Add the following:
Phyllanthus amarus
DEFINITION
Phyllanthus amarus consists of the dried aerial parts of Phyllanthus amarus Schumach. (Fam. Euphorbiaceae). It contains NLT 0.25% of lignans, calculated as the sum of phyllanthin and hypophyllanthin on the dried basis.
IDENTIFICATION
• A.
Phyllanthus amarus meets the requirements in Specific Tests for Botanic Characteristics.
• B. Thin-Layer Chromatographic Identification Test 
201
201
Standard solution A:
0.1 mg/mL of USP Phyllanthin RS in methanol
Standard solution B:
10 mg/mL of USP Powdered Phyllanthus amarus Extract RS in methanol. Sonicate for about 10 min, centrifuge, and use the supernatant.
Sample solution:
Sonicate about 0.5 g of Phyllanthus amarus, finely powdered, in 5 mL of methanol 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:
Hexane and ethyl acetate (2:1)
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 the 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 the Spray reagent, heat for 3 min at 120, and examine under visible light.
Acceptance criteria:
The chromatogram of the Sample solution exhibits a blue band in the lower third of the plate due to phyllanthin, corresponding in color and RF to that in the chromatogram of Standard solution A; a violet band due to hypophyllanthin at an RF higher than that of phyllanthin; a blue band at an RF higher than that of hypophyllanthin; and an additional violet band in the upper third of the plate. Bands detected in the chromatogram of the Sample solution correspond in position and color to bands in the chromatogram of Standard solution B. Other minor bands may be observed in the chromatograms of the Sample solution and Standard solution B.
• C. HPLC:
The chromatogram of the Sample solution obtained in the test for Content of Lignans shows a peak at a retention time corresponding to that of phyllanthin in the chromatogram of Standard solution A. Identify other lignan peaks in the chromatogram of the Sample solution by comparison with the chromatogram of Standard solution B and the reference chromatogram provided with the lot of USP Powdered Phyllanthus amarus Extract RS being used. The chromatogram of the Sample solution shows an additional peak corresponding to hypophyllanthin.
COMPOSITION
• Content of Lignans
Solution A:
Dissolve 0.14 g of potassium dihydrogen phosphate in 900 mL of water, add 0.5 mL of phosphoric acid, dilute with water to 1000 mL, mix, filter, and degas.
Mobile phase:
Acetonitrile and Solution A (4:6)
Standard solution A:
0.1 mg/mL of USP Phyllanthin RS in methanol
Standard solution B:
Sonicate a portion of USP Powdered Phyllanthus amarus Extract RS in methanol to obtain a solution having 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:
Transfer about 3.0 g of Phyllanthus amarus, finely powdered and accurately weighed, to a 250-mL flask fitted with a reflux condenser. Add 50 mL of methanol, reflux in a water bath for about 20 min, allow to settle, and decant the supernatant. Repeat until the last extract is colorless. Combine the extracts, concentrate under vacuum, and adjust the volume to 100 mL with methanol. 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 230 nm
Column:
4.6-mm × 25-cm; 5-µm packing L1
Flow rate:
1.5 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 Phyllanthus amarus Extract RS being used.
Resolution:
NLT 1.5 between the phyllanthin and hypophyllanthin peaks, Standard solution B
Tailing factor:
NMT 1.5 for the phyllanthin peak, Standard solution A
Relative standard deviation:
NMT 2.0% determined from the phyllanthin 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 Phyllanthus amarus Extract RS being used, identify the retention times of the peaks corresponding to phyllanthin and hypophyllanthin.
Separately calculate the percentages of phyllanthin and hypophyllanthin in the portion of Phyllanthus amarus taken:
Result = (rU/rS) × CS × (V/W) × F × 100
| rU | = | = peak response of the analyte from the Sample solution |
| rS | = | = peak response of phyllanthin from Standard solution A |
| CS | = | = concentration of USP Phyllanthin RS in Standard solution A (mg/mL) |
| V | = | = volume of the Sample solution (mL) |
| W | = | = weight of Phyllanthus amarus taken to prepare the Sample solution (mg) |
| F | = | = conversion factors for the analytes: 1.00 for phyllanthin; 0.75 for hypophyllanthin |
Acceptance criteria:
Add the percentages of phyllanthin and hypophyllanthin: NLT 0.25% is found on the dried basis.
CONTAMINANTS
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561:
Meets the requirements
561:
Meets the requirements
• Heavy Metals, Method III 231:
NMT 20 ppm
231:
NMT 20 ppm
• Microbial Enumeration Tests 2021:
The total aerobic bacterial count does not exceed 105 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the bile-tolerant Gram-negative bacteria does not exceed 103 cfu/g.
2021:
The total aerobic bacterial count does not exceed 105 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the bile-tolerant Gram-negative bacteria does not exceed 103 cfu/g.
• 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:
Erect annual herb, 10–60-cm high; slender stem, leaves on main stem are reduced to scales; secondary branchlets, short, extend at right angles, each carrying 15–30 leaves; leaves have a green upper surface with raised midrib and a pale green lower surface with prominent midrib and secondary veins, simple, alternate, 3–11 mm long, 1.5–6 mm wide, short petiolate, elliptical-oblong to obovate, apex obtuse and often with small pointed tip, margin entire, base often slightly asymmetric; flowers minute, yellowish, greenish, or whitish, unisexual, axillary on secondary branchlets, 1–2 and sometimes 3 per axil, first 1–2 internodes of each branchlet bear 1–2 male flowers, the rest have male and female flowers; fruits are flattened spherical capsules, straw color, 3-loculed, about 2 mm in diameter; seeds usually 2 per locule, light brown, about 0.9-mm long, triangular with 6–7 longitudinal ribs and many transverse striations on the back. Pharmacopeial article is green to yellowish-green masses composed mostly of leaves, branchlets, and stem fragments; taste bitter.
Histology
Transverse section of stems:
Epidermal layer; about 15 layers of cortex cells, thick wall, contain chloroplast, some contain calcium oxalate crystals, inner 7–10 layers are made of thick-wall cells interrupted at regular interval by parenchyma cells; a layer of parenchyma cells containing starch grains; phloem 7–10 layers of thin-wall cells; groups of xylem vessels; pith, multilayer of thin-wall cells, few contain calcium oxalate crystals.
Transverse section of branchlets:
The transverse section is round; 6–8 layers of cortex, thick-wall cells, most contain chloroplast and a few calcium oxalate crystals, after 3–4 layers there is a layer of cells containing starch grains, followed by 2–3 layers of fiber cells interrupted by cortex parenchyma; phloem 5–7 layers of thin-wall cells; groups of xylem vessels; pith, multilayer of thin-wall cells, containing chloroplasts.
Transverse section of leaves:
Upper and lower epidermis; a single layer of palisade cells, which occupy nearly half of the space between each epidermis; 3–5 layers of parenchyma cells, a few contain crystals of calcium oxalate; vascular bundles are also present.
• Loss on Drying 731:
Dry 1.0 g of finely powdered Phyllanthus amarus at 105 for 2 h: it loses NMT 12.0% of its weight.
731:
Dry 1.0 g of finely powdered Phyllanthus amarus at 105 for 2 h: it loses NMT 12.0% of its weight.
• Articles of Botanical Origin, Total Ash 561:
NMT 8.0%, determined on 1.0 g of finely powdered Phyllanthus amarus
561:
NMT 8.0%, determined on 1.0 g of finely powdered Phyllanthus amarus
• Articles of Botanical Origin, Acid-Insoluble Ash 561:
NMT 5.0%, determined on 1.0 g of finely powdered Phyllanthus amarus
561:
NMT 5.0%, determined on 1.0 g of finely powdered Phyllanthus amarus
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.
USP35
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 1410
Pharmacopeial Forum: Volume No. 36(6) Page 1620