experimental - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/9354/7/07_chapter 3.pdf3. 3.2....

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EXPERIMENTAL

3.1. PLAN OF WORK

1. LITERATURE SURVEY

2. PHYTOCHEMICAL INVESTIGATION OF PLANT EXTRACTS

2.1. Procurement of the medicinal plants from the authenticated source

2.2. Preparation of the plant aqueous ,hydroalcoholic and methanolic extracts

.2.3 Quantitative analysis of plant

2.4. Physicochemical characterisation of the plant extract

2.5. Qualitative screening using appropriate phytochemical tests

2.6. HPTLC of the plant extracts

3. PHARMACOLOGICAL SCREENING OF PLANT EXTRACTS

3.1. In vitro screening of the plant extracts for its antioxidant activity

3.1.1. DPPH radical scavenging activity

3.1.2. Nitric oxide radical scavenging activity

3..2. Acute toxicity of plant extracts in rats and mice as per OECD guidelines (423)

3. 3. Preliminary pharmacological evaluation of plant extracts

3.3.1. Activity cage model: Evaluation of spontaneous locomotar activity

3. 3.2. Elevated plus maze test: Evaluation of anxiogenicity

3.3.3. Hole board test: Evaluation of exploratory activity

3.3.4. Forced swim test (Chronic fatigue syndrome)

3. 3.5. Tail suspension test (Gravitational stress)

3.4. Evaluation of anti stress activity of plants MK and OS by using stress models

3.4.1. Weight loaded forced swim test

3.4.2. Cold restraint stress model

3.4.3. Milk induced leukocytosis

3.4.4. Restraint stress model

3.4.5. Foot shock model

3.4.6. Electro convulsive shock

3.4.7. Sleep deprivation model

3.4.8. Cold swim test

4. Formulation development

4.1. Pre formulation studies

4.2. Formulation of MK and OS extracts

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4.3. Evaluation of stability of formulations

4.4. Evaluation of In Vivo efficacy of formulations

5. Isolation and structural elucidation of phyto-constituent

5.1. Separation of alkaloid fraction from extract

5.2. Isolation of carbazole alkaloid from alkaloid fraction.

5.3. Structural elucidation of isolated carbazole alkaloid

5.4. In vitro testing of Isolated carbazole alkaloid

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3.2. MATERIALS AND METHODS

Plant Materials

� .Leaves of Murraya Koenigii (L.) Syn. Beregera Koenigi i(L.)(Family: Rutaceae)

� Leaves of Ocimum Sanctum (L.) Syn. Ocimum tenuiflorum (L.) (Family: Lamiaceae)

Fresh leaves of both the plants were procured from Local vegetable Market at Thane and

were air dried in shade.

� Animals

Wistar albino rats of either sex in the weight range of 150-200 g and Swiss albino mice of

either sex in the weight range of 20-35 g were selected for the study. Animals were housed in

group of 5-6 animals in clean plastic cages containing husk bedding.

The experimental procedures and protocol for this study was reviewed and approved

(080901,091008) by the Institutional Animal Ethics Committee (IAEC) and the

pharmacological work was performed as per Committee for the Purpose of Control and

Supervision of Experimentation on Animals (CPCSEA) norms [529].

Animals were maintained under standard environmental condition of temperature (21⁰C ±

2°C), humidity (51 ± 10%) and 10 hr light and 14 h dark cycles with standard pellet diet

(Amrut brand pelleted standard feed manufactured by Nav Maharashtra Chakan Oil Ltd.

Pune , Maharashtra) and water ad libitum. The animals were allowed to acclimatize to the

animal house conditions for 8-10 days period prior to the conduct of experimentation.

� Chemicals and Drugs: (All the chemical were of analytical (AR) grade)

Acetic acid AR S.D. Fine Chemicals, Mumbai Acetic acid glacial 99-100% AR E. Merck India Ltd. Mumbai Acetic anhydride was purchased S.D. Fine Chemicals Ltd Mumbai Acetylcholine chloride (Sigma, US) Aerosil (silicon dioxide) commercial pack Ashwagandha (Ashvgandha , Himalaya drugs) Avicel pH101 Commercial pack Butanol AR S.D. Fine Chemicals, Mumbai Chloroform AR Merck Ltd Mumbai Copper sulphate Sisco Research Labs Ltd. Mumbai Di potassium hydrogen phosphate S.D. Fine Chemicals Ltd Mumbai Di sodium hydrogen phosphate S.D. Fine Chemicals Ltd Mumbai Diazepam tablets (Calmpose 5 mg) Ranbaxy Lab. Goa, India Disodium ethylenediamine tetraacetate dehydrate (EDTA) S.D. Fine Chemicals Ltd Mumbai 5,5-Dithiobis (2-nitro-benzoic acid), Sigma Dopamine hydrochloride (Himedia, India)

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DPPH (Di phenyl picryl hydrazyl) S.D. Fine Chemicals Ltd Mumbai Ethanol AR S.D. Fine Chemicals, Mumbai Ethyl acetate AR S.D. Fine Chemicals, Mumbai Ferrous chloride Sisco Research Labs Ltd. Mumbai Ferrous sulphate Sisco Research Labs Ltd. Mumbai Folin ciocalteau reagent Merck Ltd Mumbai Formaldehyde Rankem, Mumbai Formic acid AR S.D. Fine Chemicals, Mumbai Glucose S.D. Fine Chemicals, Mumbai Hydroxylamine hydrochloride S.D. Fine Chemicals Ltd Mumbai Imipramine hydrochloride tablets (sigma,US ) Lead acetate Sisco Research Labs Ltd. Mumbai L-Glutathione reduced 99% pure (Sigma, US) Magnesium stearate S.D.fine chemicals Malonaldehyde bis (Phenylimine) dihydrochloride from Aldrich Chem Methanol AR S.D. Fine Chemicals, Mumbai Methyl Isobutyl ketone Merck Ltd Mumbai n-Butanol Merck Ltd Mumbai n-Heptane 95% LR S.D. Fine Chemicals, Mumbai Norepnephrine hydrochloride (Sigma, US) Piracetam tablets (Neurocetam 400 mg): Microlabs Pondichery India Potassium iodide S.D. Fine Chemicals Ltd Mumbai Serotonin hydrochloride (Sigma, US) Silica 60/120 Merck Mumbai Silica gel 60 F254 plates Merck Mumbai Sodium acetate anhydrous E Merck India Ltd. Mumbai Sodium bicarbonate E. Merck India Ltd. Mumbai Sodium carbonate E. Merck India Ltd. Mumbai Sodium carboxy methyl cellulose Loba Chemie, Mumba Sodium chloride S.D. Fine Chemicals Ltd Mumbai Sodium dihydrogen phosphate S.D. Fine Chemicals Ltd Mumbai Sodium hydroxide pellets E. Merck India Ltd. Mumbai Sodium lauryl sulphate Research lab fine chem. Industries Ltd. Sodium nitrate S.D. Fine Chemicals Ltd Mumbai Sodium nitropruside S.D. Fine Chemicals Ltd Mumbai Sodium starch glycollate West coast laboratories Ltd. Sodium sulfite anhydrous S.D. Fine Chemicals Ltd Mumbai Sulphuric acid Rankem, Mumbai Thio-barbituric acid AR Sisco Research Lab Ltd. Mumbai Thiourea AR S.D. Fine Chemicals, Mumbai Toluene AR Merck, Mumbai Trichloroacetic acid S.D. Fine Chemicals, Mumbai Tween 80 Research lab fine chem. Industries Ltd.

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� Biochemical Kits

Cholesterol (CHOD-PAP method) kit was purchased from Erba Diagnostics Transasia Mumbai. Triglyceride (GPO-TRINDER method) kit was purchased from Erba Diagnostics Transasia Mumbai. Total protein (Biuret method) kit was purchased from Erba Diagnostics Transasia Mumbai. Glucose (GOD-POD method) kit was purchased from Erba Diagnostics Transasia, Mumbai. LDH-P(SL) was purchased from AGAPEE Diagnostics ,Kerala. UREA-L (BERTHELOT method)was purchased from AGAPEE Diagnostics ,Kerala. � Major Instruments Used

Camag Linomat IV (Switzerland) Camag TLC Scanner 3 (Switzerland) Cook’s pole (Medicraft India) Capsule filling Machine. Kripson Pharma Machine. Cell Counter(ABACUS) Densitometer D 60 DESAGA ProQuant Digital actophotometer (Techno India) Disintigration Test apparatus (IP/USP/STD) Adinath Industries Mumbai Dissolution Apparatus Electrolab Model 8L Double beam spectrophotometer JASCO V-530 (Japan) Electro-convulsiometer (Medicraft India) Elevated plus maze Erba Chem 5 Plus Bioanalyser (Transasia, India) Heating water bath (Remi Scientific labs Mumbai) I.R.Specrometer Perkin Elmer Spectra XI Morris water maze Muffle furnace (Thermolab, India) Refrigerated Centrifuge (Lab enterprises, India) Rotary vacuum evaporator (Medica Instruments Mumbai India) Shuttle box (Medicraft India) Spectro flourimeter (Jasco Japan) Tissue homogeniser (Remi Scientific Mumbai) Vaccum oven Vortex mixer (Remi Scientific labs Mumbai)

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METHODS

3.3. PHYTOCHEMICAL INVESTIGATION

3.3.1. Procurement of the medicinal plants authentification and

preparation of extracts The selected plants namely the leaves of the Murarya Koenigii (MK), and leaves of Ocimum

sanctum (OS) were procured from the local market. The whole plant of Murarya Koenigii,

and Ocimum sanctum were authenticated from Agharkar Research Centre Pune.and Blatter

Herbarium St. Xavier college Mumbai. with voucher specimen (20537/SKW-2549) deposited

in Blatter herbarium for the future reference The leaves of Murraya Koenigii, and Ocimum

sanctum were air dried in shade and then powdered .

The dried powdered leaves of Murraya Koenigii, and Ocimum sanctum were weighed and

extracted using 50 % ethanol (hydroalcoholic mixture) at 60°C and methanol at 50°C in

soxhlet apparatus and distilled water (aqueous extract) at 100°C for 18 h by hot reflux

extraction method The aqueous , hydroalcoholic and methanolic plant extracts of MK and OS

were then filtered and concentrated using rotary vacuum evaporator. The dried plant extracts

were stored in amber colored wide mouth bottles under refrigeration (4°C) and were used for

phytochemical and pharmacological investigations.

The plant extracts throughout the study were abbreviated as Murraya Koenigii aqueous

extract (MKAQ), Murraya Koenigii hydroalcoholic (MKHA), Murraya Koenigii methanolic

(MKM). Ocimum sanctum aqueous (OSAQ) Ocimum sanctum hydroalcoholic (OSHA)

Ocimum sanctum methanolic (OSM).

3.3.2. DETERMINATION OF QUANTITATIVE DATA [530]

1 Physicochemical characterization of the plant extract

Physicochemical parameters such as color, consistency, pH and percent yield (% w/w). were

determined for all plant extracts

2. Determination of ash values [531]

� .Total ash [531]

The air dried crude drug (2 to 3 g) was weighed accurately in a tarred silica dish and

incinerated at a temperature not exceeding 450�C until free from carbon. It was then allowed

to cool in a desiccators and later weighed. The procedure was repeated till it gave a constant

Experimental

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weight. The percentage of ash was calculated with respect to the air-dried drug. An average

value of three readings was determined.

� Acid insoluble ash [531]

Ash insoluble in hydrochloric acid is the residue obtained after extracting the total ash with

hydrochloric acid. The ash obtained from above procedure mentioned in the total ash was

boiled with 25 ml of 2 M hydrochloric acid for 5 min and the insoluble matter was collected

on an ash less filter paper. The insoluble matter was washed with hot water, ignited, cooled in

a desiccator and weighed. The percentage of acid-insoluble ash was calculated with reference

to the air-dried drug. An average value of three readings was determined.

� Water soluble ash [531]

Water soluble ash is that part of the total ash content which is soluble in water. It is good

indicator of either previous extraction of the water soluble salts in the drug or incorrect

preparation. Thus it is the difference in weight between the total ash and the residue obtained

after treatment of total ash with water.

Accurately weighed quantity of the total ash was boiled for 5 min with 25 ml of water. The

insoluble matter was collected on an ash less filter paper and washed with hot water. It was

then ignited for 15 min at a temperature not exceeding 450�C. The weight of the insoluble

matter was subtracted from the weight of the ash, and the difference in weight represented the

water-soluble ash. The percentage of water-soluble ash was calculated with respect to the air-

dried drug. An average of three readings was determined.

3. Determination of solvent extractive values [531]

� Water-soluble extractive[531]

Accurately weighed quantity of 5 g of powdered crude drug was added to 50 ml of water at

80˚C in a stoppered flask. The stoppered flask was allowed to stand for 10 min. To the cooled

contents of the stoppered flask, 2 g of kieselguhr was added and filtered. The filtrate (5 ml)

was transferred to a tarred evaporating dish of 7.5 cm in diameter. The filtrate was evaporated

on a water bath and the drying was continued for 30 min. Finally the filtrate was dried in a

steam oven for 2 h and the residue was weighed. The percentage of water-soluble extractive

was weighed with reference to the air–dried drug. An average of three readings was

determined.

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� Ethanol soluble extractive[531]

The coarsely powdered 5 gm of the air dried drug was macerated with 100 ml of ethanol in a

closed flask for 24 hours. The content of the closed flask was frequently shaken during the

first 6 h and allowed to stand for 18 h. Thereafter, it was rapidly filtered taking precautions

against loss of ethanol and 25 ml of the filtrate was evaporated to dryness in tarred flat-

bottomed shallow dish. Finally it was dried at 105˚C and weighed. The percentage of ethanol-

soluble extractive was calculated with reference to the air-dried drug. An average of three

readings was determined.

4. Loss on drying [531]

Loss on drying was estimated by accurately weighing 5 g of the air dried powdered crude

drug in a dried weighing bottle. The loaded bottle was placed in a hot air over and was heated

to about 100˚C -105˚C. The stopper of the weighing bottle was removed and was also left in

the oven. The sample was dried to a constant weight and after the drying was completed, the

hot air oven was opened and the bottle was closed promptly. The bottle was allowed to cool

to room temperature in a desiccator before weighing. The bottle and the contents were

weighed and the loss of drying was estimated.

3.3.3 PRELIMINARY QUALITATIVE SCREENING USING APPROPRIATE

PHYTOCHEMICAL TESTS [532].

The preliminary phytochemical investigations were conducted employing various

phytochemical tests and the presence of various phytochemical constituents were detected.

Test for carbohydrates

A small quantity of aqueous and hydroalcoholic plant extracts were dissolved in 5 ml of

distilled water and filtered.

a. Molisch’s test- The filtrate was tested with alcoholic solution of � napthol and sulphuric

acid. A purple coloured ring indicated the presence of carbohydrates.

b. Fehling’s test- The filtrate was treated with equal quantity of Fehling A (Copper sulphate)

and Fehling B (Sodium potassium tartarate) and solution was heated. Brick red precipitate

indicates the presence of sugars.

c. Barfoed’s test- Formation of red colour within 2 min after addition of the reagent indicates

the presence of monosaccharides.

d. Benedict’s test- The filtrate was heated with this reagent for 2 min. Formation of red

precipitate indicates the presence of reducing sugars.

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e. Selwinoff’s test- The filtrate was heated with this reagent for 1-2 min. The formation of

red colour of the solution indicated the presence of ketohexose like fructose.

2. Tests for non-reducing sugars

The aqueous and hydroalcoholic plant extracts which did not give response to Fehling’s and

Benedict’s tests confirmed the presence of non-reducing sugars. The presence of non-

reducing sugars was also indicated by positive Fehling’s and Benedict’s tests by the

hydrolysed test solution.

3. Tests for non-reducing polysaccharides

In this test, 3 ml of test solution of extract was mixed with few drops of dilute iodine solution.

The blue colour of the solution confirmed the presence of non-reducing polysaccharides.

4. Test for gums and mucilage:

About 1 ml of extract was added slowly to about 25 ml of alcohol with constant stirring.

Formation of a precipitate indicates the presence of gums and mucilage

5. Test for resins

The extract was dissolved in alcohol and diluted it 10 times with water, turbidity formed

indicated the presence of resins.

6. Test for proteins

Biuret test (General test): To 3 ml extract solution 4% sodium hydroxide and few drops of

1% copper sulfate solution was added. The appearance of violet or pink colour indicated the

presence of proteins.

7. Tests for amino acids

a. Ninhydrin test (General test): The extract and 3 drops of 5% ninhydrin solution were

heated in a boiling water bath for 10 min. Purple or bluish colour indicated the presence of

amino acids.

b. Millon’s reagent: The extract was heated with 3 drops of Millon’s reagent. The dark red

colour solution confirmed the presence of tyrosine.

8. Test for glycosides

A small portion of the extract was hydrolyzed by boiling with dilute hydrochloric acid for

few min and hydrolysate was subjected to following tests.

a. Libermann-Burchard test - Chloroform solution of hydrolysate was treated with acetic

anhydride and sulphuric acid. Formation of blue or blue–green colour indicated the presence

of steroidal saponins whereas red, pink or violet colour indicated the presence of triterpenoids

saponins.

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b. Legal’s test - The hydrolysate was dissolved in pyridine and solution of sodium

nitroprusside was added to it and made alkaline. Formation of pink or red colour indicated the

presence of cardiac glycosides.

c. Borntrager’s test - An organic solvent like ether or chloroform was added to the

hydrolysate and the contents were shaken. The organic layer was shaken and treated with

solution of ammonia. The development of pink colour indicated the presence of

anthraquinone glycosides.

9. Test for Saponin Glycosides

Foam test: About 1 ml of extract was diluted with water to 20 ml and shaken in a graduated

cylinder for 15 min. A 1 cm layer of foam indicates presence of saponins.

10. Test for flavonoids

Shinoda test: A small piece of magnesium ribbon was added to the alcoholic solution of the

extract followed by drop wise addition of concentrated hydrochloric acid. The green blue

colour indicates the test is positive.

11. Test for alkaloids

A small portion of solvent free extract was stirred with few drops of dilute hydroalcoholic

acid and filter. The filtrate was tested with following reagents.

a. Dragendrof reagent (Potassium bismuth iodide) - To 2-3 ml filtrate, few drops of the

reagent was added. Orange brown precipitate is formed.

b. Mayer’s reagent (Mercury potassium iodide) – To 2-3 ml filtrate, few drops of the

reagent added gives cream precipitate.

c. Hager’s reagent (Saturated picric acid)- With 2-3 ml of filtrate the reagent gives yellow

precipitate.

d. Wagner’s reagent (Iodine reagent)-With 2-3 ml of filtrate the reagent gives reddish brown

precipitate.

12. Test for phenolic compounds and tannins

A small quantity of extract was diluted with water and tested with following reagent.

a) Dilute ferric chloride (FeCl3) solution (5%) Intense blue, green, red or purple colour

indicates the presence of phenolic compounds. An appearance of violet colour indicates

the presence of tannins.

b) Acetic acid solution: Forms red colour solution indicating presence of phenolic

compounds

c) Solution of gelatin (1%) containing 10% sodium chloride (NaCl)- Precipitate indicates

positive test for tannins.

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d) Lead acetate solution (10%)-Gives buff coloured precipitate for phenolic compounds.

3.3.4. High performance thin layer chromatography (HPTLC) of the plant extracts

[533,534]

The aqueous , hydroalcoholic and methanolic extracts of the leaves of MK and OS were

subjected to HPTLC profile to determine the solvent system giving the best resolution. The

extracts were spotted on HPTLC plates; air dried and subsequently run in the suitable solvent

system. The chromatographic parameters considered for the HPTLC studies were as follows:

Stationary phase - Precoated silica gel 60 F254 plates

Plate size - 10 cm x 10 cm

Thickness - 0.2 mm

Applicator - Camag Linomat IV

Band width - 7 mm

Spotting volume - 1 – 10 �l

Development chamber - Camag Twin Trough

Solvent front - 8 cm

Scanner - Camag

Densitometric conditions:

i. � 254 mode - Absorption Reflection (Deuterium lamp)

ii � 366 mode - Fluorescence Reflection ( Mercury lamp )

A Camag Linomat IV applicator was used for spotting the extracts on the precoated silica gel

60 F254 plates. Microsyringe was used for spotting the extract in a band of 7 mm on the

plates. The spots were allowed to air dry. The plates were developed using a Camag twin

trough chamber saturated with the solvent system, which gave the best resolution of spots for

the extract. The plates were run in the various solvent systems. The different solvents used in

different combinations for developing HPTLC system were Hexane Methanol, chloroform

Formic acid,acetic acid, water, toluene and ethyl acetate etc. (Refer to Table no 3.1 for the

solvent systems and spraying agents used for HPTLC of each extract)

The developed plates were air dried and then scanned using a Camag 3 Scanner under

ultraviolet (UV) 254 nm absorption mode and 366 nm fluorescence mode. The photographic

images were obtained under UV 254 nm absorption mode and 366 nm fluorescence mode.

The plates were sprayed with suitable spraying reagent and were heated in hot air oven at

125ºC and were observed for their spectra at the wavelengths of 254 nm.

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3.4. IN-VITRO EFFICACY EVALUATION

Dried plant aqueous and hydroalcoholic extracts were dissolved in the respective solvents at

the stoc0.8ml 0fk concentration of 1mg/ml. The appropriate dilutions of the stock solutions

were made and used for the in vitro antioxidant assays.

3.4.1 DPPH radical scavenging activity [535].

Method: To 0.1 ml of methanolic solution of DPPH an equal volume of test compound was

added at different concentrations in methanol. Equal volume of methanol was added to

control. Above mixture was kept for incubation at room temperature for 20 minutes.

Absorbance was recorded at 517 nm. Scavenging capacity was measured by monitoring the

decrease in absorbance at 517 nm. The anti oxidant activity of test drug was expressed as

IC50.

Percentage Inhibition was calculated by using following formula;

Percentage Inhibition = (Absorbance of Control – Absorbance of Test/ Absorbance of

Control) X100

3.4.2. Nitric oxide radical scavenging activity [536].

Method: Nitric oxide generated from sodium nitroprusside in aqueous solution at

physiological pH interacts with oxygen to produce nitrite ions, which were measured by the

Griess reaction. The reaction mixture (3 ml) containing sodium nitroprusside (10mM) in

phosphate-buffered saline and various concentrations of MK and OS Aqueous ,

hydroalcoholic and methanolic extracts at different concentrations were incubated at 25ºC for

150 min. A 0.5-ml aliquot of the incubated sample was removed and 0.5 ml Griess reagent

(1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride in 2% phosphoric acid )

was added. The absorbance of the purple chromophore formed during diazotization of nitrite

with suphanilamide and subsequent coupling with napthylethelene diamine was measured at

546 nm.

Percent inhibition was calculated by comparing the absorbance values of test samples as per

the following formula:

Inhibition (%) = {(Control – test) / Control }x 100

.

Experimental

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3.5 ACUTE TOXICITY STUDY [537].

It refers to those adverse effects occurring within a short time following oral administration

of a single dose of a substance or multiple doses given within 24 hours.

The acute toxicity study in rats and mice was performed as per the OECD guidelines (No

423) to evaluate the undesirable effects or toxicity of plant extracts.

Species : Wistar rats Species : Swiss Albino mice

Sex : Male and Female Sex : Male and Female

Body Weight : 150-180 g Body weight : 20-25 g

Method [538].

Rats and Mice of either sex were divided into the groups of 3 animals per group. A single

dose oral study was conducted to determine the acute toxic category of the plant extracts used

in the study. The acute toxicity study was conducted as per Organisation for Economic Co-

operation and Development (OECD) 423 guidelines. Each rat was fasted overnight and

mouse 4 h prior to dosing with free access to water. Following the period of fasting, the

animals was weighed and the plant extracts (MKAQ, MKHA,MKM ,OSAQ,OSHA and OSM

) were administered in the form of suspension at a dose of 5 mg/kg p.o. initially and were

observed for mortality. If mortality was observed in two out of three animals then the dose

administered was considered as toxic dose. However if mortality was observed in any one

animal out of the three then the same dose was repeated again to confirm the toxic effect. If

no mortality was observed then the higher doses of 50, 300 2000 mg/kg p.o. were employed

for further toxicity studies.

The mice/rats were then critically observed for clinical signs, gross behavioral changes,

morbidity and mortality. These behavioral observations were made daily for a period of 14

days. Simultaneously, various other parameters and effect on reflexes were made daily for a

period of 14 days.

� Righting Reflex (RR): The ability of the mouse to regain its normal posture when placed

on its back, on a flat surface, within 30 sec was noted.

� Pinna Reflex (PR): Using a fine hair or thread, the external auditory meatus was

stimulated. The pinna reflex was considered abolished if no response was obtained on this

stimulation.

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� Corneal Reflex (CR): To induce contraction of the orbicular occulis muscle, a fine hair or

thread was used to touch the surface of the cornea and the conjunctiva of both the eyes.

The reflex was considered abolished when either eye elicits no reflex for 1 second.

� Hyperesthesia was monitored by measuring body temperature of animals by using

thermometer.

� Motor activity was measured by testing the movement of animals on inclined surface.

� Muscle relaxation was observed by putting the animals on cage lid and tilting it so as to

hang the animal freely in air thereby assessing grip strength.

� Animals were observed for occurrence of clonic convulsion ,tonic extension ,straub

reaction ,pilo-erection ,muscle spasm ,catatonia for 1 hour,2 hours , 4 hours,8 hours and

24 hours after administration of extracts

� Body Weight: The mean group body weight of the control and test group animals were

recorded on 0th, 7th and 14th day respectively.

� Any unusual abnormalities like changes in fur of the animals, or any abnormal behavior

was noted.

� Mortality was observed every day.

After observing mortalities and behavioral profile for the stipulated time, the maximal safe

dose for the study was noted. Further, in accordance with the OECD guidelines, the doses for

the study were narrowed down.

DOSE CALCULATION [539]:

Doses for extracts of MK and Os were calculated as shown in a Table no 3.1

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98

3.6 PRELIMINARY NEUROPHARMACOLOGICAL SCREENING OF MK AND OS

EXTRACTS. (Refer Figure 3.1-3.4)

Drug treatments:

Albino mice (20-30 gm) of both the sexes were pretreated with vehicle ( equi volume 0.1%

Na CMC), MKAQ (50,100 and 200mg/kg p.o) ,MKHA (50,100 and 200mg/kg p.o) and

MKM (50,100 and 200mg/kg p.o) and OSAQ (50,100 and 200mg/kg p.o) ,OSHA (50,100

and 200mg/kg p.o) and OSM (50,100 and 200mg/kg p.o) extracts suspended in 0.1% Na

CMC for 7 days. Standard Diazepam( 1mg/kg i.p) was administered on 7th day of treatment

30 minutes prior to the preliminary neuro pharmacological evaluation by using following

models:

Experimental groups for Acto Photometer ,Elevated Plus Maze (EPM )and Hole board

(HB) models

The experimental animals used were albino mice which were divided in to 20 groups and

each group consisted of eight animals as follows:.

Group I : Rats received 0.1% Na CMC in vehicle (Vehicle control group)..

Group II : Rats were treated with standard Diazepam (1mg/kg i.p.) (Standard Group)

Group III -V : Rats were treated with MKAQ at doses of 50,100 and 200 mg/kg resp p.o.

Group VI - VIII :Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o.

Group IX -XI: Rats were treated with MKM at doses of 50,100 and 200 mg/kg resp. p.o.

Group XII -XIV : Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg resp. p.o.

Group XV -XVII: Rats were treated with OSHA at doses of 50, 100 and 200 mg/kg resp. p.o.

Group XVIII-XX : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp. p.o.

3.6 1 ACTOPHOTOMETER [542].

Method:

On 7th day 1 hr after administration of last dose of MK and OS extracts and 30 minutes after

I.P. administration of Standard Diazepam 1mg/kg effect on loco motor activity was observed

in Acto-photometer.

Each animal was placed individually at the centre of the Acto-photometer and was allowed to

explore the instrument for initial 5 seconds .There after the spontaneous motor activity score

in terms of photo cell count was measured during a period of later 5 min.

Experimental

99

3.6.2 ELEVATED PLUS MAZE TEST (EPM) [543].

Method:

On 7th day of treatment 1 hr after administration of last dose of MK and OS extracts and 30

minutes after I.P. administration of Standard Diazepam 1mg/kg animals were tested on EPM.

Each animal was placed individually at the centre of the elevated maze facing towards the

open arm and was allowed to explore the maze for initial 5 seconds .There after the number

of entries in the open arm of the elevated maze and duration of stay in open arm during a

period of 5 min were noted in sound proof area.

3.6.3 HOLE BOARD APPARATUS (HB) [544].

Method:

On 7th day 1 hr after administration of last dose of MK and OS extracts and 30 minutes after

I.P. administration of Standard Diazepam 1mg/kg each animal was placed individually in the

apparatus and was allowed to explore the apparatus for initial 5 seconds. Thereafter number

of head dips throughout the hole and total time spent with head dipped were noted during a 5

min test session.

3.6.4 .FORCED SWIM TEST (FSW) [545].

Drug treatment:

Albino mice (20-30 gm) of both the sexes were pretreated with vehicle (equi volume) 0.1%

Na CMC), MKAQ (50,100 and 200mg/kg p.o) ,MKHA (50,100 and 200mg/kg p.o) and

MKM (50,100 and 200mg/kg p.o) and OSAQ (50,100 and 200mg/kg p.o) ,OSHA (50,100

and 200mg/kg p.o) and OSM (50,100 and 200mg/kg p.o) extracts suspended in 0.1%Na

CMC were administered for 7 days. On7th day 1 hr after administration of of MK and OS

extracts and 30 minutes after I.P. administration of standard Imipramine 10 mg/kg (i.p.)

animals were forced to swim.

Experimental groups for FSW models

Albino mice (20-35g) of both the sex were divided randomly in to 20 groups and each group

consisted of eight animals as follows:

Group I : Rats received 0.1% Na CMC in vehicle (Vehicle control group)

Group II : Rats were treated with standard Imipramine (10 mg/kg .p.o.) (Standard Group)

Group III -V : Rats were treated with MKAQ at doses of 50,100 and 200 mg/kg resp p.o

Group VI -VIII :Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o.

Experimental

100

Group IX -XI: Rats were treated with MKM at doses of 50,100 and 200 mg/kg resp.p.o.

Group XII-XIV : Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg resp. p.o.

Group XV-XVII: Rats were treated with OSHA at doses of 50, 100 and,200 mg/kg resp. p.o.

Group XVIII-XX : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp.p.o.

Method:

A day before the start of the treatment mice were placed individually in to plexy glass

container (25x 12 x 25 cm) containing water at room temperature (250C + 30C). The height of

water level was maintained to 15 cm and kept constant throughout the experiment. The

animals were left to swim in water for 15 min and then removed, dried with towel and

returned to their home cages. The study was initiated 24 hrs after pretest session. On 1st and

7th day of treatment animals were forced to swim in the container for 10 minutes. The

duration of immobility was recorded during last 5 min of 10 min test swimming session. A

rat was judged immobile when it floated in an upright position making only small movements

to keep head above water. Immobility was observed on 1st and 7th day of treatment. Following

all swim session animals were removed from cylinder dried with towels and returned to their

home cages.

3.6.5. TAIL SUSPENSION TEST (TS) [546].

Drug treatment:

Animals in respective groups were pretreated with vehicle (equi volume) 0.1% Na CMC),

standard Imipramine (10 mg/kg p.o) MKAQ (50,100 and 200mg/kg p.o) ,MKHA (50,100 and

200mg/kg p.o) and MKM (50,100 and 200mg/kg p.o) and OSAQ (50,100 and 200mg/kg p.o)

,OSHA (50,100 and 200mg/kg p.o) and OSM (50,100 and 200mg/kg p.o) extracts suspended

in 0.1% Na CMC successively for 7 days. On 7th day of treatment 1 hr after administration of

last dose of MK and OS extracts and 30 minutes after I.P. administration of Standard

Imipramine 10 mg/kg (i.p) animals were subjected to TS

Experimental groups for TS models

Albino mice (20-35g) of both the sex were divided randomly in to 20 groups and each group

consisted of eight animals as follows:



Group III -V : Rats were treated with MKAQ at doses of 50,100 and 200 mg/kg resp p.o

Group VI -VIII :Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o.

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101

Group IX -XI: Rats were treated with MKM at doses of 50,100 and 200 mg/kg resp.p.o.

Group XII-XIV : Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg resp. p.o.

Group XV-XVII: Rats were treated with OSHA at doses of 50, 100 and,200 mg/kg resp. p.o.

Group XVIII-XX : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp.p.o.

Method:

In TS test each animal was suspended on the edge of a shelf 58 cm above a table top by

adhesive tape placed approximately 1 cm from the tip of the tail. The duration of mobility

was measured in later 5 min of total 10 min duration from hanging. Mice were considered

immobile when they hang passively and completely motionless for at least 1 min.

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102

3.7.EVALUATION OF ANTI STRESS ACTIVITY OF MK AND OS EXTRACTS.

3.7.1. WEIGHT LOADED FORCED SWIM TEST MODEL (WFS) [547].

Drug treatments:

Albino mice (20-35 gm) were divided in to 21 groups each containing 8 animals. Animals

were Pre-treated with vehicle (Equi volume 0.1% Na CMC), standards Ashwagandha

(100mg/kg p.o)( Himalaya Ashvgandha) , Imipramine (10mg/kg p.o) (Sigma aldrich) and

extracts MKAQ (50,100 and 200mg/kg p.o) ,MKHA (50,100 and 200mg/kg p.o) and MKM

(50,100 and 200mg/kg p.o) and OSAQ (50,100 and 200mg/kg p.o) ,OSHA (50,100 and

200mg/kg p.o) and OSM (50,100 and 200mg/kg p.o) suspended in 0.1% Na CMC daily for a

period of 21 days.

Experimental groups for WFS models Albino mice (20 -35gm) of both the sexes were

divided randomly into 21 groups each containing eight mice as follows



Group III : Rats were treated with standard Ashwagandha (100 mg/kg p.o.) (Standard Group)

Group IV -VI : Rats were treated with MKAQ at doses of 50,100 and 200 mg/kg resp p.o.

Group VI I- IX :Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o.

Group X -XII: Rats were treated with MKM at doses of 50,100 and 200 mg/kg resp. p.o.

Group XIII-XV : Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg resp. p.o.

Group XVI-XVIII: Rats were treated with OSHA at doses of 50, 100 and 200 mg/kg resp.

p.o.

Group XIX-XXI : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp. p.o

Method:

Animals in all groups except vehicle control group were subjected to the Weight loaded

forced swimming test on 21st day of treatment ,1 hour after the treatment with respective

vehicle, Standards and extracts The forced swimming capacity of mice till exhaustion was

measured in acrylic plastic pool (65 × 45 ) filled with water to a depth of 40 cm [548]. The

temperature of the water was maintained at 34ºc ± 1ºc. Each mouse was loaded with a tin

wire (8 % body weight) which was attached to the tail and was introduced in water . This

arrangement forced the mouse to maintain continuous rapid leg movement [549]. Swimming

time to exhaustion was regarded as time spent by the mouse floating in water with struggling

and making necessary movements until exhausting its strength and drowning Exhaustion was

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103

determined as a failure to swim and the mice were assessed to be exhausted when they failed

to rise to the surface of water to breathe within a 7 seconds period [550]. The swimming time

to exhaustion was used as the index of the forced swimming. It is reported that a period of

longer than 7 seconds resulted in frequent drowning, and less than 5 s reduced the

reproducibility of the test. After removing the animal from water it was dried blood was

withdrawn from retro-orbital plexus under light ether anaeshesia. whole blood samples were

collected. Blood serum was prepared by centrifugation at 3000 rpm, 4⁰C for 10 min. The

levels of serum corticosterone, glucose, triglyceride , lactate dehydrogenase (LDH) and

Blood urea nitrogen (BUN) values were determined by using commercial kits (Erba

diagnostics and Agappe diagnostics). After blood withdrawal animals were sacrificed

humanely by cervical dislocation and gastronemius muscle from thigh of hind limb of mouse

and liver were isolated and were homogenized in TCA (2.5%) and kept in refrigerator at 4⁰c

for analysis of glycogen.

Analysis of blood biochemical parameters Alterations in the following biochemical

parameters were estimated using commercially available kits (Erba Diagnostics)

� Estimation of serum glucose levels (GLU)

� Estimation of Serum Triglyceride levels (TG)

� Estimation of serum Lactate dehydrogenase (LDH)

� Estimation of blood urea nitrogen (BUN)

� Estimation of Muscle Glycogen content

� Estimation of liver Glycogen content

3.7.2. COLD RESTRAINT STRESS (CRS) [551].

Drug treatments: Albino wistar rats (150-200g) of bot the sex were divided randomly in to

21 groups each containing 8 animals . Animals in respective groups were pre-treated with

vehicle (equi volume0.1% Na CMC) Diazepam (1mg/kg p.o) (Calmpose 5mg Ranbaxy Lab.

LTD), Ashwagandha (100mg/kg p.o)(Ashvagandha Himalaya drugs ) and extracts MKAQ

(50,100 and 200mg/kg p.o) ,MKHA (50,100 and 200mg/kg p.o) and MKM (50,100 and

200mg/kg p.o) and OSAQ (50,100 and 200mg/kg p.o) ,OSHA (50,100 and 200mg/kg p.o)

and OSM (50,100 and 200mg/kg p.o) suspended in 0.1% NaCMC daily for a period of 14

days.

Experimental

104

Experimental groups for CRS models

Wistar rats were divided randomly into 21 groups, each containing eight animals as follows:

Group I: Rats received 0.1% Na CMC in vehicle (Vehicle control group).

Group II: Rats were treated with standard Diazepam (1 mg/kg .p.o.) (Standard Group).

Group III: Rats were treated with standard Ashwagandha (100 mg/kg p.o.) (Standard Group)

Group IV -VI: Rats were treated with MKAQ at doses of 50,100 and 200 mg/kg resp p.o

Group VII- IX: Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o.

Group X -XII: Rats were treated with MKM at doses of 50,100 and 200 mg/kg resp.p.o.

Group XIII-XV: Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg resp. p.o.

Group XVI-XVIII: Rats were treated with OSHA at doses of 50, 100 and 200 mg/kg resp.

p.o.

Group XIX-XXI: Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp.p.o.

Method : In this study on 10th,12th ,13th ,and 14th days of treatment ; one hour after

administration of Vehicle /extract/standard to the rats in respective groups except the rats in

vehicle control group were immobilized by placing them individually in plastic restraint

boxes (19.5cm × 6.5 cm ) which were then placed in refrigerator at 4–7 ⁰C for 4 h every day

successively for 4 days . On 14th day of treatment at the end of 4 hours of cold restraining ,

animals were freed from restrainers and blood was withdrawn from retro orbital plexus under

light ether anaesthesia.. Blood serum was prepared by centrifugation at 3000 rpm, 4 ◦C for 10

min. The levels of serum corticosterone, glucose, triglyceride were determined by using

commercial kits (Erba diagnostics) After withdrawal of blood the animals were humanely

sacrificed by cervical dislocation; the stomach was isolated from each animal and cut open

along the greater curvature and inner surface of stomach was examined for lesions. With the

help of a transparent plastic sheet, placed over the stomach, the outline of stomach and the

areas of erosions were traced [552] The ulcer index was calculated using the formula:

Ulcer index = 10/X

Where, X = Total mucosal area

Total ulcerated area

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105

3.7.3. MILK INDUCED LEUKOCYTOSIS (MLK) [553]. Drug treatments: The respective groups were treated with vehicle (0.1% equi volume)

Diazepam (1mg/kg p.o) (Calmpose 5mg Ranbaxy Lab. LTD), Ashwagandha (100mg/kg p.o)

(Ashvagandha Himalaya drugs ) and extracts MKAQ (50,100 and 200mg/kg p.o) ,MKHA

(50,100 and 200mg/kg p.o) and MKM (50,100 and 200mg/kg p.o) and OSAQ (50,100 and

200mg/kg p.o) ,OSHA (50,100 and 200mg/kg p.o) and OSM (50,100 and 200mg/kg p.o)

suspended in 0.1% Na CMC were administered daily for a period of 14 days.

Experimental groups for MLK model :

Wistar rats (150-200g) of both the sex were divided randomly into 21 groups each containing

eight rats as follows:


Group II : Rats were treated with standard Diazepam (1 mg/kg .p.o.) (Standard Group)

Group III : Rats were treated with standard Ashwagandha (100 mg/kg p.o.) (Standard Group)

Group IV -VI : Rats were treated with MKAQ at doses of 50,100 and 200 mg/kg resp p.o.

Group VI I- IX :Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o.

Group X -XII: Rats were treated with MKM at doses of 50,100 and 200 mg/kg resp.p.o.

Group XIII-XV : Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg resp. p.o.

Group XVI-XVIII: Rats were treated with OSHA at doses of 50, 100 and,200 mg/kg resp.

p.o.

Group XIX-XXI : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp.p.o.

Method:

The animals in respective groups were treated with vehicle/ extracts / standards daily for a

period of 14 days. On 13th day of study blood was withdrawn by puncturing retro orbital

plexus for determination of WBC count before administration of milk and then one hour after

administration of vehicle/ extracts / standards to the respective groups the S.C. injection of

raw cow milk (4 ml/kg sc) was administered to all groups. Twenty-four hours later i.e. on 14th

day of treatment 1 hour after administration of vehicle/ extracts / standards blood was

withdrawn from the retro orbital plexus of the animals and the white blood cell count was

computed using cell counter (ABACUS). After blood withdrawal animals were sacrificed

humanely under ether anaesthesia by cervical dislocation and thymus gland and spleen were

isolated , washed with isotonic saline solution and weights of wet tissues were recorded.

Experimental

106

3.7.4. RESTRAINT STRESS MODEL (RS) [554]

Drug treatments : Vehicle (0.1% equi volume) Diazepam (1mg/kg p.o) (Calmpose 5mg

Ranbaxy Lab. LTD), Ashwagandha (100mg/kg p.o)(Ashvagandha Himalaya drugs )

Piracetam (200 mg/kg p.o.) (Neurocetam 400 mg) and extracts MKAQ (50,100 and 200

mg/kg p.o) ,MKHA (50,100 and 200 mg/kg p.o) and MKM (50,100 and 200 mg/kg p.o) and

OSAQ (50,100 and 200 mg/kg p.o) ,OSHA (50,100 and 200 mg/kg p.o) and OSM (50,100

and 200 mg/kg p.o)were administered to the respective groups dailyt for a period of 14 days

one hour prior to the application of restraint stress.

Experimental groups for RS:

Wistar rats (150-200 g) of both sex were divided randomly into twenty three groups , each

containing eight rats as follows:

Group I : Rats received 0.1% Na CMC in vehicle (Equi volume) (Vehicle control group).

Group II : Rats received 0.1% Na CMC in vehicle and subjected to restraint stress (stress

control group.

Group III : Rats were treated with standard Ashwagandha (100 mg/kg .p.o.) and subjected to

restraint stress (Standard Group).

Group IV : Rats were treated with standard Diazepam (1 mg/kg .p.o.) and subjected to


Group V : Rats were treated with standard Piracetam (200 mg/kg p.o.) and subjected to


Group VI- VIII: Rats were treated with MKAQ at doses of 50,100 and 200 mg/kg

respectively p.o and subjected to restraint stress.

Group IX-XI: Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o. and

subjected to restraint stress.

Group XII-XIV: Rats were treated with MKM at doses of 50,100 and 200 mg/kg respectively

.p.o. and subjected to restraint stress.

Group –XV-XVII : Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg

respectively. p.o. and subjected to restraint stress.

Group XVIII-XX: Rats were treated with OSHA at doses of 50, 100 and,200 mg/kg

respectively. p.o and subjected to restraint stress.

Group XXI-XXIII : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg

respectively .p.o and subjected to restraint stress.

Experimental

107

Method:

Animals were trained to establish transfer latency (TL) on EPM and step down latency (SDL)

before subjecting them to restraint stress. All groups except vehicle control group were

subjected to immobilization stress for 2 hours/day daily for a period of 14 days 1hour after

the respective treatments.

The restraint stress was applied by placing each rat in a plastic restrainer. The Plastic

restraint tubes were equipped with air holes and an adjustable end plate, which helped to

account for differences in body weight and length and secured the rat within the tube. Once

the rat was inserted in their respective plastic restrainer the end plate was lowered down to

compress the animal in restrainer and was screwed tightly to immobilize the animal. It’s tail

movements were restricted by pasting adhesive tape to further restrict the change in posture.

Then the cohort was placed in a temperature control and sound proof room separate from the

main animal colony. Individual animal was restrained inside cylindrical plastic restrainer

(19.5cm × 6.5 cm ) daily for 2 hrs .The immobilization procedure was followed with the

cognition studies on 1st ,7th and 14th day of study. Blood was collected from retro orbital

plexuses immediately at the end of restraining procedure periodically on 1st ,7th and 14th day

of study, centrifuged at 4⁰c at 3000 rpm ×15 min and the serum was separated. The serum

was used for estimation of various biochemical parameters such as corticosterone , glucose

,triglyceride and cholesterol. Animals were sacrificed at the end of the study period (14th day)

by cervical dislocation. Tissues like brain and adrenal glands were removed , rinsed in

isotonic saline solution and their weights were recorded. Brain was isolated and immediately

homogenized for estimation of catecholamines

Cognitive assessment:

A. Elevated Plus maze [555].

The elevated plus maze consisted of two opposite open arms (50 x 10 cm) crossed with two

closed arms of the dimension with 40 cm high wall. The arms were connected with central

square 10 x 10 cm to give the maze the shape of plus sign. The maze was elevated 50 cm

above the floor and kept in dimly lit room. Rat was placed individually on one far end of an

open arm and the time taken to enter one of the closed arms was recorded as the transfer

latency (TL). A day before giving stress and drug treatment the rat was given five trials at 10

min interval. The transfer latency was usually established by this time. Transfer latency (TL)

Experimental

108

was recorded on 1st, 7th and 14th day of the study in order to assess the retention of memory of

learned task

B. Step down inhibitory avoidance Test [556].

The animals were trained for one way step down inhibitory task using a 50×25×25 cm

plywood box with a Perspex wall front and a floor consisting of 1 mm bronze bars spaced 10

mm apart. The left end of the grid was covered with 5 cm high, 25 cm wide and 7.5 cm long

wood platform. A rat was placed on the platform with it’s all four limbs on the platform and

allowed to step down. Twenty-four hours later the animals were gently held by the body and

lowered onto the platform , at which point a timer was activated to measure the latency to

step down (i.e. placing all four paws on the grid) and on stepping down it received

intermittent foot shock (6mA) through the grid floor of 5 second duration until the animal

climbed back on the platform. The rat was given three trials with inter trial interval of 30 min.

for 3 days until the latency of the step down had stabilized prior to subjecting them to chronic

restraint stress. In the test session on 1st, 7th and 14th day of stress and treatment the test was

repeated to record step down latency (SDL) to assess acquisition and retention of memory of

learned task. During testing sessions 300 seconds ceiling was imposed, latencies >300 s were

counted as 300 sec. Retention of memory for each animal was calculated in seconds (cut off

point 300 s)

Biochemical estimations :

Alterations in the following biochemical parameters were estimated using commercially

available kits (Erba Diagnostics)

� Serum glucose levels

� Serum triglyceride levels

� Serum cholesterol levels

� Estimation of serum Corticosterone was performed in Harmone lab, Mumbai by radio-

immuno assay method

Estimation of catecholamines: At the end of the study after blood withdrawal and cognitive studies animal were killed

humanely by cervical dislocation. Brains were isolated cleaned with isotonic saline solution

and were immediately homogenized for catecholamine estimations.

Experimental

109

Histopathological estimation :

Immediately after decapitation the adrenal glands were weighed and fixed in the 10%

formalin solution (prepared in 0.9% normal saline). The fixed adrenal gland samples were

given for further histopathological investigations at Biodiagnostic Centre Parel, Mumbai.

3.7 5. FOOT SHOCK STRESS (FS) [557].

Drug treatments : Vehicle (0.1% equi volume) Diazepam (1mg/kg p.o) (Calmpose 5mg

Ranbaxy Lab. LTD), Ashwagandha (100mg/kg p.o) (Ashvagandha Himalaya drugs )

Piracetam (200 mg/kg p.o.) () and extracts MKAQ (50,100 and 200mg/kg p.o) ,MKHA

(50,100 and 200mg/kg p.o) and MKM (50,100 and 200mg/kg p.o) and OSAQ (50,100 and

200mg/kg p.o) ,OSHA (50,100 and 200mg/kg p.o) and OSM (50,100 and 200mg/kg p.o)

suspebded in 0.1% NaCMC were administered per orally to the animals in respective groups

daily for a period of 14 days one hour prior to foot shock stress.

Experimental groups for Foot shock models

The wistar rats (150-200 gm) were divided randomly in to 22 groups, each consisted of eight

animalsa sfollows:

Group I : Rats received equi volume 0.1% Na CMC in vehicle (Vehicle control group)

Group II : Rats received equi volume 0.1% Na CMC in vehicle and subjected to foot shock

stress (stress control group)


foot shock stress (Standard Group)

Group IV : Rats were treated with standard Imipramine (10 mg/kg .p.o.) and subjected to

foot shock stress (Standard Group)

Group V VII: Rats were treated with MKAQ at doses of 50,100 and 200 mg/kg resp p.o and

subjected to foot shock stress

GroupVIII-XI: Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o.

and subjected to foot shock stress

Group XI-XIII: Rats were treated with MKM at doses of 50,100 and 200 mg/kg resp.p.o. and

subjected to foot shock stress

Group –XIV-XVI : Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg resp.

p.o. and subjected to foot shock stress

Group XVII-XIX: Rats were treated with OSHA at doses of 50, 100 and,200 mg/kg resp. p.o


Experimental

110

Group XX-XXII : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp.p.o


Method:

The method of ‘Armario et al’ was used.. The animals in each group except vehicle control

group were subjected daily to 1 hr of foot shock through a grid floor in a wooden chamber for

14 days . The duration of each shock (2 mA) and interval between the shocks were randomly

programmed between 3 sec, 5 sec, and 10 sec 110 sec respectively to make the received

shock unpredictable. Animals were sacrificed on day 14 one hr after last shock procedure on

completion of the other test procedures such as behavioural despair test and evaluation of

retention of memory in shuttle box. Blood was collected from retro orbital plexus under light

ether anesthesia at different intervals on 1st, 7th and 14th day of study. Blood collected was

centrifuged at 4⁰c at 3000 rpm for 5 min and the serum was separated. The serum was used

for estimation of various biochemical parameters At the end of study animals in all groups

were humanely sacrificed by cervical dislocation and brain tissues were dissected out for

estimations of concentration of hydrolysed Acetyl choline.

Behavioural Despair test [558].

Behavioral despair is an experimental model of endogenous depression. Rats were forced to

swim in individually in polypropylene vessel (45 x 40 x 30 cm) with a water level 20 cm by

ensuring that rat’s feet did not touch the floor of the vessel and it could not climb out of it. On

14th day after receiving the foot shocks rats were allowed to swim in water for 10 minutes.

The total duration of immobility (DOI) was measured in last 5 minutes. The rat was judged to

be immobile whenever it remained floating passively in water in a slightly bunched but

upright position with its head just above surface of water with minimum limb movements to

keep it floating in the water.

Active avoidance test [559]

The apparatus consists of a rectangular box 50X 15 cm with 40 cm high metal walls and a

grid floor. The box was divided by a wall with a guillotine door (10X10 cm) in to two

compartments . Animals were trained for an active avoidance task before subjecting them to

stress During training the animal was placed in the right electrified compartment of a shuttle

box (Techno) and allowed to acclimatize for 5min.The guillotine door was closed and animal

was placed in to a starting area. There after 20 seconds the animal was subjected to light

Experimental

111

stimulus (conditional stimulus) and the gullitone door was simultaneously opened which was

followed by electric shock. (1mA 50 Hz) given through the grid floor (Unconditioned

stimulus) 5 seconds later in illuminated area and continued till animal escapes to the dark

compartment. If the escape response occurs then both shock and light signal were terminated.

Once the animal runs to the safe area it was left there for 10 seconds The procedure started

again and was repeated till the animal achieves the criteria of 9 avoidances out of 10

consecutive trials The avoidance response was characterized by escape to the adjoining safe

chamber during conditioned stimulus, failure to escape during unconditioned stimulus within

15 s was assessed as escape failure which is postulated to indicate depression. The test was

repeated on first and 14th day after the stress procedure and escape failures for 10 consecutive

trials were noted down.

Biochemical estimations:

� Estimation of Serum corticosterone was performed in Harmone lab, Mumbai by radio-

immuno assay method

� Serum glucose levels were estimated using commercially available kits (Erba

Diagnostics)

Tissue estimations:

Brains were isolated to estimate levels of hydrolyzed Acetyl choline by using Augusteiin

method. At the end of the study the animals were sacrificed by cervical decapitation under

light anesthesia. Immediately after decapitation whole brain was removed from the skull. Wet

tissue weight was recorded. The whole brain was transferred to a glass homogenizer

containing 10 volumes of normal saline (0.9% sodium chloride solution) and homogenized in

ice bath. Brain homogenate was centrifuged at 3000 rpm for 15 min. The supernatant was

collected and was used as a source of enzyme for assay.

3.7.6. SLEEP DEPRIVATION MODEL (SD) [560].

Drug treatments:

Wistar rats were divided into 23 groups randomly each containing eight rats. Reference

standard Piracetam (200 mg/kg p.o), Ashwagandha(100mg/kg p.o) Diazepam (1mg/kg p.o.

and extracts MKAQ (50, 100 and 200mg/kg p.o.) MKHA (50, 100 and 200mg/kg p.o.),MKM

(50, 100 and 200mg/kg p.o.),OSAQ (50, 100 and 200mg/kg p.o.) ,OSHA(50, 100 and

200mg/kg p.o.) and OSM (50, 100 and 200mg/kg p.o.) were suspended in 0.1% sodium

carboxy methyl cellulose solution and administered per orally daily for 14 days.

Experimental

112

Experimental groups for sleep deprivation model :

Wistar rats (150-200 gm) were divided randomly in to 22 groups each consisted of eight

animals as follows:


Group II : Rats received equi volume 0.1% Na CMC in vehicle and subjected to sleep

deprivation stress (stress control group


sleep deprivation stress (Standard Group)


sleep deprivation stress (Standard Group)

Group V -VII: Rats were treated with MKAQ at doses of 50,100 and 200 mg/kg resp p.o and

subjected to sleep deprivation stress

GroupVIII-XI: Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o.

and subjected to sleep deprivation stress


subjected to sleep deprivation stress


p.o. and subjected to sleep deprivation stress

Group XVII-XIX: Rats were treated with OSHA at doses of 50, 100 and 200 mg/kg resp. p.o




Method:

Animals in respective groups were treated with vehicle /standard / extract for 15 days. On 9th

day of treatment, after an hour of respective treatments with Vehicle/Standards/Extracts each

animal was placed in Cook’s pole apparatus and allowed to explore for 180 seconds. Then

animals were subjected to learn avoidance trials for next 2 days. On 12th day 1 hour after

administration of vehicle/ standards /extracts to the animals in respective groups they were

subjected to sleep deprivation stress. Animals in all groups except vehicle control group were

subjected to sleep deprivation of 72 hours. Rats were deprived of sleep by placing individual

rat on narrow rectangular platform (9 cm long x 7 cm widex6 cm height) cm inside the plastic

cage filled with water to 1 cm below the platform for 72 hrs. The water inside the tanks was

changed daily; rats had food and water ad lib. At the end of sleep deprivation period blood

was withdrawn from retro orbital plexus and animals were tested for retention of memory of

Experimental

113

learned task in cooke’s pole apparatus. During which latency period (LP) to climb on the rod

,total period of retention (RP) on the rod and no of successful climbing attempts on the rod

were measured .At the end the animals were humanely sacrificed by cervical dislocation and

brain tissues were isolated ,rinsed with isotonic saline solution and were weighed and

homogenized for estimation of 5HT levels in brain.

Cooks pole climbing test

The apparatus described by Cook and Widely (1957) was used to study the efficacy of the

drug on acquisition of learning and its retention in rats. The training and testing of the rats is

conducted in a 25 × 25 × 40 cm chamber that is enclosed in a dimly lit, sound attenuating

box. Scrambled shock was delivered to the grid floor of the chamber. A 2.8-kHz speaker and

a 28-V light are situated on top of the chamber. A smooth stainless-steel pole, 2.5 cm in

diameter, is suspended by a counterbalance weight through a hole in the upper center of the

chamber. A response was recorded when a rat jumps on the pole as a latency period to climb

on rod. On first day animal was subjected to electric shock of 1.5mA which was given

through electrified grid floor and repeated scrambled shocks were delivered through the grid

floor, for 15 sec. with a gap of 5 seconds till the animal climbed up on the rod. When the

animal fell down on the grid application of shock (1.5mA) was started immediately. Cutoff

time was 300 seconds. Animals failing to climb on the rod within 300 seconds were

eliminated from study. Animals were trained till they learn to climb on the rod within first 30

seconds of observation period.

Biochemical estimations

� Estimation of Serum corticosterone was performed in Harmone lab, Mumbai by radio-

immuno assay method

� Estination of following biochemical parameters in serum were performed using

commercially available kits (Erba Diagnostics)

� Serum glucose

� Serum Triglycerides

� Serum cholesterol

Tissue homogenate estimations

Brains were isolated to estimate levels of 5HT in rat brains by spectro fluorimetric assay

method. At the end of the study animals from different groups were sacrificed by cervical

dislocation. The whole brain was dissected out from the skull. The weight of wet brains were

recorded. The isolated brains were then immediately homogenized and used for estimation.

Experimental

114

3.7.7. ELECTROCONVULSIVE SHOCK MODEL (ECS) [561].

Drug treatments : Wistar rats (150-200gm) of both the sex were divided randomly in to 23

groups Animals were trained in water maze before subjecting them to an electro convulsive

shock stress. After the training on water maze animals were treated with reference standard

Piracetam (200 mg/kg p.o), Ashwagandha (100mg/kg p.o) ,Diazepam (1mg/kg p.o. and

extracts MKAQ (50, 100 and 200mg/kg p.o.) MKHA (50, 100 and 200mg/kg p.o.), MKM

(50, 100 and 200mg/kg p.o.),OSAQ (50, 100 and 200mg/kg p.o.) ,OSHA(50, 100 and

200mg/kg p.o.) and OSM (50, 100 and 200mg/kg p.o.) suspended in 0.1% sodium carboxy

methyl cellulose solution administered per orally successively for 15 days,1hour before the

application of ECS as per the following protocol.

Experimental groups for Electro convulsive shock method :

The wistar rats (150-200 gm) were divided randomly in to 22 groups , each consisted of eight

animals as follows:


Group II : Rats received equi volume 0.1% Na CMC in vehicle and subjected to ECS stress

(stress control group.


ECS stress (Standard Group).


ECS stress (Standard Group).


subjected to ECS stress.

Group VIII-XI: Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o.

and subjected to ECS stress.


subjected to ECS stress.


p.o. and subjected to ECS stress.





Experimental

115

Method:

In this model rats were subjected to chronic electro convulsive shock by means of electro-

convulsometer using pineal electrodes. Shocks of increasing intensities were given

successively for a period of 15 days for a fixed duration of 0.1 second as per following

protocol:

Day 1: 15 mA

Day 2: 25 mA

Day 3: 35 mA

Day 4 : 45 mA

Day 5 : 55 mA

Day6 : 65 mA

Day7 : 75 mA

Day8 : 85 mA

Day9 : 95 mA

Day10 : 105 mA

Day11 : 115 mA

Day12 : 125 mA

Day13 : 135 mA

Day14 : 145 mA

Day 15 : 155 mA

Immediately after the stress procedure on 1st 7th and 15th day of study blood was withdrawn

from retro orbital plexus of animals in all groups including vehicle control group and

collected blood samples were centrifuged at 4⁰c 3000 rpm. for 5 minutes After withdrawal of

blood the animals were tested for retention of memory of learned task in water maze by

recording the latency period (LP) required to reach the platform. Finally on 15th day after the

blood withdrawal and evaluation on water maze the animals were sacrificed humanly by

cervical dislocation and brain tissues were isolated for histopathological studies and for

estimations.

Morri’s Water Maze Test (MWM) [562]

Morris water maze (MWM) was a circular pool (90 cm in diameter and 50 cm in height)

filled with tapped water (25⁰c,40 cm deep). The pool was located at one end of a small

rectangular room with a variety of visual clues, distal (on the wall) and proximal (on the rim

of the pool).. The circular pool of water maze was divided into 4 quadrants: east, south, west

Experimental

116

and north. A white platform (4 cm in diameter and 30 cm in height) was centered in the

northeast position and submerged below the water surface so that it was invisible at water

level and the platform was fixed in the same place in the 4 training days. Latency to reach the

platform were recorded manually.

The training in MWM consisted of 4 trials /day. In water maze tests, the rat was placed in the

water facing the perimeter of the pool at the predetermined randomly selected quadrant

(north, south east and west).For subsequent trials rat was released in each of other three

quadrants .for each trial rat swam in the pool until climbing on to the platform or for 60

seconds If the rat did not find the platform within 60 seconds , he was led by experimenter to

the platform. The rat was allowed to remain on the platform for 15 seconds and then was

removed from the pool , dried in a clean cage without bedding and returned to it’s home cage

The pool was strained of feces and stirred gently between each swim session to disrupt any

scent left behind from the rat’s path taken to find the platform. Eight rats were trained at a

time and all eight rats received their first training trial before the second training trial for that

day occurred (Inter trial interval of 15 minutes). The training continued for 4 days. Animals

that do not go to the visible platform on training and testing trials in the allotted time of 120 s

were given LP of 120 seconds. Animals those refused to search for the hidden platform

during training and floated on water were removed from the study.

Biochemical Estimations :

� Estimation of Serum corticosterone was performed in Harmone lab, Mumbai by

radio-immuno assay method

� Estination of glucose in serum was performed using commercially available kits (Erba

Diagnostics)

Estimations in brain homogenates:

At the end of the study animals from each group were sacrificed by cervical decapitation

under light anesthesia. Immediately after decapitation, whole brain was removed from the

skull the weight of whole brain was recorded. Six brains from each group were homogenized

in 10 ml of 0.9% saline solution. Homogenates were used for the estimation of following

parameters:

� Estimation of lipid peroxidation

� Estimation of levels of Catalase

� Estimation of levels of GSH

Experimental

117

Histopathological Investigations Immediately after decapitation the whole brain was removed from the skull. Two isolated

brains from each group were fixed in the 10% formalin solution prepared in 0.9% normal

saline. The fixed brain samples were given immediately for further histopathological

investigations at Biodiagnostic Centre Parel, Mumbai.

3.7.8. COLD SWIM TEST (CST)[563].

Drug Treatments: Wistar rats were divided into 23 groups randomly , each containing eight

rats .Reference standard Piracetam (200 mg/kg p.o), Ashwagandha(100mg/kg p.o) Diazepam

(1mg/kg p.o. and extracts MKAQ (50, 100 and 200mg/kg p.o.) MKHA (50, 100 and

200mg/kg p.o.),MKM (50, 100 and 200mg/kg p.o.),OSAQ (50, 100 and 200mg/kg p.o.)

,OSHA(50, 100 and 200mg/kg p.o.) and OSM (50, 100 and 200mg/kg p.o.) were suspended

in 0.1% sodium carboxy methyl cellulose solution and administered per orally successively

for 7 days

Method:

In cold swim test 24 hours before the start of treatment rats were placed individually in to

plastic container and were left to swim in water for 15 min and then removed, dried with

towel and returned to their home cages. The study was initiated 24 hrs after pretest session.

Vehicle /extracts /standards were administered to the animals 1 hour prior to the cold swim

test. In CST animals were introduced in a water tank (length 100 cm, width 40 cm and depth

60cm ) filled with the water (15-20⁰c) up to the level of (40 cm) ensuring that limbs and tail

of animal do not touch to the bottom of plastic tank and were allowed to swim for 1 hours

continuously. In the beginning session of 1 hour swimming animals were observed for initial

10 minutes in which DOI was measured in later 5 minutes period of observation, then at the

end of swimming session DOI was measured for another 5 minutes. Animal was considered

immobile when it stops paddling four limbs and floats on the surface of water with minimum

movements just to keep it’s head above the surface of water. Before introducing another

animal in swimming tank the water was replaced with a fresh cold water. Following all swim

sessions animals were removed from cylinder dried with towels and returned to their home

cages. On 7th day after the completion of swim session blood was withdrawn from retro-

orbital plexus and was separated by centrifugation at 4⁰c , 3000 rpm for 5 minutes The serum

was separated and was used for estimation of serum corticosterone ,glucose , cholesterol and

triglyceride levels.

Experimental

118

Biochemical estimation in Serum;

1. Estimation of Serum corticosterone was performed in Harmone lab, Mumbai by radio-

immuno assay method

2. Estimation of following biochemical parameters in serum were performed using

commercially available kits ( Erba Diagnostics)

� Serum Glucose levels � Serum Triglyceride levels � Serum, cholesterol levels

Experimental groups for cold swim test

The wistar rats (150-200 gm) were divided randomly in to 22 groups each consisted of eight

animals as follows


Group II : Rats received equi volume 0.1% Na CMC in vehicle and subjected to CST (stress

control group)


CST (Standard Group)


CST (Standard Group)


subjected to CST

Group VIII-XI: Rats were treated with MKHA at doses of 50,100 and 200 mg/kg resp. p.o.

and subjected to CST


subjected to CST


p.o. and subjected to CST



Group XX-XXII : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp. p.o


Experimental

119

3.7.9. BIOCHEMICAL ESTIMATIONS FROM SERUM

1. Estimation of serum glucose levels (GOD-POD METHOD) [564].

Glucose was estimated by Trinder’s method. The accurate measurement of glucose in body

fluids is important in the diagnosis and management for diabetes, hypoglycemia, adrenal

dysfunction and various other conditions. The causes of increased serum glucose

concentrations include postprandial blood collection, diabetes mellitus, hyper-

adrenocorticism, morbidity, exogenous glucocorticoids, and glucagon. The clinical signs of

hypoglycemia include confusion, lethargy, ataxia and seizure, which may progress to loss of

consciousness and death. The principle involved in the estimation of glucose involves the

oxidation of glucose to gluconic acid and hydrogen peroxide (H2O2) in the presence of

glucose oxidase. The formed hydrogen peroxide interacts with 4–aminoantipyrine (4AAP)

and 4-hydroxy benzoic acid (4HBA) in the presence of peroxidase to form quinoneimine dye.

The intensity of the pink color formed is proportional to the glucose concentration and

absorbance was measured at 510 nm.

Principle:

Glucose + O2 + H2O Glucose oxidase Gluconic acid + H2O2

H2O2 + 4 HBA + 4 AAP Peroxidase Quinoeimine Dye +2 H2O

4AAP : 4- Aminoantipyrine

4HBA : 4- Hydroxy benzoic acid

Assay procedure for glucose estimation

Blank Standard Test

Working Reagent 1000 μl 1000 μl 1000 μl

Distilled water 20 μl -- --

Standard -- 20 μl --

Test -- -- 20 μl

The serum was mixed well with the working reagent and was incubated at 37�C for 15 min.

The blank was aspirated followed by standard and tests for glucose estimation. The

absorbances of the test and standard samples were noted at 510 nm against reagent blank.

Experimental

120

2. Estimation of serum triglycerides levels [564].

The measurement of triglycerides is important in the diagnosis and management of

hyperlipidaemias. These diseases can be genetic or secondary to other disorders including

nephrosis, diabetes mellitus and endocrine disturbances. Elevation of triglycerides has been

identified as a risk factor for atherosclerotic disease.

The principle involved in the estimation of triglyceride involves the conversion of

triglycerides into glycerol and free fatty acids in the presence of lipase. The glycerol and

adenosine triphosphate (ATP) in the presence of glycerol kinase (GK) forms glycerol-3-

phosphate. The glycerol-3-phosphate is oxidized to dihydroxyacetone phosphate (DAP) and

hydrogen peroxide (H2O2) in the presence of glycerol phosphate oxidase (GPO). The

hydrogen peroxide (H2O2), 4 aminoantipyrine (4-AAP) and 3, 5-Dichloro-2-hydroxybenzene

sulfonate (DHBS) forms quinoneimine dye in the presence of peroxidase. The intensity of

chromogen (Quinoeimine) formed is proportional to the triglycerides concentration in the

sample when measured at 510 nm.

Triglycerides + H2O Glycerol + Fatty acids

Glycerol + ATP Glycerol-3-phosphate + ADP

Glycerol-3-phosphate +O2 Dihydroxy acetone phosphate +H2O2

H2O2 + 4-AAP + 3, 5- DHBS Quinoneimine dye + 2 H2O

LPL: Lipoprotein Lipase, GK: Glycerol Kinase,

GPO: Glycerol Phosphate Oxidase,

3, 5 DHBS: 3, 5-Dichloro-2-hydroxybenzene sulfonate

4-AAP: 4-aminoantipyrine

The intensity of chromogen (Quinoneimine) formed is proportional to the triglycerides

concentration in the sample when measured spectrophotometrically.

Assay procedure for serum triglyceride estimation

Blank Standard Test




Test -- -- 10 μl

LPL G K, Mg++

GPO

Peroxidase

Experimental

121


The blank was aspirated followed by standard and tests. The absorbance of the test and

standard samples were noted at 510 nm.

3. Estimation of serum cholesterol levels (CHOD-PAP method) [564].

The measurements of serum cholesterol levels are useful in evaluation of the risk of the

coronary arterial occlusion, atherosclerosis, myocardial infarction, liver function, biliary

function, intestinal absorption, thyroid function and adrenal disease.

The principle involved in the estimation of cholesterol involves the conversion of cholesterol

ester to cholesterol and fatty acid in the presence of cholesterol esterase (CE). Cholesterol is

oxidized to Cholest-4-en-3-one and hydrogen peroxide (H2O2) in the presence of cholesterol

oxidase. The phenol and 4-aminoantipyrine (4AAP) present in the cholesterol reagent kit

interacts with the hydrogen peroxide in the presence of peroxidase (POD) present in the

cholesterol reagent kit and forms the Quinoeimine. The absorbance of Quinoneimine so

formed is directly proportional to cholesterol concentration.

Cholesterol esters + H2O cholesterol + fatty acids

Cholesterol + O2 4-cholestenone + H2O2

2 H2O2 + 4- Aminophenazone + phenol 4 H2O + Quinoneimine

C E: Cholesterol esterase, CHOD: Cholesterol oxidase

Method:

Blank Standard Test Working Reagent 1000 μl 1000 μl 1000 μl Distilled water 20 μl -- -- Standard -- 20 μl -- Test -- -- 20 μl


The blank was aspirated followed by standard and tests. The absorbance of the test and

standard samples were noted at 510 nm.

4. Estimation of lactate dehydrogenase (ldh) (AGAPPE DIAGNOSTICS) [565]

Clinical significance:

This enzyme is found in all organ cells, but especially plentiful in cardiac and muscle, liver,

kidney and RBC’s. LDH is found in the form of iso-enzymes based on their electrophoretic

mobility with each iso-enzymes being primarily from different organs.

C E CHOPeroxidase

Experimental

122

Elevated levels are found in myocardial infarction, liver diseases hemolytic anaemias,

pernicious anaemia, leukemia and pulmonary diseases. Elevation in acute MI reaches a peak

in 48-72 hrs. Belonged elevations, (10-14 days) are useful in late diagnosis of the condition.

Kinetic determination of lactate dehydrogenase according to the following reaction

. LDH

Pyruvate + NADH + H+-------------�L-lactate + NAD+

Assay procedure for LDH :

Reagent composition:

LDH-P(S.L) R1 2X8ml/ 2x16ml

Tris buffer (pH 7.4) 80mmol/L

Pyruvate 1.6mmol/L

Sodium Chloride 200mmol/L

LDH –P(S.L) R2 2x2ml/2x4ml

NADH 240μmol/L

Preparation and stability of working reagent:

4 volumes of Reagent1 (R1) was mixed with 1 volume of Reagent2 (R2).

The working reagent is stable for 21 days at 2-8ºC.

Note:

Discard the working reagent if the blank absorbance is less than 1.0 at 340 nm.

Assay procedure: for LDH:

Working Reagent : 100μL

Sample : 10μL

The contents in test tube were mixd and incubated at 37ºC for 1 minute. the change in

absorbance was measure per Minute ( ∆ OD/min) during 3 minutes.

Calculation:

LDH –P avtivity (U/L) = ( ∆ OD/min) x 16030

5. Estimation of blood urea nitrogen – (BRETHELOT) [565].

Urea is the major Nitrogen – containing metabolic product of protein catabolism in humans,

accounting for more than 75% of the non- protein nitrogen eventually excreted. It is

synthesized in the liver from the ammonia produced as a result of deamination of amino

acids. Urea is transported by blood to the kidneys from where it is excreted. Increased urea

Experimental

123

levels are found in liver diseases, urinary obstructions, shock, congestive heart failure and

burns. Decreased urea levels are found in malnutrition, hepatic failure and pregnancy.

Urea is converted quantitatively to ammonia and CO2 in the prescence of urease. The

ammonium ions react with phenolic chromogen and hypochlorite to give a green coloured

complex. The intensity of the colour formed is measured at 578 nm and is directly

proportional to the concentration of urea in the test specimen.

Urease

Urea + H2O --------------� Ammonia + CO2

Ammonia + Phenolic Chromogen + Hypochlorite -----------� Green coloured complex

Reagents:

2x50 ml 2x 100 ml

Urea (Enzyme Reagent) 2 2

Urea (Chromogen Reagent) 2 2

Urea Diluent (Buffer) 2 2

Urea Standard (50mg/dl) 1 1

Preparation and stability of working reagent:

For 2x 50 ml and 2x 100 ml

The contents of vial of 1- Urea were transferred to the bottle of 3- Urea Diluent . 1-Urea vial

was rinsed properly with 3- Urea Diluent and mixed gently. These working reagents were

stored at 2-8 ºC when not in use. 2-Urea reagent and Urea Standard were ready to use.

The working reagent is stable for 180 days at 2-8ºC in a tightly capped bottle.

Method

Pippeted into clean and dry test tubes labelled as Blank (B), Standard(S) and Test(T):

ADDITION

SEQUENCE

BLANK STANDARD TEST

Working Reagent 1000μl 1000μl 1000μl

Distilled Water 10μl - -

Urea Standard - 10μl -

Specimen - - 10μl

All the reagents in test tubes were mixed well and incubated for 5 mins at 37ºC

2- Urea Reagent 1000μl 1000μl 1000μl

Mix well and incubate for 5 mins at 37ºC.

Experimental

124

The absorbance was measured of standard (Abs. S) and Test sample (Abs. T) against the

blank at 578 nm. Colour is stable for 45 min when protected from light, so absorbance was

measured within the period.

Calculations:

Urea conc. in mg/dl of Test Specimen = Abs T ÷ Abs S x50

Blood Urea Nitrogen (BUN) in mg/dl = Urea (in mg/dl) x 0.467

6. Estimation of serum total protein levels [566].

Total protein was estimated by Biuret method. The majority of the plasma proteins are

synthesized in the liver. The serum total protein and albumin are the two plasma proteins that

are measured. The serum total protein concentration is in direct proportion to the serum

albumin concentration. Total protein increases in dehydration, multiple myeloma and chronic

disease. Total protein decreases in renal diseases and terminal liver failure. The principle

involved in the estimation of total protein involves the interaction of the peptide bonds of

protein with copper II ions in alkaline solution to form blue-violet complex. In biuret

reaction, each copper ion complexes with 5 to 6 peptide bonds. Tartarate is added as a

stabilizer and iodide is used to prevent auto-reduction of the alkaline copper complex. The

color formed is proportional to the protein concentration and is measured at 546 nm.

Method:

Blank Standard Test




Test -- -- 20 μl


The blank was aspirated followed by standard and tests for protein estimation. The

absorbances of the test and standard samples were noted at 546 nm against reagent blank.

Experimental

125

3.7.10. ESTIMATIONS OF TISSUE HOMOGENATES

1. Estimation of glycogen content in liver and gastrocnemius muscle [567].

Analysis of tissue glycogen contents

Because glycogen is insoluble in ethanol, it can be precipitated in anhydrous ethanol.

Glycogen is dehydrated by 98% H2S04 in order to produce furfural derivatives, which then

produce a blue compound after reacting with anthrone. The OD of the color compound can be

determined by photometry

Reagents:-

1. Anthrone reagent. A solution containing 0.05 % anthrone, 1per cent thiourea, and 72 %

by volume H2S04 is used. For each liter of reagent, 720 ml. of concentrated H2S04 was

added to 280ml of distilled water cautiously. In a flask 500 mg. of purified anthrone, 10

gm. of highest purity thiourea, and 1 liter of the 72 %t H2SO4 were added. The mixture

was warmed to 80-90ºc with occasionally shaking the flask to mix the contents and

overheating of the mixture was avoided. The reagent was cooled and stored in a

refrigerator. This reagent can be kept for at least 2 weeks in a refrigerator.

2. 5 % trichloroacetic acid.

3. 95 %ethanol.

4. Glucose

(a) Stock solution. 100 mg. of dry, highest purity glucose was dissolved in 100 ml. of

saturated benzoic acid solution.

(b) Working standard. 5 ml. of the stock solution placed in a 100 ml. volumetric flask and

volume was made up with saturated benzoic acid solution. 2 ml.of this solution, containing

0.1 mg. of glucose, was used as a standard. The ODs of glucose at different concentrations

(50, 25, 12.5, 6.25, 3.12, and 1.6 mg/ml) were 0.88, 0.4, 0.26, 0.10, and 0.05, respectively.

Method:-

200 mg tissue was homogenized in 20 ml of 5 % TCA. Filtered to obtain 10 ml of filtrate.2

ml. of the trichloro acetic acid filtrate is pipetted into a 15 ml. centrifuge tube. To each tube

added 5 ml of 95 %ethanol with careful blowing to effect thorough mixing.. The tubes were

capped with clean rubber stoppers and allowed to stand overnight at room temperature. After

precipitation was complete, the tubes were centrifuged at 3000 r.p.m. for 15 minutes. The

clear liquid was gently decanted from the packed glycogen and the tubes were allowed to

drain in an inverted position for 10 minutes. The glycogen was dissolved by addition of 2 ml.

of distilled water, the water being added in a manner that will wash down the sides of the

Experimental

126

tube. If the glycogen did not dissolve instantly, the tubes were sonicated until solution is

complete. A reagent blank was prepared by pipetting 2 ml. of water into a clean centrifuge

tube. A standard was prepared by pipeting 2 ml. of standard glucose solution, containing 0.1

mg. of glucose, into a similar tube .At this point 10 ml. of anthrone reagent was delivered into

each tube. The stream of anthrone reagent was directed into the center of the tube and was

sufficient to insure good mixing. As each tube received anthrone reagent, it was tightly

capped and placed in a cold tap water bath. After all tubes had reached the temperature of the

cold water, they were immersed in a boiling water bath to a depth a little above the level of

the liquid in the tubes for 15 minutes and then removed to a cold water bath and cooled to

room temperature. The tubes and stoppers were wiped dry and the contents of each tube were

transferred to a cuvette and read at 620 nm after adjusting the spectrophotometer (V-530,

Jasco Co., Japan) with the reagent blank. Note: care was taken to avoid introduction of lint or

contaminating carbohydrate in to the anthrone reaction.

Calculation:-

mg. of glycogen / 100 gm. of tissue= DU/DS x 0.1 x volume of extract/ gm. of tissue x 100 x

0.9

where DU = optical density of the unknown, DS = optical density of the standard,

0.1= mg. of glucose in 2 ml. of standard solution, 0.9 = factor for converting glucose value to

glycogen value.

2. Estimation of Norepinephrine, Dopamine and 5-hydroxy tryptamine in

brain [568]

It is a simple but precise method for chemical estimation of catecholamines in brain and other

organs. The method involves extraction of catecholamines into butanol followed with

extraction in an aqueous phase by lowering the solubility of the substance in butanol by

addition of heptane The addition of heptane to butanol precipitates out water dissolved in this

phase thereby increasing the volume of the acid aqueous phase .Then the catecholamines are

converted to a fluorescent derivative by oxidation with iodine.

After oxidation at pH 5, the products of both the amines show high fluorescence, but with

change of pH to 3 fluorescence exhibited after oxidation of norepinephrine was negligible

compared to that of epinephrine. This difference in oxidation at the two pH serves as a

convenient means of differential assay of the two catecholamines. Dopamine does not get

easily oxidized as Epinephrine and NE Hence NE can be differentially assayed in presence of

Experimental

127

Epinephrine using both pH 3 and 5. NE yields the maximum value of fluorescence by heating

for 2 min in boiling water. After heating, it is quite stable for 60 min at room temperature.

The fluorescence of dopamine reaches a maximum on further heating which allows the

estimation of Dopamine using same sample but heating for another additional;40 minutes.

Reagents [569]:

The stock standard was diluted when needed with 0.02 M acetic acid to a concentration of

5μg/ml.

1.Acidified Butanol :Concentrated HCI (0.85 ml) was dissolved in 1L. n-butanol

2.n-Heptane (SD Fine Chemical Company),

3.0.1 N and 10 N Hydrochloric acid,

4.5 N Sodium hydroxide,

5.0.2 N and 5 N Acetic acid,

6.0.1 N Iodine: 1 .27 g iodine was dissolved in 100 ml of absolute ethanol.

7.0.1 M EDTA : Disodium ethylenediamine tetraacetate dihydrate (37.2 g) is dissolved in

1 M sodium acetate and made up to a volume of 1L. The pH is adjusted to a pH of 6.7-7

by the addition of NaOH

8.Alkaline sulfite: 25% (w/v). 0.5 g of anhydrous sodium sulfite was dissolved in 2 ml

distilled water. 18 ml 5 N sodium hydroxide was added and mixed well. This solution was

prepared just prior to use.

9.O-Phthalaldehyde (OPT) 4 mg% : OPT dissolved in 10 N hydrochloric acid. (Chemical

Company)

10.Alumina Neutral chromatographic grade alumina is washed with HCl and water

11.5-HT hydrogen oxalate () diluted to 1000μg/ml (as free base) with 0.1 N HCl,

12.NE hydrochloride () diluted to 1000 μg/ml(as free base) with 0.1 N HCl,

13.DA hydrochloride (Calbiochem) diluted to 1000 μg/ml (as free base)with 0.1 N HCl.

The stock standard was diluted when needed with 0.02 M acetic acid to a concentration of

5μg/mI.

Standard curve of Norepinephrine hydrochloride[568].

1. The stock solution of norepinephrine hydrochloride was prepared at a concentration of 100

μg / ml in 0.1N HCl solution. The solution remains stable in a refrigerator at 2- 80 C for

several weeks. Working standards were prepared in the range of 0.1 to 1.6 μg /ml by

appropriate dilutions of the stock with 0.1N HCl. 1ml of acetate buffer (pH 3) was added to

each of these dilutions. Then 0.1ml of iodine reagent was added. After 2 minutes the excess

iodine was removed by addition of 0.2 ml of alkaline sulfite solution. The contents of the tube

Experimental

128

were thoroughly mixed after each addition. Two minutes later 0.2 ml of 5 N acetic acid added

and mixed well. All tubes were placed in a boiling water bath for 2 min, cooled in tap water

and read for NE fluorescence. Excitation was 380 nm, and emission was 480 nm

(uncorrected). the blank fluorescence was measured. The “blank” was a sample treated as

above, except that the order of addition of iodine and alkaline sulfite reagents was reversed,

i.e., omitting the step of oxidation. The solution was activated at 380 nm and the resulting

fluorescence was measured at 480 nm in a spectro-fluorimeter. Standard curve of

fluorescence vs. concentration was plotted. The fluorescence of norepinephrine extracted

from the rat brain using solvent extraction method was extrapolated and concentration of

norepinephrine from each rat brain was determined. The concentrations of norepinephrine

were expressed as μg/gm of the weight of rat brain.

Standard curve of Dopamine hydrochloride[568].

The stock solution of dopamine hydrochloride was prepared at a concentration of 100 μg /ml

in 0.1N HCl solution. The solution remains stable in a refrigerator at 2- 80 C for several

weeks. Working standards were prepared in the range of 0.25 to 4 μg /ml by appropriate

dilutions of the stock solution with 0.1N HCl. To these dilutions 1ml of acetate buffer (pH 3)

was added to each of these dilutions. Then 0.1ml of iodine reagent was added. After 2

minutes the excess iodine was removed by addition of 0.2 ml of alkaline sulfite solution. The

contents of the tube were thoroughly mixed after each addition. Two minutes later 0.2 ml of 5

N acetic acid added and mixed well and heated in a boiling water bath for an additional 45

min and cooled in tap water. And were used for estimation of DA The “blank” was a sample

treated as above, except that the order of addition of iodine and alkaline sulfite reagents were

reversed, i.e., omitting the step of oxidation. The dilutions were activated at 320 nm and the

resulting fluorescence was measured at 375 nm with a spectro-fluorimeter. Standard curve of

fluorescence vs. concentration was plotted. The fluorescence of the dopamine extracted from

the rat brain using solvent extraction method was extrapolated and concentration of dopamine

from each rat brain was determined. The concentration of dopamine was expressed as μg /gm

of the weight of rat brain.

Standard curve of 5-Hydroxy Tryptamine Creatinine sulphate (5-HT)[568]

The stock solution of 5-HT was prepared at a concentration of 10 μg / ml in 0.1N HCl. The

solution remains stable in a refrigerator at 2-80 C for several weeks. Working standards were

prepared in the range of 0.1 to 0.5 μg / ml by appropriate dilutions of the stock with 0.1N

HCl. 1.2 ml of OPT were added and mixed well. All tubes were placed in a boiling water

bath for 10 min, cooled in tap water Hence the above dilutions were directly measured with

Experimental

129

activation maxima at 355 nm and fluorescence maxima at 470 nm. Blank was measured using

0.1 N HCl and was subtracted from the standard absorbance. Standard curve of fluorescence

vs. concentration was plotted. The fluorescence of 5-HT extracted from the rat brain using

solvent extraction method was extrapolated and concentration of 5-HT from each rat brain

was determined. The concentration of 5-HT was expressed as μg / gm of the weight of rat

brain.

Method for fluorometric analysis of NE, DA and 5HT [568]

Isolation of catecholamines:

Brain tissues were homogenized in a glass homogenizer with 10ml of chilled acidified

butanol. This process was carried out in the cold ice bath. The mixture was centrifuged and 5

ml of the supernatant butanol layer was transferred to a glass-stoppered centrifuge tube

containing 10 ml of n-heptane and 5 ml of water. The mixture was shaken for 2 min and

centrifuged. 5 ml of the aqueous phase was transferred to a glass-stoppered centrifuge tube

containing 0.2 g of alumina. By the addition of 1 ml of 2 M sodium acetate pH was adjusted

to 7.5 and mixture was shaken gently for 5 min to adsorb the catecholamines. The mixture

was centrifuged and the supernatant fluid was removed, which contains most of the

interfering substances, by aspiration. The catecholamines were eluted from the alumina by

gentle shaking for 5 min with 3 ml of 0.1 M acetic acid. The pH of the elute was maintained

4. When the catecholamines are eluted with smaller volumes of acetic acid, the strength of

acetic acid was increased to ensure that the pH will be about 4. Elutes (0.3ml) were

transferred to the test tubes to which 3ml of butanol was added which was centrifuged at

3000 rpm for 5 minutes. 2.5ml of the supernatant fluid were transferred to tubes containing

1.6 ml of 0.2 N acetic acid and 5 ml of heptane. All tubes were placed on a vortex mixer for

30 seconds and centrifuged at 3000 rpm for 5 min. The organic supernatant phase was

discarded. 0.2ml of the aqueous phase was transferred to tubes (for continuation of 5-HT

analysis) and 1 ml was transferred to tubes for the analysis of NE and DA

5-HTanalysis:

1. To samples and a reagent blank (consisting of 0.2 ml 0.1 N hydrochloric acid), 1.2 ml of

OPT were added and well mixed.

2. All tubes were placed in a boiling water bath for 10 min, cooled in tap water

3. The fluorescence was read in the spectro photo fluorometer (JASCO,Japan) Excitation was

355 nm, emission was 470 nm (uncorrected).

Experimental

130

NE and DA analysis.:

1. To all tubes was added 0.2 ml EDTA and mixed well.

2. To reagent blank (1 ml of 0.2 N acetic acid), 0.2 ml alkaline sulfite was added and mixed

well, then 0.1 ml of 0.1 N iodine was added and mixed well. Finally 0.2 ml 5 N acetic acid

was added and again mixed well.

4. To all remaining tubes 0.1 ml of 0.1 N iodine was added and mixed well. Two minutes

later 0.2 ml alkaline sulfite was added and mixed well. Two minutes later 0.2 ml of 5 N acetic

acid added and mixed well.

5. All tubes were placed in a boiling water bath for 2 min, cooled in tap water and read for

NE fluorescence. Excitation was 380 nm, and emission was 480 nm (uncorrected).

6. All solutions were returned to their original test tubes and reheated in a boiling water bath

for an additional 40 min and cooled in tap water. And were used for estimation of DA

7. For DA estimation , excitation was 320 nm, and emission was 375 nm (uncorrected).

3. ESTIMATION OF LEVELS OF HYDROLYZED ACETYLCHOLINE [ACH] IN

BRAIN [570,571]

This assay was performed with the view of studying the effect of Extracts on Choline esterase

enzyme. As Ach plays a very important role in retention of memory thus assay was

performed to explore the mechanism by which extracts might be preventing stress induced

amnesia and to investigate whether the test drugs act through inhibition of cholinesterase.

Collection of brain samples

On 14th day of the study rats from each group were euthanized by cervical dislocation. The

whole brain was immediately removed and chilled in ice-cold phosphate buffer. After

washing in ice-cold phosphate buffer it was homogenized in 5 ml of phosphate buffer in glass

Teflon homogenizer. The brain homogenate was then evaluated for enzyme activity using

Augustinsson’s (1957) [575] method of analysis

Standard Curve of Ach

Aliquots of 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ml of Ach in buffer were added to different test tubes.

Phosphate buffer was added to each tube to give a volume of 1 ml. 2ml of Alk hyd was added

to each tube by shaking vigorously. After not less than a minute, 1 ml of HCL solution (pH of

1.2± 0.2) and 1 ml of FeCl3 solution were added. The absorbance of the color in each tube

was read at 540 nm. The control tube (with 0 ml of Ach) was used to adjust the zero of the

instrument. A plot of hydrolysed concentration of Ach (mg/ml of) vs absorbance was

obtained.

Experimental

131

Estimation of inhibition of choline esterase activity

Acetylcholine being a acetic acid ester of choline it reacts with alkaline hydroxylamine

hydrochloride solution to form acetahydroxamic acid. Acetahydroxamic acid then reacts with

ferric chloride to form a reddish-purple complex. The intensity of color of the complex

formed is proportional to the concentration of Acetylcholine present in sample.. This method

was developed by Hestrin [570]. It is suitable for determination of choline esters in blood,

erythrocytes, plasma, serum or any other tissue. A specific amount of Ach is added to all test

tubes. Due to addition of tissue homogenate hydrolysis of Ach takes place by Choline Estrase

present in the tissue homogenate. Which would render the Ach unsuitable for reaction with

alkaline hydroxide. Thus, the color developed would indicate the amount of unhydrolyzed

acetylcholine whose concentration can be determined from standard curve. The hydrolyzed

amount of Ach can be calculated from the amount of ACH added before addition of brain

homogenate. The concentration of hydrolyzed acetylcholine would thus indicate the extent of

activity of enzyme acetyl choline esterase present in tissue homogenate.

Method:

Reagents

1. Hydroxylamine. Hydroxylamine hydrochloride, 2 M. The solution

should be stored in the cold.

2. Alkali. Sodium hydroxide, 3.5 N.

3. Acid. Concentrated hydrochloric acid, sp. gr. 1.18, diluted with 2

ml of water.

4. Ferric chloride, 0.37 M, in hydrochloric acid, 0.1 N.( Reagent

, ferric (FeCl,.GHyO), crystals, of Merck and company

5. Standard solution. Acetylcholine chloride, 0.004 M, in sodium acetate

solution, 0.001 N, of pH 4.5. This standard may be kept in the

refrigerator for a fortnight without measurable loss

Three types of test tubes were prepared

Tube 1: served as control that contained 1 ml of buffer (instead of Acetylcholine solution)

and other reagents.

Tube 2: served as test that contained 1 ml of Acetylcholine solution, 0.1 ml of homogenate

and was kept for incubation at 37°C ± 1°C for 1hr.

Experimental

132

Tube 3: served as Std that contained 1 ml of Acetylcholine solution and 0.1 ml of

homogenate was added after the addition of alkaline hydroxide which itself was added after

incubation at 37ºC±1ºC for 1 hr.

After the incubation period, 2ml of alkaline hydroxide was added by shaking vigorously to

tubes 1 and 2. After not less than a minute, 1 ml of HCL solution (pH of 1.2 ± 0.2) and 1 ml

of FeCl3 were added to all three test tubes.The resultant mixtures were centrifuged and the

absorbance of the supernatant was read at 540 nm.

Std was used to correct the determination of non-enzymatic hydrolysis of acetylcholine since

the homogenate was added after incubation. The control tube (with 1 ml of buffer) was used

to adjust the zero of the instrument.

The amount of Ach in Test and Std was calculated from the standard plot. The amount of

acetylcholine in Test would give us the unhydrolyzed amount of acetylcholine. Hence Std

(OD)-test (OD) would give us the hydrolyzed amount of Ach which indicates acetyl choline

esterase enzyme activity

4. Esimation of anti oxidant activity in rat brain .

4.1. Esimation of lipid peroxidation [572]: Lipid peroxidation refers to the oxidative degradation of lipids . It is the process in which free

radicals "steal" electrons from the lipids in cell membrane resulting in cell damage. This

process proceeds by a free radical chain reaction mechanism. It most often affects poly

unsaturated fatty acids because they contain multiple double bonds in between which lie

methylene -CH2- groups that possess especially reactive hydrogens. As with any radical

reaction, the reaction consists of three major steps: initiation, propagation, and termination.

The test is available for the quantification of the end-products of lipid peroxidation, to be

specific,malondialdehyde (MDA) [576]. The most commonly used test is called a TBARS

Assay (thiobarbituric acid reactive substances assay). Thiobarbituric acid reacts with

Experimental

133

malondialdehyde to yield a pink coloured species that absorbs at 532 nm. Thus the extent of

lipid peroxidation was assayed by estimating the thiobarbituric acid-reactive substances

formed in brain homogenate

Method:

0.1ml of brain homogenate was added in a tube containing 0.1ml (8.1%) sodium dodecyl

sulphate, 0.75ml of 20% acetic acid and 0.75ml of 0.8% Thio barbituric acid (TBA) aqueous

solution .the volume in each test tube was made to 2 ml with distilled water and heated on a

water bath at 95⁰c for 60 minutes. After 60 minutes of incubation period volume in each tube

was made to 2.5 ml and then 2.5 ml of butanol : pyridine (5:1) was added in each tube . The

reaction mixture was vortexed and centrifuged at 4000 rpm for 10 minutes .The organic layer

was removed and absorbance was read at 532 nm in UV spectrophotometer. The inhibition of

lipid per oxidation was calculated by comparing the results of the test with those of blank.

The MDA equivalents of the sample were calculated in terms of thio barburic acid consumed

/mg protein

4.2. Estimation of Catalase [573].

Catalase activity was measured by the method of Patterson and his colleagues (Patterson et

al., 1984).The method is based on the photoelectric measurement of the color intensities of

the product of the interaction between hydrogen peroxide and titanium reagent.

Method:

The reaction mixture contained 0.1ml enzyme source (brain homogenate), 1 ml phosphate

buffer (pH 6.5) and 0.1 ml of H2O2.The enzyme reaction was stopped by the addition of 1 ml

of the titanium reagent. The intensity of the yellow color of Ti-H2O2 complex was measured

at 410 nm against blank which was prepared in similar without the brain homogenate.

The enzyme activity was expressed as μmol H2O2 consumed per milligram protein per

minute. Thus change in OD as compared with blank represents the amount of H2O2

consumed by enzyme which indirectly reflects the activity of enzyme

4.3. Estimation of Reduced Glutathione [574].

Thiols in glutathione react with Ellman reagent compound, cleaving the disulfide bond to

give 2-nitro-5-thiobenzoate (NTB-), which ionizes to the NTB2- dianion in water at neutral

and alkaline pH. This NTB2- ion has a yellow color. The NTB2- is quantified in a

spectrophotometer by measuring the absorbance of visible light at 412nm

Experimental

134

Method: Protein free supernatant was obtained by addition of equal volume of 10% TCA to 1 ml of

tissue homogenate and centrifuged at 5000 rpm for 10 min. 0.3ml of the supernatant was

taken and made up to 1ml with phosphate buffer (pH=8). 2ml freshly prepared 0.6mM DTNB

was added to the tubes and intense yellow color formed was recorded at 412nm. Standard

curve was prepared by using various aliquots from GSH stock solution. The unknown GSH

concentration was obtained from standard graph. GSH was recorded as nmoles/mg protein

3.7 11. STATISTICAL ANALYSIS

The data presented was analyzed for statistical significance using Graph pad software version

(Prism 4 Demo). One-way ANNOVA followed by post hoc Dunnett’s test for comparison

with stress control and vehicle control. The difference was considered to be moderately

significant with p < 0.05, significant at p<0.01 and highly significant at p<0.001 respectively.

Experimental

135

3.8. ANALYTICAL METHOD DEVELOPMENT

For carrying out analysis of extracts MKM and OSHA present in formulations FM1-04 and

FO1-04 respectively analytical methods were developed. Two techniques namely ultra-violet

(UV) spectroscopic and high performance thin layer chromatographic (HPTLC) techniques

were standardised.

1.U.V. Spectroscopy: Method of Assay: Capsule blends of FM1-04 was extracted with known quantities of

methanol and filtered through whatman filter paper �max was determined on

spectrophotometer ( Jasco Japan ).A stock solution of 0.1 mg/ml was prepared and aliquots of

0.05,0.1,0.2,04,0.6,0.8,1.6ml were pipetted out in test tubes and were diluted to make a final

volume of 5 ml The absorbance was determined at varying concentrations ranging from 1-16

�g/ml to obtain the linearity plot. During stability studies at various sampling intervals the

capsule contents of FM1-04 were extracted with 100 ml of methanol filtered through

whatman filter paper and 1ml of filtrate was diluted to 10ml From this 0.1 ml was pipetted

out in a test tube and volume was made up of 5ml with methanol and absorbance was

recorded at wavelength 280 nm.

Similarly capsule contents of FO1-04 were extracted with 50% ethanol filtered through

whatmann filter paper and �max was determined a stock solution of 1mg/ml was prepared

Aliquots of 0.1,0.2,0.4,0.8,1.6 and ,2ml were pipetted out in test tubes and volume was made

up to 10 ml and absorbance were recorded for concentration of 10-200 �g/ml and analyzed

spectro-photometrically to develop linearity plot. During stability studies at different

sampling intervals the contents of the capsule FO1-04 were extracted in 100ml of 50%

ethanol filtered 1ml of filtrate was diluted to 10ml in volumetric flask from which 1ml of

aliquot was pipetted out in a test tube and volume was made up to 10 ml with 50% ethanol

and absorbance was measured at wavelength 205 nm Absorbances were measured in

duplicate and then concentrations were calculated from the standard curve.

2. High Performance thin Layer Chromatography HPTLC: [575]

Method of Assay : Capsule blend containing accurately weighed quantity of extract MKM

was prepared and extracted with 100ml of methanol and filtered From the filtrate a stock

solution of 10 mg/ml was prepared It was then spotted onto TLC plates (Merck) and run in

the solvent system developed for the pure extract namely Toluene :Ethyl acetate :formic acid

(9.3:0.7:0.1). The peak areas were obtained by spotting varying concentrations (10-50 μg/ml)

of the extract and running in the solvent system which were used to plot the standard graph.

Experimental

136

Capsule blend containing accurately weighed quantity of extract OSHA was prepared and

extracted with 100ml of 50 % ethanol and filtered From the filtrate a stock solution of 10

mg/ml was prepared It was then spotted onto TLC (Merck) plates and run in the solvent

system developed for the pure extract namely n Hexane :chloroform :methanol (6.5:3.5:0.5).

The peak areas were obtained by spotting varying concentrations (10-50 μg/ml) of the extract

and running in the solvent system which were used to plot the standard graph.

3. Dissolution [576].:

Method: Dissolution testing is an important parameter for the development of new products.

This is due to the fact that in vitro dissolution data can predict the in vivo performance of the

dosage form. USFDA guidelines recommend that dissolution testing should be carried out

under mild test conditions. An aqueous medium with a pH range of 1.2 to 6.8 has been

suggested for the dissolution studies. USP suggests the application of test for uncoated tablets

to be used for hard gelatin capsule. we evaluated our capsules using the Apparatus I with

baskets rotating at speed of 50 rpm and studied the dissolution profile over a period of 120

minutes. The evaluation has been carried out at pH 1.2, pH 6.8 and in water using a UV

spectroscopic method.

Capsule blend of FM1-04 containing accurately weighed quantity of the MKM extract was

prepared and extracted with 20ml methanol filtered through a whatman filter paper From

which a stock solution of 1mg/ml was prepared which was used to make various dilutions

and absorbance were measured for concentrations ranging from 10-250 μg/ml at pH=7 and

10-800 μg/ml for pH 1.2 and 6.8 .The absorbance was determined at varying concentrations

to obtain the linearity plot. Capsule blend of FO1-04 containing accurately weighed quantity

of the OSHA extract was prepared and extracted with 10ml methanol filtered through a

whatman filter paper From which a stock solution of 1mg/ml was prepared which was used to

make various dilutions and absorbance were measured for concentrations ranging from 10-

320 μg/ml at pH=7 and 10-200 μg/ml for pH 1.2 and 5-100 μg/ml at pH 6.8 .The absorbance

was determined at varying concentrations to obtain the linearity plot.

Experimental

137

3.9. FORMULATION DEVELOPMENT AND EVALUATION :

3.9.1. Preformulation study [577]

Compatibility between extract and excipients were tested by combining extract with various

excipients and were exposed to U.V light. The mixtures were observed visually for any

physical changes. Physical mixtures of the MKM extract of leaves of Murraya Koenigii and

OSHA extract of the leaves of Ocimum sanctum with various excipients in the ratio 1:1 was

made and subjected to high temperature at 40⁰c and and were also kept at room temperature.

These samples were observed for any physical changes for period of 14 days. The samples

exposed to UV light were observed for any physical changes for a period of 24 hours.. The

excipients used included microcrystalline cellulose, DCP, lactose, sodium lauryl sulphate,

Tween 80, magnesium stearate, talc.

A. Formulation development trials Weighed quantities of the MKM extract of Murraya koenigii was dissolved in methanol and

were mixed thoroughly with Avicel PH 101. The blend was dried in a hot air oven at 40°C.

The dried blend was then passed through 20# sieve. The other excipients were passed through

60# sieve and mixed with the dried blend. The blend was then filled in size ‘0’ orange/black

capsules. Similarly weighed quantity of the OSHA extract of Ocimum sanctum was dissolved

in 50% ethanol. Solution of the extract was mixed thoroughly with Avicel PH 101. The blend

was dried in a hot air oven at 40°C. The dried blend was then passed through 20# sieve. The

other excipients were passed through 60# sieve and mixed with the dried blend. The blend

was then filled in size ‘0’ orange/black capsules. Three trial batches with different excipients

at variable ratios were taken for both the extracts namely MKM and OSHA.(Refer Table Nos

3.2 and 3.3 for formulae)

The efficiency of the formulated trial batches of FM1 and FO1 capsules was evaluated by

carrying out dissolution studies in three different media namely pH 1.2, distilled water and

pH 6.8 buffer.

B. Dissolution studies of trial batches [578]

Dissolution was performed using USP Dissolution Apparatus type I (Basket type). 900 ml of

the dissolution medium maintained at 37�C � 0.5�C was used. The basket was adjusted to a

speed of 50 rpm. Aliquots of 5 ml were withdrawn at intervals of 15, 30, 45, 60, 90 and 120

min The solution was filtered by Whatman filter paper and absorbance of the solutions was

noted and the release profile of the capsule was obtained.

Experimental

138

3.9.2 FORMULATION OF FM1-04 AND FO1-04 CAPSULES

Based on the results of the trial batches a larger stability batch of batch size 1000 capsules

was manufactured (Refer to Table no: 3.4 and 3.5 for formulae of stability batches). The

capsule blend was evaluated for the various powder characteristics before filling into

capsules.

3.9.3. EVALUATION OF POWDER CHARACTERISTICS FOR FM1-04 AND FO1-04

Bulk Density[579]: Bulk density of the powder primarily depends on particle size

distribution, shape and tendency of the particles to adhere together. Bulk density is defined as

the mass of powder divided by the bulk volume. 5 g of blend was allowed to flow gradually

into a measuring cylinder. The initial volume occupied by the blend was noted and used for

calculating bulk density.

Bulk density (gm/cc) = Wt. of blend (g) / untapped volume of capsule blend (cc)

Tapped density [579]: The measuring cylinder was tapped until no further reduction in

volume was observed. The final volume after tapping was noted. Tapped Bulk Density was

obtained as weight of blend divided by the tapped volume.

Tapped density (gm/cc) = Wt. of blend (g) / tapped volume of capsule blend (cc)

Moisture content [580]: The moisture content of the capsule blend was estimated by It was

determined accurately by weighing 2 g of capsule blend from 10 capsules in a dried weighing

bottle. The loaded bottle was placed in a hot air oven and was heated to about 100⁰c -105⁰c,

the stopper of the weighing bottle was removed and was left in the oven to dry the sample to

a constant weight and after drying is completed , the hot air oven was opened with a prompt

closure of the bottle and was allowed to cool to room temperature in a dessicator before

weighing The bottle was weighed along with the contents

The blend was then manually filled in size ‘0’ empty orange/black hard gelatin capsules and

was packed in glass bottles along with a desiccant silica gel bag. The capsules were then

evaluated initially and at each sampling station as per protocol.

3.9.4 STABILITY STUDIES

1. Stations for evaluation of fm1-04 and fo1-04 [581]

As per the ICH guidelines, samples were stored at 25�C/60%RH (long term studies),

30�C/65%RH (intermediate studies) and 40�C/75%RH (accelerated stability studies). The

samples kept at 30�C/65%RH are to be evaluated only if accelerated stability studies reveal a

significant change in the physicochemical parameters of FM1-04 and FO1-04.

Experimental

139

Samples stored at. 25�C/60%RH (long term studies) were evaluated for predefined

parameters after 0, 1, 2, 3, 6 and 12 months and samples stored at 40�C/75%RH (accelerated

stability studies).were evaluated after 0, 1, 2, 3 and 6 months .

2. Parameters evaluated during stability stidies [581]

� Appearance

The capsules were examined externally for changes in capsule color and appearance. The

capsule shells were opened and the contents examined visually for physical changes.

� .Moisture content

The moisture content of the capsules was monitored from time to time in order to find the

tendency of the filled capsules to take up moisture. It was determined accurately by weighing

2 g of capsule blend from 10 capsules in a dried weighing bottle. The loaded bottle was

placed in a hot air oven and was heated to about 100⁰c -105⁰c, the stopper of the weighing

bottle was removed and was left in the oven to dry the sample to a constant weight and after

drying is completed , the hot air oven was opened with a prompt closure of the bottle and was

allowed to cool to room temperature in a desiccators before weighing The bottle was weighed

along with the contents.

� .Weight variation

Intact capsules (20) were individually weighed and the average weight was determined. The

test requirements are met if none of the individual weights are less than 90% or more than

110% of the average.

� .Content uniformity

The contents of 10 capsules were assayed by UV method and the average content was

determined. The test requirements are met if nine out of ten are within the limits.

� Disintegration time (D.T.)

Disintegration time was determined using USP Basket rack assembly. Six capsules were

sampled randomly were tested for the disintegration time.

� .Assay

a. UV Spectroscopy

The contents of five capsules of FM1-04 were pooled and an amount equivalent to 200 mg of

the extract was accurately weighed, dissolved in 10 ml of methanol. It was then filtered

Experimental

140

through Whatman paper, the 1ml of filtrate was further diluted to 100ml from which 0.4 ml

of solution was transferred to the test tube and final volume of 5ml was made with methanol

and absorbance of this solution was noted at wavelength 280 nm. The readings were recorded

in duplicate. The corresponding concentrations were obtained from the equation in linearity

studies.

The contents of five capsules of FO1-04 were pooled and an amount equivalent to 200 mg of

the extract was accurately weighed, dissolved in 10 ml of 50% ehanol. It was then filtered

through Whatman paper, the 1ml of filtrate was further diluted to 10ml from which 0.4 ml of

solution was transferred to the test tube and final volume of 10 ml was made up with

methanol and absorbance of this solution was noted at wavelength 205 nm. The readings

were recorded in duplicate. The corresponding concentrations were obtained from the

equation in linearity studies.

b. Assay by HPTLC method

The capsule contents of 5 capsules of FM1-04 on 0 month and 12 month were pooled and

weighed to the quantity equivalent to that of 200mg of extract and dissolved in 20 ml

methanol filtered and filtrate was used for spotting onto TLC plates. The plates were then run

in the solvent system Toluene :Ethyl acetate :formic acid (9.3:0.7:0.1 Peak areas were

determined and the capsule content of the initial and 12 month samples were estimated by

using the equation obtained in linearity studies

The contents of FO1-04 were 10 ml of 50% ethanol. It was then filtered, The capsule contents

of five capsules of FO1-04 on 0 month and 12 month were pooled and an amount equivalent

to 200 mg of the extract was accurately weighed, dissolved in 20 ml of 5% ethanol filtered

through Whatman paper and filtrate was used for spotting on to TLC plates. The plates were

then run in the solvent system n Hexane :chloroform :methanol (6.5:3.5:0.5). From

Chromatogram peak areas were determined and the capsule content of the initial and 12

month samples were estimated by using the equation obtained in linearity studies

� Dissolution

Dissolution was performed using USP dissolution Apparatus I (Basket Type)

The FM1-04 capsules developed in-house were then subjected to dissolution studies under

specified conditions. 5ml of the medium was withdrawn at intervals of 15.30,60,90 and 120

minutes and analyzed spectrophotometrically. The contents of the capsule dissolved were

then calculated from the standard curves.

Experimental

141

The FO1-04 capsules developed in-house were then subjected to dissolution studies under

specified conditions. 10ml of the medium was withdrawn at intervals of 15, 30, 60, 90 and

120 minutes and analyzed spectrophotometrically. The contents of the capsule dissolved were

then calculated from the standard curves.

3.9.5. IN-VIVO EFFICACY OF FM1-04 AND FO1-04

Restraint Stress Drug treatment :

Vehicle (equi volume of 0.1%Na CMC),Ashwagandha (100 mg/kg p.o ), FM1-04 (200mg/kg

p.o) and FO1-04 (200mg/kg p.o) were suspended in 0.1%Na CMC and were administered

per orally1 hour before subjecting them to the restraint stress .

Experimental groups for In-vivo efficacy of FM1-04 AND FO1-04:

Wistar Rats (150-250 g ) of either sex were divided randomly in to 4 groups each containing

6 animals. Animals were subjected to restraint stress 1 hour after administration of vehicle

/standard /formulations.

Group I : Rats received equi volume 0.1% Na CMC in vehicle p.o(Vehicle control group)

Group II : Rats were received equi volume 0.1% Na CMC p.o.in vehicle and subjected to

restraint stress (stress control group)

Group III-: Rats were treated with Standard Ashwagandha (100mg/kg) p.o.

Group IV: Rats were treated with FM1-04 at doses 200 mg/kg resp.p.o. and subjected to

restraint stress

Group V: Rats were treated with FO1-04 at doses 200 mg/kg resp.p.o. and subjected to

restraint stress

Method [554]:

Restraint stress (RS) model was used to carry out In vivo testing of FM1-04 and FO1-04. The

restraint stress was applied by placing each rat in a Plexiglas tube. The Plastic restraint tubes

are equipped with air holes and an adjustable endplate, which helps to account for differences

in body weight and length and secures the rat within the tube. Once the rats were secured in

their respective Plastic restrainer, the cohort was placed in a temperature control room

separate from the main animal colony. Individual animal was restrained inside cylindrical

restrainer (19.5 cm × 6.5 cm ) daily for 2 hrs and this process was repeated continuously for

14 days. Blood was collected from retro orbital plexuses immediately at the end of restraining

periodically on 1st ,7th and 14th day of study, centrifuged at 4⁰c at 3000 rpm for 15 min and

Experimental

142

the serum was separated. The serum was used for estimation of various biochemical

parameters such as corticosterone , glucose ,triglyceride and cholesterol.

3.10. ISOLATION AND STRUCTURAL ELUCIDATION AND EFFICACY

EVALUATION OF ISOLATED PHYTOCONSTITUENT

3.10.1. Extraction and separation of alkaloid fractions [582] :

Method: Murraya koenigii leaves were collected from local market at thane and were air

dried. The dried leaves were powdered and powder was passed through 40 # and extracted

with methanol. The 20 gm of extract was put in a beaker to which 100ml methanol was added

and pH was adjusted to 2.5 by gl.acetic acid. The solution was kept overnight. 24 hours later

the solution of extract MKM was filtered. Filtrate was collected and solvent was evaporated

under reduced pressure and paste was obtained. To the paste 30 ml of water was added and

pH was adjusted to 2 by adding gl.acetic acid . The aqueous phase formed was filtered and

the filtrate was subjected to liquid liquid extraction with solvent Methyl Isobutyl Ketone

(MIBK.). On separation of two phases the upper MIBK layer was collected and solvent

(MIBK) was evaporated in vacuum oven to obtain the residue. The resultant MIBK fraction

was spotted on TLC run in a solvent systems Toluene : Ethyl acetate (9:1) and was spread

with Dragendorff reagent. This separated fraction was used as an alkaloid fraction (MK1F) to

load on column for isolation of an alkaloid (MK1)

3.10.2. ISOLATION OF MK1 FROM ALKALOID FRACTION (MKF1) [583,584]

Method :

A vertical column of length 1 meter and diameter 13 cm with a Sintered glass disk at the

bottom was used for column chromatography. Silica (Merck) 60/120# was used as a

adsorbent 250 Gm of silica was weighed activated in oven at 200.⁰C and slurry was made by

adding solvent Chloroform (AR). The slurry was poured uniformly in to the column. After

the adsorbent had settled a piece of cotton was placed on the top of column and then the

supernatant liquid was run off until the level falls to about 1cm above the top layer of silica.

Then alkaloid fraction (MKMF1) (1 G) was loaded on the top layer of silica column. The

level of chloroform was never allowed to fall down the level of silica by periodically

maintaining the liquid column of fresh chloroform solvent on top of silica. Elutes (5ml) were

collected by maintaining drop rate of 36-38 drops /minute which was followed by

development of TLC run in mobile phase Toluene . ethyl acetate (9:1) and which was

observed under short UV ( wave length 254 nm) for identification of spots. On identification

of spot for the phyto constituent Rf was measured .There after column chromatography was

Experimental

143

repeated successively and elutes of MK1 were pooled together and were dried in vacuum

oven and yield was recorded. Isolated MK1 was re-crystallized by dissolving in solvent n

Hexane and was used for carrying out various physical methods required for structural

elucidation.

3.10.3. Structural elucidation [585].:

The structural elucidation of the isolated alkaloid MK1 was carried out by following various

physical techniques:

� UV spectroscopy [586]

UV can be used to measure λmax. which can be used as a preliminary parameter for

identification of component.

Method: 0.1mg of MK1 was dissolved in 10 ml chloroform (AR) and spectra was recorded

in range of 200 to 400nm on Spectrophotometer (JASCO,JAPAN). The U.V spectrum

obtained was used to determine λmax values.

� I.R.Spectroscopy [587]

IR spectrum thus provides one of the simplest and reliable method for structural elucidation.

Method: 0.1 gm of MK1 was triturated with KBR (1:10) and discs was prepared under

unhydrous conditions. I.R spectrum was recorded within a range of 4000 to 667 cm-.

� HPTLC [575]

HPTLC provides the means not only for flexible screening procedures and qualitative

analysis but also for quantitative determinations. HPTLC fingerprint is obtained at low cost

and high speed.

Method:

Stationary phase - Precoated silica gel 60 F254 plates

Plate size - 10 cm x 10 cm

Thickness - 0.2 mm

Applicator - Camag Linomat IV

Band width - 7 mm

Spotting volume - 1 – 10 �l

Development chamber - Camag Twin Trough

Solvent front - 8 cm

Experimental

144

Scanner - Camag

Densitometric conditions:

i. � 254 mode - Absorption Reflection (Deuterium lamp)

ii � 366 mode - Fluorescence Reflection ( Mercury lamp )

0 1 mg of MK1 was dissolved in 10 ml of chloroform and was spotted on the precoated silica

gel 60 F254 plate by using Camag Linomat IV applicator. Micro syringe was used for

spotting the extract in a band of 7 mm on the plates. The spots were allowed to air dry. The

plates were developed using a Camag twin trough chamber saturated with the solvent system,

which gave the best resolution of spots for the extract. The plates were run in the toluene

:ethyl acetate (9:1) solvent system.The developed plates were air dried and then scanned

using a Camag 3 Scanner under ultraviolet (UV) 254 nm absorption mode fluorescence

mode.

� NMR Spectrum [588]

NMR was carried out to investigate molecular structure and molecular interactions.

Method: 50 mg sample of MK1 sample was submitted to carry out 1H NMR and 13C NMR

by using CDCL3 solvent at SAIF department, IIT Mumbai.

� Mass spectrum [589]

Mass spectrum was used to confirm the molecular weight if isolated constituent.

Method: 5 mg sample of MK1 was submitted to SAIF Department of IIT Mumbai for

recording MS spectrum.

3.10.4. IN VITRO TESTING OF ISOLATED PHYTOCONSTITUENT MK1

1. Assay of DPPH radical scavenging activity.[590]

Method: DPPH radical scavenging activity was measured in a reaction mixture containing

0.1ml of 1mM DPPH radical solution, 0.8 ml of 99% methanol and 0.1ml sample solution

which were mixed and incubated for 20 minutes. Scavenging capacity was measured by

monitoring the decrease in absorbance at 517 nm. The anti oxidant activity of test drug was

expressed as IC50.

IC50 was defined as the concentration of test compound required for inhibition of formation

of DPPH radicals by 50%



Control) X100

Experimental

145

2. Thiobarbituric acid reactive substances (TBARS ) Assay.[590]

Method: Rat was sacrificed by decapitation. Brain was isolated and washed with ice cold

normal saline solution and homogenized in glass Teflon homogenizer with 10 ml of ice cold

5mM potassium phosphate buffer (pH=7.4). different concentrations of test drugs were added

to 3 ml of tissue homogenate and lipid peroxidation was initiated by adding 0.1ml of 15 mM

FeSo4 solution. After incubation of this mixture at room temperature for 30minutes 0.1ml of

brain homogenate was added in a tube containing 0.1ml (8.1%) sodium dodecyl sulphate,

0.75ml of 20% acetic acid and 0.75ml of 0.8% Thio barbituric acid (TBA) aqueous solution

.the volume in each test tube was made to 2 ml with distilled water and heated on a water

bath at 95⁰c for 60 minutes. After 60 minutes of incubation period volume in each tube was

made to 2.5 ml and then 2.5 ml of butanol : pyridine (5:1) was added in each tube . The

reaction mixture was vortexed and centrifuged at 4000 rpm for 10 minutes .The organic layer

was removed and absorbance was read at 532 nm in UV spectrophotometer.



Control) X100

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