Panax ginseng Steamed Root and Rhizome Powder

Panax ginseng Steamed Root and Rhizome Powder

Proposed For Development Version 0.1

Panax ginseng Steamed Root and Rhizome Powder


 

DEFINITION

The article consists of the dried root and rhizome of cultivated Panax ginseng C.A.Mey. (Family Araliaceae) collected in the fall, steamed until the material externaly reddish-brown and translucent, reduced to a fine or very fine powder. It contains NLT 1.1% of total ginsenosides, calculated as the sum of ginsenoside Rg1 (C42H72O14), ginsenoside Re (C48H82O18), ginsenoside Rf (C42H72O14), ginsenoside Rb1 (C54H92O23), ginsenoside Ro (C48H76O19), ginsenoside Rc (C53H90O22), ginsenoside Rb2 (C53H90O22), and ginsenoside Rd (C48H82O18), on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

Panax ginseng Root

Panax quinquefolius Root

Panax notoginseng Root

 

CONSTITUENTS OF INTEREST

Ginsenosides: Ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Ro, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Yellowish-white

Microscopic: Resin canal fragments contain yellow masses of secretion. Clusters of calcium oxalate are 20–68 μm in diameter with acute angles. Cork cells are subsquare or polygonal with fine, wavy, sinuous walls in the surface view. Reticulated and scalariform vessels are 10–56 μm in diameter. Abundant in gelatinized starch granules.

• B. Thin-Layer Chromatography

Standard solution A: 0.5 mg/mL of USP Ginsenoside Rg1 RS in methanol

Standard solution B: Sonicate about 50 mg of USP Panax ginseng Steamed Root and Rhizome Dry Extract RS in 10 mL of 20% methanol for 20 min and centrifuge. Transfer 5 mL of the supernatant to a solid phase extraction column containing L1 packing with a sorbent mass-to-column volume ratio of 200 mg/5 mL. The column was previously conditioned as described in the note. Wash the column with 2 mL of water at a rate of 1 drop/s and discard the water solution. Elute the column with 1.0 mL of methanol, collect the methanol elution into a 1-mL volumetric flask, and mix.

[Note—Initially condition the solid phase extraction column with 5 mL of methanol and then with 3 mL of water at a rate of 1 drop/s. Do not allow the column to dry.]

Sample solution: Sonicate about 500 mg of Panax ginseng Steamed Root and Rhizome Powder in 10 mL of 20% methanol for 20 min and centrifuge. Transfer 5 mL of the supernatant to a solid phase extraction column containing L1 packing with a sorbent mass-to-column volume ratio of 200 mg/5 mL. The column was previously conditioned as described in the note under Standard solution B. Wash the column with 2 mL of water at a rate of 1 drop/s and discard the water solution. Elute the column with 1.0 mL of methanol, collect the methanol elution into a 1-mL volumetric flask, and mix.

Chromatographic system

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

Adsorbent: Use a suitable chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates).

Application volume: 3 µL, as 8-mm bands

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

Temperature: About 25º

Developing solvent system: Methylene chloride, anhydrous ethanol, and water (60: 45: 6.5)

Developing distance: 6 cm

Derivatization reagent: 10% Sulfuric acid in alcohol. [Note—Slowly add sulfuric acid to ice-cold ethanol.]

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 at 105° for 5 min, and examine immediately under visible light and UV light at 366 nm.

System suitability: Under visible light, Standard solution B exhibits: in the lower-third section, three reddish-violet bands due to ginsenoside Rb1 and Ro (co-elute), ginsenoside Rb2, and ginsenoside Rc in order of increasing RF; in the middle-third section, three reddish-violet bands due to ginsenoside Rd, ginsenoside Re, and ginsenoside Rf in order of increasing RF; and in the upper-third section, a most intense reddish-violet band corresponding in RF and color to the band of ginsenoside Rg1 in Standard solution A.

Under UV light at 366 nm, Standard solution B exhibits: in the lower-third section, three blue fluorescent bands due to ginsenoside Rb1 and Ro (co-elute), ginsenoside Rb2, and ginsenoside Rc in order of increasing RF; in the middle-third section, three bands due to ginsenoside Rd (blue fluorescent), ginsenoside Re (pinkish-violet), and ginsenoside Rf (pinkish-violet) in order of increasing RF; and in the upper-third section, a most intense pinkish-violet band corresponding in RFand color to the band of ginsenoside Rg1 in Standard solution A.

Acceptance criteria: Under visible light, the Sample solution exhibits: in the lower-third section, three reddish-violet bands corresponding in RFand color to the bands due to ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rc (distinction from Panax notoginseng Root and Rhizome) in Standard solution B; in the middle-third section, three reddish-violet bands corresponding in RF and color to the bands due to ginsenoside Rd, ginsenoside Re, and ginsenoside Rf (distiction from Panax quinquefolius Root and Rhizome) in Standard solution B; and in the upper-third section, a most intense reddish-violet band corresponding in RF and color to the band of ginsenoside Rg1 in Standard solution A and Standard solution B.

Under UV light at 366 nm, the Sample solution exhibits: in the lower-third section, three blue fluorescent bands corresponding in RFand color to the bands due to ginsenoside Rb1, ginsenoside Rb2, and ginsenoside Rc (distinction from Panax notoginseng Root and Rhizome) in Standard solution B; in the middle-third section, three bands corresponding in RFand color to the bands due to ginsenoside Rd (blue fluorescent), ginsenoside Re (pinkish-violet), and ginsenoside Rf (pinkish-violet, distinction from Panax quinquefolius Root and Rhizome) in Standard solution B; and in the upper-third section, a most intense pinkish-violet band corresponding in RF and color to the band of ginsenoside Rg1 in Standard solution A and Standard solution B.

C. HPLC">

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

Acceptance criteria: The chromatogram of the Sample solution exhibits a peak with a retention time corresponding to ginsenoside Rg1 in Standard solution A and peaks for ginsenoside Re, ginsenoside Rf (distinction from Panax quinquefolius Root and Rhizome and Panax notoginseng Root and Rhizome, they do not contain ginsenoside Rf), ginsenoside Rb1, ginsenoside Ro, ginsenoside Rc, ginsenoside Rb2 (distinction from Panax notoginseng Root and Rhizome, which does not contain ginsenosides Ro, Rc, and Rb2), and ginsenoside Rd corresponding to the retention times for the same ginsenosides in Standard solution B. The intensity of the peak between ginsenoside Ro and ginsenoside Rc is much smaller than that for the peak of ginsenoside Rc; the intensity for the peaks of ginsenoside Rg1, ginsenoside Rb1, and ginsenoside Ro are similar and most intense in the HPLC chromatogram (distinction from Panax ginseng Root and Rhizome, in which the peak between ginsenoside Ro and ginsenoside Rc is clearly observed; and the peak of ginsenoside Rb1 is smaller than the peak of ginsenoside Ro).

 

ASSAY

• Content of Ginsenosides

Solution A: 0.01% Phosphoric acid in water

Solution B: Acetonitrile

Mobile phase: See Table 1.

Table 1

Time

(min)

Solution A

(%)

Solution B

(%)

0 82 18
25 79 21
35 72 28
75 68 32
75.5 0 100
80.5 0 100
81 82 18
88 82 18

Solvent: Methanol and water (7:3)

Standard solution A: 0.15 mg/mL of USP Ginsenoside Rg1 RS in Solvent

Standard solution B: 5 mg/mL of USP Panax ginseng Steamed Root and Rhizome Dry Extract RS in Solvent. Before injection, pass through a nylon filter of 0.22-μm or finer pore size.

Sample solution: Accurately transfer about 500 mg of Panax ginseng Steamed Root and Rhizome Powder to a 100-mL glass-stoppered conical flask. Add 25 mL of Solvent, sonicate for 30 min (565 W, 37 KHz), and filter. Repeat the extraction one more time. Rinse the flask with 15 mL of Solvent and filter into the same flask. Evaporate under reduced pressure to dryness. Dissolve the residue with Solvent and quantitatively transfer the solution to a 10-mL volumetric flask. Dilute with Solvent to volume and mix. Before injection, pass through a nylon filter of 0.22-μm or finer pore size. Discard the first portion of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 203 nm

Column: 4.6-mm × 15-cm; 2.7-μm packing L1 (similar to Agilent™ C18, 120Å)

Column temperature: 40 ± 1°

Flow rate: 1.2 mL/min

Injection volume: 20 µL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Chromatogram similarity: The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Panax ginseng Steamed Root and Rhizome Dry Extract RS being used.

Resolution: NLT 1.5 between ginsenoside Rg1 and ginsenoside Re peaks, Standard solution B

Tailing factor: NMT 2.0 for the ginsenoside Rg1 peak, Standard solution A

Relative standard deviation: NMT 2.0% for the ginsenoside Rg1 peak, Standard solution A

Analysis

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

[Note—Protect from light. The Standard solutions and Sample solution are stable for 24 h at room temperature.]

Using the chromatograms of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Panax ginseng Steamed Root and Rhizome Dry Extract RS being used, identify the retention times of the peaks corresponding to ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Ro, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd in the Sample solution. [Note—The approximate relative retention times of the analytes are provided in Table 2.]

Table 2

Analyte

Approximate Relative Retention

Time

Conversion

Factor

Ginsenoside Rg1 1.00 1.00
Ginsenoside Re 1.04 1.00
Ginsenoside Rf 1.75 0.83
Ginsenoside Rb1 2.37 1.27
Ginsenoside Ro 2.44 1.03
Ginsenoside Rc 2.56 1.22
Ginsenoside Rb2 2.78 1.22
Ginsenoside Rd 3.23 1.02

 

Separately calculate the percentages of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Ro, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd in the portion of Panax ginseng Steamed Root and Rhizome Powder taken:

 

Result = (rU/rS) × CS × (V/W) × F ×100

 

rU   = peak area of the relevant analyte from the Sample solution

rS   = peak area of ginsenoside Rg1 from Standard solution A

CS  = concentration of USP Ginsenoside Rg1 RS in Standard solution A (mg/mL)

V    = volume of the Sample solution (mL)

W   = weight of Panax ginseng Steamed Root and Rhizome Powder taken to prepare the Sample solution (mg)

F    = conversion factor for the analytes as provided in Table 2

 

Calculate the total ginsenosides by adding the percentages of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Ro, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd.

Acceptance criteria: NLT 1.1% on the dried basis

 

CONTAMINANTS

• Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 0.3 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

• Articles of Botanical Origin, Aflatoxins <561>: Meets the requirements

 

SPECIFIC TESTS

• Articles of Botanical Origin, Alcohol-Soluble Extractives, Method 2 <561>: NLT 3.0%

• Loss on Drying <731>

Sample: 2 g of Panax ginseng Steamed Root and Rhizome Powder

Analysis: Dry the Sample at 105° for 5 h.

Acceptance criteria: NMT 12.0%

• Articles of Botanical Origin, Total Ash <561>

Analysis: 3.0 g of Panax ginseng Steamed Root and Rhizome Powder

Acceptance criteria: NMT 4.5%

• Articles of Botanical Origin, Acid-Insoluble Ash <561>: NMT 1.0%

 

ADDITIONAL REQUIREMENTS

• Packaging and Storage: Preserve in well-closed containers, protected from light and moisture, and store at controlled 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 Ginsenoside Rg1 RS

USP Panax ginseng Steamed Root and Rhizome Dry Extract RS

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Other Versions

Final Authorized Version 1.0
Proposed For Comment Version 0.2