Add the following:Malabar-Nut-Tree, Leaf
Malabar-Nut-Tree, Leaf, also known in commerce as vasaka, consists of the dried leaves of Justicia adhatoda L., also known as Adhatoda vasica Nees (Fam. Acanthaceae). It contains NLT 0.6% of vasicine, calculated on the dried basis.
• A. Malabar-Nut-Tree Leaf meets the requirements in Specific Tests, Botanic Characteristics.
• B. Thin-Layer Chromatographic Identification Test
Standard solution A: 0.5 mg/mL of USP Vasicine RS in methanol. Sonicate to dissolve if necessary.
Standard solution B: 50 mg/mL of USP Powdered Malabar-Nut-Tree, Leaf Extract RS in methanol. Sonicate for about 15 min, centrifuge, and use the supernatant.
Sample solution: Transfer about 2.0 g of Malabar-Nut-Tree, Leaf, finely powdered and accurately weighed, to a 250-mL round-bottom flask fitted with a reflux condenser. Add 50 mL of methanol, reflux on a water bath for 15 min, cool to room temperature, and decant the supernatant. Repeat until the last extract is colorless. Combine the extracts, filter, concentrate under vacuum, and adjust the volume to 25 mL, using methanol. [NoteSave the filtrate for use in the test for Content of Vasicine. ]
Adsorbent: Chromatographic silica gel with an average particle size of 1015 µm (TLC plates)
Application volume: 10 µL, as 4-mm bands
Developing solvent system: A mixture of ethyl acetate, methanol, and ammonia (8: 2: 0.2)
Samples: Standard solution A, Standard solution B, and Sample solution
Apply the Samples as bands to a suitable thin-layer chromatographic plate (see Chromatography 621, Thin-Layer Chromatography). Use a saturated chamber. Develop the chromatograms until the solvent front has moved up about three-fourths of the length of the plate. Remove the plate from the chamber, dry, and examine under UV at 254 nm.
Acceptance criteria: The chromatogram of the Sample solution exhibits a quenching zone at an RF value of approximately 0.35 for vasicine, corresponding to a zone in the chromatogram of Standard solution A. The chromatogram of the Sample solution also exhibits an additional quenching zone at an RF value of approximately 0.53 for vasicinone, corresponding to a similar zone in the chromatogram of Standard solution B. Other minor zones may be observed in the Sample solution and Standard solution B chromatograms.
• C. HPLC: The chromatogram of the Sample solution from the test for Content of Vasicine shows a main peak at a retention time corresponding to that of vasicine in the chromatogram of Standard solution A. Identify other peaks in the Sample solution by comparison with the chromatogram of Standard solution B and the reference chromatogram provided with the lot of USP Powdered Malabar-Nut-Tree, Leaf Extract RS being used. The Sample solution shows an additional peak corresponding to vasicinone.
• Content of Vasicine
Buffer solution: Dissolve 1.36 g of anhydrous potassium dihydrogen phosphate in 900 mL of water. Add 2.0 mL of phosphoric acid. Dilute with water to 1000 mL, and filter.
Mobile phase: Buffer solution, acetonitrile, and tetrahydrofuran (92:5:3)
Standard solution A: 0.1 mg/mL of USP Vasicine RS in methanol. Sonicate to dissolve if necessary.
Standard solution B: 5.0 mg/mL of USP Powdered Malabar-Nut-Tree, Leaf Extract RS in methanol. Sonicate for about 15 min. Before injection, pass through a membrane filter of 0.45-µm or finer pore size.
Sample solution: Dilute the Sample solution, prepared as directed in Identification test A (1:5), with methanol. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, and discard the first part of the filtrate.
Detector: UV 280 nm
Column: 4.6-mm × 25-cm; 5-µm packing L10
Flow rate: 1.0 mL/min
Injection size: 20 µL
Samples: Standard solution A and Standard solution B
[NoteThe approximate relative retention times of the vasicine and vasicinone peaks are 1.00 and 1.23, respectively. ]
Chromatogram similarity: The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Malabar-Nut-Tree, Leaf Extract RS being used.
Resolution: NLT 2.0 between the vasicine and vasicinone peaks, Standard solution B
Relative standard deviation: NMT 2.0% determined from the vasicine peak in repeated injections, Standard solution A
Samples: Standard solution A, Standard solution B, and Sample solution
Using the chromatogram of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Powdered Malabar-Nut-Tree, Leaf Extract RS being used, identify the retention times of the peaks corresponding to vasicine and vasicinone.
Calculate the percentage of vasicine in the portion of Malabar-Nut-Tree, Leaf taken:
Result = (rU/rS) × CS × (V/W) × 100
Acceptance criteria: NLT 0.6% on the dried basis
• Heavy Metals, Method III 231: NMT 20 ppm
• Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561: Meets the requirements
• Microbial Enumeration Tests 2021: 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.
• Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
• Botanic Characteristics
Macroscopic: Leaves, simple, green to dull grayish brown, minutely pubescent, lanceolate to ovate-lanceolate, tapering base and slightly acuminate apex, 1020 cm long, 310 cm broad, petioles 28 cm long, and 810 pairs of reticulate lateral veins; characteristic odor, and bitter taste. Pharmacopeial article consists of dry, brittle, and greyish brown leaves.
Transverse section of leaves: Upper epidermal cells uniform in size and sinuous in outline, while lower epidermal cells vary in size and are less wavy; 46 layers of collenchymal cells below epidermis in the midrib region; shows two layers of palisade cells with elongated cystoliths, which are absent in epidermal cells (a diagnostic feature that is not seen in the leaves of Ailanthus excelsa, an adulterant for Malabar-Nut-Tree, Leaf); globules of oil dispersed in palisade and spongy layers; numerous stomata diacytic or anomocytic; glandular and non-glandular trichomes are visible on both epidermal layers.
• Loss on Drying 731: Dry 1.0 g of finely powdered Malabar-Nut-Tree, Leaf at 105 for 3 h: it loses NMT 12.0% of its weight.
• Articles of Botanical Origin, Total Ash 561: NMT 21%, determined on 1.0 g of finely powdered Malabar-Nut-Tree, Leaf
• 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, following the official name, the part of the plant contained in the article.
• USP Reference Standards 11USP35
USP Powdered Malabar-Nut-Tree, Leaf Extract RS
USP Vasicine RS
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USP35NF30 Page 1376Pharmacopeial Forum: Volume No. 36(6) Page 1614