Salvia miltiorrhiza Root and Rhizome Dry Extract

Salvia miltiorrhiza Root and Rhizome Dry Extract

Final Authorized Version 1.0

Salvia miltiorrhiza Root and Rhizome Dry Extract


 

DEFINITION

The article is prepared from the dried roots and rhizomes of Salvia miltiorrhiza Bunge (Family Lamiaceae), collected in spring or fall, by extraction with hydroalcoholic mixtures. The ratio of starting crude plant material to Dry Extract is 10:1. It contains NLT 90.0% and NMT 110.0% of the labeled amount of tanshinones, calculated as the sum of cryptotanshinone, tanshinone I, and tanshinone IIA; and NLT 90.0% and NMT 110.0% of the labeled amount of salvianolic acid B; all calculated on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

Salvia bowleyana Dun

Salvia przewalskii Maxim.

Salvia trijuga Diels

Salvia yunnanensis C. H. Wight

 

CONSTITUENTS OF INTEREST

Diterpenoid quinones: Tanshinone I, tashinone IIA, and cryptotanshinone

Phenolic acids: Salvianolic acid, lithospermic acid, and rosmarinic acid

 

IDENTIFICATION

• A. Thin-Layer Chromatography

Standard solution A: 1.0 mg/mL each of USP Tashinone IIA RS and USP Salvianolic Acid B RS in methanol

Standard solution B: 50 mg/mL of USP Powdered Chinese Salvia Extract RS in methanol. Sonicate for 15 min, centrifuge, and use the supernatant.

Sample solution: 50 mg/mL of Dry Extract in methanol. Sonicate for 15 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, Si 60 F254)

Application volume: 5 µL, 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: Toluene, dichloromethane, ethyl acetate, methanol, and formic acid (4:6:8:1:4)

Developing distance: 6 cm

Derivatization reagent: 20 mL sulfuric acid in 180 mL methanol

Analysis 

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

Apply the Samples as bands to a suitable HPTLC plate. Use saturated chambers. Develop the chromatograms in Developing solvent system, remove the plate and dry. Treat with Derivatization reagent, heat for 5 min at 100°, and examine under visible light.

System suitability: Under visible light, the chromatogram of Standard solution B exhibits two dark brown and two pink bands in lower half of the chromatogram, with the intense pink band similar in RF and color to the salvianolic acid B band in the chromatogram of Standard solution A. About seven bands in upper half of the chromatogram with increasing RF: a pink, a light purple, a light brown, a purple, a yellow-brown, and an intense purple band similar in RF and color to the tanshinone IIA band in the chromatogram of Standard solution A.

Acceptance criteria: Under visible light, the lower half of the chromatogram of the Sample solution exhibits an intense pink band corresponding in color and RF to the band due to salvianolic acid B in the chromatogram of Standard solution A. The upper half of the chromatogram of the Sample solution exhibits an intense purple band close to solvent front corresponding in color and RF to the band due to tanshinone IIA in the chromatogram of Standard solution A. Nine additional bands corresponding to similar bands in the chromatogram of Standard solution B: two dark brown bands and a pale pink band in lower half and a pink, a light purple, a light brown, a purple, and a yellow-brown band in upper half of the chromatogram of Sample solution.

• B. HPLC

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

Acceptance criteria: The chromatogram of the Sample solution exhibits the most intense peak at a retention time corresponding to that of tanshinone IIA in the chromatogram of Standard solution A. The Sample solution chromatogram exhibits two additional peaks corresponding to tanshinone I and cryptotanshinone, of lesser intensity and accounting for about half of the total tanshinones content.

• C. HPLC

Analysis: Proceed as directed in the Assay for Content of Salvianolic Acid B.

Acceptance criteria: The chromatogram of the Sample solution exhibits the most intense peak at a retention time corresponding to that of salvianolic acid B in the chromatogram of Standard solution A.

 

ASSAY

Content of Tanshinones

Solution A: 0.02% Phosphoric acid in water

Solution B: Acetonitrile

Mobile phase: See Table 1.

 

Table 1

Time
(min)

Solution A
(%)

Solution B
(%)

0

39

61

6

39

61

20

10

90

20.5

39

61

25

39

61

 

 

[Note—Proceed under subdued light or use low-actinic glassware. The Standard solutions and Sample solution are stable for 24 h at room temperature.]

Standard solution A: 0.02 mg/mL of USP Tanshinone IIA RS in methanol

Standard solution B: 2.0 mg/mL of USP Powdered Chinese Salvia Extract RS in methanol. Sonicate for 15 min, and pass through a membrane filter of 0.45-µm or finer pore size. Discard the first few mL of the filtrate.

Sample solution: 2.0 mg/mL of Dry Extract in methanol. Sonicate for 15 min, and pass through a membrane filter of 0.45-µm or finer pore size. Discard the first few mL of the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 270 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1 (similar to Zorbax Extend C18)

Column temperature: 25±1°

Flow rate: 1.0 mL/min

Injection volume: 10 µL

System suitability

Samples: Standard solution A and Standard solution B

[Note—The approximate relative retention times of the different peaks for cryptotanshinone, tanshinone I, and tanshinone IIA are 0.75, 0.79, and 1.00, respectively.]

Suitability requirements 

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

Resolution: NLT 1.5 between the cryptotanshinone and tanshinone I peaks, Standard solution B

Tailing factor: NMT 2.0 for the tanshinone IIA peak, Standard solution A

Relative standard deviation: NMT 2.0% determined from the tanshinone IIA peak in repeated injections, 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 Powdered Chinese Salvia Extract RS being used, identify the retention times of the peaks corresponding to different tanshinones in the Sample solution chromatogram.

Separately calculate the percentages of cryptotanshinone, tanshinone I, and tanshinone IIA in the portion of Dry Extract taken:

 

Result = (rU/rS) × (CS/CU) × F × 100

 

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

rS      = peak area of tanshinone IIA from Standard solution A

CS    = concentration of tanshinone IIA in Standard solution A (mg/mL)

CU    = concentration of Dry Extract in the Sample solution (mg/mL)

F      = conversion factor for analytes; 1.18 for crytotanshinone, 1.31 for tanshinone I, and 1.00 for tanshinone IIA

Calculate the content of tanshinones as the sum of the percentages of cryptotanshinone, tanshinone I, and tanshinone IIA.

Calculate the percentage of the labeled amount of tanshinones in the Dry Extract:

 

Result = (P/L) × 100

 

P    = content of tanshinones as determined above (%)

L    = labeled amount of tanshinones (%)

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

• Content of Salvianolic Acid B

Solution A: 0.1% Phosphoric acid in water

Mobile phase: Solution A and acetonitrile (78:22)

[Note—The Standard solution and Sample solution are stable for 12 h at room temperature.]

Solvent: Methanol and water (8:2)

Standard solution: 0.1 mg/mL of USP Salvianolic Acid B RS in Solvent

Sample solution: 2 mg/mL of Dry Extract in methanol. Sonicate for 15 min, and pass through a membrane filter of 0.45-µm or finer pore size. Discard the first few mL of the filtrate.

Chromatographic system 

(See Chromatography <621>, System Suitability.)

Mode: LC

Detector: UV 286 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1 (similar to Zorbax SB C18)

Column temperature: 25±1°

Flow rate: 1.2 mL/min

Injection volume: 10 µL

System suitability 

Sample: Standard solution

Suitability requirements 

Tailing factor: NMT 2.0 for the salvianolic acid B peak

Relative standard deviation: NMT 2.0%, determined from the salvianolic acid B peak in repeated injections

Analysis 

Samples: Standard solution and Sample solution

Using the chromatogram of the Standard solution, identify the retention time of the peak corresponding to salvianolic acid B in the Sample solution chromatogram.

Calculate the percentage of salvianolic acid B in the portion of Dry Extract taken:

 

Result = (rU/rS) × (CS/CU) × 100

 

rU    = peak area of salvianolic acid B from the Sample solution

rS    = peak area of salvianolic acid B from Standard solution A

CS   = concentration of salvianolic acid B in Standard solution A (mg/mL)

CU   = concentration of Dry Extract in the Sample solution (mg/mL)

Calculate the percentage of the labeled amount of salvianolic acid B in the Dry Extract taken:

 

Result = (P/L) × 100

 

P     = content of salvianolic acid B as determined above (%)

L     = labeled amount of salvianolic acid B (%)

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

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

Microbial Enumeration Tests <61>: The total aerobic bacterial count does not exceed 104 cfu/g and the total combined molds and yeasts count does not exceed 102 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 g of Dry Extract

Analysis: Dry at 105° for 5 h.

Acceptance criteria: NMT 8.0%

 

ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers, protected from light and moisture. Store at controlled room temperature.

Labeling: The label states the Latin binomial and, following the official name, the part(s) of the plant from which the article was derived. It meets other labeling requirements under Botanical Extracts <565>.

USP Reference Standards <11>

USP Aflatoxins RS

USP Powdered Chinese Salvia Extract RS

USP Salvianolic Acid B RS

USP Tanshinone IIA RS

Other Versions

Proposed For Comment Version 0.2