Piper methysticum Rootstock Powder

Piper methysticum Root and Rhizome Powder

Proposed For Development Version 0.1

Piper methysticum Rootstock Powder

 


 

DEFINITION

 

The article consists of the dried rootstock (rhizome) of Piper methysticum G. Forst (Family Piperaceae) reduced to powder or very fine powder. It contains not less than 4.5% of kavalactones, calculated as the sum of methysticin, dihydromethysticin, kawain, dihydrokawain, yangonin, and desmethoxyyangonin on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

Piper wichmannii C.DC.

Piper methysticum var. wichmannii (C.DC) Lebot. 

 

CONSTITUENTS OF INTEREST

Kavalactones: Kawain (kavain), dihydrokawain, methysticin, dihydromethysticin, yangonin, and demethoxyyangonin

Chalconoides: Flavokavain A-C 

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Pale yellow brown powder with a slight, agreeable odor and a bitter, pungent taste.

Microscopic: Fragment of parenchyma cells containing reddish or yellowish brown oleoresin; elongated, lignified, and pitted parenchyma of the xylem and medullary rays; fragments of vessels with scalariform thickening; groups of moderately thickened, slightly lignified fibers; large, thin-walled parenchymatous cells of the pith; occasional fragments of thin-walled cork; and numerous starch granules, simple or compounded, of 2-3 lenicular to obconic components. Crystals of methysticin that polarize light in the red to violet range are observed; calcium oxalate crystals are absent.

B. Thin-Layer Chromatography

Standard solution A: 0.5 mg/mL of USP Kawain RS in toluene

Standard solution B: 50 mg/mL of USP Powdered Kava Extract RS in methanol. Sonicate for 10 min, centrifuge and use supernatant.

Sample solution: Mix about 1.0 g of Piper methysticum Rootstock Powder with 5 mL of methanol and sonicate for 10 min. Centrifuge and use supernatant.

Chromatographic system

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

Adsorbent: Use a suitable chromatographic material with an average particle size of 5 µm (HPTLC plates, Si 60 F254).

Reagent for impregnation: Dissolve 8 g of caffeine in 200 mL of dichloromethane

Application volume: 2 µL as 8-mm bands

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

Developing solvent system: tert-Butyl ether and cyclohexane (70:30)

Developing distance: 6 cm

Derivatization reagent: Anisaldehyde reagent: a mixture of 170 mL of ice-cooled methanol with 20 mL of glacial acetic acid, 10 mL of sulfuric acid, and 1 mL of anisaldehyde

Analysis

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

Dip the plate in the Reagent for impregnation. Dry at the room temperature for 5 min, then heat at 80° for 5 min. Apply the Samples as bands to a suitable HPTLC plates. Develop the chromatogram in an unsaturated chamber, remove the plate from the chamber, and dry. Treat with Derivatization reagent, heat at 100° for 4 min, and examine under UV 366 and visible light.

System suitability: Under UV 366 nm, in the lower half of the chromatogram, the Standard solution B exhibits an orange band corresponding to kawain similar in RF and color to the band observed in the chromatogram of Standard solution A; below kawain, with decreasing RF, a bright blue fluorescent band and a yellow/orange band due to yangonin and methysticin, respectively; above kawain, a pale blue band corresponding to desmethoxyyangonin; one yellowish green band above desmethoxyyangonin band and one bright blue band below methysticin band. In upper half of the chromatogram, with increasing RF, a green, a pale yellow, a black, a white, and a orange bands appear.

Under visible light, in the lower half of the chromatogram, the Standard solution B exhibits a red band corresponding to kawain similar in RF and color to the band observed in the chromatogram of Standard solution A; below kawain, with decreasing RF, a brown band due to methysticin. Above kawain band, with increasing RF, three yellow and one purple bands appear.

Acceptance criteria: Under UV 366 nm, in the lower half of the chromatogram, the Sample solution exhibits an orange band corresponding to kawain similar in RF and color to the band observed in the chromatogram of Standard solution A; below kawain, with decreasing RF, a bright blue fluorescent band and a yellow/orange band due to yangonin and methysticin, respectively; above kawain, a pale blue band corresponding to desmethoxyyangonin; one yellowish green band above desmethoxyyangonin band and one bright blue band below methysticin band. In upper half of the chromatogram, with increasing RF, a green, a pale yellow, a black, a white, and a orange bands appear.

Under visible light, in the lower half of the chromatogram, the Standard solution B exhibits a red band corresponding to kawain similar in RF and color to the band observed in the chromatogram of Standard solution A; below kawain, with decreasing RF, a brown band due to methysticin. Above kawain band, with increasing RF, three yellow and one purple bands appear.

C. HPLC

Analysis: Proceed as directed in the Assay for Content of Kavalactones

Acceptance criteria: The chromatogram of the Sample solution exhibits peaks at the retention times corresponding to the peaks due to methysticin, dihydromethysticin, kawain, dihydrokawain, yangonin, and demethoxyyangonin of the Standard solution B.

 

ASSAY

• Content of Kavalactones

Mobile phase: 0.1% Phosphoric acid, acetonitrile, and isopropyl alcohol (64:20:16).

Solvent: Methanol and water (70:30)

Standard solution A: 0.2 mg/mL of USP Kawain RS in methanol

Standard solution B: Transfer a quantity of USP Powdered Kava Extract RS, accurately weighed, to a suitable volumetric flask, and dilute with Solvent to obtain a solution having known concentration of about 0.2 mg of kawain per mL, sonicate to dissolve. Pass through a membrane filter of 0.45-μm of pore size before injection.

Sample solution: Transfer about 750 mg of Piper methysticum Rootstock Powder to a 50 mL volumetric flask, dilute with Solvent to volume, and sonicate for 60 min. Pass through a membrane filter of 0.45-μm of pore size before injection.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 220 nm

Column: 4.6-mm × 25-cm; L7 (similar to Waters YMCbasic S-5)

Flow rate: 0.6 mL/min

Injection volume: 5 µL

System suitability

Sample: Standard solution A and Standard solution B

Suitability requirements

Chromatogram similarity: The chromatogram of Standard solution B is simiar to the reference chromatogram provided witht the lot of USP Powdered Kava Extract RS being used.

Resolution: NLT 1.8 between desmethoxyyangonin and yangonin

Relative standard deviation: NMT 2.0%

Analysis

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

Using the chromatograms of Standard solution A and Standard solution B, and the reference chromatogram provided with the lot of USP Powdered Kava Extract RS being used, identify the retention time of the peaks corresponding to methysticin, dihydromethysticin, kawain, dihydrokawain, yangonin, and desmethoxyyangonin in the Sample solution.

Separately calculate the percentage of methysticin, dihydromethysticin, kawain, dihydrokawain, yangonin, and desmethoxyyangonin in the portion of Piper methysticum Rootstock Powder taken:

 

Result = (rU/rS) × (CS/W) × 5000 x F

 

rU   = response from the Sample solution

rS   = response from the Standard solution

CS  = concentration of the Standard solution A (mg/mL)

W   = weight of Piper methysticum Powder taken to prepare the Sample solution, (mg)

F    = conversion factor for the analytes as provided in Table 1

 

Table 1

Analyte

Approximate relative retention time

Conversion factor

 Methysticin

0.80

0.66

 Dihydromethysticin

0.94

1.65

 Kawain

1.00

1.00

 Dihydrokawain

1.09

1.70

 Yangonin

1.20

0.88

 Desmethoxyyangonin

1.31

0.90

 

Acceptance criteria: NLT 4.5% of kavalactones

 

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 0.1 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 <2022>: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli

• Articles of Botanical Origin, Test for Aflatoxins <561>: Meet the requirements

 

SPECIFIC TESTS

• Articles of Botanical Origin, Alcohol-Soluble Extractives, Method 1 <561>: NLT 10.0%

• Articles of Botanical Origin, Water-Soluble Extractives, Method 2 <561>: NLT 20.0%

• Loss on Drying <731>:

Analysis: Dry 1 g Piper methysticum Rootstock Powder at 105° for 2 h.

Acceptance criteria: NMT 12%

• Articles of Botanical Origin, Total Ash <561>: NMT 8%

• Articles of Botanical Origin, Acid-Insoluble Ash <561>: NMT 1%

 

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 part(s) of the plant contained in the article.

• USP Reference Standards <11>

USP Kawain RS

USP Powdered Kava Extract RS

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Other Versions

Final Authorized Version 1.0
Proposed For Comment Version 0.2