Vitex negundo Leaf Powder Extract

Vitex negundo Leaf Dry Extract

Proposed For Development Version 0.1

Vitex negundo Leaf Powder Extract


 

DEFINITION

The article is prepared from dried leaves of Vitex negundo L. (Family Lamiaceae) by extraction with hydroalcoholic mixture. It contains NLT 90.0% and NMT 110.0% of labeled amount of iridoid glycosides as sum of agnuside and negundoside, on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

Vitex agnus-castus L.

Vitex rotundifolia L.f

Vitex trifolia L.

 

CONSTITUENTS OF INTEREST

Iridoid glycosides: Negundoside, nishindaside, and agnuside

 

IDENTIFICATION

B. Thin-Layer Chromatography

Standard solution A: 0.5 mg/mL of USP Negundoside RS in methanol

Standard solution B: 50 mg/mL of USP Powdered Vitex negundo Extract RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant

Sample solution: Vitex negundo Leaf Powder Extract at the concentration equivalent to 0.5 mg/mL of negundoside according to the label claim. Sonicate if necessary.

Chromatographic system

Adsorbent: Use a suitable chromatographic material with an average particle size of 5 µm (HPTLC plates)

Application volume: 10 µL of Standard solution A, Standard solution B, and Sample solution; as 10-mm bands

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

Developing solvent system: A mixture of ethyl acetate, water, acetic acid (80:10:10)

Developing distance: 8 cm

Derivatization reagent: Anisaldehyde-sulfuric acid reagent

Analysis 

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

Apply the samples as bands to a suitable high performance thin-layer chromatographic plate and dry in air (see <621> Chromatography). Develop the chromatograms in a saturated chamber, remove the plate from the chamber, dry, treat with Derivatization reagent, heat at 105° till the colored bands appear and examine under visible light.

System Suitability: Under visible light, the chromatogram of Standard solution B exhibits ten bands in the chromatogram. These include, with increasing RF; two weak gray bands, one strong red band, one yellow-brown band, one strong black band, one strong red-pink band, one pale purple band, one pale blue-purple band, and two black bands; the red-pink band with RF in the middle of chromatogram corresponds to the band due to negundoside in the chromatogram of Standard solution A

Acceptance criteria: Under visible light, the chromatogram of Sample solution exhibits a red-pink band in the middle of chromatogram corresponds to the band due to negundoside in the chromatogram of Standard solution A, and the following bands, with increasing RF, correspond to similar bands in the chromatogram of Standard solution B; two weak gray, one strong red, one yellow-brown, one strong black, one pale purple, one pale blue-purple and two black bands in the chromatogram.

C. HPLC

Analysis: Proceed as directed in the test for Content of Iridoid Glycosides

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

 

ASSAY

• Content of Iridoid Glycosides

Solution A: 0.14 g of Potassium phosphate monobasic in 900 mL of water, add 0.5 mL of o-phosphoric acid, complete to 1 L with water, and mix.

Solution B: Acetonitrile

Mobile phase: See Table 1

 

Table 1

 

 

 

Time (min)

Solution A (%)

Solution B (%)

0.01

90

10

10

85

15

20

80

20

23

80

20

25

85

15

30

90

10

35

90

10

 

Standard solution A: 1.0 mg/mL of USP Negundoside RS in methanol

Standard solution B: Heat 2 g of USP Powdered Vitex negundo Extract RS in 75 mL of methanol at 70° in water bath for 10 min, centrifuge and use supernatant.

Sample solution: Vitex negundo Leaf Powder Extract in methanol at a concentration equivalent to 1.0 mg/mL negundoside according to the label claim. Sonicate if necessary. Before injection, pass through a membrane filter of 0.45-µm or finer size. Discard first few mL of the filtrate.

Chromatographic system

(See <621> Chromatography, System Suitability.)

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; L1 (similar to Hibar 250-4.6 HPLC Column, Lichrospher 100 RP-18)

Flow rate: 1.5 mL/min

Injection volume: 20 µL

System suitability

Sample: Standard solution A and Standard solution B

Suitability requirements

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

Resolution: 17.6 between negunsodie and agnuside peaks, Standard solution B

Tailing factor: NMT 1.5

Relative standard deviation: NMT 2.0%

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 Vitex negundo Extract RS being used, identify retention time of the peaks corresponding negundoside and agnuside. The approximate relative retention times of the peaks for negundoside and agnuside are 0.7 and 1.0, respectively.

Separately calculate the percentage of negundoside and agnuside in the portion of Vitex negundo Leaf Powde Extract taken:

 

Result = (rU/rS) × (CS/CU) × 100

 

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

rS          = peak area of negundoside in the Standard solution A

CS         = concentration of negundoside in the Standard solution A (mg/mL)

CU         = concentration of Vitex negundo Leaf Powder Extract in the Sample solution (mg/mL)

Calculate the content of iridoid glycoside as the sum of the percentage of agnuside and negundoside.

Calculate the percentage of the labeled amount of iridoid glycosides in the Powdered Extract:

 

Result = (P/L) × 100

 

P          = content of negundoside and agnuside as determined above (%)

L          = labeled amount of iridoid glycosides: negundoside and agnuside (%)

Acceptance criteria: 90.0%-110.0% of the labeled amount of negundoside and agnuside, on the dried basis.

 

CONTAMINANTS

Elemental Impurities—Procedures <233>

Acceptance Criteria:

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 1.0 µg/g

Lead: NMT 10.0 µg/g

Mercury: NMT 1.0 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.

• 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>:

Analysis: Dry 1.0 g powdered Vitex negundo Leaf Powder Extract at 105° for 2 h.

Acceptance criteria: 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 of the plant contained in the article.

• USP Reference Standards <11>

USP Powdered Vitex negundo Extract RS

USP Negundoside RS

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Other Versions

Final Authorized Version 1.0
Proposed For Comment Version 0.2