CHAPTER – III MATERIALS AND METHODOLOGYshodhganga.inflibnet.ac.in/bitstream/10603/88817/3/chapter3.pdf · Materials and Methodology A B C Figure 6: Plant of Aloe vera (barbadensis)

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.


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.


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


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,


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


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,


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).


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


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


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


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.


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


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.


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.


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.


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.


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


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

→←


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.


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).


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.


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.


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:


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.


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,


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


AV after-withdrawal of

D2

10. OS Recovery

X

Treated with OS for 30 days after removal of D1 MCZ


OS after-withdrawal of

D1

11. OS Recovery

XI

Treated with OS for 30 days after removal of D2 MCZ


OS after-withdrawal of

D2


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CHAPTER – III MATERIALS AND METHODOLOGYshodhganga.inflibnet.ac.in/bitstream/10603/88817/3/chapter3.pdf · Materials and Methodology A B C Figure 6: Plant of Aloe vera (barbadensis)