WWuster ldquoThe cobras of the genus Naja in IndiardquoHamadryadvol 23 pp 15ndash32 1998
[2] P E P Deraniyagala ldquoThe taxonomy of the cobras of south-eastern Asia part 2rdquo Spolia Zeylanica vol 29 pp 205ndash232 1961
[3] T M Davidson S Schafer and J Killfoil ldquoCobrasrdquo Wildernessand Environmental Medicine vol 6 no 2 pp 203ndash219 1995
[4] M H H B Asad M T Razi T Khan et al ldquoCoagulopathies inNaja naja karachiensis (black Pakistan cobra) bites and its effect
on coagulation tests upon storage of platelet poor plasmardquo ActaPoloniae Pharmaceutica vol 69 pp 1031ndash1034 2012
[5] R C Hider E Karlsson and S Namiranian ldquoSeparation andpurification of toxins from snake venomsrdquo in InternationalEncyclopedia of Pharmacology andTherapeutics 11 A L HarveyEd section 134-snake toxins pp 1ndash34 Pergamon Press NewYork NY USA 1991
[6] M H H B Asad M T Razi Durr-e-Sabih et al ldquoAnti-venompotential of Pakistani medicinal plants inhibition of anticoagu-lation activity of Naja naja karachiensis toxinrdquo Current Sciencevol 105 pp 1419ndash1424 2013
[7] D Mebs ldquoToxicity in animals Trends in evolutionrdquo Toxiconvol 39 no 1 pp 87ndash96 2001
[8] S D Aird ldquoOphidian envenomation strategies and the role ofpurinesrdquo Toxicon vol 40 no 4 pp 335ndash393 2002
[9] T Chijiwa Y Yamaguchi T Ogawa et al ldquoInterisland evolutionofTrimeresurus flavoviridis venomphospholipaseA
2isozymesrdquo
Journal of Molecular Evolution vol 56 no 3 pp 286ndash293 2003[10] B Lomonte Y Angulo and L Calderon ldquoAn overview of lysine-
49 phospholipase A2 myotoxins from crotalid snake venomsand their structural determinants of myotoxic actionrdquo Toxiconvol 42 no 8 pp 885ndash901 2003
[11] B LDhananjaya B S Vishwanath andC JMDrsquoSouza ldquoSnakevenom nucleases nucleotidases and phosphomonoesterasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 155ndash171 CRC Press Boca Raton Fla USA 2009
[12] S PMackessy ldquoPhosphodiesterases ribonucleases anddeoxyri-bonucleasesrdquo in Enzymes from Snake Venom G S Bailey Edpp 361ndash404 Alaken Fort Collins Colo USA 1998
[13] B L Dhananjaya A Nataraju R Rajesh et al ldquoAnticoagu-lant effect of Naja naja venom 5∘nucleotidase demonstration
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
12 BioMed Research International
through the use of novel specific inhibitor vanillic acidrdquoToxicon vol 48 no 4 pp 411ndash421 2006
[14] B L Dhananjaya and C J M DrsquoSouza ldquoThe Pharmacologicalrole of phosphatases (acid and alkaline phosphomonoesterases)in snake venoms related to release of purinesmdasha multitoxinrdquoBasic and Clinical Pharmacology and Toxicology vol 108 no 2pp 79ndash83 2011
[15] K S Girish D K Jagadeesha K B Rajeev and K KemparajuldquoSnake venom hyaluronidase an evidence for isoforms andextracellular matrix degradationrdquo Molecular and Cellular Bio-chemistry vol 240 no 1-2 pp 105ndash110 2002
[16] NH Tan and S Y Fung ldquoSnake venomL-amino acid oxidasesrdquoin Handbook of Venoms and Toxins of Reptiles S P MackessyEd pp 221ndash235 CRC Press Boca Raton Fla USA 2009
[17] D Mebs Venomous and Poisonous Animals A Handbookfor Biologists Toxicologists and Toxinologists Physicians andPharmacists CRC Press Boca Raton Fla USA 2002
[18] NAAbdoon andA J Fatani ldquoCorrelation between blood pres-sure cytokines and nitric oxide in conscious rabbits injectedwith Leiurus quinquestriatus quinquestriatus scorpion venomrdquoToxicon vol 54 no 4 pp 471ndash480 2009
[19] K S Latimer E A Mahaffey and K W Prasse Duncanand Prassersquos Veterinary Laboratory Medicine Clinical PathologyWiley-Blackwell New York NY USA 5th edition 2003
[20] J E Harkness P V Turner S V Woude and C L WhelerHarkness and Wagnerrsquos Biology and Medicine of Rabbits andRodents Wiley-Blackwell New York NY USA 5th edition2010
[21] F S Apple ldquoThe specificity of biochemical markers of cardiacdamage a problem solvedrdquo Clinical Chemistry and LaboratoryMedicine vol 37 no 11-12 pp 1085ndash1089 1999
[22] HO AlkadiM A Noman A K Al-Thobhani F S Al-Meklafiand Y A Rajarsquoa ldquoClinical and experimental evaluation of theeffect of Khat-induced myocardial infarctionrdquo Saudi MedicalJournal vol 23 no 10 pp 1195ndash1198 2002
[23] M H H B Asad G Murtaza S Siraj et al ldquoEnlisting the sci-entifically unnoticed medicinal plants of Pakistan as a source ofnovel therapeutic agents showing anti-venom activityrdquo AfricanJournal of Pharmacy and Pharmacology vol 5 no 20 pp 2292ndash2305 2011
[24] M H H B Asad D E Sabih B A Chaudhary et al ldquoAnti-hemolytic (anti-venom) activity of Pakistani medicinal plantsuponNaja naja karachiensis venominduced hemolysisrdquo Journalof Animal and Plant Sciences In press
[25] E B Ezenwanne and R E Ucheya ldquoA study of the serumconcentrations of some hepatic enzymes in doses of aqueousleaf extract of Vernonia amygdalina in rabbitsrdquo InternationalJournal of Animal and Veterinary Advances vol 4 pp 80ndash832012
[26] Z A Mirakabadi S M Khatoonabadi S H Teimourzadehand G H H Sabiri ldquoSerum enzymes studies in scorpion(Hemiscorpius lepturus) dose related envenomation in rabbitsrdquoArchives of Razi Institut vol 65 pp 83ndash89 2010
[27] C J Green J Knight S Precious and S Simpkin ldquoKetaminealone and combined with diazepam or xylazine in laboratoryanimals a 10 year experiencerdquo Laboratory Animals vol 15 no2 pp 163ndash170 1981
[28] S S Hasson A A Al-Jabri T A Sallam M S Al-Balushiand R A A Mothana ldquoAntisnake venom activity of Hibiscusaethiopicus L against Echis ocellatus and Naja n nigricollisrdquoJournal of Toxicology vol 2010 Article ID 837864 8 pages 2010
[29] M Tahir Razi M H H B Asad T Khan et al ldquoAnti-haemorrhagic potentials of Fagonia cretica against Naja najakarachiensis (black Pakistan cobra) venomrdquo Natural ProductResearch vol 25 no 20 pp 1902ndash1907 2011
[30] M Chethankumar and L Srinivas ldquoGangliosides as potentialinhibitors of Naja naja venom PLA2 (NV-PLA2) inducedhuman erythrocyte membrane damagerdquo African Journal ofBiochemistry Research vol 2 no 1 pp 8ndash14 2008
[31] T R Rahmy and K Z Hemmaid ldquoHistological and histochem-ical alterations in the liver following intramuscular injectionwith a sublethal dose of the Egyptian cobra venomrdquo Journal ofNatural Toxins vol 9 no 1 pp 21ndash32 2000
[32] B Adzu M S Abubakar K S Izebe D D Akumka and K SGamaniel ldquoEffect of Annona senegalensis rootbark extracts onNaja nigricotlis nigricotlis venom in ratsrdquo Journal of Ethnophar-macology vol 96 no 3 pp 507ndash513 2005
[33] M A Huffman ldquoAnimal self-medication and ethno-medicineexploration and exploitation of the medicinal properties ofplantsrdquo Proceedings of the Nutrition Society vol 62 no 2 pp371ndash381 2003
[34] B W Segelke D Nguyen R Chee N H Xuong and E ADennis ldquoStructures of two novel crystal forms ofNaja naja najaphospholipase A
2lackingCa2+ reveal trimeric packingrdquo Journal
of Molecular Biology vol 279 no 1 pp 223ndash232 1998[35] Q Lu JM Clemetson andK J Clemetson ldquoSnake venoms and
hemostasisrdquo Journal of Thrombosis and Haemostasis vol 3 no8 pp 1791ndash1799 2005
[36] S Ehrenforth J F Schenk and I Scharrer ldquoLiver damageinduced by coumarin anticoagulantsrdquo Seminars in Thrombosisand Hemostasis vol 25 no 1 pp 79ndash83 1999
[37] M Nandave S K Ojha S Joshi S Kumari and D SArya ldquoCardioprotective effect of Bacopa monneira againstisoproterenol-induced myocardial necrosis in ratsrdquo Interna-tional Journal of Pharmacology vol 3 no 5 pp 385ndash392 2007
[38] H V Patel A A Vyas K A Vyas et al ldquoHeparin and heparansulfate bind to snake cardiotoxin sulfated oligosaccharides asa potential target for cardiotoxin actionrdquo Journal of BiologicalChemistry vol 272 no 3 pp 1484ndash1492 1997
[39] S Yingprasertchai S Bunyasrisawat and K Ratanabanang-koon ldquoHyaluronidase inhibitors (sodium cromoglycate andsodium auro-thiomalate) reduce the local tissue damage andprolong the survival time of mice injected with Naja kaouthiaand Calloselasma rhodostoma venomsrdquo Toxicon vol 42 no 6pp 635ndash646 2003
[40] M Schneemann R Cathomas S T Laidlaw A M El NahasR D G Theakston and D A Warrell ldquoLife-threateningenvenoming by the Saharan horned viper (Cerastes cerastes)causingmicro-angiopathic haemolysis coagulopathy and acuterenal failure clinical cases and reviewrdquo QJMmdashMonthly Journalof the Association of Physicians vol 97 no 11 pp 717ndash727 2004
[41] J Meier and K Stocker ldquoEffects of snake venoms on hemosta-sisrdquoCritical Reviews inToxicology vol 21 no 3 pp 171ndash182 1991
[42] N Marsh D Gattullo P Pagliaro and G Losano ldquoThe gaboonviper Bitis gabonica hemorrhagic metabolic cardiovascularand clinical affects of the venomrdquo Life Sciences vol 61 no 8pp 763ndash769 1997
[43] M H Pipelzadeh A Jalali M Taraz R Pourabbas and AZaremirakabadi ldquoAn epidemiological and a clinical study onscorpionism by the Iranian scorpion Hemiscorpius lepturusrdquoToxicon vol 50 no 7 pp 984ndash992 2007
[44] M H H B Asad Durr-e-Sabih B A Choudary A F Asad GMurtaza and I Hussain ldquoCompensatory effects of medicinal
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
BioMed Research International 13
plants of Pakistan upon prolongation of coagulation assaysinduced by Naja naja karachiensis biterdquo Current Science vol106 pp 870ndash873 2014
[45] S R BaquarMedicinal and Poisonous Plants of Pakistan PrintasKarachi Pakistan 1st edition 1989
[46] I Archetti C Tittarelli M Cerioli R Brivio G Grilli and ALavazza ldquoSerum chemistry and hematology values in commer-cial rabbits preliminary data from industrial farms in northernItalyrdquo in Proceedings of the 9th World Rabbit Congress pp 1147ndash1152 Verona Italy 2008
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology
Submit your manuscripts athttpwwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anatomy Research International
PeptidesInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporation httpwwwhindawicom
International Journal of
Volume 2014
Zoology
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Molecular Biology International
GenomicsInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioinformaticsAdvances in
Marine BiologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Signal TransductionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Evolutionary BiologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Biochemistry Research International
ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Genetics Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Virolog y
Hindawi Publishing Corporationhttpwwwhindawicom
Nucleic AcidsJournal of
Volume 2014
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Enzyme Research
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
International Journal of
Microbiology