Amla Dry Extract

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 labeled amount of tannins, calculated on the dried basis. It may contain suitable added substances. Description. A beige to off-white powder.

Identification
A. 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 l of each of solution as bands of 10 mm by 2 mm. Allow the mobile phase to rise up to 8 cm above the line of application. Dry the plate in air and examine under ultraviolet light at 254 nm. Spray the plate with a anisaldehyde- sulphuric acid reagent solution. Heat at 110 for 10 minutes and examine the plate under 365 nm and under day light. The chromatogram obtained with the test solution shows a band corresponding to the band obtained using reference solution indicating the presence of gallic acid.

Tests
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 content (2.3.43). Not more than 5.0 per cent. Heavy metals (2.3.13). 1.0 g complies with the limit test for heavy metals. Method B (20 ppm). Microbial contamination (2.2.9). Complies with the microbial contamination tests. Gallic acid (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. Chromatographic system - a stainless steel column 25 cm x 4.6 mm packed with octadecylsilane bonded porous silica (5m), - mobile phase: - flow rate. 0.7 ml per minute - spectrophotometer set at 272 nm. - injection volume 20 l Time Mobile phase A Mobile phase B (in min.) (per cent v/v) (per cent v/v) 25 95 5 30 0 100 40 0 100 42 95 5

Inject the reference solution. The test is not valid unless the relative standard deviation for the replicate injections is not more than 2.0 per cent. Inject 20 l of the test solution. Calculate the content of the gallic acid in extract. Assay Test Solution. Weigh accurately about 2 g of the extract, add 50ml 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 500ml volumetric flask, taking care that particles of the material are not carried on to the cotton. 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 6th 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. Procedure. Pipette out the above extract 25ml into a 250ml conical flask and add 25ml of indigo sulphonic acid solution. [Preparation of Indigo sulphonic acid solution: Dissolve 1 g of indigo carmin in 25ml of concentrated sulphuric acid, and another 25 ml of concentrated sulphuric acid and transfer it into a 1000 ml volumetric flask

containing 500 ml distilled water, dilute this solution to 1000ml [Very carefully dilute because it is a exothermic reaction]. Titrate, with constant stirring, against 0.1 M potassium permanganate solution. End point is a golden yellow colour. Run a blank test by titrating 25ml of indigo sulphonic acid in 25 ml of water and calculate the content of total tannins. Each ml of 0.1 M potassium permanganate solution is equivalent to 0.004157g of tannin compound calculated as tannic acid. Storage. Store in clean and airtight container purged with nitrogen, protected from heat and moisture.

Amla Extract

RS

TEST

RS

TEST

RS

TEST

(Under UV light at 254 nm)

(Under UV light at 365 nm after spraying with anisaldehyde-sulphuric acid reagent)

(Under day light after spraying with anisaldehyde-sulphuric acid reagent)

TLC Chromatogram of Amla Extract

Amla Extract

mAU 325 272nm,8nm (1.00) 300 275 250 225

Gallic acid RS
200 175 150 125 100 75 50 25 0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

Minutes

HPLC Chromatogram of gallic acid RS

275 mAU 272nm,8nm (1.00)

250

225

200

175

150

Gallic acid
125

100

75

50

25

0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

Minutes

HPLC Chromatogram of Amla Extract