Centella asiatica Triterpenes
DEFINITION
Centella asiatica Triterpenes is a fraction enriched in Centella asiatica triterpenes derivatives. It is prepared from Centella asiatica Extract using suitable solvents or other means. It contains NLT 90.0% of triterpene derivatives, calculated on the anhydrous basis, as the sum of two or more of the following: madecassoside, asiaticoside B, asiaticoside, madecassic acid, terminolic acid, and asiatic acid.
IDENTIFICATION
• A. 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:
Transfer an amount of Centella asiatica Triterpenes, equivalent to about 5 mg of triterpene derivatives, to a centrifuge tube. Add 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.
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. The Sample solution shows additional bands corresponding to some or all of the following triterpene derivatives: a violet band due to madecassoside at an RF lower than that of asiaticoside, a violet band in the upper third of the plate due to asiatic acid, and a violet band due to madecassic acid at an RF lower than that of asiatic 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.
• B. 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 some or all of the following: 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:
0.2 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 solution:
About 1.0 mg/mL of Centella asiatica Triterpenes in methanol; sonicate if necessary. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first few mL of the filtrate.
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 the 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 triterpene derivatives in the portion of Centella asiatica Triterpenes taken:
Result = (rU/rS) × (CS/CU) × 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) |
| CU | = | = concentration of Centella asiatica Triterpenes in the Sample solution (mg/mL) |
| F | = | = conversion factors for analytes: 1.00 for asiaticoside, 1.017 for madecassoside, 1.017 for asiaticoside B, 0.526 for madecassic acid, 0.526 for terminolic acid, and 0.509 for asiatic acid |
Acceptance criteria:
Add the percentages of different triterpene derivatives: NLT 90.0% on the anhydrous basis.
CONTAMINANTS
• Heavy Metals, Method III 231:
NMT 20 ppm
231:
NMT 20 ppm
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561:
Meets the requirements
561:
Meets the requirements
• Microbial Enumeration Tests 2021:
The total aerobic microbial count does not exceed 103 cfu/g. The total combined yeast and mold count does not exceed 102 cfu/g.
2021:
The total aerobic microbial count does not exceed 103 cfu/g. The total combined yeast and mold count does not exceed 102 cfu/g.
• Microbiological Procedures for Absence of Specified Microorganisms 2022:
Meets the requirements of the tests for absence of Escherichia coli
2022:
Meets the requirements of the tests for absence of Escherichia coli
SPECIFIC TESTS
• Water Determination, Method I 921:
NMT 5%
921:
NMT 5%
• Other Requirements:
Meets the requirements of the test for Residual Solvents under Botanical Extracts 565
565
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in well-closed containers, protected from light and moisture, and store at controlled room temperature.
• Labeling:
The label states the Latin binomial and, following the official name, the part of the plant from which the article was derived.
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Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Maged H. Sharaf, Ph.D.
Principal Scientific Liaison 1-301-816-8318 |
(DS2010) Monographs - Dietary Supplements |
| 2021 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 2022 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1236
Pharmacopeial Forum: Volume No. 36(4) Page 946