Vitex negundo Leaf

Vitex negundo Leaf

Proposed For Development Version 0.1

Vitex negundo Leaf



The article consists of dried leaves of Vitex negundo L. (Family Lamiaceae), collected in summer. It meets the Acceptance criteria under the Assay.



Agnus-castus negundo (L.) Carriére

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

Vitex nogondo L. ap. Bojer

Vitex trifolia var. foliolis obtuse crenatis Lam.



Vitex agnus-castus L.

Vitex rotundifolia L.f

Vitex trifolia L.



Bengali: Nirgundi, nishinda

Chinese: 黄荆

English: Chinese chastetree, five-leaf chastetree

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

Pinyin: Huang jing, huang jing ye



Iridoid glycosides: Negundoside, nishindaside, and agnuside



• 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.


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; the cells of the lower epidermis much smaller, stomata and trichomes plenty, 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, and a continuous ring of pericycle fibers lie in the center of the parnechyma tissue, and a small vascular strand also lies under each wing; pith cells lignified, pitted embedded with groups of phloem bundles.

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, finely powdered, 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


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.


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.



• 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 (%)























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, finely powdered and accurately weighed, 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%


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 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 taken to prepare the Sample solution (mg)

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



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



• 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, finely powdered, at 105° for 2 h.

Acceptance criteria: NMT 5%

• Articles of Botanical Origin, Total Ash <561>

Analysis: 3 g of Vitex negundo Leaf, finely powdered

Acceptance criteria: NMT 8% 

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

Analysis: 6 g of Vitex negundo Leaf, finely powdered

Acceptance criteria: NMT 1%



• 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 Vitex negundo Leaf Powdered 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