Centella asiatica
DEFINITION
Centella asiatica consists of the dried aerial parts of Centella asiatica (L.) Urb. [Syn: Hydrocotyle asiatica L.] (Fam. Apiaceae). It is also known in commerce as gotu kola. It contains NLT 2.0% of triterpene derivatives, calculated on the dried basis.
IDENTIFICATION
• A.
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 Centella asiatica, finely powdered, 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.
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 Centella asiatica, finely powdered and 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 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 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 Centella asiatica taken:
Result = (rU/rS) × CS × (V/W) × D × F × 100
| rU | = | = peak response of the triterpene derivative(s) from the Sample solution |
| rS | = | = peak response(s) 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 Centella asiatica used to prepare 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
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 bacterial count does not exceed 105 cfu/g, the total combined yeasts and molds count does not exceed 103 cfu/g, and the bile-tolerant Gram-negative bacteria count does not exceed 103 cfu/g.
2021:
The total aerobic bacterial count does not exceed 105 cfu/g, the total combined yeasts and molds 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
2022:
Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
SPECIFIC TESTS
• Botanic Characteristics
Macroscopic:
Stem is slender, yellowish-brown, with long internodes, rooting at nodes; leaves are grayish-green, simple, alternate or grouped together at the nodes, reniform or oblong-elliptic, have palmate venation, usually with 7 veins, apex obtuse, margin crenate, base cordate, variable in size, 1–4 cm long, 2–4 cm and sometimes up to 7 cm wide, young leaves show a few trichomes on the lower surface and adult leaves are glabrous; petioles are long, grooved, base wider and sheathing; the inflorescence, if present, is a single umbel and consists of 3 flowers, rarely 2 or 4; the flowers are very small (about 2 mm), pentamerous, and have an inferior ovary; the fruit, brownish-gray, orbicular cremocarp, up to 5 mm long, is very flattened laterally and has 7–9 prominent curved ridges. Pharmacopeial article is green to yellowish-green masses composed mostly of leaves and stems fragments; odor slight; taste slightly bitter to sweet.
Histology
Transverse section of stems:
Epidermal layer, subrounded or subsquare cells; 2–4 layers of collenchyma cells; 6–8 layers of thin-wall parenchyma cells with intercellular spaces; 6–7 collateral vascular bundles, xylem vessels radially arranged, slightly lignified fiber groups occurring outside the phloem; pith large, composed of thin-wall parenchyma cells; secretory canals, composed of 5–7 secretory cells, observed in cortex and medullary rays
Transverse section of leaves:
Upper and lower epidermis; mesophyll composed of parenchyma cells, some contain crystals of calcium oxalate; 2–3 layers of collenchyma present in the midrib region next to both epidermal layers; vascular bundles in the center with xylem on the ventral side and phloem on the dorsal side. Transverse section of petioles has a U shape, showing an upper and a lower epidermis, followed by 2–3 layers of collenchyma next to both epidermal layers; a broad parenchymatous zone, some cells contain crystals of calcium oxalate; 7 vascular bundles forming a U shape in the parenchymatous zone, the two present in the projecting arms being less developed
• Articles of Botanical Origin, Foreign Organic Matter 561:
NMT 7.0%, of which NMT 5.0% is of underground organs and NMT 2% is of other foreign matter
561:
NMT 7.0%, of which NMT 5.0% is of underground organs and NMT 2% is of other foreign matter
• Loss on Drying 731:
Dry 1.0 g of finely powdered Centella asiatica at 105 for 2 h: it loses NMT 12.0% of its weight.
731:
Dry 1.0 g of finely 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 finely powdered Centella asiatica
561:
NMT 12%, determined on 1.0 g of finely 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).
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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 1232
Pharmacopeial Forum: Volume No. 36(4) Page 941