Vitex negundo Leaf

Vitex negundo Leaf

Final Authorized Version 1.0

Vitex negundo Leaf



The article consists of the dried leaves of Vitex negundo L. (Family Lamiaceae) collected in summer. It contains NLT 0.8% of iridoid glycosides calculated as the sum of agnuside and negundoside on the dried basis.



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: 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; 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: 0.5 mg/mL of USP Negundoside RS in methanol

Standard solution B: 50 mg/mL of USP Vitex negundo Leaf Dry 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 of methanol for 10 min, centrifuge, and use supernatant.

Chromatographic system

(See Chromatography <621>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 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, acetic acid, and water (80:10:5)

Developing distance: 6 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 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 visible light, Standard solution B exhibits six bands in the lower half and about five purple bands in the upper half. The bands in the lower half include the following bands, with increasing RF: two weak gray-brown, one pink, one dark-brown, one strong pink, and one pink. The strong pink band RF corresponds to the band due to negundoside in Standard solution A

Acceptance criteria: Under visible light, the Sample solution exhibits a strong pink band in the lower half that corresponds to the band due to negundoside in Standard solution A, and a dark-brown band below the strong pink band due to agnuside. 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.


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

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



• Content of Iridoid Glycosides

Solution A: Dissolve 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


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 Vitex negundo Leaf Dry Extract RS in 75 mL of methanol at 70° in a water bath for 10 min, centrifuge, and use supernatant.

Sample solution: Transfer 5 g of Vitex negundo Leaf, finely powdered and accurately weighed, to a 250-mL beaker. Add 75 mL of methanol and heat at 70° in a water bath for 10 min. Repeat extraction three more times with 50 mL of methanol. Collect all filtrates, concentrate, and transfer to a 100-mL volumetric flask. Mix well and 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.

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

Tailing factor: NMT 1.5, Standard solution A

Relative standard deviation: NMT 2.0%, Standard solution A


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 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 Standard solution A

CS   = concentration of negundoside 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)

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

Add the percentages of agnuside and negundoside.

Acceptance criteria: NLT 0.8% on the dried basis



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, 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>

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, 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 parts of the plant contained in the article.

USP Reference Standards <11>

USP Negundoside RS

USP Vitex negundo Leaf Dry Extract RS


Other Versions

Proposed For Comment Version 1.1
Proposed For Comment Version 0.2
Proposed For Development Version 0.1