Powdered Centella asiatica
DEFINITION
Powdered Centella asiatica is Centella asiatica reduced to a powder or very fine powder. It contains NLT 2.0% of triterpene derivatives, calculated on the dried basis.
IDENTIFICATION
•  A. Powdered Centella asiatica meets the requirements for Specific Tests, Botanic Characteristics.
•  B. Thin-Layer Chromatographic Identification Test
Standard solution A:  0.5 mg/mL of USP Asiaticoside RS in methanol
Standard solution B:  10 mg/mL of USP Powdered Centella asiatica Extract RS in methanol. Sonicate for about 10 min, centrifuge, and use the supernatant.
Sample solution:  About 0.5 g of Powdered Centella asiatica in 5 mL of methanol. Sonicate for 10 min, centrifuge, and use the supernatant.
Adsorbent:  Chromatographic silica gel with an average particle size of 10–15 µm (TLC plates) or with an average particle size of 5 µm (HPTLC plates)
Application volume:  10 µL (TLC plates) or 4 µL (HPTLC plates)
Developing solvent system:  A mixture of methylene chloride, methanol, and water (14:6:1)
Spray reagent:  A solution of 10% sulfuric acid in methanol.[Note—Prepare fresh. ]
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Apply the samples as bands to a suitable thin-layer chromatographic plate (see Chromatography 621). Use a saturated chamber. Develop the chromatograms until the solvent front has moved up about three-fourths of the plate. Remove the plate from the chamber, dry, spray with Spray reagent, heat for 3 min at 120, and examine under visible light.
Acceptance criteria:  The Sample solution chromatogram exhibits a violet band in the lower third of the plate due to asiaticoside, corresponding in color and RF to that in Standard solution A; a violet band due to madecassoside at an RF lower than that of asiaticoside; and two additional violet bands in the upper third of the plate due to asiatic acid and madecassic acid. Bands detected in the Sample solution correspond in position and color to bands in Standard solution B. Other minor bands may be observed in the Sample solution and Standard solution B.
•  C. HPLC Identification Test: The Sample solution chromatogram from the test for Content of Triterpene Derivatives shows a peak at the retention time corresponding to that of asiaticoside in Standard solution A. Identify other triterpene derivative peaks in the Sample solution by comparison with the chromatogram of Standard solution B and the reference chromatogram provided with the lot of USP Powdered Centella asiatica Extract RS being used. The Sample solution shows additional peaks corresponding to madecassoside and asiaticoside B (these two peaks may co-elute), madecassic acid, terminolic acid, and asiatic acid.
COMPOSITION
•  Content of Triterpene Derivatives
Solution A:  Dilute 3 mL of phosphoric acid with water to 1000 mL, mix, filter, and degas.
Solution B:  Acetonitrile
Mobile phase:  See the gradient table below.
Time
(min)
Solution A
(%)
Solution B
(%)
0 78 22
65 45 55
66 5 95
75 5 95
76 78 22
85 78 22
Standard solution A:  0.05 mg/mL of USP Asiaticoside RS in methanol
Standard solution B:  Sonicate a portion of USP Powdered Centella asiatica Extract RS in methanol to obtain a solution with a concentration of about 5.0 mg/mL. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first few mL of the filtrate.
Sample stock solution:  Transfer about 1.0 g of Powdered Centella asiatica, accurately weighed, to a Soxhlet apparatus. Add 100 mL of methanol, extract for 8 h, cool, and dilute with methanol to 100 mL. Pass through a membrane filter of 0.45-µm or finer pore size, discarding the first few mL of the filtrate. [Note—Use a thimble of a suitable size such that the volume of methanol used in the Soxhlet extraction is at least twice the volume of the thimble. ]
Sample solution:  Dilute 5.0 mL of Sample stock solution with methanol to 10 mL.
Chromatographic system 
Mode:  LC
Detector:  UV 200 nm
Column:  4.6-mm × 25-cm; 5-µm packing L1
Flow rate:  1.0 mL/min
Injection size:  10 µL
System suitability 
Samples:  Standard solution A and Standard solution B
Suitability requirements 
Chromatogram similarity:  The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Centella asiatica Extract RS being used.
Tailing factor:  Between 0.8 and 2.0 for the asiaticoside peak, Standard solution A
Resolution:  NLT 1.5 between the madecassic acid and terminolic acid peaks, Standard solution B
Relative standard deviation:  NMT 2.0% determined from the asiaticoside peak in repeated injections, Standard solution A
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution. [Note—Standard solution A, Standard solution B, and Sample solution are stable for 48 h at room temperature. ]
Using the chromatograms of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Powdered Centella asiatica Extract RS being used, identify the retention times of the peaks corresponding to different triterpene derivatives. The approximate relative retention times of the different triterpene derivatives are provided in the following table.
Analyte Approximate Relative
Retention Time
Madecassoside 0.71
Asiaticoside B 0.72
Asiaticoside 1.00
Madecassic acid 2.40
Terminolic acid 2.44
Asiatic acid 3.12
Separately calculate the percentages of the sum of madecassoside and asiaticoside B (these two peaks may co-elute), asiaticoside, the sum of madecassic acid and terminolic acid, and asiatic acid in the portion of Powdered Centella asiatica taken:
Result = (rU/rS) × CS × (V/W) × D × F × 100
rU== peak response(s) of the triterpene derivative(s) from the Sample solution
rS== peak response of asiaticoside from Standard solution A
CS== concentration of USP Asiaticoside RS in Standard solution A (mg/mL)
V== final volume of Sample stock solution (mL)
W== weight of Powdered Centella asiatica used to prepare the Sample stock solution (mg)
D== dilution factor to prepare the Sample solution from the Sample stock solution
F== conversion factors for analytes: 1.00 for asiaticoside, 1.017 for the sum of madecassoside and asiaticoside B, 0.526 for the sum of madecassic acid and terminolic acid, and 0.509 for asiatic acid
Acceptance criteria:  Add the percentages of different triterpene derivatives: NLT 2.0% on the dried basis.
CONTAMINANTS
•  Heavy Metals, Method III 231: NMT 20 ppm
•  Microbial Enumeration Tests 2021: The total aerobic bacterial count does not exceed 105 cfu/g, the total combined yeast and mold count does not exceed 103 cfu/g, and the bile-tolerant Gram-negative bacteria count does not exceed 103 cfu/g.
•  Microbiological Procedures for Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
SPECIFIC TESTS
•  Botanic Characteristics: Greenish-gray to greenish-brown in color; odor slight; taste slightly bitter to sweet. Under a microscope, factions of epidermal cells of the leaves with irregular striated cuticle, showing anisocytic, some paracytic, and rarely anomocytic stomata; epidermal cells of young leaves showing unicellular, occasionally multicellular, non-glandular trichomes; secretory canals composed of 5–7 secretory cells; parenchyma cells, some showing prisms or rosettes of calcium oxalate; bundles of narrow septate fibers from the stem; spiral vessels; fragments of the fruits, layers of wide cells in a parquetry arrangement, annular vessels, parenchyma cells containing simple or compound starch granules
•  Loss on Drying 731: Dry 1.0 g of Powdered Centella asiatica at 105 for 2 h: it loses NMT 12.0% of its weight.
•  Articles of Botanical Origin, Total Ash 561: NMT 12%, determined on 1.0 g of Powdered Centella asiatica
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed containers, protected from light and moisture, and store at room temperature.
•  Labeling: The label states the Latin binomial and, following the official name, the parts of the plant contained in the article. The label states that the article is exempted from the requirements of the General Notices with respect to the pregnancy and lactation statement (section 10.40.50, Labeling Botanical-Containing Products).
•  USP Reference Standards 11
USP Asiaticoside RS Click to View Structure
USP Powdered Centella asiatica Extract RS
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