Amla Juice Powder. Arjuna Dry Extract. Identification. Tests
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1 HERBS AND HERBAL PRODUCTS Amla Juice Powder Amla Dry Extract is obtained from the dried fruit pericarp of Phyllanthus emblica Linn. (Emblica officinalis Gaertn, Family- Euphorbiaceae) fruits by cold pressed juice and spray dried. Amla Dry Extract contains not less than 40.0 per cent w/w of the stated amount of tannins, calculated on the dried basis. It may contain suitable added substances. Description. A beige to off-white powder. Determine by thin-layer chromatography (2.4.17) coating the plate with silica gel GF254 Mobile phase. A mixture of 15 volumes of ethyl acetate, 1.5 volume of methanol and 1 volume of water. Test Solution. Dissolve 500 mg of the extract under examination with 10 ml of water and filter it into a clear solution. Reference solution. A 0.01 per cent w/v solution of gallic acid RS in water. Apply to the plate 10 μl of each solution as bands of 10 mm by and examine in ultraviolet light at 254 nm. Spray the plate with a anisaldehyde- sulphuric acid reagent solution. Heat the plate at 110 for 10 minutes and examine the plate under 365 nm and in day light. The chromatographic profile of the test solution is similar to that of the reference solution. Water - soluble extractive (2.6.3). Not less than 85.0 per cent. Total Ash (2.3.19). Not more than 15.0 per cent. Water (2.3.43). Not more than 5.0 per cent. heavy metals. Method B (20 ppm). Gallic acid. Determine by liquid chromatography(2.4.14). Not more than 2.0 per cent. Test Solution. Dissolve about 50 mg of the extract under examination or quantity equivalent to 10 mg of gallic acid RS in water, by frequently shaking and make it up to 50.0 ml and filter. Reference solution. A 0.01 per cent w/v solution of gallic acid RS in water. a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded porous silica (5μm), mobile phase: filtered and degassed gradient mixtures of 0.1 per cent formic acid in water and methanol, flow rate. 0.7 ml per minute, a linear gradient programme using the conditions given below, spectrophotometer set at 272 nm, Time Buffer solution Acetonitrile (min) (per cent v/v) (per cent v/v) relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject 20 μl of the test solution. Calculate the content of the gallic acid in extract. Assay. Weigh accurately about 2 g of the extract, add 50 ml boiling water and heat it on a water bath for 30 minutes with frequent stirring. Allow the solution to settle and carefully decant it through a piece of cotton wool to a 500 ml volumetric flask. Repeat the extraction for 5 times (5 x 50ml of boiling water). To confirm that all tannins have been extracted, add 3-4 drops of ferric ammonium sulphate solution (8 per cent w/v in water) to 5ml of the extract. Blue colour will not be produced, if all tannins have been extracted. If blue colour develops extract again with 2 x 50 ml of boiling water and check with ferric ammonium sulphate again. Cool the extracts and make up to the mark with water. Pipette out the above extract 50 ml into a 250 ml conical flask and add 50 ml of indigo sulphonic acid solution, prepared by dissolving 1 g of indigo carmin in 50 ml of concentrated sulphuric acid, add 500 ml of water and dilute this solution to ml. Titrate, with 0.1 M potassium permanganate solution using indigo sulphonic acid as indicator until a golden yellow colour is produced. Carry out a blank titration. 1 ml of 0.1 M potassium permanganate solution is equivalent to g of tannin compound calculated as tannic acid. Arjuna Dry Extract Arjuna Dry Extract is obtained from the Terminalia arjuna Wight & Arn (Fam. Combretaceae) bark or wood by extraction with methanol or any other suitable solvent. Arjuna Dry Extract contains not less than 60.0 per cent stated 1
2 HERBS AND HERBAL PRODUCTS IP 2010 amount of the arjunolic acid on the dried basis. It may contain suitable added substances. Description. A light brown to beige powder with or without red tinge. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel GF254. Mobile phase. A mixture of 92 volumes of chloroform and 8 volumes of methanol. Test Solution. Dissolve 50 mg of the extract under examination with 50.0 ml of methanol and filter it into a clear solution. Reference solution. A 0.1 per cent w/w solution of arjunolic acid RS in the methanol. Apply to the plate 5 μl of each solution as bands of 10 mm by and spray with anisaldehyde sulphuric acid reagent solution. Heat the plate at 110 for 10 minutes and examine the plate in ultraviolet light at 365 nm and day light. The chromatographic profile of the test solution is similar to that of the reference solution. Ethanol - soluble extractive (2.6.2). Not less than 80.0 per cent. Total Ash (2.3.19). Not more than 5.0 per cent. Loss on drying (2.4.19). Not more than 5.0 per cent determined Test Solution. Shake a quantity of the extract under examination containing about 50 mg of arjunolic acid in 50.0 ml of the methanol, filter. Reference solution. A 0.1 per cent w/v solution of arjunolic acid RS in methanol. a stainless steel column, 25 cm x 0.46 cm packed with octadecylsilane bonded to porous silica (5 μm), mobile phase: a mixture of 35 volumes of 5 mm â- Cyclodextrin and 65 volumes of methanol, flow rate. 1.0 ml per minute, spectrophotometer set at 205 nm, than 2.0 per cent. Calculate the content of the arjunolic acid in the extract. Ashwagandha Dry Extract Ashwagandha Dry Extract is obtained from the Ashwagandha (Withania somnifera) rhizomes by extraction with methanol or alcohol or any other suitable solvent. Ashwagandha Dry Extract contains not less than 0.25 per cent of the stated amount of withaferin A, not less than 7.0 per cent and not more than 10.0 per cent w/w of the total withanolides calculated as the sum of free withanolides and glycowithanolides, with not less than 1.0 per cent of alkaloids, calculated on the dried basis. Description. A pale brown to light brown powder. Determine by thin layer chromatography (2.4.17) coating the plate with silica gel GF254. Mobile phase. A mixture of 5 volumes of toluene, 5 volumes of ethyl acetate and 1 volume of formic acid. Test Solution. Dissolve about 200 mg of the extract under examination with 10 ml of methanol and filter. Reference solution. A 0.05 per cent w/v solution of withaferin A RS in methanol. Apply to the plate 10 μl of each solution as bands of 10 mm x and examine in ultraviolet light at 254 nm. Spray the plate with anisaldehyde-sulphuric acid reagent solution. Heat the plate at 110 for 10 minutes and examine the plate in 365 nm and under day light. The chromatographic profile of the test solution is similar to that of the reference solution. Water - soluble extractive (2.6.3). Not less than 70.0 per cent. Sulphated Ash (2.3.18). Not more than 10.0 per cent. Loss on drying (2.4.19). Not more than 5.0 per cent, determined 2
3 Total alkaloids. Test solution. Shake about 20 g of the extract with 400 ml of a solution containing 4 volumes of ether and 1 volume of ethanol. Add 20 ml of 5 per cent v/v ammonia solution into 1000 ml conical flask and shake for one hour. Decant and filter through cotton. Transfer the filtrate into a separator. Wash the residue with 2 x 50 ml of ether alcohol mixture (4:1).To the combined ether alcohol solution, add 100 ml of 0.5 M sulphuric acid. Shake well and allow to separate. Collect the lower layer into another separator. To the ether alcohol layer; add 100 ml of 0.25 M sulphuric acid and alcohol mixture (4:1) and extract. Continue the extraction with 3 x 80 ml of the 0.25 M sulphuric acid and ether alcohol mixture (4:1) until aqueous layer is colourless. Combine the acid solution and wash with 40 ml of chloroform followed by 2 x 20 ml of chloroform. Wash, the combined chloroform layer, with acid alcohol mixture. Discard the chloroform layer. Combine the acid alcohol solutions and make it alkaline with 5 per cent v/v ammonia solution and add 10 ml excess. Extract with 3 x 100 ml of chloroform. Add 2 ml of 0.1 M hydrochloric acid to 0.5 ml of the extract, remove the chloroform by evaporation, transfer the aqueous residue to a test tube and add 0.05 ml of potassium mercury - iodide solution; not more than a very faint opalescence is produced. If the opalescence is more, the extraction is not complete. Extract further with 2 x 100 ml of chloroform. Combine the chloroform extract and wash with 20 ml of distilled water. Filter the chloroform layer through cotton plug into a tared beaker. Wash the residue with a little chloroform, transferring the chloroform layer to the same tared beaker. Evaporate on a water bath. Add 5 ml of alcohol to the residue and dry to constant weight at 105º. Finally weigh the residue and calculate the content of total alkaloids. Total withanolides. Shake about 5 g of dry extract with 25 ml of methanol and 25 ml of water in a separating funnel. It is defatted by extraction with hexane (4 x 50 ml). This fraction is discarded. The remaining aqueous methanolic solution is extracted with hexane and then with ether (5 x 25 ml). The ether extracts are combined, washed twice with water and dried under vacuum to give the free withanolides content. Finally weigh the residue and calculate the content of free withanolides. Glycowithanolides. The above remaining aqueous methanolic solution is extracted with chloroform- methanol (2:1) (3 times). The chloroform- methanol extracts are combined and evaporated on water bath and dried under vacuum at 80º to constant weight. Finally weigh the residue and calculate the content of glycowithanolides. Calculate the content of total withanolides as the sum of free withanolides and glycowithanolides. Test solution. Dissolve a quantity of the extract under examination containing about 25 mg of withaferin A in 25.0 ml of water, filter. Reference solution. A 0.1 per cent w/v solution of withaferin A RS in methanol. a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (10 μm), mobile phase: a mixture of 65 volumes of 0.1 per cent v/ v solution of orthophosphoric acid and 35 volumes of acetonitrile, flow rate. 1.0 ml per minute, spectrophotometer set at 220 nm, than 2.0 per cent. Calculate the content of the withaferin A in the extract. Belladonna Tincture Belladonna Tincture obtained from Belladonna leaf of one or more of the cultivated varieties of Atropa belladonna Linn. or of A. acuminate (Fam. Solanaceae) or a mixture of both species. Belladonna Tincture contains not less than per cent and not more than per cent of total alkaloids, calculated as hyoscyamine, C 17 H 23 NO 3. The alkaloids consists mainly of hyoscyamine together with small quantity of hyoscine. Description. A clear green or brownish green liquid. A. Determine by thin-layer chromatography (2.4.17), coating Mobile phase. A mixture of 10 volumes of anhydrous formic acid, 10 volumes of water, 30 volumes of methyl ethyl ketone and 50 volumes of ethyl acetate. Test solution. Evaporate to dryness 10 ml of the tincture under examination in a water-bath at 40 under reduced pressure. Dissolve the residue in 1.0 ml of the methanol. Reference solution. Dissolve 1 mg of chlorogenic acid RS and 2.5 mg of rutin RS in 10 ml of the methanol. 3
4 Apply to the plate 40 μl of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 15 cm. Dry the plate in air, heat the plate at 110 for 10 minutes. Spray the warm plate with 1 per cent v/v solution of diphenylboric acid aminoethyl ester in methanol. Allow to cool and spray with 5 per cent v/v solution of macrogol 400 in methanol, allow the plate to dry in air for 30 minutes and examine in ultraviolet light at 365 nm. The chromatographic profile of the test solution is similar to that of the reference solution. S B. Atropine. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 3 volumes of strong ammonia solution, 7 volumes of water and 90 volumes of acetone. Test solution. To 15.0 ml of the tincture under examination, add 15 ml of 0.05 M sulphuric acid and filter. Add 1 ml of strong ammonia solution to the filtrate, with two quantities, each of 10 ml, of peroxide-free ether, separate the ether layer by centrifugation if necessary, dry the combined ether extracts over anhydrous sodium sulphate, filter and evaporate to dryness on a water-bath. Dissolve the residue in 0.5 ml of methanol. Reference solution. Dissolve 50 mg of hyoscyamine sulphate in 9 ml of methanol and 15 mg of hyoscine hydrobromide in 10 ml of methanol, separately. Mix 1.8 ml of the hyoscine hydrobromide solution and 8 ml of the hyoscyamine sulphate solution. Apply to the plate 20 μl and 40 μl of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 10 cm. Dry the plate at 105 for 15 minutes, spray with potassium iodobismuthate solution, dry the plate and spray with sodium nitrile solution until the plate is transparent. Examine the plate after 15 minutes in day light. The chromatogram obtained with the test solution corresponds to the chromatogram obtained with the reference solution. Ethanol (2.3.45). Method III, 64 to 69 per cent v/v. Assay. Evaporate 50.0 g of the tincture under examination to a volume of about 10 ml. Transfer quantitatively to a separating funnel, with the minimum volume of ethanol (70 per cent v/ v). Add 5 ml of strong ammonia solution and 15 ml of water. Extract with three quantities, each of 40 ml of a mixture of 1 volume of dichloromethane and 3 volumes of peroxide-free ether, carefully to avoid emulsion, until the alkaloids are completely extracted. Combine the dichloromethane and ether extracts, concentrate the solution to a volume of about 50 ml by heating on a water-bath. Transfer the resulting solution quantitatively to a separating funnel, rinsing with peroxidefree ether. Add a quantity of peroxide-free ether equal to at least 2.1 times the volume of the solution to produce a layer having a density well below that of water. Extract the resulting solution with minimum of three quantities, each of 20 ml of 0.25 M sulphuric acid until the alkaloids are completely extracted. Separate the layers by centrifugation if necessary and transfer the layers to a separating funnel. Make the combined layers alkaline with strong ammonia solution and extract with minimum of three quantities, each of 30 ml of dichloromethane until the alkaloids are completely extracted. Combine the dichloromrthane and ether extracts, add 4 g of anhydrous sodium sulphate and allow to stand for 30 minutes with occasional shaking. Decant the dichloromethane and filter. Wash the sodium sulphate with three quantities, each of 10 ml of dichloromethane. Combine the dichloromethane and ether extracts, evaporate to dryness on a water-bath. Heat the residue in an oven at 105 for 15 minutes. Dissolve the residue in a few ml of dichloromethane, evaporate to dryness on a water-bath and heat the residue in an oven at 105 for 15 minutes again. Dissolve the residue in a few ml of dichloromethane. Add 20.0 ml of 0.01 M sulphuric acid and remove the dichloromethane by evaporation on a waterbath. Titrate the excess of acid with 0.02 M sodium hydroxide using methyl red mixed solution as indicator. 1 ml of 0.01 M sulphuric acid is equivalent to g of total alkaloids calculated as hyoscyamine. Calculate the content of total alkaloids with reference to the dried material. Storage. Store protected from light and moisture. Bhibhitaki Aqueous Extract Bhibhitaki Dry Extract is obtained by extracting Bhibhitaki (Terminalia belerica) fruit with water. Bhibhitaki Dry Extract contains not less than 90.0 per cent w/w and not more than per cent w/w of the stated amount of gallic acid and ellagic acid. Usual strength. A 5.0 per cent w/w gallic acid and 0.5 per cent w/w ellagic acid. Description. A light brown to dark brown powder. Determine by thin-layer chromatography (2.4.17), coating the plate with silica gel G. Mobile phase. A mixture of 35 volumes of benzene, 10 volumes of methanol, 4 volumes of acetone and 1 volume of water. Test solution. Dissolve 500 mg of the extract under examination in 20 ml of methanol under reflux at 80 o on a water bath and filter. Reference solution. A 0.01 per cent w/v solution of gallic acid RS in methanol. Apply to the plate 10 μl of each solution as bands 10 mm by 2 4
5 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air and examine in ultraviolet light at 254 nm. The chromatographic profile of the test solution is similar to that of the reference solution. Acid insoluble ash (2.3.19). Not more than 4.0 per cent. Loss of drying (2.4.19). Not more than 5.0 per cent, determined on 1.0 g by drying in an oven at 105. Test solution. Dissolve about 200 mg of the extract under examination or a quantity containing 10 mg of gallic acid and 1 mg of ellagic acid in 80 per cent v/v methanol and dilute to ml with water and filter. Reference solution (a). A 0.01 per cent w/v solution of gallic acid RS in 80 per cent v/v methanol in water. Reference solution (b). A per cent w/v solution of ellagic acid RS in 80 per cent v/v methanol in water. a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5 μm), mobile phase: A mixture of acetonitrile and a 0.1 per cent v/v orthophosphoric acid in water, a linear gradient programme using the conditions given below, flow rate. 1 ml per minute, spectrophotometer set at 252 nm for ellagic acid and at 271 nm for gallic acid, a 20 μl loop injector. Time Buffer solution Acetonitrile (in min) (per cent v/v) (per cent v/v) Inject reference solution (a) and (b). The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution, reference solution (a) and (b). Calculate the content of gallic acid and ellagic acid in the extract. Coleus Dry Extract Coleus Dry Extract is obtained from the Coleus (Coleus Forskohlii Willd. Briq., Family- Lamiaceae) roots by extraction with alcohol or methanol or any other suitable solvent. Coleus Dry Extract contains not less than 10.0 per cent w/w to not more than 12.0 per cent w/w of the stated amount of forskolin, calculated on the dried basis. It may contain suitable added substances. Description. A light brown to brown colour powder. A. Determine by thin-layer chromatography (2.4.17), coating Mobile phase. A mixture of 40 volumes of ethyl acetate and 60 volumes of hexane. Test Solution. Dissolve about 500 mg of the extract under examination with 10 ml methanol, filter. Reference solution. A 0.1 per cent w/v solution of forskolin RS in methanol. Apply to the plate 10 μl of each solution as bands of 10 mm by and spray the plate with a anisaldehyde-sulphuric acid reagent solution. Heat the plate at 110 for 10 minutes and examine in ultraviolet light at 365 nm and in day light. The chromatographic profile of the test solution is similar to that of the reference solution. Ethanol-soluble extractive (2.6.2). Not less than 30.0 per cent. Water-soluble extractive (2.6.3). Not less than 30.0 per cent. Total Ash (2.3.19). Not more than 10.0 per cent. Loss on drying (2.4.19). Not more than 6.0 per cent, determined Test Solution. Dissolve a quantity of the extract under examination containing about 50 mg of forskolin in 50.0 ml of methanol,filter. Reference solution. A 0.1 per cent w/v solution of forskolin RS in the methanol. a stainless steel column packed with octadecylsilane 5
6 bonded to porous silica 0.46 cm x 25 cm of dimension. mobile phase: a mixture of 50 volumes of water and 50 volumes of acetonitrile, flow rate. 1 ml per minute, spectrophotometer set at 210 nm, than 2.0 per cent. Calculate the content of the forskolin in the extract. Curcuminoids Dry Extract Curcuminoids Dry Extract is a partially purified natural complex of diaryl heptanoid derivatives isolated from the Curcuma longa L. (Fam. Zingiberaceae), with acetone or ethanol or any other suitable solvent. Curcuminoids Dry Extract contains not less than 95.0 per cent and not more than per cent of the stated amount of total curcuminoids calculated on the dried basis, as the sum of curcumin, demethoxycurcumin and bisdemethoxycurcumin. It contains not less than 70.0 per cent to not more than 80.0 per cent of curcumin, not less than 15.0 per cent to not more than 25.0 per cent of demethoxycurcumin and not less than 2.5 per cent to not more than 6.5 per cent of bisdemethoxycurcumin. Description. An orange yellow crystalline powder. A. Determine by thin layer chromatography (2.4.17) coating Mobile phase. A mixture of 10 volumes of chloroform, 1.0 volume of methanol, 0.5 volume of glacial acetic acid and 0.5 volume of formic acid. Test Solution. Dissolve about 50 mg of the extract under examination with ml methanol, filter. Reference solution. A solution containing 0.01 per cent w/v each of the bisdemethoxycurcumin RS1, demethoxycurcumin RS2, curcumin RS in the methanol. Apply to the plate 10 μl of each of solution as bands of 10 mm by 2 mm. Allow the mobile phase to rise 8 cm. Dry the plate in air and examine in ultraviolet light at 254 nm. Spray the plate with a anisaldehyde- sulphuric acid reagent solution. Heat the plate at 110 for 10 minutes and examine the plate in 365 nm and day light. The chromatographic profile of the test solution is similar to that of the reference solution. Sulphated ash (2.3.18). Not more than 1.0 per cent. Loss on drying (2.4.19). Not more than 2.0 per cent determined Assay. Total curcuminoids Determine by liquid chromatography (2.4.14). Test Solution. Dissolve a quantity of the extract under examination containing about 50 mg curcumin RS3 in ml of tetrahydrofuran. Dilute 5 ml of this solution to 50 ml with the mobile phase. Reference solution. A solution containing 0.01 per cent w/v each of bisdemethoxycurcumin RS1, demethoxycurcumin RS2, curcumin RS in ml of tetrahydrofuran. Dilute 5ml of this solution to 50 ml with the mobile phase. a stainless steel column, 25 cm x 0.46 mm packed with octadecylsilane bonded to porous silica (5μm), mobile phase: a mixture of 40 volumes of 0.1 per cent w/v solution of citric acid and 60 volumes of tetrahydrofuran, flow rate. 1.0 ml per minute, spectrophotometer set at 420 nm, than 2.0 per cent. The relative retention time with reference to curcumin for bisdemethoxycurcumin is about 1.3 and for demethoxycurcumin is about Calculate the content of the bisdemethoxycurcumin, demethoxycurcumin and curcumin in extract. Fenugreek Fenugreek is dried, ripe seeds of Trigonella foenum-graecum L. Description. The seed is hard, flattened, brown or reddishbrown and more or less rhomboidal with rounded edges. The widest surfaces are marked by a groove that divides the seed 6
7 into 2 unequal parts. The smaller part contains the radicle; the larger part contains the cotyledons. They have a strong aromatic characteristic odour. A. Macroscopic Seed is rhomboidal with rounded edges. It is 3 mm to 5 mm long, 2 mm to 3 mm wide and 1.5 mm to 2 mm thick. The widest surfaces are marked by a groove that divides the seed into two unequal parts. The smaller part contains the radicle; the larger part contains the cotyledons. B. Reduce to a powder. The powder is yellowish-brown. Examine under a microscope using chloral hydrate solution. The powder shows fragments of the testa in sectional view with thick cuticle covering lageniform epidermal cells, with an underlying hypodermis of large cells, narrower at the upper end and constricted in the middle, with bar-like thickenings of the radial walls; yellowish-brown fragments of the epidermis in surface view, composed of small, polygonal cells with thickened and pitted walls, frequently associated with the hypodermal cells, circular in outline with thickened and closely beaded walls; fragments of the hypodermis viewed from below, composed of polygonal cells whose bar-like thickenings extend to the upper and lower walls; parenchyma of the testa with elongated, rectangular cells with slightly thickened and beaded walls; fragments of endosperm with irregularly thickened, sometimes elongated cells, containing mucilage. C. Determine by thin-layer chromatography (2.4.17), coating Mobile phase. A mixture of 30 volumes of water and 70 volumes of methanol. Test solution. To 1.0 g of the powdered substance under examination, add 5.0 ml of the methanol. Heat on a water-bath at 65 for 5 minutes, cool and filter. Reference solution. Dissolve 3 mg of trigonelline hydrochloride in 1.0 ml of methanol. Apply to the plate 20 μl of each solution as bands 10 mm by 2 mm. Allow the mobile phase to rise 10 cm. Dry the plate in air and examine in ultraviolet light at 254 nm and 365 nm, spray with potassium iodobismuthate solution. Heat the plate at 105º for 10 minutes and examine in day light. The chromatographic profile of the test solution is similar to that of the reference solution. It also shows in its upper half, a broad light brownish-yellow zone (triglycerides). Swelling Power. Not less than 6.0, determined on the powdered substance under examination, determined by the following method. Transfer 1g to a 100 ml stoppered cylinder containing 90 ml of water, shake well for 30 seconds and allow to stand 24 hours, shaking gently on three occasions during this period. Add sufficient water to produce 100 ml, mix gently for 30 seconds, avoiding the entrapment of air, allow to stand for 5 hours and measure the volume of mucilage. Repeat the determination three times. The average of four determinations is not less than 40 ml. Ash (2.3.19). Not more than 5.0 per cent. Loss on drying (2.4.19). Not more than 12.0 per cent, determined on 1 g by drying in an oven at 105º. Garcinia Aqueous Extract Garcinia Dry Extract is obtained from the Garcinia (Garcinia cambogia, Family-Clusiaceae) fruit rinds by extraction with water or any other suitable solvent. Garcinia Dry Extract contains not less than 50.0 per cent w/w to not more than 60.0 per cent w/w of the stated amount of hydroxy citric acid, and calcium not less than 18.0 per cent calculated on the dried basis. It may contain suitable added substances. Description. A pale brown to brown powder. Total Ash (2.3.19). Not more than 10.0 per cent. heavy metals. Method B (20 ppm). Loss on drying (2.4.19). Not more than 6.0 per cent determined Assay. hydroxy citric acid Determine by high performance liquid chromatography (2.4.14). Test Solution. Dissolve 200 mg of the extract under examination or quantity equivalent to 100 mg of hydroxy citric acid RS, add 2 ml of 3 M hydrochloric acid and add 10 ml of water, sonicate it to dissolve, make up to volume ml with water and mix. Reference solution. A 0.1 per cent w/v solution of hydroxy citric acid RS with 2 ml of 3 M hydrochloric acid in water. a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded to porous silica (5μm). mobile phase: 0.1 per cent ortho- phosphoric acid in water, filter and degas. flow rate. 0.6 ml per minute 7
8 spectrophotometer set at 210 nm. a 20 μl loop injector. relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject the test solution and the referene solution. Calculate the content of the hydroxy citric acid in extract. Calcium Test solution. Weigh accurately about 0.2 g of the sample and transfer into a 500 ml conical flask. Procedure. Dissolve in 2 ml of 3 M hydrochloric acid and add 200 ml of water. Add 15 ml of 1 M Sodium hydroxide and titrate with 0.05 M EDTA using hydroxy naphthol blue (50 mg) as indicator. The end point is the appearance of deep blue colour. Calculate the content of calcium. Each ml of 0.05 M EDTA solution is equivalent to g of calcium. Storage. Store in clean and airtight container, protected from heat and moisture. Tulasi Dry Extract Tulasi dry Extract is obtained from the Tulasi (Ocimum sanctum Linn, Fam. Laminaceae) leaves by extraction with methanol or other any suitable solvent. Tulasi Extract contains not less than 2.0 per cent and not more than 3.0 per cent of the stated amount of the ursolic acid, calculated on the dried basis. It may contain suitable added substances. Description. A pale green powder. A. Determine by thin layer chromatography (2.4.17) coating Mobile phase. A mixture of 95 volumes of chloroform and 5 volume of methanol. Test Solution. Dissolve about 200 mg of the extract under examination with 10.0 ml methanol, filter. Reference solution. A 0.02 per cent w/v solution of ursolic acid RS in the methanol. Apply to the plate 10 μl of each solution as bands of 10 mm by and spray with anisaldehyde-sulphuric acid reagent solution. Heat the plate at 110 for 10 minutes and examine in ultraviolet light at the plate at 365 nm and in day light. The chromatographic profile of the test solution is similar to that of the reference solution. Alcohol-soluble extractive (2.6.2). Not less than 15.0 per cent. Water-soluble extractive (2.6.3). Not less than 30.0 per cent. Total ash (2.3.19). Not more than 10.0 per cent. Loss on drying (2.4.19). Not more than 6.0 per cent determined Test Solution. Dissolve about 500 mg of the extract under examination in 100 ml of the methanol. Reference solution. A 0.03 per cent w/v solution of ursolic acid RS in the methanol. a stainless steel column, 25 cm x 0.46 mm packed with octadescylsilane bonded to porous (0.5 μm), mobile phase: a mixture of 30 volumes of methanol and 70 volumes of acetonitrile, flow rate. 0.6 ml per minute, spectrophotometer set at 210 nm, a 20 μl loop injector. than 2.0 per cent. Calculate the content of the ursolic acid in extract. Storage. Store in a container purged with Nitrogen under the refrigerated condition, protected from heat and moisture. 8
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