Materials and Methodology
CHAPTER – III
MATERIALS AND METHODOLOGY
3.1. MATERIALS
3.1.1. Experimental Animals
The Swiss albino mice (Musculus albinus) (Figure 4) were selected for this
present study as the experimental animal. They were obtained from College of
Veterinary Science and Animal Husbandry, Rasalpura, Mhow, Indore (M.P.) after
obtaining the approval from Institutional Animal Ethical Committee (IAEC) vide
proposal no. CPCSEA/118/2014 dated 20/12/012 and were kept in the animal house
of IPS Academy, Indore (CPCSEA Reg. No.465/01/ab dated 24th August, 2001).
Mice, both male and female weighing 20-28 gm 6-8 weeks old were used for the
experiments. Animals were kept in the laboratory under constant temperature of 22
± 20C, relative humidity of 50± 10 % and 12 hour photoperiod. Commercial pellet
diet & water were fed ad libitum. The animals were kept in sterilized cages with dry
husk padding and were fed daily with standard balanced diet (certificate enclosed).
Figure 4: Swiss albino mice
3.1.2.1. Test Toxicant
Mancozeb (commercial grade of 75% wettable powder) was made available
from Indofill Chemical Company, Mumbai. The powder of mancozeb has been
diluted in distilled water and dimethyl sulfoxide as per the dose selected after
conducting the lethal dose (LD) 50 toxicity tests. The low dose was selected as
4.156 mg/kg/body weight (bw) and the high dose was selected as 6.650 mg/kg/bw.
The doses of mancozeb will be administered by oral gavage in albino mice.
Materials and Methodology
The technical details of the test pesticide are:
General Name: Mancozeb
3.2.1.2. IUPAC nomenclature
Manganese ethylene-bis (dithiocarbamate) (polymeric)
complex with zinc salt.
3.2.1.3. Chemical name
1, 2-ethaznediybis (carbamodithio) (2- )] manganese zinc salt
3.2.1.4. Empirical formula / molecular formula
(C4H6N2S4Mn)x . (C4H4N2S4Zn)
3.2.1.5. Structural formula
Figure 5. Structure of Mancozeb (MCZ) and Ethylenethiourea (ETU)
3.2.1.6. Half life
In water, mancozeb can be quickly hydrolyzed with a half-life of less than 2
days. In another study, the half-lives for mancozeb (MCZ) in water at pH 5-9 were
reported as less than 1 day (Lyman and Lacoste, 1975). Mancozeb is of low soil
persistence with half-lives of less than 2 and 8 days in aerobic and anaerobic soils,
respectively. In general mancozeb has half life = 1-7 days.
Materials and Methodology
Mancozeb has negligible vapour pressure; hence it has low potential to
volatilize into the air. The identified hydrolysis degradates are Ethylenethiourea
(ETU), Ethyleneurea (EU) and Ethylene Bis-isothiocyanate sulfide (EBIS).
Ethylene-bis-dithiocarbamate (EBDC) possibly degrades via the oxidation to form
Ethylene Bis-isothiocyanate sulfide; the major metabolites being Ethylenethiourea
and Ethyleneurea. Under aerobic conditions, the metabolites breakdown further to
produce CO2. The primary concern with mancozeb (MCZ) is its spontaneous
degradation to Ethylenethiourea (ETU) in the presence of water and oxygen.
3.1.3. EXPERIMENTAL PLANTS
For the proposed work, A. vera and O. sanctum plants were selected as the
experimental herbal plants. The plants were identified by Department of Botany,
Holkar Science College, Indore and Ashtang Ayurvedic College, Lokmanya Nagar,
Kesharbagh Road, Indore (M.P.). These plants were then cultivated to obtain
sufficient quantity for preparing the extracts.
3.1.3.1. Aloe Vera
A. Scientific Classification
Kingdom : Plantae
Clade : Angiosperms
Order : Asparagales
Family : Aloaceae / Asphodelaceae
Subfamily : Asphodeloideae
Genus : Aloe
Species : Aloe vera/ barbadensis Miller
Common Names : Hindi : Gheekumari, Ghee Kunvar, Gwarpattha;
Sanskrit: Ghritkumari; Marathi: Khorpad.
The Species name vera means “True” Or “Genuine”.
In the past Aloe vera has also been assigned to Families Aloaceae and
Liliaceae or Lily Family
Materials and Methodology
A B
C Figure 6: Plant of Aloe vera (barbadensis).
A. vera is a succulent and mucilaginous species found only in cultivation,
having no naturally occurring populations. A. vera (Figure 6) is a stemless or very
short-stemmed plant growing to 60–100 cm (24–40 inches) tall, spreading
by offsets. It does not have a stem and its green leaves resemble blades or a sword
coming out from a central point. The leaves are thick and fleshy, green to grey-
green, with some varieties showing white flecks on their upper and lower stem
surfaces. The margin of the leaf is serrated and has small white teeth. They have
orange flowers which bloom in the summertime.
The leaf is protected by a thick, green epidermis layer (skin or rind), which
surrounds the mesophyll. Immediately beneath the rind are located the vascular
bundles- the xylem, phloem and large pericyclic tubules that contains the yellow leaf
exudates commonly referred to as “aloes,” “sap,” or “latex” (Boudreau and Beland,
Materials and Methodology
2006). The parenchyma or pulp, the major part of the leaf by volume contains the
clear mucilaginous gel possessing precious healing property and medicinal value
(Femenia, et al. 1999; Femenia, et al. 2003).
The medicinal uses of A. vera are amazing, as the benefits are truly
significant. However, more and better trial data are needed to define the clinical
effectiveness of this popular herbal remedy more precisely (Oogler, 1999 and Feily
and Namazi, 2009).
B. Active Compounds (Chemical Composition)
More than 75 potentially active constituents present in A. vera have been
identified so far reported. It contains vitamins, minerals, saccharides, amino acids,
anthraquinones, enzymes, lignin, saponins, and salicylic acids. A. vera leaves
exudate contains phytochemicals under study for possible bioactivity, such as
acetylated mannans, polymannans, C-glycosides, anthrones, anthraquinones such as
emodin, barbaloin and various lectins (King, et al. 1995; Eshun and He, 2004 and
Boudreau and Beland, 2006;). Barbaloin (polyphenolic compound) appear to be
responsible for its bitter taste and cathartic effect (Dagne, et al. 2000). A. vera also
contains products of the isoprenoid pathway, including carotenoids, steroids,
terpenes, and phytosterols (Samman, et al. 1998).
The active gel components include polysaccharides, particularly mannose-
containing polysaccharides, cellulose, and pectic polysaccharides. Juice or gel from
the inner leaf parenchyma contains very small amounts of barbaloin. However, since
there are a number of other potentially active compounds in the plant, it is possible
that the biological activities of A. vera result from the synergistic action of a variety
of compounds, rather than from a single defined component (Dagne, et al. 2000 and
Hamman, 2008). Equally, the potential for constituents to exhibit antagonistic and
competitive activities also influences the overall biological activity of A. vera
preparations (Hamman, 2008).
3.1.3.2. Ocimum Sanctum
Ocimum sanctum also known as Ocimum tenuiflorum is commonly known as
“Tulsi” in Indian subcontinent. It is a tiny perennial herb or small shrub that grows
Materials and Methodology
one to two feet tall with numerous medicinal benefits bestowing upon mankind. This
small shrub has many branches with strongly scented green leaves which are ovate
and slightly toothed (Figure 7). The flowers are purplish to white leaves are of two
types, one with green leaves and other with purple leaves (Wikipedia, 2011).
A. Scientific Classification
Kingdom : Plantae
Division : Angiosperms Class : Magnoliopsida
Order : Lamiales Family : Lamiaceae
Genus : Ocimum Species : sanctum
Binomial name : Ocimum sanctum L. Common Names : Holy Basil, Tulsi, Tulasi, Madura-tala.
Figure 7: Plant of Ocimum sanctum.
B. Active Compounds (Chemical Composition)
Many active components have been identified in O. sanctum L. in which
Eugenol (1-hydroxy-2-methoxy-4- allyl benzene), have been found and largely
responsible for the therapeutic potentials. The leaf volatile oil contains eugenol (also
called eugenic acid), urosolic acid, carvacrol linalool, limatrol, caryophyllene,
Materials and Methodology
methyl carvicol (also called estragol) and anthocyans; while the seed volatile oil
have fatty acids and sitosterol, sugars like xylose and polysaccharides.
The stem and leaves of holy basil contain other variety of constituents
including saponins, flavonoids, triterpenoids, and tannins which also may have
biological activity. Phenolic acids such as rosmarinic acid, propanoic acid, apigenin,
cirsimaritin, isothymusin and isothymonin present in tulsi exhibits antioxidant and
antiinflammatory activities. Two water-soluble flavonoids: Orientin and Vicenin
have shown to provide protection against radiation-induced chromosomal damage in
human blood lymphocytes. The leaves contain ascorbic acid and carotene as well.
The present day information about the chemical properties is based on the
various studies that have been done in different parts of the world and it is likely that
chemical constituents may be varying due to edaphic and geographic factors. Zhang,
et al., (2009) reported that the main components of O. basilicum are: linalool, (Z)-
cinnamic acid methyl ester, cyclohexene, alpha- cadinol, 2,4-diisopropenyl-1-
methyl-1-vinylcyclohexane, 3,5-pyridine-dicarboxylic acid, 2,6-dimethyl-diethyl
ester, beta-cubebene, guaia-1(10),11-diene, cadinene, (E)-cinnamic acid methyl ester
and beta-guaiene.
3.2. METHODOLOGY
3.2.1. Acute Toxicity Test and Mancozeb Intoxication
The acute toxicity lethal dose 50 (LD50) test of mancozeb was carried out to
define the range of the lethal dose and the safe range for the extract. Preliminary
toxicity test were conducted according to the Organisation for Economic Co-
operation and Development (OECD) guideline for Acute Oral Toxicity. Thirty six
(36) Albino mice of both sexes weighing 18 – 25 g were randomly divided into 6
groups of 6 mice each. The groups of mice were treated with mancozeb diluted in
distilled water and dimethyl sulfoxide as adjuvant by oral compulsion. Deaths within
a period of 24 hours were recorded and the median lethal dose LD50 of the extract
was determined according to the method of Weil (104). Simultaneously breeding
was done to continue with the experimentation (Figure 7A).
Materials and Methodology
The LD50 of the aqueous dilution was calculated to be 33.250 mg/kg body
weight (bw). Two doses were selected for the experimental studies; the low dose
was taken as 4.156 mg/kg body weight (D1) and the high dose was taken as 6.650
mg/kg body weight. All the doses used in this study were carefully chosen to
exclude the lethal range (Figure 7: B, C, D).
A B
C D Figure 7: Maintenance of Swiss albino mice in the laboratory.
3.2.2. Preparation of extract of the test plants
Leaves of Aloe vera and Ocimum sanctum were selected for the experiment
and used to prepare the aqueous extract.
3.2.1. Aloe vera extract (Aloe barbadensis Mill.): By method of Bhaya and Saini,
2008 with little modifications is as follows:-
The rinds of fresh medium of Aloe barbadensis leaves were removed and
colourless parenchyma containing aloe gel was exposed and scraped out. It was then
ground in electric blender and centrifuged to remove fibers. The supernatant dried in
shade, powdered and extracted with double distilled water by refluxing for 36 hours
(12 hours × 3) at 800C. The prepared extract was evaporated in vacuum so as to
Materials and Methodology
make it in form of powder. This extract was re-dissolved in double distilled water at
a dose of 400 mg/kg body weight just before oral administration.
3.2.2. Tulsi extract (Ocimum sanctum Linn): By method of Mahdi, et al., 2003
adopted with little modifications is as follows:-
Leaves of O. sanctum (OS) were identified and collected regularly from a
local garden at Department of Botany, Holkar Science College, Indore. The leaves
were washed and dried in the shade, then ground to a powder in the laboratory
grinder. The shed dried powder of O. sanctum was refluxed for 24 hour with double
distilled water at 1000C, cooled and filtered. The solvent was removed under
reduced pressure and evaporated using a lyophiliser into powder (Ganasoundari, et
al. 1998). The powder was stored in refrigerator until further use. The aqueous
extract was prepared using the powder in distilled water at a dose of 250 mg/kg
body weight.
All the herbal extracts as well as mancozeb dosages were administered by
oral gavage in albino mice for 6 consecutive days a week and were continued for 30
days.
3.3. COLLECTION OF BLOOD SAMPLES AND SEPARATION OF SERUM
The requirements for the above procedure included test animal, anaesthetic
agent, towel, and cotton, 19 to 25G needle with 1 to 5 ml syringe, surgical blade,
plastic disposable bag and blood sample collection tubes. All the disposable syringes
used in the experiment were manufactured by Bectan Dickinson and Company,
Rutherford, New Jersey, USA. Blood from the experimental and control mice were
collected by cardiac puncture under mild anaesthesia. 2ml syringe with 20 gauge
needle was preferably inserted in the ventricle either by a thoracotomy, the left side
of the chest, through the diaphragm, or from the top of the sternum just behind the
xiphoid cartilage and slightly left of the middle.
The mouse was held by the scuff of skin above the shoulders with head up
and its rear legs down. The needle was inserted 5mm from the centre of the thorax
towards the animal’s chin 5-10 mm deep at 25-300 degrees away to draw the blood.
About 0.1 - 1 ml of blood could be obtained depending on the size of the mouse and
whether the heart was pumping. Some portion of the blood samples were kept in
Materials and Methodology
anticoagulant tubes for some of the parameter testing while some were kept in cold
overnight for clotting after which centrifuged to carefully pipette out the clear serum
and stored at -200C until required (Figure 8)
A B
Figure 8: Terminal blood collection by cardiac puncture in anesthetized mice in the laboratory.
The serum was separated from the whole blood by leaving the sample for 1
hour at 37°C to clot in the vial collected without anticoagulant. Using a glass pasteur
we carefully loosen the clot from the sides of the tube without lysing the red cells as
they cannot then be separated from the serum. Then the sample was centrifuged at
4000 rotation per minute for 20 minutes at 4°C to obtain the serum. The serum was
removed from the clot by gently pipetting off into a clean tube using a glass pasteur.
Then the samples were labelled with the group number, date and day. The sample
was stored at -20°C for future use (Figure 9).
.
Figure 9: Collection of blood sample in vials
Materials and Methodology
3.4. PREPARATION OF TISSUE
After completion of the proposed dose schedules, the animals were sacrificed
and target organs (kidney and liver) were removed from the dissected body. The
organs were washed in 5% saline water and fixed in 10% formalin solution
according to method of Humanson (1979) for histopathological and histochemical
analysis. Blocks were prepared by usual procedure of dehydration and paraffin
embedding. Sections were cut at 5µ thickness and stained with Harri’s
Haematoxylin and counter stained with Eosin (double staining) in the routine
procedure (McManus, et al. 1964). For general histochemical analysis Bromophenol
Blue was used for staining proteins, Periodic Acid Schiff’s reagent for staining
carbohydrates and Sudan Black B for staining lipids.
3.5. PROTOCOL OF THE PARAMETERS
3.5.1. Assay of Haematological Parameters
A. Haemoglobin Cynamet Method.
The blood samples were collected from the animals for haematological
investigations to determine of haemoglobin level recommended by Cynamet
haemoglobin method (Crosby, et al.1954).
Principle: Haemoglobin is a metallic chromoprotein present in blood and can be
determined by measurement of its colour, its power of combining with oxygen or
carbon monoxide or by its iron content. It is converted to cyanmethaemoglobin
when diluted in a solution containing potassium cyanide and potassium ferricyanide.
The absorbance of the solution is then measured in a photoelectric colorimeter at a
wavelength of 540 nm or with a yellow-green filter. The intensity of the colour is
proportional to Haemoglobin concentration.
Hb+ ferricyanide → methhaemoglobin → cyanmethaemoglobin (HiCN) (stable)
Drabkin’s Reagent: It contains 100 mg Potassium cyanide, 400 mg Potassium
Ferricyanide, 280 mg Potassium Dihydrogen phosphate, 1ml nonidet.
0.20µl of blood are added to 5ml of Drabkin’s Reagent and mixed well.
Absorbance is read against blank after 10 minutes at 540 nano meter.
Materials and Methodology
Calculations:
O.D. test Hb (g/dl) = ---------------------- 15
O.D. standard Where, Hb= haemoglobin, g = gram, dl= decilitre, O.D. = optical density. Using the formula data was recorded for Hb gm per decilitre (gm/dl)
B. Red Blood Cell (RBC) count by Haematocytometer.
The number of RBCs in a known volume of diluted blood counted and the
number of cells in one cubic millimeter of undiluted blood calculated from it.
Requirements:-
R.B.C. diluting fluid (Hayem’s fluid is commonly used). It is composed of
0.5 gram Sodium chloride, 2.5 gram Sodium sulphate, 0.25 gram Mercuric
perchloride, 100 ml distilled water.
Blood was mixed with diluting fluid and charged the counting chambers.
RBC counting area was focussed under high power and then counting was done. At
least 5 square each having 16 smallest squares (preferably 4 corners and 1 central)
counted.
Calculation:
Total RBC/cubic millimeter (cu. mm) = No. of red cells counted dilution
Dilution = 1:200 (i.e. 200). Blood is diluted from
0.5 to 101 mark i.e., 200 times.
Area of one small square = 1/400 square millimeter
Area of 5 medium sized = 80 areas = 80/400 square millimeter = 1/5 square millimeter
Squares i.e. 5x16
Depth of fluid = 1/10 millimeter
No. of red cells counted = N
Area of the central square = 1 square millimeter
Materials and Methodology
Hence total red blood cells (cubic millimeter) = [( )][( / ) ( / )]
= N 200 50
= N 10,000
If the number of RBC in the five squares is N, then 1 cubic millimeter of
blood will contain N50. Therefore, the number of RBC in one cu.mm of blood is
N50200= N10000.
C. Total Leukocyte Count (TLC) by Haematocytometer.
Principle
A sample of whole blood was mixed with a weak acid solution that lyses non
nucleated RBCs. Following adequate mixing, the specimen was introduced into a
counting chamber where the white blood cells (leukocytes) in a diluted volume were
counted.
Reagent: Turke’s Fluid
It contains 1.5 ml glacial acetic acid, 1ml 1% aqueous solution of gentian violet, 98
ml distilled water.
Blood was mixed with diluting fluid and charged in the haematocytometer. It
was then focussed under low-power magnification and counting was done in the
four 1 square millimeter corner areas. All the white cells lying within the square and
those touching the upper and right-hand centre lines were not counted.
Calculation:
Area of each square = 1 square millimeter
Area of four squares-14 = 4 square millimeter
Volume of the four squares 41/10 = 4/10
If the no. of WBCs in the four squares –N, then 1 cubic millimeter of blood will
contain –N10/4. Since blood was diluted 20 times
Therefore, the number of WBC in 1 square millimeter of blood = N10/420=N50.
Materials and Methodology
D. Erythrocyte Sedimentation Rate (ESR) by Wintrobe’s Method (Dacie and Lewis, 1975).
Principle: When anti-coagulated whole blood was allowed to stand in a narrow
vertical tube for a period of time, the RBCs under the influence of gravity settle out
from the plasma. The rate at which they settle was measured as the number of
millimeters of clear plasma at the top of the column after one hour (mm/hr).
Procedure:
EDTA anti-coagulated 2-3 millimeter litre blood without extra diluents was
drawn into the Wintrobe’s tube of 3 nanometer internal diameter and 110 nanometer
length upto the mark ‘0’ by pasteur pipette. The Wintrobe tube is vertically kept
stand. The rate of fall of red blood cells was measured in millimeters after one hour
of sedimentation and length of column of plasma was noted.
3.5.2. ASSAY OF BIOCHEMICAL PARAMETERS
A. Total Protein Estimation by Method of Lowry, et al. (1951).
Blood from each experimental and control groups of mice was collected by
cardiac puncture with a sterile disposable syringe and allowed to clot at room
temperature for 11/2 hrs and then centrifuged to separate serum which was then used
for the estimations of total protein by Lowry, et al. (1951) method and albumin and
globulin by Schalm, et al. (1975) method.
Principle:
The estimation of proteins is based upon reaction of peptide bonds of protein
with alkaline solution of CuSO4 to produce Cu+, which reacts with the Folin reagent,
and the Folin–Ciocalteau reaction, phosphomolybdotungstate is reduced to
heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic amino
acids. The reactions result in a strong blue colour, which depends partly on the
tyrosine and tryptophan content. The optical density of which is measured at 600
nano meter. The method is sensitive down to about 0.01 milligram of protein per
millilitre, and is best used on solutions with concentrations in the range 0.01–1.0
milligram per millilitre of protein.
Materials and Methodology
Reagent: Prepared immediately before use
Reagent I- 48ml 2% Na2CO3 in 0.1 N NaOH, 1ml 1% Na K tartarate in H2O
and 1ml 0.5% CuSo4. H2O in H2O.
Reagent II- 1 part Folin- phenol (2N): 1 part water.
Standards: A stock solution of bovine serum albumin (BSA) containing 2 mg/ml
protein in distilled water was used and stored at –200C. Standards prepared by
diluting the stock solution with distilled water were as follows:
In the sample or standard NaOH was added and hydrolyzed at 1000C for 10
minutes in boiling water bath. After cooling Reagent I was added and allowed to
stand at room temperature for 10 minutes. Folin reagent was added and kept at room
temperature for 30–60 minutes. The absorbance was read at 600 nanometer and
recorded to determine the unknown protein concentrations using the calibration
curve.
Calculations:
Protein (gram per 100 millilitre or gm/100 ml)
= concentration of standard
where, T= test sample, B= blank, S= standard sample
B. Total Cholesterol estimation by Method of Sackett, (1925).
Principle:
The Lieberann-Burchard reaction is used for estimation of cholesterol. It
forms green cholesterol complex when treated with acetic anhydride and conc.
H2SO4. It is a modification of Bloor and Knudson, (1916) method in which blood is
added to ethanol- ether mixture, which precipitates proteins and extracts the
cholesterol. The supernatant fluid obtained on centrifugation was evaporated, the
cholesterol taken up in chloroform and determined colorimetrically.
Reagents
1. Stock cholesterol-200 mg cholesterol dissolved in 100 ml chloroform.
Materials and Methodology
2. Working standard- 4 ml stock diluted to 100 ml by chloroform.
To the test “T’ tube 9 millimeter (ml) of ethanol and 3 ml ether was added
then 0.2 ml serum or plasma or blood was mixed and shaken vigorously for 1
minute. The tube was kept horizontally to precipitate for 30 minutes. It was then
centrifuged at 3000 rotation per minute (rpm). Supernatant was evaporated to
dryness then 5 ml chloroform was added. To blank only 5 ml chloroform and to
standard tube 5 ml working standard cholesterol was added. To each of the test tubes
2 ml acetic anhydride and 0.1 ml concentration, H2SO4 was mixed and kept in dark
for 15 minutes at 250C. The observations were calculated as follows:
Calculation
Cholesterol (milligram per 100 millilitre or mg/100 ml)
= concentration of standard
= 0.4.
= 200
where, T= test sample, B= blank, S= standard sample
C. Glucose estimation by Method of Folin and Wu, (1918).
Principle:
Blood was collected in sodium fluoride and was mixed with King’s isotonic
solution which caused glucose to diffuse out of the cells without causing
haemolysis. The protein free filtrate of blood was then allowed to react with alkaline
copper salt solution when cuprous oxide was formed as a precipitate in proportion to
the amount of glucose. Treatment with phosphomolybdic acid reagent causes the
formation of molybdenum blue and the intensity of blue colour is proportional to the
amount of cuprous oxide and then to the amount of glucose. The colour was
compared with that produced by a standard solution of glucose similarly treated.
Materials and Methodology
Reagents:
1. Kings’ isotonic solution-320 millilitre of 3% hydrated sodium sulphate
solution with 30ml 7 % copper sulphate solution and 10% sodium tungstate
solution.
2. Alkaline copper reagent (Folin and Wu)- 40 gram anhydrous sodium
carbonate was dissolved in water and 7.5 gram tartaric acid was dissolved. To
it a solution of 4.5gram crystalline copper sulphate dissolved in 100ml water
was added. The solution was mixed and diluted upto 1 litre with water.
3. Phosphomolybdic acid reagent (Folin and Wu)- 35 gram molybdic acid, 5
gram sodium tungstate, 200ml of 10% NaOH and 200ml of distilled water was
mixed in a large beaker. Boiled for about 45 minutes to drive out ammonia
cool and added 125 ml phosphoric acid 89% the volume was made upto 500
millilitres with water and mixed.
4. 1% Stock glucose- 1gram anhydrous glucose was mixed in saturated solution
of benzoic acid and made the volume 100ml with same solution.
5. 0.01% working standard glucose solution- the stock standard was diluted to 1
in 100 with water freshly prepared.
Protein free blood sample was obtained and 2 ml of the sample was taken in
Folin Wu tubes Distilled water and working standard glucose solution was taken in
separate tubes as blank and standard respectively. To all the tubes 2 ml of alkaline
copper sulphate solution was added and kept in water bath for 8 minutes. Thereafter,
cooled and 2 phosphomolybdic acid was added. After 1 minute it was diluted to
mark 25 ml with water and mixed. Then its reading was observed at 420 nanometer
and calculated as follows:
Calculation:
Blood sugar = milligram of glucose in standard(0.2).
Blood sugar = 100 milligram per 100 ml
where, T= test sample, S= standard sample
Materials and Methodology
2.5.3. ASSAY OF ENZYMOLOGICAL PARAMETERS
A. Serum Glutamate Pyruvate Transaminase (SGPT) by Method of UV
Kinetic Test by Bergmeyer, et al. (1978).
B. Serum Glutamate Oxaloacetate Transaminase (SGOT) by UV Kinetic Test
Method:
The substrate buffer solution for Glutamate Oxaloacetate Transaminase or
GOT (U/liter of L-asparate, 2-oxoglutarate aminotransferases; E.C.2.6.1.1)
contained L-aspirate and for Glutamate Pyruvate Transaminase or GPT (micromoles
per litre per minute) of L-alanine, 2-oxoglutarate aminotransferases; E.C.2.6.1.2) it
contained L-alanine. To 1000 millilitre of enzyme solution (LDH+NADH), 10µl of
serum was added and mixed well. It was incubated for 1minute at 200C.Then 100
microlitre (µl) of substrate was added and mixed well. Three readings on 365 nm at
the interval of one minute were taken. Mean value was taken for the final result. The
enzyme activities were expressed as GOT and GPT unit per litre by adopting the
method of Bergmeyer, et al. (1978).
Principle:
α-oxoglutarate + alanine L-glutamate + pyruvate
pyruvate +NADH +H+ lactate + NAD+
α -oxoglutarate + L-aspartate L L- glutamate +oxaloacetate
oxaloacetate + NADH + H+ L- malate + NAD+
Calculation for Serum Glutamate Pyruvate Transaminase (SGPT)
The amount of pyruvate formed per minute per liter of serum sample
= 0.4 .
standard
→←
→←
LDH
SGPT
→←
LDH
SGOT
→←
Materials and Methodology
Serum SGPT (µmoles/litre/minute) = 133.33
where, T= test sample, C= control, B= blank, S= standard sample
Calculation for Serum Glutamate Oxaloacetate Transaminase (SGOT)
The amount of pyruvate formed per minute per liter of serum sample
= 0.4 .
standard
Serum SGPT (µmoles/litre/minute) = 66.66
where, T= test sample, C= control, B= blank, S= standard sample
2.5.4. ASSAY OF IMMUNOLOGICAL TESTS
A. Differential Leucocyte Count (DLC) percentage by Method of Rajgopal and
Ramkrishnan, (1983).
This was assessed according to usual methods given by Rajgopal and
Ramkrishnan (1983). Whole blood was used to prepare blood smear on the slides.
The slides were dried and stained in 0.25% Leishman’s stain. The percentage of
lymphocytes, polymorphocytes, monocytes and basophils were calculated by
counting at least 100 cells.
B. Albumin Globulin Ratio by Method of Kabat and Mayer, (1961)
Blood from mice of each experimental and control groups was collected by
cardiac puncture and allowed to clot at room temperature for 1 hours and then
centrifuged to separate serum which was used to estimate albumin, globulin and
albumin - globulin ratio by method of Kabat and Mayer (1961).
C. Immediate Type Hypersensitivity (ITH) skin testing-Passive Cutaneous
Anaphylaxis (PCA) by Method of Overy, (1964).
The toxicant was injected intra-dermally in the abdomen and back along with
adjuvant dimethyl sulfoxide in a volume of 0.1 millilitre. The dose was chosen in
such a way as to give maximum and linear relationship. A trial experiment was
carried out to select the doses to be injected. After 4 hours 0.1 ml soluble test
antigen (volume 1:1000, weight by volume in normal saline) and a marker dye (1%
Evans blue, 20 mg/kg) were injected together intravenously in a small volume.
Materials and Methodology
After 20 minutes animals were sacrificed and skin opened by freeing from
subcutaneous attachments. The size of the lesions was evaluated with a transparent
ruler while the skin was being illuminated from the opposite side with a lamp.
However, care was taken to avoid drying of stretching of skin. The reaction greater
than 5 millimeter in diameter was considered significant.
D. Delayed Type Hypersensitivity (DTH) estimation by Method of Talwar,
(1983).
The toxicant (1:1000 weight per volume) was injected along with adjuvant
dimethyl sulfoxide in a volume of 0.03 ml into planter side of the foot-pad of
experimental mice tip of the 30 G needle. The DTH response was measured as the
increase in foot-pad thickness in the foot-pad injected with the toxicant as compared
to the control foot-pad of the same mouse. Foot-pad thickness was measured by a
calliper (Schnell Taster, Germany). Each DTH response was measured at 24 hours
and the reaction greater than 5 mm in diameter was considered significant. DTH
response was confirmed by carrying out histological examination in both foot-pads.
Fixation was done in buffered formalin, sectioned and stained with haematoxylin-
eosin stains. A positive DTH response was confirmed by observing a strong
mononuclear infiltration.
3.6. METHODS FOR HISTOCHEMICAL OBSERVATIONS
The histochemical studies in mice exposed to 4.2 mg/kg body weight and 6.7
mg/kg/body weight with mancozeb and 400 mg/kg body weight of aqueous extract
of A. vera and 250 mg/kg body weight of aqueous extract of O. sanctum herbal
drugs were carried in the kidney and the liver tissues. The specimens were preserved
using routine method in 10% formalin fixative for 4 hours, then dehydrated, cleared
and infiltrated with paraffin for making blocks.
The paraffin sections were cut at 5 µm thickness. The slides were deparaffinised
and divided for staining by Periodic Acid Schiff’s reagent (PAS), Mercuric
Bromophenol Blue (BPB) and Sudan Black for carbohydrates, proteins and lipids or
fats respectively (Mazia, et al., 1953; Pearse, 1981). Haematoxylin was used as a
counter stain in PAS-treated sections. After staining the slides were cleared and
mounted in Distyrene Plasticizer Xylene (DPX).
Materials and Methodology
The histochemical sections were examined and micro photographed using a
research light microscope Nikon, Eclipse, 50i (Japan) and ACD See 32V 2.4
Software. The photographs were taken in two magnifications 400X and 100X. The
photographs were printed with Canon MP145 printer using glossy picture papers.
A. Carbohydrates: PAS reaction
Reagents
Periodic acid -1% in distilled water
Basic fuchsin -1gm
Hydrochloric acid (HCl)-15 ml
Sodium metabisulphite (anhydrous)-1.9 gm
Activated Charcoal 2gm
Sulphurous acid solution- Potassium metabisulphite 10% 36 millilitre and
HCl (39.3 millilitre in 400 millilitre water) 30 millilitre and the solution was made
upto 600 millilitre with water.
Schiff’s reagent (Barger and Delamater, 1948): basic fuchsin was dissolved
in 400 millilitre boiling distilled water. It was then cooled to 500C and filtered. In
the filtrate 1 millilitre of thionyl chloride was added and kept in dark overnight.
Then activated charcoal was added and filtered which was stored for future use.
Staining Procedure
Deparaffinised and alcohol graded (100 to 70%) sections after washing in
water were placed in periodic acid (5 minutes) and again rinsed in distilled water.
Next kept in Schiff’s reagent (15 minutes), water (10 minutes), stained in
Haematoxylin (3 minutes), washed in water (2 minutes) Sulphurous acid (2
minutes), running water (3 minutes), Mayer’s haemalum (10 minutes), alcohols,
xylene and mounted. The periodic acid brought oxidative cleavage of the C_C bond
in 1-2 glycols of their amino or alkyl amino derivatives to form dialdehyde which
reacts with fuchsin-sulfurous acid to combine with basic fuchsin forming the
magenta colour.
Materials and Methodology
B. Proteins: Mercuric Bromophenol Blue (BPB) reaction
Reagents
HgCl2 -10 gm BPB- 100 mg
Ethanol- 95% HgCl2 and BPB were mixed in 100ml of ethanol to obtain the reagent.
Staining Procedure
Deparaffinised sections were stained in the above solution for 15 minutes
then washed in 0.5% acetic acid for 20 minutes. Next the slides were washed in
water and in 0.01M phosphate buffer of pH 7.0 for 3 minutes (the dye converts into
blue alkaline form). After this the slides were dehydrated in alcohol and cleaned in
xylol for mounting.
C. Lipids: Sudan Black B reaction (Lison and Dagnelie, 1935)
Reagents
Sudan Black B, C.I. 26150, Tri-ethyl phosphate, Mayer’s Carmalum
40 millilitre distilled water was added to 60 millilitre tri-ethyl phosphate
mixed and 1 gram of the stain was added to it. The mixture was heated to 1000C for
5 minutes and filtered. The filtrate was kept as stock solution.
Staining Procedure
After following the usual procedure the slides were kept 60% tri-ethyl
phosphate then sections were kept in Sudan Black B at 200C for 10 minutes. The
sections were then rinsed in 60% tri-ethyl phosphate for 30 seconds. Next washed
with 6 changes of distilled water and then dipped in Mayer’s Carmalum solution for
3 minutes and again washed in distilled water. The slides were then cleared and
mounted for observation.
3.7 METHODS FOR HISTOPATHOLOGICAL OBSERVATIONS
The histopathological studies in the mice exposed to 4.156 mg/kg body
weight and 6.650 mg/kg/body weight with mancozeb and 400 mg/kg body weight of
aqueous extract of A. vera and 250 mg/kg body weight of aqueous extract of O.
Materials and Methodology
sanctum herbal drugs were carried in the kidney and the liver tissues. After
completion of the proposed doses schedules the mice were sacrificed using the
Institutional Animal Ethical Committee approved method by cervical dislocation.
The mice were dissected and liver and kidney tissues were removed from the body,
cut into small sections and washed in normal saline solution.
Then the tissues were fixed in 10% formalin for 24 hours and dehydrated in
alcohol after thorough washing. Blocks were prepared by paraffin infiltration by
usual procedure of dehydration and paraffin embedding (Lillie, 1954 and 1977). The
sections were then cut at 5 µm thicknesses and were dewaxed in xylol then
dehydrated in descending series of alcohol from 100%→90%→70%→50%→30%
and then washed in distilled water for 5 minutes each. Then stained with Harri’s
Haematoxylin and counter stained with Eosin by routine procedure (McManus, 1948
and McManus, et al. 1964). Aqueous Haematoxylin stain was used in the next step
for 2-5 minutes, rinsed in water (sections turn blue).
The sections were then dehydrated through ascending series of alcohol
30%→50%→70%→90% and then counter stained in alcoholic eosin for 30 seconds.
Next sections were washed in alcohol 90%→100% cleared in xylol and mounted
with the help of Distyrene Plasticizer Xylene (DPX) and permanent slides were
prepared.
3.8. STATISTICAL ANALYSIS- SNEDECOR AND COCHRAN (1980)
Continuous observation, data collection and statistical analysis of above
parameters with herbal treated mice, pesticide treated mice and control was done as
per the method described by Snedecor and Cochran, (1980). The tests were repeated
again to compare the results obtained. The following statistical formulae were
applied for the analysis of data:
A. Standard deviation:
Standard deviation ‘S’ of the observation was calculated by the formula:
Materials and Methodology
S = ∑ (∑ )
Where, S= Standard deviation.
∑x = Sum of squares of individual values.
(∑푥) = Square of the sum of individual values.
n = Number of observations.
B. Analysis of variance:
Difference between groups for different parameters were tested for
significance, using one-way ANOVA technique (Panse and Sukhatme, 1954) for
mancozeb treated and herbal drugs treated mice to study their effects. The MSTATC
Software was used for the study. The mean values of each observation were used for
analysis of data, following the procedure of standard Completely Randomised
Designs (CRD) analysis of variance. The structure of ANOVA is given below:
Skeleton of ANOVA
Source of variation DF MS `F` test
Genotypes (v-1) M2 M2/M3
Error (r-1) (v-1) M3
Where,
v = Number of Groups
M1 = Mean sum of squares for replication, M2 = Mean sum of squares for groups
M3 = Error mean sum of squares
DF = Degree of freedom
Estimation of mean, range and critical differences:
Mean: The mean value of each parameter was worked out by dividing the totals by
the corresponding number of observations.
Range: The difference between the lowest and highest values for each character was
taken as the range, by which the minimum and maximum values are known.
Materials and Methodology
Standard deviation: The variance is the measure of the variability and it is defined
as the average of squared deviations from the mean. The standard deviation is the
square root of the variance.
Critical difference: It was calculated as follows:
Critical difference (CD) = √ {2( EMS/ r}× tedf
where EMS = Error mean square
tedf = tabulated `t` value at 5% level with (r-1) (t-1) df.
C. Correlation Coefficient
For single correlation coefficient the same software was used for the analysis
of the above study.
3.9. EXPERIMENTAL GROUPS/DESIGN
Experiments were carried out with three main groups containing albino mice:
i. Control Group: Non exposed and non- treated (Gr I)
ii. Exposed Groups: Exposed and non- treated (Gr II and III)
iii. Experimental Groups: Exposed and treated (Gr IV to XI)
Distilled water was given in control group (Group I). The low dose of
mancozeb (MCZ) (D1) was taken as 4.156 mg/kg/ body weight of mancozeb
dissolved in distilled water and dimethyl sulfoxide (Group II). Similarly high dose
of mancozeb (D2) was taken as 6.450 mg/kg/ body weight of mancozeb (Group III).
The dose of aqueous A. vera (AV) extract was taken as 400 mg/kg/ body weight and
dose of aqueous O. sanctum (OS) extract was taken as 250 mg/kg/ body weight. 1
ml of each dose was given to the mice. The dose duration was carried out for 6
consecutive days a week for 30 days.
In the simultaneous treated groups (Groups IV to VII), mancozeb and herbal
drugs was given simultaneously for 6 consecutive days a week for 30 days while in
the after withdrawal groups (Group VIII to XI), first mancozeb was given for 6
consecutive days a week for 30 days method thereafter mancozeb was withdrawn
and herbal drugs were given in the same way for the next 30 days. This study was
observed periodically after 7 days in a month in case of haematological,
Materials and Methodology
biochemical, enzymological and some immunological parameters whereas in case of
some immunological, histopathological and histochemical parameters observation
was made after 15 days in a month.
Table 1: Designing of Experiment
Sl. No Groups Group
code Treatment Group nomenclature
1
Control I Given distilled water for 30 days DW
2 MCZ Exposed II Induced with D1 MCZ for 30
days D1 MCZ
3. MCZ Exposed III Induced with D2 MCZ for 30
days D2 MCZ
4. AV Treated
Simultaneous IV
Treated with D1 MCZ simultaneously with AV for 30
days D1 MCZ+ AV
5. AV Treated
Simultaneous V
Treated with D2 MCZ simultaneously with AV for 30
days D2 MCZ+ AV
6. OS Treated
Simultaneous VI
Treated with D1 MCZ simultaneously with OS for 30
days D1 MCZ+ OS
7. OS Treated
Simultaneous VII
Treated with D2 MCZ simultaneously with OS for 30
days D2 MCZ+ OS
8. AV Recovery
VIII
Treated with AV for 30 days after removal of D1 MCZ
toxic stress given for 30 days
AV after-withdrawal of
D1
9. AV Recovery
IX
Treated with AV for 30 days after removal of D2 MCZ
toxic stress given for 30 days
AV after-withdrawal of
D2
10. OS Recovery
X
Treated with OS for 30 days after removal of D1 MCZ
toxic stress given for 30 days
OS after-withdrawal of
D1
11. OS Recovery
XI
Treated with OS for 30 days after removal of D2 MCZ
toxic stress given for 30 days
OS after-withdrawal of
D2
Materials and Methodology