Phyllanthus amarus Aerial Parts Powder

Phyllanthus amarus Aerial Parts Powder

Final Authorized Version 1.0

Phyllanthus amarus Aerial Parts Powder


 

DEFINITION

The article consists of the dried aerial parts of Phyllanthus amarus Schumach. & Thonn. (Family Euphorbiaceae), collected during fruiting stage, reduced to a powder or very fine powder. It contains NLT 0.25% of lignans calculated as the sum of phyllanthin and hypophyllanthin on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

Related Phyllanthus species including P. debilis Klein ex Willd., P. fraternus G. L. Webster, P. maderaspatensis L., P. niruri L., P. urinaria L., and P. virgatus G. Forst.

 

CONSTITUENTS OF INTEREST

Lignans: Hypophyllanthin, phyllanthin, niranthin, phyltetralin, nirtetralin, and litetralin

Alkaloids: Securinine, norsecurinine, isobubbialine, dihydronorsecurinine, tetrahydronorsecurinine, allosecurinine, and 4-methoxynorsecurinine

Hydrolysable tannins: Phyllanthusiin D, amariin, amarulone, and amarinic acid

Fatty acids: Ricinoleic acid, linolenic acid, and linoleic acid

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Greenish to greenish-brown in color

Microscopic: Fragments of epidermal cells of the leaves with wavy walls, showing anisocytic and paracytic stomata; parenchyma cells, some showing both cluster and rosette crystals of calcium oxalate, several arranged in rows, 6–15 µm in diameter, polychrome when observed under the polarized light microscope; prisms of calcium oxalate, present in parenchymatous cells, subsquare or subpolygonal in shape, 6–14 µm in diameter; polychrome when observed under the polarized light microscope; fragments of epidermis of stem, polygonal in surface view, stomata anisocytic or anomocytic; fragments of epidermis of pericarp, subrounded or subpolygonal in surface view, walls thickened at the corners, showing anomocytic stomata, often linked with vessels; sclerenchymatous cells of testa, elongated-polygonal or subrectangular in surface view, with fine and dense pit canals, bright yellowish-white when observed under the polarized light microscope, fragments of narrow fibers from the stem; vessels, mainly spiral and annular, 2–10 µm in diameter.

• B. Thin-Layer Chromatography

Standard solution A: 1.0 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 Aerial Parts Powder in 5 mL of methanol for 10 min, centrifuge, and use the supernatant.

Chromatographic system

(See Chromatography <621>, Thin-Layer Chromatography.

Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)

Application volume: 8 µL each of Standard solution A and Standard solution B, and 4 µL of Sample solution, as 8-mm bands

Relative humidity: Condition the plate to a relative humidity of about 33% using a suitable device.

Temperature: 25°

Developing solvent system: Hexane and ethyl acetate (2:1)

Developing distance: 6 cm

Derivatization reagent: A solution of 10% sulfuric acid in methanol. [Note—Prepare fresh. Keep alcohol cold over ice, carefully and gradually add sulfuric acid.]

Analysis 

Samples: Standard solution A, Standard solution B, and Sample solution

Apply the Samples as bands to a suitable HPTLC plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry. Treat with Derivatization reagent, heat for 3 min at 120°, and examine under UV light at 366 nm and visible light.

System suitability: Under UV light at 366 nm, the chromatogram of Standard solution B exhibits, in the lower-third section, three bands that are clearly separated: a dark band corresponding in color and RF to the phyllanthin band of Standard solution A; a dark band due to hypophyllanthin at an RF higher than that of phyllanthin; and a dark band at an RF higher than that of hypophyllanthin. The last band is the most intense band in the chromatogram. The chromatogram also exhibits a light gray band at about the middle. Under visible light three bands clearly separated in the lower-third section are observed: two brown bands, due to phyllanthin and hypophyllanthin, and a brownish-violet band (most intense) at an RF higher than that of hypophyllanthin.

Acceptance criteria: Under UV light at 366 nm, the chromatogram of the Sample solution exhibits a band due to phyllanthin corresponding in color and in RF to the band of Standard solution A. The Sample solution exhibits a dark band at an RF higher than that of hypophyllanthin, corresponding to a similar band of Standard solution B. It also exhibits four red bands: one below the band corresponding to phyllanthin, one at an RF similar to that of hypophyllanthin, one at about the middle just above the faint dark band seen with Standard solution B, and the fourth and most intense red band observed above. Under visible light, the following bands correspond to similar bands of Standard solution B: a brown band due to hypophyllanthin, at an RF higher than that of phyllanthin; a brownish-violet band at an RF higher than that of hypophyllanthin; and a brownish-violet band at about the middle. Additional bands detected in the Sample solution chromatogram include two olive-green bands that correspond to two red bands under UV 366 nm: one at an RF below that of phyllanthin and the other at about the middle with the higher RF

• C. HPLC

Analysis: Proceed as directed in the Assay for Content of Lignans.

Acceptance criteria: The chromatogram of the Sample solution exhibits peaks at the retention times corresponding to the peaks due to phyllanthin, hypophyllanthin, and niranthin of Standard solution B. The most abundant peak is that of phyllanthin.

 

ASSAY

• 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, and filter.

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 Aerial Parts Powder 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 and filter. Concentrate the filtrate under vacuum and dilute with methanol to 100 mL. 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

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 230 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1 (similar to Luna C18 and Inertsil ODS-3)

Column temperature: 25 ± 1°

Flow rate: 1.5 mL/min

Injection volume: 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 1.0 between 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

[NoteStandard 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 Phyllanthus amarus Extract RS being used, identify the peaks corresponding to phyllanthin, hypophyllanthin, and niranthin. The approximate relative retention times, relative to phyllanthin, are provided in Table 1.

 

Table 1

Analyte

Relative Retention Times

Phyllanthin

1.00

Hypophyllanthin

1.08

Peak eluting before Niranthin

1.43

Niranthin

1.48

 

Separately calculate the percentages of phyllanthin and hypophyllanthin in the portion of Phyllanthus amarus Aerial Parts Powder 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 phyllanthin in Standard solution A (mg/mL)

V          = volume of the Sample solution (mL)

W         = weight of Phyllanthus amarus Aerial Parts Powder taken to prepare the Sample solution (mg)

F          = conversion factors for the analytes; 1.00 for phyllanthin, 0.75 for hypophyllanthin

Calculate the content of lignans as the sum of the percentages of phyllanthin and hypophyllanthin.

Acceptance criteria: NLT 0.25% on the dried basis

 

CONTAMINANTS

Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 0.5 µg/g

Lead: NMT 5.0 µg/g

Mercury: NMT 0.2 µg/g

Articles of Botanical Origin, General Method for Pesticide Residues Analysis <561>: Meets the requirements

Microbial Enumeration Tests <61>: 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.

Tests for Specified Microorganisms <62>: Meets the requirements of the tests for the absence of Salmonella species and Escherichia coli

 

SPECIFIC TESTS

Loss on Drying <731>

Sample: 1.0 g of Phyllanthus amarus Aerial Parts Powder

Analysis: Dry the Sample at 105° for 2 h.

Acceptance criteria: NMT 12.0%

Articles of Botanical Origin, Total Ash <561>

Analysis: 2.0 g of Phyllanthus amarus Aerial Parts Powder

Acceptance criteria: NMT 8.0%

Articles of Botanical Origin, Acid-Insoluble Ash <561>

Analysis: 2.0 g of Phyllanthus amarus Aerial Parts Powder

Acceptance criteria: NMT 5.0%

 

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 the parts of the plant from which the article was derived.

USP Reference Standards <11>

USP Phyllanthin RS

USP Powdered Phyllanthus amarus Extract RS

Other Versions

Proposed For Comment Version 0.2