Polygonum multiflorum Root Dry Extract

Polygonum multiflorum Root Dry Extract

Proposed For Development Version 0.1

Polygonum multiflorum Root Dry Extract

 


DEFINITION

The article is prepared from the dried root of Polygonum multiflorum Thunb. (Family Polygonaceae), collected in autumn or winter, by extraction with a mixture of alcohol and water (7:3). The ratio of starting crude plant material to Dry Extract is about 5:1. It contains NLT 90.0% and NMT 110.0% of the labeled amount of 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside (C20H22O9); NLT 90.0% and NMT 110.0% of the labeled amount of total anthraquinones, calculated as the sum of emodin-8-O-β-d-glucoside (C21H20O10), physcion-8-O-β-d-glucoside (C22H22O10), emodin (C15H10O5), and physcion (C16H12O5); and NLT 90.0% and NMT 110.0% of the labeled amount of anthraquinone glycosides, calculated as the sum of emodin-8-O-β-d-glucoside and physcion-8-O-β-d-glucoside; all on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

Pteroxygonum giraldii Damm. et Diels

Polygonum cillinerve (Nakai) Ohwi

Cynanchum bungei Decne.

 

CONSTITUENTS OF INTEREST

Stilbenes: 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside

Anthraquinones: Emodin-8-O-β-d-glucoside, physcion-8-O-β-d-glucoside, emodin, and physcion

 

IDENTIFICATION

• A. Thin-Layer Chromatography

Standard solution A: 0.5 mg/mL of USP 2,3,5,4'-Tetrahydroxystilbene -2-O-β-d-glucoside RS and 0.1 mg/mL of USP Emodin RS in methanol

Standard solution B: 60 mg/mL of USP Polygonum multiflorum Root Dry Extract RS in methanol. Sonicate for 15 min, centrifuge, and use the supernatant.

Sample solution: 60 mg/mL of Polygonum multiflorum Root Dry Extract in methanol. Sonicate for 15 min, centrifuge, and use the supernatant.

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: 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 A: Ethyl acetate, methanol, and acetic acid (4: 1: 0.2)

Developing solvent system B: Hexanes and ethyl acetate (3:2)

Developing distance

Developing solvent system A: 3.5 cm

Developing sovent system B: 7 cm

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 containing Developing solvent system A to 3.5 cm, and remove the plate from the chamber. After drying the plate in a hood, develop the chromatograms in a saturated chamber containing Developing solvent system B to 7 cm. Remove the plate from the chamber and dry in a hood. Examine under UV light at 366 nm.

System suitability: Under UV light at 366 nm, Standard solution B exhibits, in the lower-half section, a bright blue band corresponding to the band of 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside in Standard solution A, an orange-red band due to emodin-8-O-β-d-glucoside right below the bright blue band, a blue band right below the orange-red band, and another orange-red band below the blue band; the first three bands are clearly separated. In the upper-half section, Standard solution B exhibits two yellow bands, one corresponding to the band of emodin in Standard solution A, and another band due to physcion above emodin.

Acceptance criteria: Under UV light at 366 nm, the Sample solution exhibits, in the lower-half section, a bright blue band corresponding to the band of 2,3,5,4'-tetrahydroxystllbene-2-O-β-d-glucoside (distinguished from Polygonum cillinerve root, Pteroxygonum giraldii root, and Cynanchum auriculatum root) in Standard solutions A and B, an orange-red band corresponding to the band of emodin-8-O-β-d-glucoside in Standard solution B right below the bright blue band, a blue band right below the orange-red band, and another orange-red band below the blue band, all corresponding to similar bands in RF and color to Standard solution B. In the upper-half section, the Sample solution exhibits two yellow bands (distinguished from Pteroxygonum giraldii root and Cynanchum auriculatum root), one corresponding to the band of emodin in Standard solutions A and B, and another band corresponding to a similar band in Standard solution B due to physcion. The Sample solution exhibits additional minor bands corresponding to similar bands in Standard solution B.

• B. Anthraquinones HPLC Profile

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

Acceptance criteria: The Sample solution exhibits a peak with a retention time corresponding to emodin in Standard solution A; the peaks related to anthraquinones for emodin-8-O-β-d-glucoside, physcion-8-O-β-d-glucoside, and physcion correspond to the same anthraquinones in Standard solution B and the reference chromatogram provided with the lot of USP Polygonum multiflorum Root Dry Extract RS being used. The Sample solution exhibits additional peaks corresponding to similar peaks in Standard solution B. The content ratio for emodin-8-O-β-d-glucoside to emodin is about 1.3: 6; the content ratio for physcion-8-O-β-d-glucoside to physcion is about 1.2: 5.

C. Stilbene glycosides HPLC Profile

Analysis: Proceed as directed in the Assay for Content of 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside.

Acceptance criteria: The Sample solution exhibits only one principal peak with a retention time corresponding to 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (distinguished from Polygonum cillinerve root, which shows three principal peaks and Polygonum cuspidatum root for which the pricipal peak corresponds to polydatin) in the Standard solution.

 

ASSAY

• Content of 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside

Solution A: 0.1% Formic acid in water

Solution B: Acetonitrile

Mobile phase: See Table 1.

Table 1

Time
(min)

Solution A
(%)

Solution B
(%)

0

83

17

10

80

20

20

70

30

25

0

100

30

0

100

31

83

17

40

83

17

 

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

Solvent: Methanol and water (7:3)

Standard solution: 0.25 mg/mL of USP 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside RS in methanol

System suitability solution: 0.25 mg/mL of USP 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside RS and 0.25mg/mL of USP Polydatin RS in methanol

Sample solution: Accurately transfer about 60 mg of Polygonum multiflorum Root Dry Extract into a suitable stoppered conical flask and accurately add 10 mL of methanol. Weigh the filled flask with a precision of ± 0.1 mg and then sonicate for 15 min. After cooling to room temperature, adjust to the initial weight by adding methanol. Before injection, pass through a membrane filter of 0.45-μm or finer pore size, and discard the first portion of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 320 nm

Column: 4.6-mm × 25-cm; packing L1 (similar to Shimadzu ODS C18 and Aglient Zorbax Stable Bond C18)

Column temperature: 35 ± 5°

Flow rate: 1.0 mL/min

Injection volume: 10 µL

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 1.5 between the peaks of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside and polydatin, System suitability solution [Note: The approximate relative retention times of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside and polydatin are 1.00 and 0.95 respectively]

Tailing factor: NMT 1.5 for 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside

Relative standard deviation: NMT 2.0% for 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside

Analysis

Samples: Standard solution and Sample solution

Using the chromatogram of the Standard solution, identify the retention time of the peak for 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside in the Sample solution.

Calculate the percentage of 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside in the portion of Polygonum multiflorum Root Dry Extract taken:

 

Result = (rU/rS) × CS × (V/W) × 100

 

rU   = peak area of 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside from the Sample solution

rS   = peak area of 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside from the Standard solution

CS  = concentration of USP 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside RS in the Standard solution (mg/mL)

V    = volume of the Sample solution (mL)

W   = weight of Polygonum multiflorum Root Dry Extract taken to prepare the Sample solution (mg)

 

Calculate the percentage of the labeled amount of 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside in the portion of Polygonum multiflorum Root Dry Extract taken:

 

Result = (P/L) × 100

 

P   = content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside determined above (%)

L   = labeled amount of 2,3,5,4'-tetrahydroxystilbene-2-O-β-d-glucoside (%)

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

• Content of Anthraquinones

Solution A: 0.1% Formic acid in water

Solution B: Acetonitrile

Mobile phase: See Table 2.

Table 2

Time
(min)
Solution A
(%)
Solution B
(%)
0 70 30
3 70 30
8 50 50
10 0 100
13 0 100
14 70 30
19 70 30

 

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

Solvent: Methanol

Standard solution A: 0.01 mg/mL of USP Emodin RS in methanol

Standard solution B: 6 mg/mL of USP Polygonum multiflorum Root Dry Extract RS in methanol, sonicate for 15 min, centrifuge, and pass through a membrane filter of 0.45-μm or finer pore size.

Sample solution: Accurately transfer about 60 mg of Polygonum multiflorum Root Dry Extract into a suitable stoppered conical flask and accurately add 10 mL of methanol. Weigh the filled flask with a precision of ± 0.1 mg and then sonicate for 15 min. After cooling to room temperature, adjust to the initial weight by adding methanol. Before injection, pass through a membrane filter of 0.45-μm or finer pore size, and discard the first portion of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 4.6 mm × 15 cm; 5-μm packing L1 (similar to Shimadzu ODS C18, Aglient Zorbax Stable Bond C18, and Thermo Syncronis C18)

Column temperature: 30 ± 5°

Flow rate: 1.0 mL/min

Injection volume: 10 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 Polygonum multiflorum Root Dry Extact RS being used.

Resolution: NLT 1.5 betwen the peak of emodin-8-O-β-d-glucoside and the peak after it, Standard solution B

Tailing factor: NMT 1.5 for the peak of emodin, Standard solution A

Relative standard deviation: NMT 2.0% for emodin, Standard solution A

Analysis

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

Using the chromatograms of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Polygonum multiflorum Root Dry Extract RS being used, identify the retention times of the peaks corresponding to emodin, emodin-8-O-β-d-glucoside, physcion-8-O-β-d-glucoside, and physcion. The approximate relative retention times are provided in Table 3.

Table 3

Analyte Approximate Relative Retention Times Conversion Factor
Emodin-8-O-β-d-glucoside 0.55 1.15
Physcion-8-O-β-d-glucoside 0.68 1.18
Emodin 1.00 1.00
Physcion 1.08 1.04

 

Separately calculate the percentages of emodin-8-O-β-d-glucoside, physcion-8-O-β-d-glucoside, emodin, and physcion in the portion of Polygonum multiflorum Root 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 emodin from the Standard solution A

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

V    = volume of the Sample solution (mL)

W   = weight of Polygonum multiflorum Root Dry Extract taken to prepare the Sample solution (mg)

F    = conversion factor for the analytes as provided in Table 3.

 

Calculate the content of total anthraquinones as the sum of emodin-8-O-β-d-glucoside, physcion-8-O-β-d-glucoside, emodin, and physcion.

Calculate the content of total anthraquinone glycosides as the sum of emodin-8-O-β-d-glucoside and physcion-8-O-β-d-glucoside.

 

Calculate the percentage of the labeled amount of total anthraquinones in the portion of Polygonum multiflorum Root Dry Extract taken:

 

Result = (P/L) × 100

 

P  = content of total anthraquinones determined above (%)

L  = labeled amount of total anthraquinones (%)

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

 

Calculate the percentage of the labeled amount of total anthraquinone glycosides in the portion of Polygonum multiflorum Root Dry Extract taken:

 

Result = (P/L) × 100

 

P  = content of total anthraquinone glycosides determined above (%)

L  = labeled amount of total anthraquinone glycosides (%)

Acceptance criteria: 90.0%–110.0% 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.

Absence of 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

Loss on Drying <731>

Sample: 1 g of Polygonum multiflorum Root Dry Extract

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

Acceptance criteria: NMT 10.0%

Articles of Botanical Origin, Total Ash <561>

Analysis: 3.0 g of Polygonum multiflorum Root Dry Extract

Acceptance criteria: NMT 7.0%

 

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 from which the article was prepared. It meets other labeling requirements under Botanical Extracts <565>.

USP Reference Standards <11>

USP Emodin RS

USP Polygonum multiflorum Root Dry Extract RS

USP 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside RS

 

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Other Versions

Final Authorized Version 1.0
Proposed For Comment Version 0.2