สารบัญ

          Centella Dry Extract is prepared from the powdered Centella by extraction with ethanol.  It contains not less than 90.0 per cent and not more than 110.0 per cent of the labelled amounts of asiaticoside (C₄₈H₇₈O₁₉) and madecassoside (C₄₈H₇₈O₂₀); the labelled amounts of asiaticoside and madecassoside are not less than 3.0 per cent and 4.0 per cent, respectively, calculated on the dried basis.

Description  Pale greenish yellow to brownish yellow powder, slightly hygroscopic.

Packaging and storage  Centella Dry Extract shall be kept in tightly closed containers, protected from light, and stored in a cool and dry place.

Labelling  The label on the container states (1) the amounts of asiaticoside and madecassoside; (2) the expiration date.

Identification 

          A. The chromatogram of the Assay preparation shows several peaks, two of which correspond to those of the Standard preparation, as obtained in the Assay.

          B. Carry out the test as described in the “Thin-Layer Chromatography” (Appendix 3.1), using silica gel G as the coating substance and a mixture of 60 volumes of chloroform,  28 volumes of methanol and 4 volume of water as the mobile phase and allowing the solvent front to ascend 8 cm above the line of application.  Apply separately to the plate as bands of 8 mm, 15 µL each of the following three solutions.  For solution (A), dissolve 50 mg of the sample in 4 mL of methanol.  Solution (B) contains 0.1 mg per mL of asiaticoside in methanol and solution (C) contains 0.1 mg per mL of madecassoside in methanol.  After removal of the plate, allow it to dry in air.  Spray the plate with anisaldehyde TS and heat at 105° for  5 minutes.  The chromatogram obtained from solution (A) shows a greenish blue band and  a violet band, corresponding to the asiaticoside and the madecassoside bands from solutions (B) and (C), respectively.  Several other bands of different colours are observed. 

Loss on drying  Not more than 10.0 per cent w/w after drying at 105° to constant weight (Appendix 4.15).

Assay  Carry out the determination as described in the “Liquid Chromatography” (Appendix 3.5). 

          Mobile phase A  Use acetonitrile, containing 6.0 per cent v/v of methyl tert-butyl ether and 0.01 per cent v/v of trifluoroacetic acid.  Make adjustments if necessary.

          Mobile phase B  Prepare a 0.01 per cent v/v solution of trifluoroacetic acid.  Make  adjustments if necessary.

          Standard preparation  Dissolve an accurately weighed quantity of asiaticoside and madecassoside in methanol to obtain a solution having known concentrations of about 100 µg of asiaticoside and 100 µg of madecassoside per mL.

          Assay preparation  Transfer an accurately weighed quantity of Centella Dry Extract, containing about 5 mg of asiaticoside, to a 50-mL volumetric flask.  Dissolve and dilute with methanol to volume and mix.  Centrifuge, if necessary.  Pass a portion of the clear solution through a filter having a 0.45 µm or finer porosity.       

          Chromatographic system The chromatographic procedure may be carried out using (a)  a stainless steel column (15 cm × 4.6 mm) packed with octadecylsilane chemically bonded to porous silica or ceramic microparticles (4 µm), polar end-capped (Synergi Hydro RP C₁₈ or equivalent is suitable.), equipped with a similarly packed guard column, maintained at  a temperature of 30º, (b) Mobile phase at a flow rate of 1.0 mL per minute (the retention time is about 60 minutes for the main asiaticoside peak.), and (c) an ultraviolet photometer set at  210 nm.  The step gradient of mobile phases is as follows:

          To determine the suitability of the chromatographic system, chromatograph Standard preparation, and record the peak response as directed under Procedure: the relative standard deviation for replicate injections is not more than 2.0 per cent, the column efficiency determined from the asiaticoside peak is not less than 2000 theoretical plates, the symmetry factors for the madecassoside peak and for the asiaticoside peak are not more than 1.5, the relative retention times are about 0.6 for madecassoside and 1.0 for asiaticoside, and the resolution factors, R, between the madecassoside and its adjacent peaks and between the asiaticoside and its adjacent peaks are not less than 1.5.

          Procedure  Separately inject equal volumes (about 20 µL) of Standard preparation and Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the madecassoside and asiaticoside peaks.

          Calculation  Calculate the contents of C₄₈H₇₈O₁₉ and C₄₈H₇₈O₂₀ in the portion of the Extract taken, using the declared contents of C₄₈H₇₈O₁₉ and C₄₈H₇₈O₂₀ in asiaticoside and madecassoside, respectively.

Other requirements  Complies with the requirements described under “Extracts”  (Appendix 1.16H).