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RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1625
EFFECT ON CNS ACTIVITY AND ANALGESIC ACTIVITY OF
PARTHENIUM HYSTEROPHORUS L BY METHANOLIC
EXTRACT AT AERIAL PART IN SWISS ALBINO MICE
1Abdullah Al Mamun*,
2Ahsan Habib,
3Md. Ershad Alam,
4Md. Mahfuzur Rahman,
5 Most. Shamima Parvin
1,2,3Bangladesh University, Department of pharmacy, Mohammodpur,Dhaka-1207,BANGLADESH.
4 Gono Bishwabidyalay, BANGLADESH 5 Stamford University, BANGLADESH
Corresponding Author:
Abdullah Al Mamun
Bangladesh University
Department of pharmacy
Mohammodpur, Dhaka-1207, BANGLADESH
Email: mamunal007@yahoo.com
Phone: +8801712477029
International Journal of Innovative
Pharmaceutical Sciences and Research www.ijipsr.com
Abstract
In the present study, the analgesic and CNS depressant effect of the aerial part of the methanol extract of
Parthenium hysterophorus was investigated. The methanolic extracts of Parthenium hysterophorus were
evaluated for its analgesic activity in mice by using acetic acid-induced writhing, formalin induced Licking test,
hot plate and tail-flick tests and central nervous system (CNS) depressant effect by using rodent behavioral
models, such as hole cross, open field; thiopental sodium induced sleeping time tests and Tail suspension test
for its anti-depressant properties. The results in acetic acid-induced writhing test and formalin induced licking
test for antinociceptive activity, the methanolic extracts at 2.5mg/kg and 5mg/kg dose exhibited significant
(p<0.001) reduction of writhing response and formalin induced licking response in a dose dependent manner. In
Open Field Test and Hole Cross Test , for anti-depressant activity, both dose levels of 2.5mg/kg and 5 mg/kg
exhibited significantly (p<0.001) reduction of movement of mice and also in tail suspension test and thiopental
sodium induced sleep time test, the methanolic extract of Parthenium hysterophorus showed significant
antidepressant activity compared to standard. These results provide in vivo evidence that aerial parts of
Parthenium hysterophorus in general have significant Analgesic and antidepressant effects.
Key words: Analgesic, Anti-depressant properties, Writhing response.
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1626
INTRODUCTION
Definition of medicinal plants
A considerable number of definitions have been proposed for medicinal plants. According to the
WHO, “A medicinal plant is any plant which, in one or more of its organs, contains substances
that can be used for therapeutic purposes, or which are precursors for chemo-pharmaceutical
Semi-synthesis.” When a plant is designated as ‘medicinal’, it is implied that the said plant is
useful as a drug or therapeutic agent or an active ingredient of a medicinal preparation.
“Medicinal plants may therefore be defined as a group of plants that possess some special
properties or virtues that qualify them as articles of drugs and therapeutic agents, and are used
for medicinal purposes.
“The plants that possess therapeutic properties or exert beneficial pharmacological effect on the
animal body are generally designated as medicinal plant.” (Ghani, 2005)
History of medicinal plant
The earliest known medical document is a 4000-years-old Sumerian clay tablet that recorded
plant remedies for various illnesses. As evident from the papyrus Ebers (written in about 1500
BC), the ancient Egyptians possessed a good knowledge of the medicinal properties of hundreds
of plants. The pun-tsao containing thousands of herbal cures attributed to Shennung, chain’s
legendary emperor who lived 4500 years ago. The earliest mention of the medicinal use of plants
in the Indian subcontinent is found in Rig Veda (4500-1600 BC), which noted that the Indo-
Aryans used the Soma plant as a medicinal agent. The Badianus Manuscript is an illustrated
document that reports the traditional medical knowledge of the Aztecs (Levetin and McMahon
2003).
Development of drugs from medicinal plants
In recent past years more than 20,000 medicinal plants have been identified. Phytochemical and
pharmacological studies of some of these plants have already resulted in the discovery and
development of many important drugs. Some of the glaring examples of such drugs of plant
origin include the following: Anti- malarial drug from Cinchona species, sedative analgesic
drugs from Papaversomniferum.
Development of drugs from medicinal plants is often an elaborate, laborious, time-consuming
and expensive exercise. Careful phytochemical analysis and pharmacological and clinical tests
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1627
are pre-requisites for developing drugs from medical plants. The stages involved in the
development exercise may be summarised in the following way:
a) Selection and correct identification of the proper medicinal plant and its extraction with a
suitable solvent.
b) Detection of biological activity of the crude extract and establishment of a bioassay system to
permit the identification of the active fraction.
c) Fractionation of the crude extracts by using Physico-chemical procedures, and monitored by
biological tests, identification and separation of active fractions.
d) Isolation of the active constituents by chromatographic or other techniques and purification
of the isolated compounds by repeated chromatography and crystallisation
e) Establishment of the chemical structures of the pure compounds by various Physico-chemical
techniques and determination of their biological activity by various pharmacological and
toxicological tests
f) Production of the drug in appropriate dosage form.
Plant Biography:
Domain: Eukaryota
Kingdom: Plantae
Phylum: Spermatophyta
Class: Magnoliatae
Order: sterales
Family: Asteraceae
Genus: Parthenium
Species: Parthenium hysterophorus L.
Plant Description
Parthenium hysterophorus is a much-branched, short-lived (annual), upright (erect) herbaceous
plant that forms a basalrosette of leaves during the early stage of growth. It usually grows 0.5-1.5
m tall, but can occasionally reach up to 2 m or more in height.
Mature stems are greenish and longitudinally grooved, covered in small stiff hairs (hirsute), and
become much branched at maturity.
The alternately arranged leaves are simple with stalks (petioles) up to 2 cm long and form a
basalrosette during the early stages of growth. The lower leaves are relatively large (3-30 cm
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1628
long and 2-12 cm wide) and are deeply divided (bi-pinnatifid or bi-pinnatisect). Leaves on the
upper branches decrease in size and are also less divided than the lower leaves. The undersides
of the leaves, and to a lesser degree their upper surfaces, are covered with short, stiff hairs that
lie close to the surface (they are appressedpubescent). Numerous small flower-heads (capitula)
are arranged in clusters at the tips of the branches (in terminalpanicles). Each flower-head
(capitulum) in borne on a stalk (pedicel) 1-8 mm long. These flower-heads (4-5 mm across) are
white or cream in colour and have five tiny 'petals' (rayflorets) 0.3-1 mm long. They also have
numerous (12-60) tiny white flowers (tubularflorets) in the centre and are surrounded by two
rows of small green bracts (an involucre). Colour changes to light brown when seeds are mature
and about to shed. Flowering can occur at any time of the year, but is most common during the
rainy seasons. Five small 'seeds' (achenes) are usually produced in each flower-head (capitulum).
These achenes (1.5-2.5 mm long) consist of a black seed topped with two or three small scales (a
pappus) about 0.5-1 mm long, two straw-coloured papery structures (actually dead
tubularflorets), and a flat bract.
Fig 1: Photographs of P. hysterophorus
MATERIALS AND METHOD
Preparation of Plant Extract for Experiments
Plants Collection
The leaves of Parthenium Hysterophorus L were collected from the Karwan bazar, Dhaka,
Bangladesh. The time of collection was January, 2013 at the daytime.
Drying of the plant sample
The stem and other adulterants were removed at first. Then the leaves were washed with water to
get the fresh sample. Then the collected samples were dried under shade at room temperature for
five days.
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1629
Grinding of the dried sample
The dried samples were grounded to coarse powder with Blender Machine (NOWAKE, JAPAN)
a and powdered samples were kept in clean closed glass container. During grinding of sample,
the grinder was thoroughly cleaned to avoid contamination with any other substance that was
grounded previously. The dried grinded powder was weighed by rough balance.
Extraction of Dried Powder Sample
150g of the dried powder was taken in a 500ml beaker. Then methanol was added to the powder
with continuous stirring until the powders were soaked properly. Then the mixer was
continuously stirred after few hours, and the beaker was kept for three days. At fourth day the
extract was collected and filtered using a sterilized cotton filter. The volume of the extract was
reduced by using “Rotary Evaporator”. Then this small volume of extract was dried at room
temperature by normal air flow. After drying, 7.69g of dried extract was obtained from 150g of
powder. This extract was used for investigation.
Overall Extraction Process:
The Flow Chart of the extraction process shown below-
Identification of plants/plants part
Collection of plants at suitable time and session
Drying of plant parts in a suitable size
Grinding
Powders were collected and stored in a cool and dry place
Preparation of extracts with methanol
Filtrations were done
Tests were performed to observe the pharmacological effects of the extract.
Fig.2: Flow Chart of the Overall extraction process of Parthenium hysterophorus
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1630
Table 1: Procedure of Chemical Group Test
Sample Test solution Observation Inference
Test for Alkaloids:
# 2 ml solution of the
extract and 0.2 ml of
dilute hydrochloric acid
0.1 ml of Mayer’s
reagent.
Yellowish buff
colored precipitate
was obtained.
Presence of alkaloid
# 2 ml solution of the
extract and 0.2 ml of
dilute hydrochloric acid.
0.1 ml of
Dragendroff’s reagent.
Orange brown
precipitate was
observed.
Presence of alkaloid
# 2 ml solution of the
extract and 0.2 ml of
dilute hydrochloric acid.
0.1 ml of iodine
solution (Wagner’s
reagent).
Reddish brown
precipitate was
obtained.
Presence of alkaloid
# 2 ml solution of the
extract and 0.2 ml of
dilute hydrochloric acid.
0.1 ml of picric acid
solution (Hager’s
reagent).
Yellowish precipitate
was obtained. Presence of alkaloid
Test for Steroids:
# 10 mg extract dissolved
in 1 ml chloroform.
1 ml sulfuric acid.
Chloroform layer
acquired reddish
brown color and acid
layer showed green
fuorescence.
Presence of steroid
Tests for Flavonoids:
# 10 ml of solution of
extract hydrolyzed with
10%sulfuric acid. This
was extracted with ether
and divided into three
portions.
a) 1 ml dilute
Ammonia solution
b)1 ml dilute sodium
carbonate solution
c) 1 ml dilute sodium
hydroxide solution
a) Greenish yellow
color was obtained.
b) Pale yellow color
was obtained.
c) Yellow color was
obtained.
a) Presence of flavonoids
b) Presence of
Flavonoids
c) Presence of
Flavonoids
Tests for Reducing
Sugars:
# 5 ml solution of extract.
5 ml Fehling’s A and
B solution. Boiled for
5 minutes on a boiling
water bath.
Brick red colored
precipitate was not
observed.
Presence of reducing
sugars
# 5 ml solution of extract.
5 ml Benedict’s
reagent and boiled for
5 minutes on a boiling
water bath.
Brick red colored
precipitate was not
observed.
Presence of reducing
sugars
#5 ml solution of extract.
2 drops of 5% alpha-
Naphthol solution
(Freshly prepared and
added 1 ml of sulfuric
acid on the sides of the
test tube.)
Violet colored ring
was not formed at the
junction of two
liquids.
Presence of reducing
sugars
Tests for Tannins:
# 5 ml solution of extract.
1 ml of 10% Lead
acetate solution. Yellow precipitate. Presence of tannins
# 5 ml solution of extract.
1 ml of 10%
potassium dichromate
solution.
Yellowish brown
precipitate was not
obtained.
Presence of tannins
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1631
Test for Glycosides:
small amount of extract
1 ml of water and a
few drops of sodium
hydroxide solution
A yellow color is
formed Presence of glycosides
Test for Saponins
# 1 ml solution of the
extract
Diluted to 20 ml with
distilled water
Shake during 15
minutes and form 1
centimeter foam layer
Presence of saponins
Test for
Carbohydrates
# 2 ml of extract
2 ml of conc. sulfuric
acid
A red or reddish
violet ring is formed Presence of carbohydrate
Test for resins
1 mg of extract
5 to 10 ml of acetic
anhydride and .005 ml
of sulfuric acid
A bright purplish
color, rapidly
changing to violet is
produced
Presence of resins
RESULT AND DISCUSSION
Hot plate Test:
The hot-plate was used to measure the response latencies. It is also called thermal nociception
test method (Eddy and leimbach et al., 1953). In these experiments, the hot plate was maintained
as at 50± 5 0
C. the reaction time was recorded for animals pre-treated with DMSO (10ml/kg 30
min before orally) as control or Methanolic Extract of Perthenium hysterophorus (MEPH)
(2.5mg/kg, and 5mg/kg orally, 30 min before), morphine (5.0 mg/kg intraperitonially, 15 min
before) which was used as positive control group. Animals were placed into the hot plate
chamber and the time of latency was defined as the time period between the zero point, when the
animal was placed on the hot- plate surface, and the time when animal licked its paw or jumped
off to avoid thermal pain. The latency period of response was taken as the index of
antinociception and was determined at pretreatment, 30, 60, 90 and 120 min after the
administration of the test drugs and standard, in order to minimize the damage on the animal
paws; the cut-off time was taken as 20s.
.
Fig. 3: Hot plate Test
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1632
Hot plate Test:
In the hot plate test there were no significant difference in the antinociceptive effect of 2.5mg/kg
and 5 mg/kg MEPH in the Mean ± SEM value for nociception. The antinociceptive effect of
MEPH is dose dependent. MEPH considerably increase the animal reaction time to the heat
stimulus. The increase was significant in all cases beginning from 60 min compared to control (p
< .05) but the dose of 2.5mg/kg p.o. significantly increased the reaction time beginning from 60
min compared to control (p < .01). Morphine (5 mg/kg) markedly increased pain latency (p <
.001) in all case from 30 min.
Table 2: Effect of whole plant extract of Parthenium hysterophorus on Hot plate Test
Treatment Dose Response times (in seconds)
Pretreatment 30 min 60 min 90 min 120 min
Control
(DMSO)
10
ml/kg 4.80±.267 5.31±0.393 7.24±0.275 7.53±0.180 7.76±0.170
Positive
control
(Morphine)
5
mg/kg ٭0.466±14.73 ٭157.±12.95 ٭0.183±11.23 ٭9.49±0.373 268.5.74±
Group I
(Extract)
2.5
mg/kg
7.37±0.711
٭ ٭0.241±11.60 ٭0.220±9.49 ٭0.121±8.56 ٭7.21±0.152
Group II
(Extract)
5
mg/kg ٭6.68±0.441
٭7.58±0.213
٭8.67±0.523
٭237.9.62±
٭12.57±0.588
Each value is presented as the mean ± SEM (n=5). MEPH= methanolic extract of Parthenium
hysterophorus. DMSO= Dimethyl sulfoxide
٭ Dunnett p< .001. Dunnett test as compared to control.
Tail Flick test:
The procedure is based on the observation that the morphine like drugs selectively prolongs the
reaction time of the typical tail withdrawal reflex in mice (D’amour & Smith, 1941). The animals
of the control, positive control and test groups were treated with DMSO (10ml/kg, P.o.),
Morphine (5 mg/kg, i.p.) and test samples at the dose 2.5mg/kg, p.o. and 5 mg/kg. P.o.
respectively. 1 to 2 cm of the tail of the mice was immersed in warm kept constant at 55 0
C. The
reaction time was the time taken by the mice to deflect their tails. The first reading was discarder
and the reaction time was recorded as a mean of the next three reading. A latency period of 20 s
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1633
was defined as complete analgesic and the measurement was stopped when the latency period
exceeded to avoid injury to mice. The latent period of the tail- flick response was determined at
pretreatment, 30, 60, 90 and 120 min after the administration of the test drugs and standard.
Fig. 4 Tail Flick test
Tail Immersion test
The tail-immersion test results showed an antinoceptive activity. There were no significant
difference in the antinoceptive effect of 10 and 2.5mg/kg of MEPH when compared with
reference drugs. MEPH at dose of both 2.5mg/kg, 5mg/kg p.o. significantly increase tail flick
latency period beginning from 30 min compared to control (p < .001) and also Morphine (5
mg/kg) markedly increased tail flick latency compared to control (p < .001) in all case from 30
min.
Table 3: Effect of whole plant extract of Parthenium hysterophorus extract on Tail
Immersion test
Treatment Dose Response times (in seconds)
Pretreatment 30 min 60 min 90 min 120 min
Control
(DMSO)
10
ml/kg 1.18±0.34 1.64±0.08 2.75±0.20 2.89±.028 3.24±0.11
Positive
control
(Morphine)
5
mg/kg 1.28±0.13 4.39±0.04* 4.88±0.15* 5.94±0.18* 7.09±0.07*
Group I
(Extract)
2.5
mg/kg 1.17±0.08 2.75±0.12* 4.05±0.15* 4.68±0.14* 5.19±0.15*
Group II
(Extract)
5
mg/kg 1.15±0.02 2.87±0.20* 4.40±0.17* 4.96±0.12* 5.88±0.18*
Each value is presented as the mean ± SEM (n=5). MEPH= methanolic extract of Parthenium
hysterophorus.
.Dunnett p< .001 respectively. Dunnett test as compared to control٭
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1634
Formalin Induced licking test:
The animals were divided into control, positive control and test groups with five mice in each
group. Mice were orally pretreated with MEPH (2.5mg/kg and 5 mg/kg p.o.), and the control
group received similar volume of vehicle (DMSO, 10 ml/kg, p.o.) 60 min prior the nociceptive
agent. Morphine (10 mg/kg. i.p.) was used as the reference drug and administered 15 min before
the nociceptive agent. Animals received 20 µl of a 2.5% formalin solution (0.92% formaldehyde)
made up in saline, injected intraplantarly in the ventral surface of the right hand paw. Animal
were observed from 0 to 05 min (neurogenic phase) and 15-30 min (inflammatory phase) and the
number of licking of the injected paw was counted and considered as indicative of nociception
(Santos ARS et al., 1999 and Santos ARS et al., 1997).
Fig. 5: Half Writhing Given by Mice
Fig. 6: Full Writhing Given by Mice
Formalin Induced licking test
The results show that the methanolic extract from Parthenium hysterophorus(2.5mg/kg and 5
mg/kg orally) caused a significant dose dependent inhibition of both the neurological (0-5 min.)
and inflammatory (15-30 min.) phase of formalin induced licking. However, their
antinociceptive effects were significantly compared to control (p< .001) in both the early and late
phase and also Morphine (5 mg/kg) markedly increased significantly compared to control (p<
.001) in both the early and late phase
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1635
Table 4: Effect of whole plant extract of Parthenium hysterophorus on Formalin Induced
licking test
Treatment Dose
Licking Number
(Mean± SEM) % Inhibition
Early Phase Late Phase Early Phase Late Phase
Control
(DMSO) 10 ml/kg 117.20±4.56 96.60±3.90 00 00
Positive control
(Morphine) 5 mg/kg 39.60±1.44* 00±00* 66.21 100
Group I
(Extract) 2.5mg/kg 88.00±2.50* 7.40±1.03* 24.91 92.33
Group II
(Extract) 5 mg/kg 90.40±2.30* 2.80±.38* 22.86 76.33
Each value is presented as the mean ± SEM (n=5). MEPH= methanolic extract of Parthenium
hysterophorus.
.Dunnett p< .001 respectively. Dunnett test as compared to control٭
Fig. 7: Graphical Presentation of the effect of whole plant extract of Parthenium
hysterophorus extract on Formalin Induced licking test (First phase Paw Licking Number)
Fig. 8: Graphical Presentation of the effect of whole plant extract of Parthenium
hysterophorus extract on Formalin Induced licking test
(Second phase Paw Licking Number)
RESEARCH ARTICLE Mamun et.al / IJIPSR / 2 (8), 2014, 1625-1637
Department of Clinical Pharmacology ISSN (online) 2347-2154
Available online: www.ijipsr.com August Issue 1636
CONCLUSION
Based on the result of the present study, it can be concluded that the all parts of crude plant
extracts of Parthenium hysterophorus. Possesses of acetic acid-induced writhing test, induced
Licking test, hot plate tail-flick tests, central nervous system (CNS) depressant test and
remarkable analgesic potential. Various phytochemical constituents like glycoside, alkaloid,
tannin and carbohydrate present in the plant, as evident from phytochemical analysis. The aerial
parts of extracts shown promising analgesic properties were satisfactory compared to respective
standard drugs. At higher dose, notable analgesic activity was observed from Hot Plate and Tail
Flick Test Dose dependant activity was also identified by all the performed pharmacological
investigations.
ACKNOWLEDGEMENT
We are thankful to Ms Sanjida Haque, department of pharmacy, Bangladesh University. We are
especially grateful to ICDDR'B and Department of Pharmacy, Stamford University Bangladesh.
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