Vitex negundo Leaf Dry Extract

Vitex negundo Leaf Dry Extract

Proposed For Comment Version 1.1

Vitex negundo Leaf Dry Extract


 

DEFINITION

Vitex negundo Leaf Dry Extract consists of the dried leaves of Vitex negundo L. (Fam. Lamiaceae), commonly known as Chinese Chaste Tree or Five-Leaf Chaste Tree, by extraction with hydroalcoholic mixture. It contains NLT 2.5% of iridoid glycosides, calculated as the 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

• A. Thin-Layer Chromatography

Standard solution A: 3 mg/mL each of USP Negundoside RS and USP Agnuside RS in methanol

Standard solution B: About 30 mg/mL of USP Vitex negundo Leaf Dry Extract RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Sample solution: Suspend about 0.1 g of Vitex negundo Leaf Dry Extract in 3 mL of methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Chromatographic system

(See Chromatograpy <621>General Prodecures, Thin-Layer Chromatography.)

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

Application volume: 10 µL each of Standard solution A, Standard solution B, and the 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, glacial acetic acid, and water (80:10:5)

Developing distance: 6 cm

Derivatization reagent: Methanol, glacial acetic acid, sulfuric acid, and p-anisaldehyde (170:20:10:1). Prepare on an ice bath and mix well.

Analysis 

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

Apply the Samples as bands to a suitable HPTLC plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry. Treat with Derivatization reagent, heat at 100° for 3 min, and examine under visible light.

System suitability: Under white light, the chromatogram of Standard solution B exhibits six bands in the lower half of the chromatogram, with increasing RF: two faint gray-brown, one pink, one dark brown, one intense pink, and one pink. The intense pink band and the dark brown band immediately below it correspond to negundoside and agnuside in Standard solution A, respectively.

Acceptance criteria: Under white light, the Sample solution exhibits an intense pink band about one-third of the chromatogram corresponding to the band due to negundoside and a dark brown band immediately below the intense pink band corresponding to agnuside in Standard solution A. A yellow band and another pink band are observed.

• B. HPLC

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

Acceptance criteria: The Sample solution exhibits peaks at retention times corresponding to those for 4-hydroxybenzoic acid, negundoside, and agnuside in Standard solution B. The most prominent peak in the chromatogram corresponds to agnuside. The peak responses by 4-hydroxybenzoic acid and negundoside are similar being about one-third of that for agnuside. A fourth major peak elutes between negundoside and agnuside with a response similar to negundoside.

 

ASSAY

• Content of Iridoid Glycosides

Solution A: Dissolve 0.14 g of monobasic potassium phosphate in 900 mL of water, add 0.5 mL of o-phosphoric acid, dilute with water to 1 L, 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: 0.1 mg/mL each of USP Negundoside RS, USP Agnuside RS, and USP Salicylic Acid Related Compound A RS (4-hydroxybenzoic acid) in methanol

Standard solution B: 3 mg/mL of USP Vitex negundo Leaf Dry Extract RS in 80% methanol in water. Sonicate with heating at 70° in a water bath for 10 min, centrifuge, and use the supernatant.

Sample solution: Transfer 30 mg of Vitex negundo Leaf Dry Extract, accurately weighed, into a 10-mL volumetric flask. Add 9 mL of 80% methanol in water and sonicate, with intermittent heating at 70° for about 10 min. Allow to equilibriate to room temperature, and adjust with 80% methanol in water to volume. Centrifuge and use the supernatant.

Chromatographic system

(See Chromatography <621>, 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

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 Vitex negundo Leaf Dry Extract RS being used.

Tailing factor: NMT 1.5, Standard solution A

Relative standard deviation: NMT 2.0%, 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 Vitex negundo Leaf Dry Extract RS being used, identify the retention times of the peaks corresponding to 4-hydroxybenzoic acid, negundoside, and agnuside.

Separately calculate the percentage of negundoside and agnuside in the portion of Vitex negundo Leaf Dry 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 Standard solution A

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

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

Acceptance criteria: NLT 2.5% 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 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: 1.0 g of Vitex negundo Leaf Dry Extract

Analysis: Dry the Sample 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 parts of the plant contained in the article.

USP Reference Standards <11>

USP Agnuside RS

USP Negundoside RS

USP Salicylic Acid Related Compound A RS (4-hydroxybenzoic acid)

USP Vitex negundo Leaf Dry Extract RS

 

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Other Versions

Final Authorized Version 1.0
Proposed For Comment Version 0.2
Proposed For Development Version 0.1