Vitex negundo Leaf Powder

Vitex negundo Leaf Powder

Proposed For Development Version 0.1

Vitex negundo Leaf Powder


 

DEFINITION

The article consists of dried leaves of Vitex negundo L. (Family Lamiaceae) reduced to the powder or very fine powder. It contains NLT 0.8% 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

• A. Botanical Characteristics

Macroscopic: Greenish brown color.

Microscopic: It shows various types of plenty of trichomes consisting of simple, unicellular, short, conical, with pointed or blunt apex, thick-walled, warty; multicellular trichomes are 2 to 7 celled, often sbruptly bent at its apex, collapsed or warty, occasionally branched; grandular trichomes are sessile with globular 4 celled or one celled head, or stalked one, with unicellular head and unicellular stalk and unicellular stalk with bicellular head; occasionally transversely cut fragments of an arc of epidermis covered with trichomes, fragments of transversely cut lamina, upper epidermal thick cells of lamina devoid of stomata, lower epidermal cells in surface view with plenty of anomocytic stomata and trichomes are present. Rectangular parenchymatous cells of the rachis containing calcium oxalate crystals, lignified pitted pith cells of the rachis, vascular strands of the rachis with pitted squarish cells of the parenthyma are seen.

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: Reflux 2 g of Vitex negundo Leaf Powder in 25 mL methanol for 10 min, centrifuge, and use supernatant.

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: Transfer 5 g of Vitex negundo Leaf powder to 250 mL beaker. Add 75 mL methanol and heat at 70° in water bath for 10 min. Repeat extraction for 3 more times with 50 mL methanol. Collect all filtrates, concentrate and transfer to 100 mL volumetric flask. Mix well and filter.

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 Powder taken:

 

Result = (rU/rS) × CS × (V/W) × F × 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)

V          = volume of the Sample solution (mL)

W         = weight of Vitex negundo Leaf Powder taken to prepare the Sample solution (mg)

F          = conversion factors for the analytes: 0.3 for negundoside, 1.0 for agnuside

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

Acceptance criteria: NLT 0.8% of 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

• Articles of Botanical Origin, Foreign Organic Matter <561>: NMT 2.0%

Articles of Botanical Origin, Alcohol-Soluble Extractives, Method 1 <561>: NLT 10%

Articles of Botanical Origin, Water-Soluble Extractives, Method 2 <561>: NLT 20%

Loss on Drying <731>:

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

Acceptance criteria: NMT 5%

• Articles of Botanical Origin, Total Ash <561>

Analysis: 3 g of Vitex negundo Leaf Powder

Acceptance criteria: NMT 8% 

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

Analysis: 6 g of Vitex negundo Leaf Powder

Acceptance criteria: NMT 1%

 

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

Proposed For Comment Version 1.1
Final Authorized Version 1.0
Proposed For Comment Version 0.2