Phyllanthus amarus Aerial Parts

Phyllanthus amarus Aerial Parts

Final Authorized Version 1.0

Phyllanthus amarus Aerial Parts


 

DEFINITION

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

 

SYNONYMS

Diasperus nanus (Hook. f.) Kuntze

Phyllanthus amarus var. baronianus Leandri

Phyllanthus nanus Hook. f.

Phyllanthus niruri var. amarus (Schumach. & Thonn.) Leandri

Phyllanthus niruri var. scabrellus (Webb) Müll. Arg.

Phyllanthus scabrellus Webb

Phyllanthus swartzii Kostel.

 

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.

 

SELECTED COMMON NAMES

African: Ahlivi (Togo); bomagua kéné, bounou, bounou honlin (Ivory Coast); hinlinwe (West Africa); mokichinento (East Africa); tsekulemegbe (Togo)
English: Carry-me-seed, black catnip, child pick-a-back, gale of wind, gulf leaf flower, hurricane weed, shatterstone, stone breaker
French: Poudre de plomb (Ivory Coast)
German: Weisse Blatt-blume, bittere blattblüte
Indian: Bhuiamla (Bengali), bhui amala (Hindi), nilanelli (Kannada), bhumyaamalaki (Sanskrit), kizhukai nelli (Tamil)
Japanese: キダチコミカンソウ (kidachi komi kansou)
Javanese: Memeniran, meniran
Malay: Dukung anak, dukung-dukung anak, amin buah, rami buah, turi hutan, meniran

Portuguese: Quebra-pedra, arrebenta pedra

Spanish: Chancapiedra, rompepiedra, sarandí blanco

Vietnamese: Diệp hạ châu đắng
 

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: Erect annual herb, 10–60 cm high; stem rounded and hairy (distinction from P. debilis and P. maderaspatensis, angular and glabrous; and P. virgatus, rounded with two protrusions and glabrous); leaves on main stem are reduced to scales (cataphyll leaves) (distinction from P. maderaspatensis and P. virgatus, absent) and turn black at maturity (distinction from P. debilis, stay green); secondary branchlets present (distinction from P. maderaspatensis and P. virgatus, absent), short, extend at right angles, each carrying 15–30 leaves; leaves, simple, alternate, 4–12 mm long, 2–5 mm wide, short petiolate, oblong (distinction from P. debilis, narrowly elliptic in upper part and cuneate at base; P. maderaspatensis, spathulate; and P. virgatus, oblong-elliptic), apex mucronate, margin entire (distinction from P. fraternus with serrate margin; and P. maderaspatensis with crenate margin and bulbous at the base), base often slightly asymmetric, have a green upper surface raised at the midrib and a pale green lower surface with prominent midrib and secondary veins; 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, each flower has five sepals (distinction from P. debilis, P. fraternus, P. maderaspatensis and P. virgatus, all with flowers having six sepals); fruits are flattened, globose spherical capsules, straw color, 3-loculed, about 2 mm in diameter; seeds usually two per locule, light brown, about 0.9 mm long, triangular with 6–7 longitudinal ribs and many transverse striations on the back. Compendial article often is green to yellowish-green mass composed mostly of leaves, branchlets, fruits, and stem fragments.

Microscopic:

Transverse section of stem: The transverse section is circular, shows epidermis covered with thick cuticle, embedded with stomata, at places bearing papilla; a narrow band of chlorenchymatous hypodermis; 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 intervals 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 branchlet: The transverse section is circular; 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 leaf: Upper epidermis, one row of subrectangular cells, mostly tangentially elongated, sometimes with convex outer walls, covered with thin cuticle, at places it is pappilose and embedded with stomata; 1–4 rows of collenchymas cells, below the upper and lower epidermis in the midrib region; a single layer of palisade cells, which occupy nearly half of the space between the upper and lower epidermis and appearing across the upper part of the midrib, some cells contain prismatic and rosette clusters of calcium oxalate; 3–5 layers of parenchyma cells, a few contain prisms of calcium oxalate; vascular bundles, collateral; meristele of the midrib consists of radiate xylem and an arch of phloem; fiber bundles, outside the phloem, with thickened walls; lower epidermis, subrounded or subrectangular cells.

Transverse section of fruit: Diagrammatic transverse section is circular in outline with inconspicuous 3-ridged margin, shows narrow pericarp surrounding three locules occupying the major portion of the fruit, each has two triangular endospermic seeds. Detailed transverse section shows the outermost layer of tangentially arranged, narrow, rectangular cells of epicarp covered with thin cuticle; a layer of hypodermis consisting of large oval, tangentially arranged parenchymatous cells; a layer of small rectangular to square, radially arranged chlorenchymatous cells; 3–4 rows of collapsed parenchymatous cells; innermost layer of the pericarp composed of radially arranged, compactly placed, large rectangular thick-wall parenchymatous cells; a layer of endocarp of radially arranged parenchymatous cells.

Transverse section of seed: Testa consists of an outer layer of lens-shaped giant cells, a sclereide layer, and an innermost layer of cells containing pigment; endosperm, wide, parenchymatous, full of oil globules, microrosette crystals of calcium oxalate and aleurone grains; cotyledons are narrow, consisting of upper and lower epidermis, enclosing mesophyll cells.

• 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, finely powdered, 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 prominent peak is that of phyllanthin. Another major lignan peak elutes before niranthin with an intensity similar to that of niranthin.

 

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, 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 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 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 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 the 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

Articles of Botanical Origin, Foreign Organic Matter <561>: NMT 2.0%

Loss on Drying <731>

Sample: 1.0 g of Phyllanthus amarus Aerial Parts, finely powdered

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, finely powdered

Acceptance criteria: NMT 8.0%

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

Analysis: 2.0 g of Phyllanthus amarus Aerial Parts, finely powdered

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 contained in the article.

USP Reference Standards <11>

USP Phyllanthin RS

USP Powdered Phyllanthus amarus Extract RS

Other Versions

Proposed For Comment Version 0.2