Berberis aristata Stem Powder

Berberis aristata Stem Powder

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Berberis aristata Stem Powder

 


 

DEFINITION

The article consists of the dried stem of Berberis aristatal DC. (Family Berberidaceae), reduced to powder or very fine powder. It contains NLT 1.0% of berberine 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. Botanical Characteristics

Macroscopic: Yellow to dark brown powder

Microscopic: Yellow; shows mostly fragments of cork cells, sieve elements, yellow-colored phloem fibers entire or in pieces, stone cells in singles or in groups, numerous prismatic crystals of calcium oxalate, xylem vessels having spiral thickening, thick-walled, lignified xylem fibers and ray cells.

 

• B. 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 0.25 g of Berberis aristata Stem Powder into a beaker and add 4 mL of 80% methanol. Sonicate for 10 min, centrifuge or filter, and use the supernatant.

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, slightly overlapping with a 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 one gray band, slightly overlapped with a pale red band, near the middle of the chromatogram with similar RF corresponding with berberine, and another gray band, below the berberine band and slightly overlapped with another pale red band, with similar RF corresponding with palmatine in Standard solution A. A pale gray band appears between the berberine and palmatine bands. 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. A number of pale yellow and pale blue bands overlaps below the palmatine band. Under white light after derivatization, two yellow bands appear near the middle of the chromatogram with the upper RF corresponding with berberine and the lower RF band corresponding with palmatine. Two additional grayish-green bands appear close to origin. A purple/brown band near the lower yellow band, which appear in Hydratis canadensis, and a red band near the brown band second from the solvent origin, which appear in Coptis chinensis, should not be observed.

 

• C. 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 in 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 0.5 g of Berberis aristata Stem Powder and accurately weighed, into a 100-mL centrifuge tube, and add 50 mL of methanol. Sonicate for 5 min, centrifuge, and transfer supernatant to a 100-mL volumetric flask. Repeat the extraction two more times by adding 20 mL of methanol, and transferring the supernatant to the same 100-mL volumetric flask. Dilute with methanol to volume.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Detector: UV 348 nm

Column: 4.6-mm x 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 Powder taken:

 

Result = (rU/rS) x CS x (V/W) 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)

V    = volume of the Sample solution (mL)

W   = weight of Berberis aristata Stem Powder taken to prepare the Sample solution (mg)

 

Acceptance criteria: NLT 1.0% 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

• Articles of Botanical Origin <561>,  Methods of Analysis, Alcohol-Soluble ExtractivesMethod 1: NLT 6.0%

• Articles of Botanical Origin <561>,  Methods of Analysis, Water-Soluble ExtractivesMethod 2: NLT 8.0%

• Loss on Drying <731>

Sample: 1 g of Berberis aristata Stem Powder 

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

Acceptance criteria: NMT 7%

• Articles of Botanical Origin <561>, Methods of Analysis, Total Ash: NMT 14%

• Articles of Botanical Origin <561>, Methods of Analysis, Acid-Insoluble Ash: NMT 5%

 

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 Berberis aristata Stem Dry Extract RS

USP Berberine Chloride RS

USP Palmatine Chloride RS

 

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Proposed For Development Version 0.1