Berberis aristata Stem Dry Extract

Berberis aristata Stem Dry Extract

Proposed For Comment Version 0.2

 

Berberis aristata Stem Dry Extract

 


 

DEFINITION

The article is prepared from the dried stem of Berberis aristatal DC. (Family Berberidaceae) by extraction with hydroalcoholic mixture. It contains NLT 90.0% and NMT 110.0% of labeled amount of berberine calculated on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

Berberis asiatica

Berberis lyceum

Coscinium fenestratum

Hydrastis canadensis

Mahonia borealis
Morinda umbellata

 

CONSTITUENTS OF INTEREST

Benzoisoquinoline alkaloid: Berberine, palmatine

 

IDENTIFICATION

• A. HPTLC for Articles of Botanical Origin <203>

Standard solution A: 1 mg of USP Berberine Chloride RS in 8 mL of methanol and 1 mg of USP Palmatine Chloride RS in 16 mL of methanol

Standard solution B: Sonicate 50 mg of USP Berberis aristata Stem Dry Extract RS in 4 mL of 80% methanol for 10 min. Centrifuge or filter, and use the supernatant or the filtrate.

Sample solution: Transfer 0.1 g of Berberis aristata Stem Dry Extract into a 100-mL volumetric flask. Add 50 mL of methanol and sonicate for 10 min. Heat in a water bath for 10 min, cool, and dilute with methanol to 100 mL. Centrifuge or filter, and use the supernatant or the filtrate.

Chromatographic system

Adsorbent: Chromatographic silica gel F254 mixture with an average particle size of 5 µm

Application volume: 10 µL each of Standard solution and Sample solution as 8-mm bands

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

Developing solvent system: Ethyl acetate, formic acid, and water (80:10:10)

Developing distance: 7 cm

Derivatization reagent: Anisaldehyde reagent: add 20 mL of acetic acid and 10 mL of sulfuric acid to 170 mL of cold methanol and mix well. After cooling to room temperature, add 1 mL of anisaldehyde to the mixture.

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. Examine under UV light at 254 and 365 nm before derivatization. Then treat with Derivatization reagent and examine under white light.

System suitability: Under UV 254 nm, the chromatogram of Standard solution B exhibits one gray band, with slightly overlapping with pale red band, near the middle of the chromatogram with similar RF corresponding with the berberine band in the chromatogram of Standard solution A. Two additional pale gray bands appear below the berberine band with a lower band corresponding in similar RF with the palmatine band in the chromatogram of Standard solution A. Under UV 365 nm, the chromatogram of Standard solution B exhibits one strong yellow band near the middle of the chromatogram with similar RF corresponding with berberine, one pale yellow band appears below the berberine band, and one less strong yellow band than the berberine band appears below the pale yellow band with similar RF corresponding with palmatine. Under white light after derivatization, one yellow band appears near the middle of the chromatogram with similar RF corresponding with berberine.

Acceptance criteria: Under UV 254 nm, the chromatogram of the Sample solution exhibits a gray band, slightly overlapped with a pale red band, near the middle of the chromatogram with similar RF corresponding with berberine, and two pale gray bands below the berberine band with lower RF corresponding with palmatine in Standard solution A. Under UV 365 nm, two bright yellow bands appear near the middle of the chromatogram with higher RF corresponding with berberine and lower RF corresponding with the palmatine band in Standard solution A. A pale yellow band appears between the berberine and palmatine bands. Under white light after derivatization, a yellow band appears near the middle of the chromatogram corresponding in RF with the berberine band in Standard solution A. A purple/brown band near the lower yellow band, which appears in Hydratis canadensis, and a red band near the brown band second from the solvent origin, which appears in Coptis chinensis, should not be observed.

 

• B. HPLC

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

Acceptance criteria: The chromatogram of the Sample solution exhibits peaks at retention times corresponding to the peaks due to berberine in Standard solution B.

 

ASSAY

• Content of Berberine

Solution A: Transfer 0.6 g of sodium 1-hexanesulfonate into a 1-L volumetric flask and add 900 mL of water to dissolve. Adjust with glacial acetic acid to a pH of 2.7 and dilute with water to volume.

Solution B: Acetonitrile

Mobile phase: See Table 1.

 

Table 1

Time
(min)

Solution A
(%)

Solution B
(%)

0

90

10

10

70

30

18

70

30

22

20

80

26

20

80

30

90

10

35 90 10

 

Standard solution A: 0.1 mg/mL of USP Berberine Chloride RS in methanol

Standard solution B: Transfer USP Berberis aristata Stem Dry Extract RS, equivalent to 10 mg of berberine, into a 100-mL volumetric flask and add 50 mL of methanol. Heat at 80° with sonication for 20 min. Cool the solution and dilute with methanol to volume. Pass through a membrane filter of 0.45-µm or finer pore size and use the filtrate.

Sample solution: Transfer 0.1 g of Berberis aristata Stem Dry Extract, accurately weighed, into a 100-mL volumetric flask and add 50 mL of methanol. Sonicate for 10 min with heating. Cool the solution and dilute with methanol to volume. Pass through a membrane filter of 0.45-µm or finer pore size and use the filtrate.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Detector: UV 348 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1

Flow rate: 1.0 mL/min

Injection volume: 20 µL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Resolution: NLT 2.0 between the berberine peak and the peak before, Standard solution B

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

Relative standard deviation: NMT 2.0% for the berberine peak in repeated injections, Standard solution A

Chromatogram similarity: The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Berberis aristata Stem Dry Extract RS being used.

Analysis

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

Using the chromatograms of Standard solution AStandard solution B, and the reference chromatogram provided with the lot of USP Berberis aristata Stem Dry Extract RS being used, identify the retention times of the peaks corresponding to berberine in the Sample solution.

Calculate the percentage of berberine in the portion of Berberis aristata Stem Dry Extract taken:

 

Result = (rU/rS) x (CS/CU) x 100

 

rU   = peak area of berberine from the Sample solution

rS   = peak area of berberine from Standard solution A

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

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

 

Calculate the percentage of the labeled amount of berberine in the portion of Dry Extract taken:

 

Result = (P/L) x 100

 

P   = content of berberine as determined above (%)

L   = labeled amount of berberine (%)

 

Acceptance criteria: 90.0%–110.0% of the labeled amount of berberine on the dried basis

 

CONTAMINANTS

• Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 0.5 µg/g

Lead: NMT 5.0 µg/g

Mercury: NMT 0.2 µg/g

• Articles of Botanical Origin <561>, Pesticide Residue Analysis: 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

 

SPECIFIC TESTS

• Loss on Drying <731>

Sample: 1 g of Berberis aristata Stem, finely powdered

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

Acceptance criteria: NMT 7%

 

ADDITIONAL REQUIREMENTS

• Packaging and Storage: Preserve in well-closed containers, protected from light and moisture, and store at 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 Berberine Chloride RS

USP Berberis aristata Stem Dry Extract RS

USP Palmatine Chloride RS

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Other Versions

Proposed For Development Version 0.1