Salvia miltiorrhiza Root and Rhizome Powder

Salvia miltiorrhiza Root and Rhizome Powder

Proposed For Comment Version 0.2

Salvia miltiorrhiza Root and Rhziome Powder


 

DEFINITION

The article consists of the dried roots and rhizomes of Salvia miltiorrhiza Bunge (Family Lamiaceae) reduced to a powder or very fine powder. It contains NLT 0.1% of tanshinone IIA; NLT 0.2% of total tanshinones calculated as the sum of cryptotanshinone, tanshinone I, and tanshinone IIA; and NLT 3.0% of salvianolic acid B; all calculated on the dried basis. It is collected in spring or fall.

 

Potential Confounding Materials

Salvia przewalskii Maxim.

Salvia yunnanensis C. H. Wight

Salvia bowleyana Dun

Salvia trijuga Diels

 

Constituents of Interest

Diterpenoid quinones: Tanshinone I, tashinone IIA, and cryptotanshinone

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

 

IDENTIFICATION

• A. Botanic Characteristics

Macroscopic: Yellowish-brown to reddish-brown in color

Microscopic: It shows fragments of cork cells, subrectangular or polygonal, containing yellowish-brown pigments, 10–150 µm in diameter; parenchymatous cells of cortex, subsquare or polygonal, containing reddish-brown pigments; stone cells, subrounded, subtriangular, subrectangular or irregular shape, some elongated, mostly 14–70 µm in diameter, up to 270 µm in length; fibers mostly in bundles, long fusiform in shape, ends oblique-sharp or blunt-round, with oblique or criss-cross striations, 10–60 µm in diameter; and reticulate and pitted vessels, up to 120 µm in diameter.

• B. Thin-Layer Chromatography

Standard solution A:  0.5 mg/mL of USP Tashinone IIA RS and 1.5 mg/mL USP Salvianolic Acid B RS in alcohol

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

Sample solution: Sonicate about 1.0 g of Salvia miltiorrhiza Root and Rhizome Powder in 5.0 mL of alcohol 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)

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 A: Ethyl acetate, chloroform, toluene, formic acid, and methanol (8:6:4:4:1)

Developing solvent system B: Solvent hexane and ethyl acetate (4:1)

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 A until the solvent front has moved up about 40% of the plate. Remove the plate and allow to dry. Develop the chromatograms in Developing solvent system B until the solvent front has moved up about three-fourths of the plate. Remove the plate and dry. Examine under visible light, and under UV light at 366 nm and 254 nm.

System suitability: Under visible light, the chromatogram of Standard solution B exhibits a pink band in the upper-third section similar in RFand color to the tanshinone IIA band in the chromatogram of Standard solution A, a yellowish-orange band below the pink band, and an orange band at about the middle of the chromatogram due to tanshinone I and cryptotanshinone, respectively.

Under UV light at 366 nm, the chromatogram of Standard solution B exhibits three blue fluorescent bands: an intense band in the lower-third section of the chromatogram corresponding in RFand color to the salvianolic acid B band in the chromatogram of Standard solution A; and two minor bands in the lower-third section of the chromatogram and above the salvianolic acid B band, due to lithospermic acid and rosmarinic acid.

Under UV light at 254 nm, the chromatogram of Standard solution B exhibits intense quenching bands at an RF corresponding to those for tanshinone IIA and salvianolic acid B in the chromatogram of Standard solution A.

 Acceptance criteria: Under visible light, the chromatogram of the Sample solution exhibits a pink band corresponding in color and RFto the band due to tanshinone IIA in the chromatogram of Standard solution A, and the following bands corresponding to similar bands in the chromatogram of Standard solution B: a yellowish-orange band in the upper-third section of the chromatogram, and an orange band at about the middle of the chromatogram.

Under UV light at 366 nm, the chromatogram of the Sample solution exhibits an intense blue fluorescent band corresponding in color and RFto the band due to salvianolic acid B in the chromatogram of Standard solution A, and the following bands corresponding to similar bands in the chromatogram of Standard solution B: two minor blue fluorescent bands in the lower-third section of the chromatogram and above the salvianolic acid B band.

Under UV light at 254 nm, the chromatogram of the Sample solution exhibits two intense quenching bands corresponding in RFto the bands due to tanshinone IIA and salvianolic acid B in the chromatogram of Standard solution A.

• C. 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.

• D. 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 having a 0.45-µm or finer pore size. Discard the first few mL of the filtrate.

Sample solution: About 0.3 g of Salvia miltiorrhiza Root and Rhizome Powder, accurately weighed, in 40 mL of methanol. Sonicate for 30 min, filter into a 50-mL volumetric flask, and wash the residue and the filter paper with a few mL of methanol. Adjust with methanol to volume, mix, and pass through a membrane filter having a 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

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. 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.

Separately calculate the percentages of cryptotanshinone, tanshinone I, and tanshinone IIA in the portion of Salvia miltiorrhiza 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 tanshinone IIA from Standard solution A

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

        = volume of the Sample solution (mL)

       = weight of Salvia miltiorrhiza Root and Rhizome Powder taken to prepare the Sample solution (mg)

         = conversion factor for the 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.

Acceptance criteria

Tanshinone IIA: NLT 0.1% on the dried basis

Total tanshinones: NLT 0.2% 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 stock solution: About 150 mg of Salvia miltiorrhiza Root and Rhizome Powder, accurately weighed, in 40 mL of Solvent. Sonicate for 30 min, filter into a 50-mL volumetric flask, and wash the residue and the filter paper with a few mL of Solvent. Adjust with Solvent to volume, mix, and centrifuge a portion.

Sample solution: Dilute a portion of the supernatant from the Sample stock solution with Solvent (1:2), mix, and pass through a membrane filter having a 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 Salvia miltiorrhiza Root and Rhizome Powder taken:

 

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

 

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

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

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

V          = volume of the Sample stock solution (mL)

W         = weight of Salvia miltiorrhiza Root and Rhizome Powder taken to prepare the Sample solution (mg)

D          = dilution factor to prepare the Sample solution from the Sample stock solution, 2

Acceptance criteria: NLT 3.0% 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 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 the absence of Salmonella species and Escherichia coli

 

SPECIFIC TESTS

Articles of Botanical Origin, Alcohol-Soluble Extractives, Method 1 <561>: NLT 15.0%

Articles of Botanical Origin, Water-Soluble Extractives, Method 2 <561>: NLT 35.0%

Loss on Drying <731>

Analysis: Dry 1 g of Salvia miltiorrhiza Root and Rhizome Powder at 105° for 2 h.

Acceptance criteria: NMT 13.0%

Articles of Botanical Origin, Total Ash <561>

Analysis: 4 g of Salvia miltiorrhiza Root and Rhizome Powder

Acceptance criteria: NMT 4.0%

Articles of Botanical Origin, Acid-Insoluble Ash <561>

Analysis: 4 g of Salvia miltiorrhiza Root and Rhizome Powder

Acceptance criteria: NMT 3.0%

 

ADDITIONAL REQUIREMENTS

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

Labeling: The label states the Latin binomial and the parts of the plant from which the article was obtained.

USP Reference Standards <11>

USP Aflatoxins RS

USP Powdered Chinese Salvia Extract RS

USP Salvianolic Acid B RS

USP Tanshinone IIA RS

 

The commenting period for this monograph has expired.

Other Versions

Final Authorized Version 1.0