Phyllanthus amarus Aerial Parts Powdered Extract

Phyllanthus amarus Aerial Parts Dry Extract

Proposed For Comment Version 0.2

Phyllanthus amarus Aerial Parts Powdered Extract


 

DEFINITION

The article is prepared from dried aerial parts of Phyllanthus amarus Schumach (Family Euphorbiaceae), collected during fruiting stage, by extraction with methanol, ethyl acetate, hexane, or a mixture of these solvents. The ratio of starting crude plant material to Powdered Extract is between 15:1 and 4:1. It contains NLT 90.0% and NMT 110.0% of the labeled amount of lignans calculated as the sum of phyllanthin and hypophyllanthin, on the dried basis. It may contain suitable added substances as carriers.

 

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, and phyltetralin

Alkaloids: Securinine, norsecurinine, and isobubbialine

 

IDENTIFICATION

• A. 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: 10 mg/mL of Powdered Extract in methanol. Sonicate for about 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 in the chromatogram 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 of the chromatogram. Similar profile is observed under visible light with three bands clearly separated in the lower-third section: two brown bands, due to phyllanthin and hypophyllanthin, and a brownish-violet band (most intense) at an RF higher than that of hypophyllanthin. The chromatogram also exhibits a brownish-violet band at about the middle of the chromatogram.  

Acceptance criteria: Under visible light, the chromatogram of the Sample solution exhibits a band due to phyllanthin corresponding in color and in RF to the band in the chromatogram of Standard solution A, and the following bands corresponding to similar bands in the chromatogram 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 of the chromatogram. Additional bands detected in the Sample solution chromatogram include two olive-green bands: one at an RF below that of phyllanthin and the other at about the middle of the chromatogram.    

Under UV light at 366 nm, the Sample solution exhibits four red bands: two at RF similar to those of the two olive-green bands detected under visible light, one at an RF similar to that of hypophyllanthin, and a red band at about the middle of the chromatogram.  It also exhibits a dark band at an RF higher than that of hypophyllanthin, corresponding to a similar band in the chromatogram of Standard solution B.

B. 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 in the chromatogram 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: Sonicate a portion of Powdered Extract 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.

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 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

[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

Niranthin

1.48

 

Separately calculate the percentages of phyllanthin and hypophyllanthin in the Extract:

 

Result = (rU/rS) × (CS/CU) × 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)

CU       = concentration of Powdered Extract in the Sample solution (mg/mL)

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

 

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

Calculate the percentage of the labeled amount of lignans in Powdered Extract:

  

Result = (P/L) × 100

 

P         = content of lignans: phyllanthin and hypophyllanthin as determined above (%)

L          = labeled amount of lignans: phyllanthin and hypophyllanthin (%)

Acceptance criteria: 90.0%–110.0% of the labeled amount of lignans on the dried basis

 

CONTAMINANTS

Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 1.0 µg/g

Lead: NMT 5.0 µg/g

Mercury: NMT 1.0 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 104 cfu/g; the total combined molds and yeasts count does not exceed 102 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>

Analysis: Dry 1.0 g of Powdered Extract at 105° for 2 h.

Acceptance criteria: NMT 7.0%

 

ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers, protected from light and moisture. 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. It meets other labeling requirements under Botanical Extract <565>.

USP Reference Standards <11>

USP Phyllanthin RS

USP Powdered Phyllanthus amarus Extract RS

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