Chaste Tree
DEFINITION
Chaste Tree consists of the dried ripe fruits of Vitex agnus-castus L. (Verbenaceae). It contains NLT 0.05% of agnuside and NLT 0.08% of casticin, calculated on the dried basis.
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identification Test
Standard solution:  100 mg of USP Powdered Chaste Tree Extract RS in 1 mL of methanol. Heat in a water bath at 60 for 10 min. Centrifuge, and use the clear supernatant.
Sample solution:  Transfer about 1 g of powdered plant material to a screw-capped centrifuge tube. Add 10 mL of methanol, heat in a water bath at 60 for 10–15 min, cool, and filter.
Adsorbent:  Chromatographic silica gel with an average particle size of 10–15 µm (TLC plates)
Application volume:  90 µL, Standard solution; 60 µL, Sample solution; in bands that are 2 cm in length
Developing solvent system:  Ethyl acetate, methanol, and water (77:15:8)
Spray reagent:  10 mg/mL of p-dimethylaminobenzaldehyde in 1 N hydrochloric acid
Analysis 
Samples:  Standard solution and Sample solution
Develop to a length of NLT 12 cm, and dry the plate in a current of air. Spray the plate with Spray reagent, and heat for 10 min at 120.
Acceptance criteria:  The Sample solution shows the following: a blue zone (at an RF value of about 0.21) due to the presence of aucubin and that corresponds in color and RF value to a similar zone for the Standard solution; a blue zone (at an RF value of about 0.44) as a result of the presence of agnuside that corresponds in color and RF value to a similar zone for the Standard solution; and one broad zone, violet in the middle, near the solvent front and that corresponds in color and RF value to a similar zone for the Standard solution. Other colored zones of varying intensities may be observed for the Sample solution.
•  B. In the test for Content of Casticin, the chromatogram of the Sample solution shows a peak at the retention time corresponding to the casticin peak in the chromatogram of the Standard solution.
COMPOSITION
•  Content of Casticin
Standard solution:  About 0.05 mg/mL of USP Casticin RS in methanol, with sonication. Pass through a cellulose membrane filter of 0.45-µm or finer pore size.
Sample solution:  Place about 1000 mg of ground plant material in a container with a stopper. Extract twice with 40 mL of methanol, using a hand homogenizer at 19,000 rpm for 2 min. Filter each supernatant, and transfer to a 250-mL round-bottom flask. Rinse the residue with methanol, and filter the resulting solution into the flask. Evaporate the combined extract to dryness. Dissolve the residue in methanol, quantitatively transfer to a 20-mL volumetric flask, and dilute with methanol to volume. Pass through a cellulose membrane filter of 0.45-µm or finer pore size.
Solution A:  Methanol
Solution B:  5.88 g/L of phosphoric acid in water
Mobile phase:  See Table 1.
Table 1
Time
(min)
Solution A
(%)
Solution B
(%)
0 50 50
0 50 50
13 65 35
18 100 0
23 50 50
Chromatographic system 
Mode:  LC
Detector:  UV 348 nm
Column:  3.1-mm × 12.5-cm; 5-µm packing L1
Column temperature:  25
Flow rate:  1 mL/min
Injection size:  10 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Tailing factor:  NMT 2.0 for the casticin peak
Relative standard deviation:  NMT 2.0% for the casticin peak, in repeated injections
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of casticin in the portion of Chaste Tree taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response of casticin from the Sample solution
rS== peak response of casticin from the Standard solution
CS== concentration of USP Casticin RS in the Standard solution (mg/mL)
CU== concentration of Chaste Tree in the Sample solution (mg/mL)
Acceptance criteria:  NLT 0.08% of casticin on the dried basis
•  Content of Agnuside
Solvent:  Methanol and water (1:19)
Standard solution:  Dissolve a quantity of USP Agnuside RS in Solvent, with sonication. Dilute with methanol to obtain a concentration of about 0.125 mg/mL. Pass through a cellulose membrane filter of 0.45-µm or finer pore size.
Sample solution:  Place about 1000 mg of ground plant material in a container with a stopper. Extract twice with 40 mL of methanol, using a hand homogenizer at 19,000 rpm for 2 min. Centrifuge, and transfer each supernatant to a 250-mL round-bottom flask. Rinse the residue with methanol, and filter the resulting solution into the flask. Evaporate the combined extract to dryness, and dissolve the residue in 2 mL of Solvent. Quantitatively transfer the solution to a solid-phase extraction cartridge packed with neutral aluminum oxide previously conditioned with 5 mL of Solvent. Connect the cartridge to a vacuum pressure not exceeding 300 mbar, and collect the eluate. Rinse the round-bottom flask with 2 mL of Solvent, pass this solution through the cartridge, apply the vacuum, and collect the eluate. Rinse the cartridge with 4 mL of Solvent, and collect the eluate. Combine the eluates from the cartridge, transfer to a 10-mL volumetric flask, and dilute with Solvent to volume.
Solution A:  Acetonitrile
Solution B:  5.88 g/L of phosphoric acid in water
Mobile phase:  See Table 2.
Table 2
Time
(min)
Solution A
(%)
Solution B
(%)
0 7 93
0.6 10 90
5 10 90
7 14 86
13 15 85
13.1 100 0
18 100 0
18.1 7 93
23 7 93
Chromatographic system 
Mode:  LC
Detector:  UV 258 nm
Column:  3.1-mm × 12.5-cm; 5-µm packing L1
Column temperature:  25
Flow rate:  1.3 mL/min
Injection size:  10 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Tailing factor:  NMT 2.0 for the agnuside peak
Relative standard deviation:  NMT 2.0% for the agnuside peak, in repeated injections
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of agnuside in the portion of Chaste Tree taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response of agnuside from the Sample solution
rS== peak response of agnuside from the Standard solution
CS== concentration of USP Agnuside RS in the Standard solution (mg/mL)
CU== concentration of Chaste Tree in the Sample solution (mg/mL)
Acceptance criteria:  NLT 0.05% of agnuside on the dried basis
CONTAMINANTS
•  Heavy Metals, Method III 231: NMT 20 ppm
•  Microbial Enumeration Tests 2021: It meets the requirements of the tests for absence of Salmonella species and Escherichia coli. The total aerobic microbial count does not exceed 106 cfu/g, the total combined molds and yeast count does not exceed 104 cfu/g, and the enterobacterial count does not exceed 1000 cfu.
SPECIFIC TESTS
•  Botanic Characteristics
Macroscopic:  Mature chaste tree fruits are spherical to ovoid, 2–4 mm in diameter, very hard, usually with a short pedicel. The fruit is reddish brown to black, slightly rough, and covered with glandular hairs. There are four grooves perpendicular to one another, and a slight depression on the apex, more evident on large fruits. The internal appearance of the fruit is yellowish. The internal structure of the fruit includes four compartments, each containing an oblong seed with a high fat content. A group of up to six spongy, light tan, immature fruits may also accompany mature fruits. The fruit is often covered by a tubular, greenish-gray, fine tomentose calyx, which is persistent and has five teeth.
Microscopic:  The exocarp is brown and narrow, consisting of parenchymatous cells with thin walls and partially lignified cells with many pitted thickenings on the inside. In surface view, the exocarp shows an epidermis of polygonal cells with irregular thickenings and glandular hairs, each with a short single-celled stalk and a four-celled head containing essential oil. The outer mesocarp consists of several layers of brown, isodiametric parenchyma cells. The inner mesocarp consists of finely pitted sclerenchymatous cells, some with moderately thickened walls, others consisting of isodiametric stone cells with small lumen. The endocarp consists of a layer of small brown sclereid cells. The seeds are small, having large cotyledons surrounded by thin-walled, large parenchymatous cells that have ribbed thickenings. The nutritive tissue and the cells of the germ contain aleuron grains and oil globules. Starch is absent. The outer epidermis of calyx is composed of polygonal cells, covered by abundant unicellular or multicellular curved trichomes. The inner epidermis of calyx is glabrous and composed of rectangular, elongated cells with slightly wavy walls.
•  Loss on Drying 731: Dry 1 g at 105 for 2 h: it loses NMT 10.0%.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed containers, and store at controlled room temperature.
•  Labeling: The label states the Latin binomial and, following the official name, the part of the plant contained in the article.
•  USP Reference Standards 11
USP Agnuside RS
USP Casticin RS
USP Powdered Chaste Tree Extract RS
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Maged H. Sharaf, Ph.D.
Principal Scientific Liaison
1-301-816-8318
(DS2010) Monographs - Dietary Supplements
2021 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
1-301-816-8339
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1-301-816-8129
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