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1. Phytochemical Investigation 1.1.Qualitative Analysis 1.1.1.

Extraction Powder the air dried plant leaves/seeds Soxhlet extraction with Petroleum ether Filter and evaporate Petroleum ether extract Marc Chloroform Filter and evaporate Chloroform extract Marc Methanol Filter and evaporate Methanolic Extract Marc Water Filter and evaporate Aqueous Extract Marc

1.2.Qualitative Chemical Tests 1.2.1. Detection of Starch Iodine test Powdered plant material will be mounted on the glass slide and then iodine solution will be added to it and kept under the microscope. Blue colour wil indicate the presence of starch.

Sodium chloride test Powdered plant material will be mounted on the glass slide and then few drops of NaCl will be added to it and kept under the microscope. Starch globules dissolves, which indicates the presence of starch. Water test Powdered plant material will be mounted on the glass slide and then few drops of water will be added to it and kept under the microscope. Starch globules ultimately gelatined on heating which indicates presence of starch. 1.2.2. Detection of Proteins Iodine test Powdered plant material will be mounted on the glass slide and then iodine solution will be added to it and kept under the microscope. Crystalloids, yellow in colour indicates presence of proteins. Picric acid test Powdered plant material will be mounted on the glass slide and then alcoholic solution of picric acid will be added to it and observed under the microscope. Crystalloids yellow in colour indicates presence of proteins. Millons reagent test Powdered plant material will be mounted on the glass slide. It will be then treated with Millons reagent, warmed and observed under the microscope. Crystalloids yellow in colour indicates presence of proteins. 1.2.3. Detection of Fixed Oil and Fats Sudan red test Powdered plant material will be mounted on the glass slide and treated with Sudan red III solution. It will be then observed under the microscope. Red colour indicates presence of fixed oil and fats. Ether, benzene and chloroform test Powdered plant material will be mounted on the glass slide and few drops of ether, benzene and chloroform will be added to it and observed under the microscope. Fats and fixed oil dissolves which indicates presence of fixed oil and fats.

1.2.4. Detection of Tannins Ferric chloride test Powdered plant material will be mounted on the glass slide and then solution of ferric chloride will be added to it and observed under the microscope. Bluish black or greenish black colour will indicate the presence of fixed oil and fats. 1.2.5. Detection of Alkaloids Extracts will be dissolved individually in dilute hydrochloric acid and filtered. The filtrates will be used to test for the presence of alkaloids. Mayers test Filtrates will be treated with Mayers reagent (saturated solution of potassium mercuric iodide). Formation of yellow cream precipitates indicates the presence of alkaloids. Wagners test Filtrates will be treated with Wagners reagent (saturated solution of iodine in potassium iodide). Formation of reddish brown precipitates indicates the presence of alkaloids. Dragendorffs test Filtrates will be treated with Dragendorffs reagent (saturated solution of potassium bismuth iodide). Formation of red precipitates indicates the presence of alkaloids. Hagers test Filtrates will be treated with Hagers reagent (saturated solution of picric acid). Formation of yellow colour precipitates indicates the presence of alkaloids. 1.2.6. Detection of Carbohydrates Extracts will be dissolved individually in 5 ml distilled water and filtered. The filtrates will be used to test for the presence of carbohydrates. Molischs test Filtrates will be treated with 2 drops of alcoholic -naphthol solution in a test tube and 2 ml of conc. sulphuric acid will be added carefully along the sides of the test tube. Formation of violet ring at the junction indicates the presence of carbohydrates. Benedicts test Filtrates will be treated with Benedicts reagent and heated on water bath. Formation of orange red precipitates indicates the presence of reducing sugars.

Fehlings test Filtrates will be hydrolysed with dil. hydrochloric acid neutralized with alkali and heated with Fehlings A and B solutions. Formation of red precipitates indicates the presence of reducing sugars. 1.2.7. Detection of Glycosides Extracts wil be hydrolyzed with dil. hydrochloric acid and then subjected to test for glycosides. Modified Borntragers test Extracts will be treated with ferric chloride solution and immersed in boiling water for about 5 minutes. The mixture will be cooled and shaken with an equal volume of benzene. The benzene layer will be separated and treated with ammonia solution. Formation of rose-pink colour in the ammoniacal layer indicates the presence of anthranol glycosides. Legals test Extracts will be treated with sodium nitroprusside in pyridine and methanolic alkali. Formation of pink to blood red colour indicates the presence of cardiac glycosides. 1.2.8. Detection of Saponins Froth test Extracts will be diluted with distilled water to 20 ml and shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins. Foam test Small amount of extract will be shaken with little quantity of water. The foam produced persisted for ten minutes, indicates the presence of saponins. 1.2.9. Detection of Phytosterols Salkowskis test Extracts will be treated with chloroform and filtered. The filtrates will be treated with few drops of conc. sulphuric acid, shaken and allowed to stand. Appearance of golden yellow colour indicates the presence of triterpenes. 1.2.10. Detection of Fixed Oils and Fats Stain test Small quantities of extracts will be pressed between two filter papers. An oily stain on filter paper indicates the presence of fixed oil.

1.2.11. Detection of Phenols Ferric chloride test Extracts will be treated with few drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenols. 1.2.12. Detection of Tannins Gelatin test To the extract, gelatin solution (1%) containing sodium chloride will be added. Formation of white precipitate indicates the presence of tannins. 1.2.13. Detection of Flavonoids

Alkaline reagent test Extracts will be treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which became colourless on addition of dilute acid, indicates the presence of flavonoids. Lead acetate test Extracts will be treated with few drops of lead acetate solution. Formation of yellow colour precipitates indicates the presence of flavonoids. Zinc hydrochloric acid reduction test To the alcoholic solution of extract, a pinch of zinc dust and conc. HCl will be added. Appearance of magenta colour after few minutes indicates presence of flavonoids. 1.3.Quantitative Analysis 1.3.1. Primary metabolite estimation Extraction of carbohydrates Total soluble sugars The dried and milled test sample 50 mg each will be macerated in a grinder with 20 ml of ethanol and left for 12 hrs. and mixtures will be centrifuged (1200 rpm) for 15 min, the supernatants will be removed and concentrated on a water-bath. The volume of these aqueous concentrates will be raised to 50 ml with distilled water (Ext. A) and processed further by following the method of Loomis and Shull for soluble sugars. However, the residual pellet obtained by centrifugation will be used for the estimation of starch.

Starch

The above residue of each test sample will be suspended in a mixture of 5 ml of 52% perchloric acid solution and 6.5 ml of distilled water, shaken vigorously (5 min) and centrifuged (2500 rpm). This step will be repeated three times and the supernatants of each sample will be pooled and the volume will be raised to 100 ml with distilled water (Ext B). Out of this (Ext. B), 1 ml aliquot will be taken separately to estimate starch quantitatively. Quantification of carbohydrates: Aliquot (1ml) of each of the test sample from Ext. A and B will be used to quantifying the total levels of carbohydrates using phenols-sulphuric acid method. A regression curve for standard sugar (glucose) will also be prepared. A stock solution of glucose (100 g/ ml) will be prepared in distilled water, out of which 0.1 to 0.9 ml will be transferred to test tube and the volume will be raised to 1 ml with distilled water. To each of these, 1 ml of 5% aqueous phenol will be added rapidly having kept in an ice chest and shaken gently. Later 5 ml of Conc. H2SO4 will be rapidly added by agitating gently during the addition of the acid subsequently, the tube will be kept on a water-bath (26 30C) for 20 min, and the optical density (ODs) of the yellow orange colors thus developed will be taken at 490 nm in a Spectrophotometer after having set it for 100% transmission against the blank. Four replicates of each sample will be run and there mean values will be calculated. A regression will be computed between its known concentrations and their respective ODs. This will be based on Beers Law. The concentration (mg/gdw) of the total soluble sugars will directly worked out from the regression curve of the standard glucose. Four replicates of each experimental sample will be taken and their mean values will be recorded. The sugar content in terms of glucose equivalent and the use of conversion factor (0.9 to convert the values of glucose to starch) will be made in each case. Extraction of Proteins A 60 mg of the dried test sample will be macerated in 10 ml of cold TCA (10%) for 30 min kept at low temperature 4 C for 24 hr and then it will be centrifuged. Each of the supernatants will be discarded and the resultant pellet will be re-suspended in 5% TCA (10 ml) and heated on a water bath at 80 C for 30 min. Each of these samples will be cooled, re-centrifuged and each time the supernatant discarded. Later the pellet will be washed with distilled water, centrifuged and each of the residues will be dissolved in 1N NaOH (10 ml) and left overnight at room temperature. Quantification of Proteins

In each of 1 ml extract, total protein content was estimated using the protocol of Lowry et al. A stock solution (1mg/ml) of bovine serum albumin will be prepared in 1 N NaOH, from which 0.1 to 0.9 ml of the solution will be dispensed separately in a test tube. After this, the volume of each will be raised to 1 ml by adding distilled water. To each test sample, 5ml of freshly prepared alkaline solution (prepared by mixing 50 ml of 2% Na2CO3 in 0.1 N NaOH and 1 ml of 0.5%CuSO4. 5H2O in 1% sodium potassium tartrate) will be added at room temperature and left undisturbed for a period of 10 min. Subsequently, to each of these mixture tubes 0.5 ml of Folin-Ciocalteau reagent will be rapidly added after half an hr, the OD of each will be measured at 750 nm using a spectrophotometer against the blank. Three replicates of each concentration will be taken and there mean values will be used to compute a regression curve. The total protein content in each sample will be calculated by referring the ODs of test sample with the standard curve of BSA. Three replicates will be examined in each case and their mean values were recorded. Extraction of Lipids One g of each of the dried and milled test sample will be macerated with 10ml distilled water. To this, 30 ml of chloroform-methanol (2: 1, v/v) will be added and mixed thoroughly. Each mixture will be left overnight at room temperature; 20 ml of chloroform and the equal volume of distilled water will be added and centrifuged. Out of the three layers, a clear lower layer of chloroform containing all lipids will be collected in pre-weighted beaker, the solvent evaporated completely and weighed, which will be taken as the weight of total lipids/g of the dried tissue sample. Extraction of Phenols Each of 200 mg dried and milled test samples will be homogenized in 80% ethanol (10 ml) for 2 hrs and left over night at room temperature. It will be centrifuged, the supernatants will be collected individually and the volume of each will be raised to 40 ml with 80% ethanol. Quantification of Phenol To estimate total phenols in each of the test sample, the protocol of Bray and Thorpe will be followed, wherein a standard curve of caffeic acid (a phenol) will be prepared. A stock solution (100 g/ml) of caffeic acid will be prepared in 80% ethanol, from which 0.1 to 0.9 ml will be transferred into test-tubes separately and the volume in each case will be raised to 1 ml with 80% ethanol. To each of these tubes, 1 ml of FolinCiocalteau reagent (prepared by diluting the

reagent with distilled water in 1:2 ratio just before use) accompanied by 2 ml of 20% Na2CO3 solution will be added and the mixture will be shaken vigorously. Each of these will be boiled on a water bath (1 min), cooled and diluted to 25 ml with distilled water. The OD will be taken at 750 nm using a spectrophotometer against a blank. Three such replicates will be taken for each concentration and the average OD will be plotted against the respective concentration to compute a regression curve. Each test sample will be processed in this similar manner, ODs will be measured and the total level of phenols will be calculated from the mean values (with reference to caffeic acid) by referring the OD of the test sample with the regression curve of the standard. 1.3.2. Secondary Metabolite Estimation 1.3.2.1.Total glycoside content ( Test for Anthraquinone Glycosides) Sample (0.3g) Add 30 ml of water, mix, weigh and reflux for 15 min

Aqueous mixture Allow to cool, weigh, adjust to the original weight with water Centrifuge at 4000 rpm for 10 min

Residue

Supernatent 1. 20 ml supernatant + 2 M HCl 0.1 ml 2. Extract with chloroform 15 ml x 3 times

Aqueous layer 1. Add 0.1 g NaHCO3, Shake for 3 min 2. Centrifuge 4000 rpm for 20 min Glycoside fraction

Chloroform layer (discard)

1. 10 ml supernatant + 20 ml 10.5 % (w/v) FeCl3.6H2O 2. Add 1 ml conc. HCl, reflux for 20 min Aglycone Extract with ether 25 ml x 3 times

Ether layer 1. Wash with 15 ml H2O x 2 times 2. Adjust to 100 ml with ether Ether layer (100 ml) Dissolve to 25 ml and evaporate to dryness Residue

Aqueous layer (discard)

Dissolve with 10 ml 0.5 % (w/v) magnesium acetate in methanol Solution containing anthraquinone glycosides Measure absorbance at 515 nm % of total anthraquinone glycosides

1.3.2.2.Total Alkaloid content One hundred mgs of finely powdered material and 40ml of 95% ethanol will be refluxed in a 100ml flask for 30min. the extract will be filtered; the residue will be washed twice with 2ml of ethanol. The washings will be added to the original filtrate and transferred into a 50ml standard flask, the volume being adjusted to the mark with 95% ethanol. 5ml of this solution will be pipetted into a test tube and ethanol will be completely removed by evaporation on a water bath. The residue will be treated with 3ml of 1N HCl and refluxed for two hours for hydrolysis. The acid will be neutralized by adding 3ml of 1N NaOH, two mo of concentrated acetic acid will be added and the contents will be transferred to a 10ml standard flask, the volume being adjusted to the mark with water. One ml of this solution will be equivalent to 1mg of dry material. Then it will be pipetted 0,1,2 and 3 of 40mcg/ml standard solution in respective seprators and the volume of each will be made up to 5ml with 20% aceticacid, to each separator 5 ml of acetate buffer and 1ml of methyl orange awill be added. After shaking for 10 sec. 5 ml of chloroform will be added. The separators will be stopped and shaken for 3 min. After standing for a few minutes chloroform layers will be withdrawn into dry test tubes, dried with small amount of anhydrous Na2SO4 and absorbances will be read on a spectrophotometer at 420 nm. 1.4.Marker Analysis using HPLC-DAD System 1.4.1. Withania sominifera