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Printed by: Le Tran Official Date: Official as of 01-May-2018 Document Type: DIETARY SUPPLEMENTS @2021 USPC
1

Table 1
Centella asiatica
Time Solution A Solution B
(min) (%) (%)
DEFINITION
Centella asiatica consists of the dried aerial parts of Centella 0 78 22
asiatica (L.) Urb. [Syn: Hydrocotyle asiatica L.] (Fam.
65 45 55
Apiaceae). It is also known in commerce as gotu kola. It
contains NLT 2.0% of triterpene derivatives, calculated on 66 5 95
the dried basis. 75 5 95
IDENTIFICATION 76 78 22
• A. Centella asiatica meets the requirements for Specific Tests,
Botanical Characteristics. 85 78 22
• B. THIN-LAYER CHROMATOGRAPHY
Standard solution A: 0.5 mg/mL of USP Asiaticoside RS in Standard solution A: 0.05 mg/mL of USP Asiaticoside RS in
methanol methanol
Standard solution B: 10 mg/mL of USP Powdered Centella Standard solution B: Sonicate a portion of USP Powdered
asiatica Extract RS in methanol. Sonicate for about 10 min, Centella asiatica Extract RS in methanol to obtain a solution
centrifuge, and use the supernatant. with a concentration of about 5.0 mg/mL. Before injection,
Sample solution: About 0.5 g of Centella asiatica, finely pass through a membrane filter of 0.45-µm or finer pore
powdered, in 5 mL of methanol. Sonicate for 10 min, size, discarding the first few mL of the filtrate.
centrifuge, and use the supernatant. Sample stock solution: Transfer about 1.0 g of Centella
Adsorbent: Chromatographic silica gel with an average asiatica, finely powdered and accurately weighed, to a

al
particle size of 10–15 µm (TLC plates) or with an average Soxhlet apparatus. Add 100 mL of methanol, extract for
particle size of 5 µm (HPTLC plates) 8 h, cool, and dilute with methanol to 100 mL. Pass
Application volume: 10 µL (TLC plates) or 4 µL (HPTLC through a membrane filter of 0.45-µm or finer pore size,
plates) discarding the first few mL of the filtrate. [NOTE—Use a
Developing solvent system: Methylene chloride, thimble of a suitable size such that the volume of methanol
methanol, and water (14:6:1) used in the Soxhlet extraction is at least twice the volume
Derivatization reagent: A solution of 10% sulfuric acid in
methanol. [NOTE—Prepare fresh.]
Analysis
ci of the thimble.]
Sample solution: Dilute 5.0 mL of Sample stock solution with
methanol to 10.0 mL.
Samples: Standard solution A, Standard solution B, and Chromatographic system
Sample solution (See Chromatography á621ñ, System Suitability.)
ffi
Apply the Samples as bands. Use a saturated chamber. Mode: LC
Develop the chromatograms until the solvent front has Detector: UV 200 nm
moved up about three-fourths of the plate. Remove the Column: 4.6-mm × 25-cm; 5-µm packing L1
plate from the chamber, dry, treat with Derivatization Flow rate: 1.0 mL/min
reagent, heat for 3 min at 120°, and examine under white Injection volume: 10 µL
light. System suitability
O

Acceptance criteria: The Sample solution chromatogram Samples: Standard solution A and Standard solution B
exhibits a violet band in the lower third of the plate due to Suitability requirements
asiaticoside, corresponding in color and R F to that in Chromatogram similarity: The chromatogram from
Standard solution A; a violet band due to madecassoside at Standard solution B is similar to the reference
an R F lower than that of asiaticoside; and two additional chromatogram provided with the lot of USP Powdered
violet bands in the upper third of the plate due to asiatic Centella asiatica Extract RS being used.
acid and madecassic acid. Bands detected in the Sample Tailing factor: Between 0.8 and 2.0 for the asiaticoside
solution correspond in position and color to bands in peak, Standard solution A
Standard solution B. Other minor bands may be observed Resolution: NLT 1.5 between the madecassic acid and
in the Sample solution and Standard solution B. terminolic acid peaks, Standard solution B
• C. HPLC: The Sample solution chromatogram from the test Relative standard deviation: NMT 2.0% determined
for Content of Triterpene Derivatives shows a peak at the from the asiaticoside peak in replicate injections,
retention time corresponding to that of asiaticoside in Standard solution A
Standard solution A. Identify other triterpene derivative Analysis
peaks in the Sample solution by comparison with the Samples: Standard solution A, Standard solution B, and
chromatogram of Standard solution B and the reference Sample solution. [NOTE—Standard solution A, Standard
chromatogram provided with the lot of USP Powdered solution B, and the Sample solution are stable for 48 h at
Centella asiatica Extract RS being used. The Sample solution room temperature.]
shows additional peaks corresponding to madecassoside Using the chromatograms of Standard solution A, Standard
and asiaticoside B (these two peaks may co-elute), solution B, and the reference chromatogram provided
madecassic acid, terminolic acid, and asiatic acid. with the lot of USP Powdered Centella asiatica Extract RS
being used, identify the retention times of the peaks
COMPOSITION corresponding to different triterpene derivatives. The
• CONTENT OF TRITERPENE DERIVATIVES approximate relative retention times of the different
Solution A: Dilute 3 mL of phosphoric acid with water to triterpene derivatives are provided in Table 2.
1000 mL, mix, filter, and degas.
Solution B: Acetonitrile
Mobile phase: See Table 1.

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Printed by: Le Tran Official Date: Official as of 01-May-2018 Document Type: DIETARY SUPPLEMENTS @2021 USPC
2

Table 2 7 cm wide, young leaves show a few trichomes on the lower

Approximate Relative surface and adult leaves are glabrous; petioles are long,
Analyte Retention Time grooved, base wider and sheathing; the inflorescence, if
Madecassoside 0.71
present, is a single umbel and consists of 3 flowers, rarely
2 or 4; the flowers are very small (about 2 mm),
Asiaticoside B 0.72 pentamerous, and have an inferior ovary; the fruit,
Asiaticoside 1.00
brownish-gray, orbicular cremocarp, up to 5 mm long, is
very flattened laterally and has 7–9 prominent curved
Madecassic acid 2.40 ridges. Pharmacopeial article is green to yellowish-green
Terminolic acid 2.44 masses composed mostly of leaves and stem fragments;
odor slight; taste slightly bitter to sweet.
Asiatic acid 3.12 Microscopic
Transverse section of stems: Epidermal layer,
Separately calculate the percentages of the sum of subrounded or subsquare cells; 2–4 layers of collenchyma
madecassoside and asiaticoside B (these two peaks may cells; 6–8 layers of thin-walled parenchyma cells with
co-elute), asiaticoside, the sum of madecassic acid and intercellular spaces; 6–7 collateral vascular bundles, xylem
terminolic acid, and asiatic acid in the portion of Centella vessels radially arranged, slightly lignified fiber groups
asiatica taken: occurring outside the phloem; pith large, composed of
thin-walled parenchyma cells; secretory canals, composed
Result = (r U/r S) × C S × (V/W) × D × F × 100 of 5–7 secretory cells, observed in cortex and
medullary rays
rU = peak areas of the triterpene derivatives from the Transverse section of leaves: Upper and lower epidermis;
Sample solution mesophyll composed of parenchyma cells, some contain

al
rS = peak area of asiaticoside from Standard solution A crystals of calcium oxalate; 2–3 layers of collenchyma
CS = concentration of USP Asiaticoside RS in Standard present in the midrib region next to both epidermal layers;
solution A (mg/mL) vascular bundles in the center with xylem on the ventral
V = final volume of Sample stock solution (mL) side and phloem on the dorsal side. Transverse section of
W = weight of Centella asiatica used to prepare Sample petioles has a U shape, showing an upper and a lower

D
stock solution (mg)
= dilution factor to prepare the Sample solution
from the Sample stock solution
ci 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
F = conversion factors for analytes: 1.00 for bundles forming a U-shape in the parenchymatous zone,
asiaticoside, 1.017 for the sum of madecassoside the two present in the projecting arms being less
ffi
and asiaticoside B, 0.526 for the sum of developed.
madecassic acid and terminolic acid, and • ARTICLES OF BOTANICAL ORIGIN, Foreign Organic Matter
0.509 for asiatic acid á561ñ: NMT 7.0%, of which NMT 5.0% are of
underground organs and NMT 2% are of other foreign
Acceptance criteria: Add the percentages of different matter
triterpene derivatives: NLT 2.0% on the dried basis. • LOSS ON DRYING á731ñ
O

Sample: 1.0 g of finely powdered Centella asiatica

CONTAMINANTS Analysis: Dry the Sample at 105° for 2 h.
• ARTICLES OF BOTANICAL ORIGIN, Limits of Elemental Acceptance criteria: NMT 12.0%
Impurities á561ñ: Meets the requirements • ARTICLES OF BOTANICAL ORIGIN, Total Ash á561ñ
• ARTICLES OF BOTANICAL ORIGIN, Pesticide Residue Analysis Sample: 1.0 g of finely powdered Centella asiatica
á561ñ: Meets the requirements Acceptance criteria: NMT 12%
• MICROBIAL ENUMERATION TESTS á2021ñ: The total aerobic • ARTICLES OF BOTANICAL ORIGIN, Acid-Insoluble Ash á561ñ:
bacterial count does not exceed 105 cfu/g, the total NMT 3.5%
combined yeasts and molds count does not exceed 103 cfu/
g, and the bile-tolerant Gram-negative bacteria count does ADDITIONAL REQUIREMENTS
not exceed 103 cfu/g. • PACKAGING AND STORAGE: Preserve in well-closed
• ABSENCE OF SPECIFIED MICROORGANISMS á2022ñ: Meets the containers, protected from light and moisture, and store at
requirements of the tests for absence of Salmonella species room temperature.
and Escherichia coli • LABELING: The label states the Latin binomial and, following
the official name, the parts of the plant contained in the
SPECIFIC TESTS article. The label states that this article is exempted from
• BOTANICAL CHARACTERISTICS the requirements of the Labeling á7ñ, Labels and Labeling for
Macroscopic: Stem is slender, yellowish-brown, with long Products and Other Categories, Botanicals, with respect to
internodes, rooting at nodes; leaves are grayish-green, the pregnancy and lactation statement.
simple, alternate or grouped together at the nodes, • USP REFERENCE STANDARDS á11ñ
reniform or oblong-elliptic, have palmate venation, usually USP Asiaticoside RS
with 7 veins, apex obtuse, margin crenate, base cordate, USP Powdered Centella asiatica Extract RS
variable in size, 1–4 cm long, 2–4 cm and sometimes up to

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