The Bark Of Madhuca Indica

Published Date: 02 Nov 2017

The bark of Madhuca indica and leaf of vitex negundo was collected and authentified by Dr. Madhav Chetty, Department of Botany, Venkateswara University; Tirupathi, India. Collected plant material was washed thoroughly with water and dried under shade.

2.2. Preparation of extract:

Dried pieces of Madhuca indica bark were powdered in a grinder and the powder was extracted with different solvents such as methanol, petroleum ether and water for 48 h by soxhlet extraction process. The extracts were evaporated to dryness at a controlled temperature (45 oC).The extracts were preserved in a refrigerator.

The leaves of vitex negundo were cleaned of extraneous matter shade dried and powdered until a constant weight was attained. The powdered leaves were macerated for 48 h in methanol; it was subjected to percolation by using methanol as a solvent. The menstrum collected and concentrated under reduced pressure. The dried extract was stored in refrigerator until ready for use

2.3. PHYTOCHEMICAL INVESTIGATIONS OF THE EXTRACTS113, 114

Madhuca indica and vitex negundo is subjected to qualitative analysis for various phytoconstituents like flavanoids, saponin glycosides, tannins, carbohydrates, reducing sugars, steroids and Amino acids.

1. Test for Saponins: 114

1. Diluted 1ml of aqueous extract separately with distilled water to 20ml and shake in a graduated cylinder for 15 min. One centimeter layer of foam indicates the presence of saponins.

2. Haemolysis test: 2ml of 1% sodium chloride solution was taken in two test tubes. To one test tube 2 ml of distilled was added and to the 2ml of 1% filtrate. Blood is obtained by pricking the thumb and 5 drops of blood were added to each tube, the contents were gently mixed and observed under microscope. Haemolysis occurs, saponins present.

2. Test for Carbohydrates:

Dissolve small quantities of extract separately with 5ml of distilled water and filtered. The filtrate was subjected to Molisch’s test

1. Fehling’s test: Extract treated with Fehling’s reagent (Fehling’s reagent A- Copper sulphate in water and Fehling’s reagent B- Sodium Potassium tartarate), red colour indicates the presence carbohydrates.

2. Barfoed’s test: Extract heated with Barford’s reagent (Copper acetate in water glacial acetate), red colour

3. Benedict’s test: Extract heated with Benedict’s reagent (Copper sulphate, Sodium citrate and Sodium carbonate in water, red colour shows the presence of carbohydrates.

4. Molisch’s test: (solution of α- napthol in alcohol)

The extract is when treated with Molisch’s reagent and conc. Sulphuric acid is passed along the sides of the test tube; a reddish violet ring shows the presence of carbohydrates.

3. Test for Flavanoids:

1. Ferric chloride test: To extracts few drops of neutral ferric chloride solution, blackish red colour is seen.

2. Lead acetate test: To extract add lead acetate solution, yellow precipitates formed that shows the presence of flavanoids.

3. Magnesium ribbon test: To extract added few fragments of magnesium ribbon and concentrated hydrochloric acid along the sides of test tubes, magenta colour shows the presence of flavanoids.

4. Zinc-hydrochloric acid test: To extracts, a pinch of zinc dust was added and concentrated hydrochloric acid along the sides of test tubes, magenta colour shows the presence of flavanoids.

4. Test of Tannins:

Diluted small quantities of aqueous extract with distilled water and subjected to

1. Ferric chloride test: Extract treated with ferric chloride solution, blue colour indicates the presence of tannins.

2. Gelatin test: Extract treated with gelatin solution, white precipitate shows that tannins are present.

3. Lead acetate test: Extract treated with lead acetate solution, yellow precipitate indicates the presence of tannins.

5. Test for Steroids

1. Libermann-Buchard test: 10 mg extract was dissolved in 1ml of chloroform, 1ml of acetic anhydride was added following the addition of 2ml of concentrated sulphuric acid from the sides of the test tube. Formation of pinkish colour indicates the presence of steroids.

2. Salkowski test: 1ml of concentrated sulphuric was added to 10mg of extract dissolved in 1ml of chloroform. A reddish brown colour exhibited by chloroform layer and green colour fluorescence by the acid layer suggests the presence of steroids.

3. Liebermann’s test: mix 3 ml of extract with 3ml of acetic anhydride. Heat and cool. Add few drops of concentrated sulphuric acid. Blue color appears.

6. Test for Proteins and Amino acids:

Dissolve small quantities of aqueous extract separately with few drops of distilled water and then subjected to

1. Millon’s test: Extract treated with Millon’s reagent (Mercuric nitrate in nitric acid), red colour.

2. Biuret test: Extract treated with sodium hydroxide and copper sulphate solution added drop wise and mixed, violet colour.

3. Ninhydrin test: Extract treated with ninhydrin reagent and ammonia, heated, violet colour.

4. Sodium bicarbonate test: Extract treated with sodium bicarbonate solution, brisk effervescence.

7. Test for Alkaloids:

A. Mayer’s Test: (solution of Potassium mercuric iodide solution)

The filtrate was treated with Mayer’s reagent; the formation of cream colour precipitate indicates presence of alkaloids.

B. Dragendroff’s Test: (solution of potassium Bismuth iodide)

The filtrate was treated with Dragendroff’s reagent; the formation of reddish brown colour precipitate indicates presence of alkaloids.

C. Wagner’s Test: (solution of Potassium iodide in Iodine)

The filtrate was treated with Wagner’s reagent; the formation of reddish brown color precipitate indicates presence of alkaloids.

D. Hager’s Test: (solution of saturated picric acid)

The filtrate was treated with Wagner’s reagent; the formation of yellow color precipitate indicates presence of alkaloids.

8. TEST FOR VOLATILE OILS:

Hydro distillate material. Separate volatile oil from distillate. The distillate

Volatile oils have characteristic odour.

Filter paper is not permanently stained with volatile oil

Volatile oils are soluble in 90% alcohol

ANIMALS:

Male Wistar rats weighing 150-175 g was selected for screening antidiabetic activity, procured two weeks prior to the study and maintained in institutional animal house, so that animals could acclimatize to the new environment. The institutional animal ethics committee permission was obtained before starting the experiments on the animals.

Swiss albino mice weighing 20-25 g and male Albino Wistar rats weighing 125-150 g were selected for screening anticonvulsant activity, procured two weeks prior to the study and maintained in institutional animal house, so that animals could acclimatize to the new environment. The institutional animal ethical committee permission was obtained before starting the experiments on the animals. Animals were housed in groups of 6-8 cages at a temperature of 250±1o C and relative humidity of 45-55%.A 12:12 dark: light cycle was followed during the experiments. Animals had free access to food and water however, food, but not water was withdrawn 8h before and during the experiments.

2.4. ACUTE ORAL TOXICITY STUDIES115

The acute oral toxicity was performed according to the OECD (Organization for Economic and cooperative development) guideline No.420.Male Wistar rats weighing 100-125g were used for the study. Animals were maintained under controlled standard animal house conditions with access to food and water ad libitum. The rats were acclimatized for 5 days and fasted overnight, food but not water was withheld. Animals were weighed; limit and main test were performed. The limit test is carried out first at 5000mg/Kg body weight for one animal and if animal dies, main test is performed. If the animals survives two more animals are dosed, if both survives the test should be terminated. First one animal is dosed with 5mg/Kg body weight. If animal dies a much lower dose is tested. If animal survives, then two more animals are dosed, after 48hrs observation of the first animal. If survives, then the main test should be terminated. If animals die, two more animals are dosed and observed. The dosing is stopped when one of the following stopping criteria is met.

1) 3 consecutive animals survive at the upper end.

2) 5 reversals occur in any 6 consecutive animals test.

3) At least 4 animals have followed the first reversal and the specified likelihood ratios exceed the critical value.

LD 50 for methanolic, petroleum ether and aqueous extracts were observed at 1000 mg/kg, 1500 mg/kg and 2000 mg/kg body weight, respectively. Therefore 1/10th of their preceding doses viz. 75 mg/kg, 125 mg/kg, 175 mg/kg body weight selected as testing doses.

Screening of anticonvulsant activity was carried out by taking 30 albino mice & rats of both sexes were randomly divided to five groups consisting of control and vitex .negundo treated groups. Mice & rats were fasted for 12 h and various doses of extract were administered orally to test groups. The mice & rats were closely observed for toxic symptoms and behavioral changes for the first 2 h after extract administration and mortality recorded within 24 h.The lethal dose that killed 50% of mice and rats was estimated after 24h.

2.5.Drugs and chemicals

Streptozotocin : Sigma Labs Ltd. U.S.A

Metformin : Gift samples from microlabs Hosur

Insulin : Torrent Pharmaceuticals Ltd.

Glucose estimation kit : RMS: Luquichem-Baddi

Cholesterol estimation kit : RMS: Luquichem-Baddi

Triglycerides estimation kit : Span Diagnostics Ltd. Surat

HDL estimation kit : Span Diagnostics Ltd

Diazepam : Gift sample from Ranbaxay

Pentylenetetrazole : Sigma-Aldrich

Picrotoxin : Sigma-Aldrich

Lithium-pilocarpine : Glenmark & FDC

Strychnine : Sigma-Aldrich

2.6.Methods:

1. Streptozotocin induced diabetes mellitus116

Male Wistar rats weighing 150-175 g were used for the study. After procuring, the animals were acclimatized for two weeks at normal laboratory condition. All animals were allowed free access to tap water and pellet diet and maintained at room temperature in polyethylene cages. Before conducting the experiment, ethical clearance was obtained from Institutional Animal Ethics Committee of Malla Reddy College of Pharmacy, Maisammaguda, Secunderabad. Diabetes was induced in 16 h fasted male Wistar rats by intraperitoneal injection of 65 mg/kg body weight of streptozotocin. Streptozotocin was dissolved in 50 mM cold sodium citrate buffer (pH 4.5) immediately before use. After 72 h, rats with marked hyperglycemic fasting blood glucose >200 mg/dL were selected and used for the study. All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in polyethylene cages.

The rats were divided into six groups consisting of six each.

Group 1: Administered vehicle serves as normal control.

Group 2: Administered streptozotocin (65 mg/kg i.p.) serves as diabetic control

Group 3: Diabetic rats treated with methanolic extract (75 mg/kg, p.o. once daily)

Group 4: Diabetic rats treated with petroleum ether extract (125 mg/kg, p.o. once daily)

Group 5: Diabetic rats treated with aqueous extract (175 mg/kg, p.o. once daily)

Group 6: Administered reference standard, Insulin (6 IU/kg, i.p.)

The animals were treated with respective drugs for 30 days. On the next day, blood was withdrawn by puncturing retro orbital plexus, serum was separated and serum glucose, cholesterol, triglycerides (TGs) and HDL levels were estimated.

2.7. STREPTOZOTOCIN- NICOTINAMIDE INDUCED DIABETES117

Diabetes was induced in 16 h fasted male Wistar rats by combined intraperitoneal injection of streptozotocin (STZ) 65 mg/kg and nicotinamide (NIC) 230 mg/kg.Streptozotocin was dissolved in 50 mM cold sodium citrate buffer (pH 4.5) immediately before use and nicotinamide was administered 15 min before administration of STZ. After 72 h, rats with marked hyperglycemic fasting blood glucose >200 mg/dL were selected and used for the study. All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in polyethylene cages.

The rats were divided into six groups consisting of six each.

Group 1: Administered vehicle serves as normal control.

Group 2: Administered streptozotocin (150 mg/kg i.p.) and nicotinamide (360 mg/kg i.p.) Serves as diabetic control.

Group 3: Diabetic rats treated with methanolic extract (75 mg/kg, p.o. once daily).

Group 4: Diabetic rats treated with petroleum ether extract (125 mg/kg, p.o. once daily).

Group 5: Diabetic rats treated with aqueous extract (175 mg/kg, p.o. once daily).

Group 6: Administered reference standard, metformin (200 mg/kg, p.o).

The animals were treated with respective drugs for 30 days. On the next day, blood was withdrawn by puncturing retro orbital plexus, serum was separated and serum glucose, cholesterol, triglycerides (TGs) and HDL levels were estimated.

Procedures for tested parameters:

2.8.Estimation of glucose

Principle:

The glucose oxidase reaction in conjugation with auxiliary reaction has been widely used for the determination of glucose in biological fluid. The method utilized in this reagent is based on the hydrogen peroxide indicator reaction which couples 4- amino antipyrine to a phenolic compound. The intensity of the coloured complex is measured at 505 nm.

Glucose + O2 + H2O Glucose oxidase Glucoronic acid + H2O2

H2O2 + Phenolic compound + 4-aminoantipyrine peroxidase Quinone imine dye + H2O

Reagent composition:

Phosphate buffer PH 7.5 :100 mmol/ L.

4-Aminophenazone :0.25 mmol/ L.

Phenol :7.0 mmol/ L.

Glucose oxidase : >15000 u/ L.

Peroxidase : > 1000 u/ L.

Stabilizers & Preservatives : Azide 0.1 %, Non-reactive Stabilizers & filters.

Procedure:

Reaction type : End Point.

Reaction slope : Increasing.

Reaction temperature : 37 0 C.

Wavelength : 505 nm.

Blank : Reagent Blank.

Sample volume : 10 µl.

Reagent volume : 1.0 ml.

Reaction time : 10 seconds.

Linearity : 400mg/dl.

Assay:

Standard /sample : 10 µl.

Reagent : 1.0 ml.

Incubate the assay mixture for 10 min at 370C.

Measure the absorbance of standard and sample against blank at 505 nm

Calculation:

Glucose (mg/dl) = Abs. of sample/Abs. of standard X 100.

2.9. Estimation of Cholesterol.

Principle:

Cholesterol esterase hydrolyses cholesterol esters into free cholesterol and fatty acids. In the second reaction cholesterol oxidase converts cholesterol to cholest-4-en-3-one and hydrogen peroxide. In presence of Peroxidase, hydrogen peroxide oxidatively couples with 4-aminoantipyrine and phenol to produce red quinoneimine dye which has absorbance maximum at 510nm (505-530nm). The intensity of the red color is proportional to the amount of total cholesterol in the specimen.

Cholesterol ester Cholesterol esterase Cholesterol + Fatty acids.

Cholesterol + O2 Cholesterol oxidase H2O2 + Cholest-4-en-3-one2H2O2..

4- Aminoantipyrine + Phenol Peroxidase Red quinoneimine Dye + H202.

Reagent composition:

Buffer; PH 7.5 : 100 mMol/ L.

Cholesterol oxidase : ≥ 100 IU/ L.

Cholesterol esterase : ≥ 150 IU/ L.

Peroxidase : ≥ 500 IU/ L.

4- Amino antipyrine ; 0.5 mMol/ L.

Phenol : ≥ 10 mMol/ L.

Procedure:

Reaction type :End point.

Reaction time :5 mins. At 37º C or 10 mins. At R.T. (25º - 30º C).

Wavelength :510nm. (505 – 530nm).

Zero setting with : Reagent blank.

Blank absorbance limit :<0.100 Abs.

Sample volume : 0.01 ml(10µl).

Reagent volume :1.0 ml.

Standard concentration :200 mg/dl.

Linearity :700 mg/ dl.

Assay:

Serum

Standard

Blank

Working reagent

0.01 ml

1 ml

0.01 ml

1 ml

-----

1 ml

The assay mixture is incubated for 5 minutes at 37º C and absorbance was measured at 510 nm against blank.

Calculation:

Total Cholesterol in mg/dl = Absorbance of sample / Absorbance of Std. x 200.

2.10.Estimation of Triglycerides

Principle:

Triglycerides are hydrolyzed by Lipoprotein Lipase (LPL)to produce Glycerol and Free Fatty Acid(FFA) .In presence of Glycerol Kinase(GK), Adenosine Triphospate(ATP) phosphorylates Glycerol to produce Glycerol 3-Phospate and Adenosine Diphospate (ADP),Glycerol 3- Phosphate is further oxidized by Glycerol 3-Phospate Oxidase (GPO) to produce Dihydroxyacetone Phosphate (DAP) and Hydrogen peroxide .In presence of Peroxidase (POD), Hydrogen Peroxide couples with 4-Aminoantipyrine (4-AAP) and 4-Chlorophenol to produce red Quinoneimine dye. Absorbance of coloured dye is measured at 505nm and is proportional to Triglycerides concentration in the sample.

Triglycerides LPL Glycerol+FFA.

Glycerol+ ATP GK Glycerol 3-Phospate+ADP.

Glycerol 3-phospate+O2 GPO DAP+H2O2 .

2H2O2 +4-AAP+4-Chlorophenol Quinoneimine dye+4H2O2 .

Reagent composition:

Pipes buffer :50 mMol/ L.

4-Chlorophenol :5 mMol/ L.

Magnesium ion :5 mMol/ L.

ATP :1.0 mMol/ L.

Lipase : ≥ 5000 U/ L.

Peroxide : ≥ 1000 U/ L.

Glycerol kinase : ≥ 400 U/ L.

4-Aminoantipyrine : ≥0.4mMol/L.

Glycerol 3-phospate oxidase : ≥ 4000 U/ L.

Preservatives : qs.

Stabiliser : qs.

Procedure:

Reaction type : End point.

Reaction time :5 mins. At 37º C or 10 mins. At R.T. (25º - 30º C)

Wavelength :505nm. (490 – 550nm).

Zero setting with :Reagent blank.

Blank absorbance limit : <0.100 Abs.

Sample volume :0.01 ml(10µl).

Reagent volume :1.0 ml (1000µ).

Standard concentration :200 mg/dl.

Linearity : 1000 mg/ dl.

Assay:

Serum

Standard

Blank

Working reagent

0.01 ml

1 ml

0.01 ml

1 ml

-----

1 ml

The assay mixture is incubated for 10 minutes at 37º C and absorbance was measured at 505 nm against blank.

Calculation:

Triglycerides in mg/dl = Absorbance of test / Absorbance of Std. x 200.

2.11.Estimation of HDL Cholesterol.

Principle:

Span Diagnostics kit was used for the estimation of serum HDL – cholesterol.

Method:

Polyethylene glycol CHOD – PAP

Endpoint Colorimetry.

Two – Reagent Chemistry with LCF (Lipid clearing Factor)

Principle:

Low and very low density lipoproteins (VLDL) are precipitated by a solution containing PEG 6000, leaving behind the high density Lipoproteins in solution.HDL Cholesterol is estimated in the supernatant by a series of enzymatic reactions Which are initiated by the oxidation of cholesterol to cholestenone by cholesterol oxidase accompanied by the formation of hydrogen peroxide to form a red colored quinoneimine.

Absorbance 505 nm is directly proportional to HDL Cholesterol concentration.

Procedure:

STEP – A: HDL – CHOLESTEROL SEPERATION

Pipette into Centrifuge tube

Quantity

Sample

Precipitating reagent

0.2 ml

0.2ml

The above were mixed well, kept at R. T. for 10 min and then centrifuged at 2000 rpm for 10 min and then centrifuged at 2000 rpm for 15 minutes to obtain a clear supernatant and proceded to step - B.

STEP – B: COLOUR DEVELOPMENT:

Pipette into tubes marked

Blank

Standard

Test

Supernatant from step A

-

-

100μl

HDL Cholesterol Standard

-

100μl

-

Cholesterol Reagent

1000μL

1000μL

1000μL

The above were mixed well, incubated at 37 o C for 10 minutes or at R.T. for 30 minutes

and absorbance were read against reagent blank at 505 nm within 60 minutes.

Programme: The basic assay parameters

Mode

End Point

Wavelength

505 nm (490 – 530 nm)

Temperature

37 o C or R.T.

Optical path Length

1 cm

Blanking

Reagent Blank

Incubation time ( minutes)

10 at 37 o C or 30 at R.T.

Sample volume

100μl

Working reagent volume

1000μl

Concentration of standard

50 mg/dL

Stability of color

1 hour

Maximum absorbance limit

2.000

Units

mg/dL

Calculation:

HDL Cholesterol (mg/dL) = Absorbance of Test x Conc of STD x 2

Absorbance of Standard

2.15.Maximum electroshock induced seizures (MES)The maximal electroshock (MES) method was performed to induce the seizures in order to screen for antiepileptic activity 118. Mice deprived of food and water ad libitum for overnight, were randomly distributed in to eight groups of six animals each. Group I served as control (vehicle treated), Group II served as standard (received Phenytoin sodium 20 mg/kg body weight); Group III and Group IV were treated with methanolic extract of vitex. negundo at 200 and 400 mg/kg body weight. The test extract were administered orally in 1% v/v Tween 80, 1hr prior to induce the convulsion and standard drug ( Phenytoin sodium 25mg/kg) was administered i.p.30 min before. Electroconvulsive shock (50 mA for 0.2 sec) was delivered through corneal electrode to induce convulsions to eight groups of mice (n=6).The various phases of convulsions which were produced are flexion, extension, clonus and stupor. Prior to delivery, current output was checked by multimeter.After the electric stimulation occurrence, the duration of phases were noted.118

2.16.Pentylenetetrazole (PTZ) induced Seizures: Mice were divided into eight groups of six animals each .Group I served as control (vehicle treated i.e.tween 80, 2%), Group II served as standard received diazepam 5mg/Kg body weight (i.p), Group III and Group IV were treated with methanolic extract as 200 & 400 mg/Kg body weight. 30 min after i.p. injection of diazepam and 60 min after oral administration of extract, 60mg/kg PTZ was injected subcutaneously. The antiepileptic activity was accessed by its ability to delay the onset of myoclonic spasms and clonic convulsions. 119

2.17.Strychnine-induced Seizures: Thirty mice were divided into five groups of six mice each. Group I received 10mL normal saline per kg body weight i.p, Group II received Diazepam 5mg/Kg body weight (i.p), Group III and Group IV were treated with methanolic extract of vitex .negundo at a dose of 200 & 400 mg/Kg body weight i.p. Thirty minutes later, mice in all the groups received 1 mg/kg body weight strychnine. Abolition of tonic extensor jerks of hind limbs was considered as an indicator that the testing drug could prevent strychnine induced convulsions. 120

2.18.Picrotoxin induced seizures: Mice were randomly allotted to the different control and test groups. Picrotoxin (5mg/Kg, i.p) was used to induce seizures. Procedure of strychnine induced convulsions was carried out for screening.120

2.19.Lithium-pilocarpine-induced seizures: Albino rats were divided randomly into five groups each containing six animals each. Convulsions were induced by administration of pilocarpine (30 mg/kg, i.p) 24 hr after lithium sulphate (3 mEq/kg i.p). Effect of methanolic extract of vitex. negundo was studied on the severity of seizures. Group I received as control, Group II received diazepam, Group III received test drug. The severity of convulsions was observed every 15 min till 90 min and thereafter 120 min, using the scoring system. No response stage 0, fictive scratching stage 1, tremors stage 2, head nodding-3, forelimb clonus stage 4, rearing and falling back stage 5. 120

FORMULATION

2.20.Extraction procedure for preparation of agnuside

The shade dried and powdered leaves (1 kg) of vitex. negundo were soaked in methanol (5L) and kept overnight. The percolate was filtered and concentrated under reduced pressure at below 500C.The extraction procedure was repeated three times more using 3L of ethanol each time. The combined extract was stirred with water (300 mL) for 1h and filtered through celite.The extract was concentrated at 50 oC and finally dried in vacuum desicator.

2.21.Isolation of agnuside

The methanol extract (50 g) of vitex. Negundo was adsorbed over silica gel (100g) to make slurry which was packed over a column of silica gel (1kg) packed in chloroform. Elution was done with chloroform followed by mixture of chloroform and methanol. Elution with 10% chloroform in methanol gave agnuside. The compounds were characterized on the basis of HPLC and IR spectra.

2.22.Materials and methods:

Agnuside extracted from vitex negundo leaf. Lactose, Microcrystalline cellulose, crossprovidone, magnesium stearate and colloidal silicon dioxide were purchased from S.D. Fine Chemicals (Mumbai, INDIA).

Determination of lambda max and preparation of calibration curve of agnuside

Preparation of standard stock solution: 100 mg of agnuside was accurately weighed and transferred into a 100 ml volumetric flask and dissolved in methanol. Then the volume was made up to 100ml.This was standard solution containing 1mg/ml i.e. (1000 µg/ml) of agnuside (stock I).

Spectrometric scanning of agnuside: From the standard solution prepared in methanol (stock I), 2ml solution was pipette out and the volume was made upto 10 ml in a 10 ml volumetric flask, and the UV scan was taken between wavelengths 250-300 nm .The blank used here was methanol and the wavelength of maximum absorption was noted at 254 nm.

Preparation of calibration curve for agnuside: From the standard stock solution of agnuside (stock solution I) 1ml was pipetted out and the volume made upto 10ml in a 10 ml volumetric flask (Stock solution II).From this stock II, again aliquot of samples pipetted out ranging from 0,2,4,6,8,10 ml into 10 ml volumetric flask and the volume was made using methanol to get concentration of 0, 20,40,60,80, 100 µg/ml.The absorbance was measured at 254 nm using methanol as blank. Plotted a calibration curve using concentration and absorbance on X and Y axis respectively.

2.23.Preparation of the Tablets:

Agnuside tablets each containing 280 mg of agnuside were prepared by direct compression method. The tablets were Prepared as per formulae given in table. Four formulations were prepared by using different concentrations of disintegrants. In this preparation initially all ingredients were sieved through sieve no 18 separately, after that all the ingredients were mixed by geometric mixing, mixture was compressed into tablets on a rotary multi-station tablet punching machine (Cadmach Machinery Co. Pvt. Ltd., Mumbai) to a hardness of 8 - 10 kg/sq. cm using 9 mm round and flat punches.

2.24.Evaluation of physical parameters:

Thickness: Thickness was determined by using digital vernier caliper. It is expressed in mm.121

Hardness: Hardness of the tablets was tested using a Monsanto Hardness Tester. It is expressed in kg/cm2. 122

Friability: Friability of the tablets was determined in a Roche Friabilator.It is expressed in %.20-pre-wieghed tablets were rotated at 25 rpm for 4 minutes. The tablets were then re-weighed after removal of fines and the percentage of weight loss was calculated.123

Weight variation: Every individual tablet in a batch should be of uniform weight and weight variation within permissible limits. To study weight variation, 20 tablets from each batch were weighed and the test was performed according to the official method.124

Uniformity of content: The drug content of the tablets was determined by taking tablets from each batch.20 tablets from each batch were accurately weighed and powdered. The powder, equivalent to 10 mg of the drug, was weighed and added into 100 ml volumetric flask. Then it was dissolved in 10 ml methanol. The volume of solution was made up to 100 ml (100 µg/ml) with phosphate buffer pH 6.8.From this solution, 1ml was withdrawn & added into 10 ml volumetric flask and the volume was made up to 10 ml with phosphate buffer pH 6.8 (10 µg/ml).Finally, the solution was filtered with Whatmann filter paper number 41 and absorbance of the resulting solution was measured at 240 nm using UV spectrophotometer.125

Disintegration time: Disintegration times were determined in Thermionic Tablet Disintegration Test Machine using dissolution fluids 0.1 N HCl, distilled water, pH 7.4 phosphate buffers.

In vitro Dissolution studies126

Drug release from the Agnuside tablets prepared was studied using 6 station dissolution test apparatus (Electro lab) employing a paddle stirrer at 50 rpm and at 371C. pH 6.8 Phosphate buffer (900 ml) used as dissolution fluid. Samples of 5 ml of each were withdrawn at different time intervals over a period of 3h. Each sample withdrawn was replaced with an equal amount of fresh dissolution medium. Samples were suitably diluted and assayed at 240nm by using an LABINDIA double beam UV - spectrophotometer. Prepared Agnuside tablets compared with Phenytoin and Diazepam Tablets (commercial) were also studied. The drug release experiments were conducted in triplicate.

Drug Excipient Compatibility Study:

Compatibility of the drug with excipients was determined by FT-IR spectral analysis, this study was carried out to detect any changes on chemical constitution of the drug after combining it with the excipients. The samples were taken for FT-IR study. IR spectra of drug in KBr pellets at moderate scanning speed between 4000-400 cm-1 was carried out using FTIR (Jasco FTIR 6100 type A). The peak values (wave number) and the possibility of functional group are shown in spectra which compare with standard value.

Drug release kinetics (Data analysis.)

The release data obtained from various batches were studied with respect to effect of drug: polymer ratio, diluents ratio. To analyze the mechanism of drug release from the formulation, the dissolution profile of all the batches were fitted to zero order, first-order, Higuchi, Korsemeyer and Peppas, to ascertain the kinetic modeling of drug release.

Zero Order: 127

In many of the modified release dosage form particularly controlled or sustained release dosage form (those dosage forms that release the drug in planned, predictable and slower than normal manner) is zero order kinetics.

Q = K t o

Where, Q is the amount of drug release at time, t and Ko is the release rate constant.

First Order: 128

Most conventional dosage form exhibits this dissolution mechanism some modified release preparations, particularly prolonged release formulation adhere to this type of dissolution pattern.

Log Q = K t 1Where Q is the percent of drug release at time, t and K is the 1release rate constant.

Stability studies: 129

The purpose of stability testing is to provide evidence on how the quality of a drug substance or product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a re-test period for the drug substance or a shelf life for the drug product and recommended storage conditions.

In general, a drug substance should be evaluated under storage conditions (with appropriate tolerances) that test its thermal stability and, if applicable, its sensitivity to moisture. The storage conditions and the lengths of studies chosen should be sufficient to cover shipment, and subsequent use.

The long term testing should cover a minimum of 12 month’s duration on at least three primary batches at the time of submission and should be continued for a period of time sufficient to cover the proposed re-test period.

Stability studies were conducted on optimized formulation ( F3) assess their stability with respect to their physical parameters, drug release characteristics and swelling index after storing them at 400C with relative humidity (RH) .

Stability storage conditions

Long term*: 25°C ± 2°C/60% RH ± 5% RH or 30°C ± 2°C/65% RH ± 5% RH (12 months).

Intermediate*: 30°C ± 2°C/65% RH ± 5% RH (6 months).

Accelerated*: 40°C ± 2°C/75% RH ± 5% RH (6 months).

*It is up to the applicant to decide whether long term stability studies are performed at 25 2°C/60% RH 5% RH or 30°C 2°C/65% RH 5% RH.

**If 30°C 2°C/65% RH 5% RH is the long-term condition, there is no intermediate condition.

If long-term studies are conducted at 25°C ± 2°C/60% RH ± 5% RH and "significant change" occurs at any time during 6 months’ testing at the accelerated storage condition, additional testing at the intermediate storage condition should be conducted and evaluated against significant change criteria. Testing at the intermediate storage condition should include all tests, unless otherwise justified. The initial application should include a minimum of 6 months’ data from a 12-month study at the intermediate storage condition.

For long-term studies, frequency of testing should be sufficient to establish the stability profile of the drug substance. For drug substances with a proposed re-test period of at least 12 months, the frequency of testing at the long term storage condition should normally be every 3 months over the first year, every 6 months over the second year, and annually thereafter through the proposed re-test period. At the accelerated storage condition, a minimum of three time points, including the initial and final time points (e.g., 0, 3, and 6 months), from a 6-month study is recommended. Where an expectation (based on development experience) exists that results from accelerated studies are likely to approach significant change criteria, increased testing should be conducted either by adding samples at the final time point or by including a fourth time point in the study design. When testing at the intermediate storage condition is called for as a result of significant change at the accelerated storage condition, a minimum of four time points, including the initial and final time points (e.g., 0, 6, 9, 12 months), from a 12-month study is recommended.

If long-term studies are conducted at 25°C ± 2°C/60% RH ± 5% RH and "significant Change" occurs at any time during 6 months’ testing at the accelerated storage condition, additional testing at the intermediate storage condition should be conducted and evaluated against significant change criteria. The initial application should include a minimum of 6 months’ data from a 12-month study at the intermediate storage condition.

In general, "significant change" for a drug product is defined as:

A 5% change in assay from its initial value; or failure to meet the acceptance criteria for potency when using biological or immunological procedures;

Any degradation product’s exceeding its acceptance criterion;

Failure to meet the acceptance criteria for appearance, physical attributes, and functionality test (e.g., color, phase separation, resuspendibility, caking, hardness, dose delivery per actuation); however, some changes in physical attributes ( e.g., softening of suppositories, melting of creams may be expected under accelerated conditions, as appropriate for the dosage form.

4. Failure to meet the acceptance criterion for pH; or

5. Failure to meet the acceptance criteria for dissolution for 12 dosage units.

"Significant change" for a drug substance is defined as failure to meet its specification

Similarity factor (f2) analysis: 130

In vitro release profile of optimized formulation (F-4) was performed under variable condition like different speed of rotation, types of dissolution, effect of storage. The similarity factor (f2) was determined using the data obtained from drug release studies. If f2 greater than 50 it is considered that the product share similar drug release behaviours.Here dissolution profile of optimized batch was compared after it subjected to various process variable effect like speed of rotation, apparatus, effect of storage. The data were analyzed by the formula shown in the following equation.

f2 – 50 log{ [1+(1/N) (Ri – Ti)2 ]-0.5 X100}

Where N = Number of time points Ri and Ti = dissolution of reference and test product at time i

2.25.Assay (by HPLC) 131

Table.7:Chromatographic parameters for assay of Agnuside by HPLC

1.

Column

C18 4.6X250mm, 5µ

2.

Flow rate

0.6 ml/min.

3.

Wavelength

254 nm

4.

Injection volume

10 µl

5.

Column oven temperature

25ºC

6.

Sample compartment temp.

25ºC

7.

Run time

30 min.

8.

Mobile phase

2%Acetic acid + 30% methanol

Procedure: An aliquot of powder equivalent to 10mg was accurately weighed and transferred to 50 mL volumetric flask containing 25 mL of methanol and then volume was made up to the mark with methanol. The flask was sonicated for 25 min to affect complete dissolution. The solution was filtered through Whatmann filter paper No. 41. Suitable aliquot of the filtered solution was transferred into a 100 mL volumetric flask and made up to the volume with mobile phase to yield the concentration of 20μg/mL. The experiment was performed six times under the chromatographic conditions described above. The peak areas were measured at 254 nm and concentration in the sample was determined by comparing the area of sample with that of the standard.