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Experimental
84
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
Experimental
85
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
Experimental
86
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)
Experimental
87
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.
Experimental
88
� 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)
Experimental
89
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
90
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.
Experimental
91
� 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.
Experimental
92
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.
Experimental
93
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.
Experimental
94
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.
Experimental
95
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
96
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.
Experimental
97
� 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
Experimental
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 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.
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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 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 : 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
Experimental
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 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 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
restraint stress (Standard Group).
Group V : Rats were treated with standard Piracetam (200 mg/kg p.o.) and subjected to
restraint stress (Standard Group).
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)
Group III : Rats were treated with standard Ashwagandha (100 mg/kg .p.o.) and subjected to
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
and subjected to foot shock stress
Experimental
110
Group XX-XXII : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp.p.o
and subjected to foot shock stress
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 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 sleep
deprivation stress (stress control group
Group III : Rats were treated with standard Ashwagandha (100 mg/kg .p.o.) and subjected to
sleep deprivation stress (Standard Group)
Group IV : Rats were treated with standard Imipramine (10 mg/kg .p.o.) and subjected to
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
Group XI-XIII: Rats were treated with MKM at doses of 50,100 and 200 mg/kg resp.p.o. and
subjected to sleep deprivation stress
Group –XIV-XVI : Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg resp.
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
and subjected to sleep deprivation stress
Group XX-XXII : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp.p.o
and subjected to sleep deprivation stress
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 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 ECS stress
(stress control group.
Group III : Rats were treated with standard Ashwagandha (100 mg/kg .p.o.) and subjected to
ECS stress (Standard Group).
Group IV : Rats were treated with standard Imipramine (10 mg/kg .p.o.) and subjected to
ECS 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 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.
Group XI-XIII: Rats were treated with MKM at doses of 50,100 and 200 mg/kg resp.p.o. and
subjected to ECS stress.
Group –XIV-XVI : Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg resp.
p.o. and subjected to ECS stress.
Group XVII-XIX: Rats were treated with OSHA at doses of 50, 100 and,200 mg/kg resp. p.o
and subjected to ECS stress.
Group XX-XXII : Rats were treated with OSM at doses of 50, 100 and 200 mg/kg resp.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 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 CST (stress
control group)
Group III : Rats were treated with standard Ashwagandha (100 mg/kg .p.o.) and subjected to
CST (Standard Group)
Group IV : Rats were treated with standard Imipramine (10 mg/kg .p.o.) and subjected to
CST (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 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
Group XI-XIII: Rats were treated with MKM at doses of 50,100 and 200 mg/kg resp.p.o. and
subjected to CST
Group –XIV-XVI : Rats were treated with OSAQ at doses of 50,100 and 200 mg/kg resp.
p.o. and subjected to CST
Group XVII-XIX: Rats were treated with OSHA at doses of 50, 100 and,200 mg/kg resp. 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
and subjected to CST
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
Working Reagent 1000 μl 1000 μl 1000 μl
Distilled water 10 μl -- --
Standard -- 10 μl --
Test -- -- 10 μl
LPL G K, Mg++
GPO
Peroxidase
Experimental
121
The serum was mixed well with the working reagent and was incubated at 37�C for 10 min.
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 serum was mixed well with the working reagent and was incubated at 37�C for 10 min.
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
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 10 min.
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
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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%
Percentage Inhibition was calculated by using following formula;
Percentage Inhibition = (Absorbance of Control – Absorbance of Test/ Absorbance of
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.
Percentage Inhibition was calculated by using following formula;
Percentage Inhibition = (Absorbance of Control – Absorbance of Test/ Absorbance of
Control) X100