Vitex negundo Leaf

Vitex negundo Leaf

Proposed For Comment Version 1.1

Vitex negundo Leaf


 

DEFINITION

The article consists of the dried leaves of Vitex negundo L. (Family Lamiaceae) collected in summer. It contains NLT 1.25% of iridoid glycosides calculated as the sum of agnuside and negundoside on the dried basis.

 

SYNONYMS

Agnus-castus negundo (L.) Carriére

Vitex agnus-castus var. negundo (L.) Kuntze

Vitex nogondo L. ap. Bojer

Vitex trifolia var. foliolis obtuse crenatis Lam.

 

POTENTIAL CONFOUNDING MATERIALS

Vitex agnus-castus L.

Vitex rotundifolia L.f

Vitex trifolia L.

 

SELECTED COMMON NAMES

Bengali: Nirgundi, nishinda

Chinese: 黄荆

English: Chinese chastetree, five-leaf chastetree

Indian: Mewri, nirgundi, nisinda, sambhalu, sawbhalu (Hindi); simali (Nepali); sambhalu, banna (Punjabi); sinduvāra, samphālika, nīla (Sanskrit); karunochchi, nocchi (Tamil)

Pinyin: Huang jing, huang jing ye

 

CONSTITUENTS OF INTEREST

Iridoid glycosides: Negundoside, nishindaside, and agnuside

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Leaf, leathery, palmately trifoliate to pentafoliate, middle leaflet largest 6–12 cm long, 1.5–3 cm wide, 1–3 cm long petiole; two lowest lateral leaflets of the pentafoliates are small and almost sessile. Leaflets, lanceolate, acute, entire or rarely crenate, pubescent and dark green on upper surface, tomentose and whitish on lower surface; venation, reticulate, lateral vein arising at an angle of 45°, almost running parallel to each other. Rachis long, circular in outline, with somewhat irregular margin covered with plenty of trichomes.

Microscopic

Transverse section: Upper epidermis of the lamina composed of polygonal tubular cells with straight, thick anticlinal walls, covered with thin cuticle and bears few trichomes; mesophyll 3–5 compact rows of palisade cells discontinuous over the midrib region, slightly narrower, reaching up to two lateral edges of a centrally located crescent-shape meristele associated with a narrow arc of parenchymatous phloem at its lower side and small islets of them on its upper side; cells of the lower epidermis much smaller, plenty of stomata and trichomes, the latter almost concealing whole lower surface of leaf, both simple and grandular trichomes of various types present, simple, unicellular; on upper epidermis, a few multicellular, uniseriate 2–6 cells often collapsed or warty cell, rarely branched; grandular trichomes, sessile with 1–4 cell globular head and with unicellular stalk and unicellular to bicellular head. Along meristeles of the mesophyll, vertical parenchymatous bands get interrupted in the palisade tissue, extend to form a continuous 2–3 layer zone underneath upper epidermis, and contain cluster crystals of calcium oxalate. Remaining parenchymatous cells throughout contain microrosette crystals of calcium oxalate and oil globules. Petiole is oval in outline with two winged projections at the upper side, covered with plenty of trichomes identical with that of the leaf, the collenchymatous hypodermis traverses throughout very narrow underneath the wings, U-shaped vascular bundle consists of xylem, phloem, a continuous ring of pericycle fibers lies in the center of the parnechyma tissue, and a small vascular strand also lies under each wing; pith cells lignified, pitted, and embedded with groups of phloem bundles.

• B. 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: Reflux mix about 0.5 g of Vitex negundo Leaf, finely powdered, in 5 mL of methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Chromatographic system

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

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

Application volume: 2 µL, 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 white 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 the negundoside and agnuside bands in Standard solution A, respectively.

Acceptance criteria: Under white light, the Sample solution exhibits an intense pink band at about one-third of the chromatogram corresponding to negundoside in Standard solution A, and a dark brown band below the strong pink band due to agnuside corresponding to a similar band in Standard solution A. The following bands, with increasing RF, correspond to similar bands in the lower half of Standard solution B: two weak gray-brown, one pink, one dark brown, one strong pink, and one pink.

• C. 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 5 g of Vitex negundo Leaf, finely powdered, and accurately weighed, into a 250-mL beaker. Add 75 mL of 80% methanol in water and heat at 70° in a water bath for 10 min. Repeat extraction three more times with 50 mL of 80% methanol in water. Collect all filtrates, concentrate under reduced pressure, and transfer to a 100-mL volumetric flask. Mix well and pass through a 0.45-µm polyethylsulfone syringe filter.

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 taken:

 

Result = (rU/rS) × CS × (V/W) × 100

 

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

rS    = peak area of negundoside and agnuside in Standard solution A

CS   = concentration of USP Negundoside RS and USP Agnuside RS in Standard solution A (mg/mL)

V     = volume of the Sample solution (mL)

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

Acceptance criteria: NLT 1.25% 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

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

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

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

Loss on Drying <731>

Sample: 1.0 g of Vitex negundo Leaf, finely powdered

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

Acceptance criteria: NMT 5%

Articles of Botanical Origin  <561>, Methods of Analysis, Total Ash

Analysis: 3 g of Vitex negundo Leaf, finely powdered

Acceptance criteria: NMT 8%

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

Analysis: 6 g of Vitex negundo Leaf, finely powdered

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 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