Panax ginseng Steamed Root and Rhizome Dry Extract

Panax ginseng Steamed Root and Rhizome Dry Extract

Proposed For Comment Version 0.2

Panax ginseng Steamed Root and Rhizome Dry Extract


 

DEFINITION

The article is prepared from the dried root and rhizome of cultivated Panax ginseng C.A.Mey. (Family Araliaceae) collected in the fall, steamed until the material is externally reddish-brown and translucent, by extraction with water or hydroalcoholic mixtures. It contains NLT 90.0% and NMT 110.0% of the labeled amount 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. HPTLC for Articles of Botanical Origin <203>

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

Standard solution B: Sonicate about 100 mg of USP Powdered Asian Ginseng Extract RS in 10 mL of 20% methanol for 20 min and centrifuge. Transfer 5 mL of the supernatant into 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 100 mg of Panax ginseng Steamed Root and Rhizome Dry Extract in 10 mL of 20% methanol for 20 min and centrifuge. Transfer 5 mL of the supernatant into 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

Adsorbent: Chromatographic silica gel mixture with an average particle size of about 5 µm

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 and dry in air. Develop in a saturated chamber, remove the plate from the chamber, and dry in air. Treat the plate 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, five reddish-violet bands, the above three due to ginsenosides 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, five blue fluorescent bands, the above three due to ginsenosides 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 ginsenosides Rb1 and Ro (co-elute), 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 ginsenosides Rb1 and Ro (co-elute), 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.

• B. 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 ginsenoside Rc peak; the intensity for the peaks of ginsenoside Rg1 and ginsenoside Rb1 are similar and most intense in the HPLC chromatogram; the peak intensity of ginsenoside Ro is similar or smaller than ginsenoside Rb1 (distinction from Standard solution B which represents Panax ginseng Root and Rhizome, in which the peak between ginsenoside Ro and ginsenoside Rc is clearly observed; and the peak intensity of ginsenoside Rb1 is smaller than 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: 10 mg/mL of USP Powdered Asian Ginseng Extract RS in Solvent. Sonicate and pass through a nylon filter of 0.22-μm pore size before injection.

Sample solution: Accurately transfer about 100 mg (adjust the amount if necessary) of Panax ginseng Steamed Root and Rhizome Dry Extract into a suitable stoppered flask. Accurately add 10 mL of Solvent and shake until the sample is totally dissolved. Pass through a nylon filter of 0.22-μm 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°

Flow rate: 1.2 mL/min

Injection volume: 20 µL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

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

Chromatogram similarity: The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Asian Ginseng Extract RS being used.

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 Powdered Asian Ginseng 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—See Table 2 for the approximate relative retention times.]

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 Dry Extract 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 Dry Extract taken to prepare the Sample solution (mg)

F    = conversion factor for the analyte (see Table 2)

 

Calculate the content of total ginsenosides as the sum of ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Ro, ginsenoside Rc, ginsenoside Rb2, and ginsenoside Rd.

 

Calculate the percentage of the labeled amount of total ginsenosides in the portion of Panax ginseng Steamed Root and Rhizome Dry Extract taken:

 

Result = (P/L) × 100

 

P   = content of total ginsenosides as determined above (%)

L    = labeled amount of total ginsenosides (%)

 

Acceptance criteria: 90.0%–110.0% on the dried basis

 

CONTAMINANTS

Articles of Botanical Origin <561>, Pesticide Residue Analysis: 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 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 Panax ginseng Steamed Root and Rhizome Dry Extract

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

Acceptance criteria: NMT 8.0%

 

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 following the official name of the plant from which the article was prepared. It meets other labeling requirements under Botanical Extracts <565>.

USP Reference Standards <11>

USP Ginsenoside Rg1 RS

USP Powdered Asian Ginseng Extract RS

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Proposed For Development Version 0.1