Ashwagandha Root is the dried mature roots of Withania somnifera (L.) Dunal (Fam. Solanaceae). It contains NLT 0.3% of withanolides, calculated on the dried basis as the sum of withanolide aglycones, calculated as withanolide A, and withanolide glycosides, calculated as withanoside IV.
• A. Thin-Layer Chromatographic Identification Test 201
Standard solution: About 200 mg of USP Powdered Ashwagandha Root Extract RS in 10 mL of methanol. Heat gently for 1015 min, centrifuge, and use the supernatant. [NoteSave the remaining volume of the supernatant for use in the test for Content of Withanolides. ]
Sample solution: Transfer about 5.0 g of Ashwagandha Root, finely powdered, to a 250-mL flask fitted with a reflux condenser. Add 50 mL of methanol, reflux on a water bath for 1015 min, cool to room temperature, and decant the supernatant. Repeat until the last extract is colorless. Combine the extracts, filter, concentrate under vacuum to about 40 mL, and adjust the volume with methanol to 50.0 mL. [NoteSave the remaining volume of the Sample solution for use in the test for Content of Withanolides. ]
Adsorbent: 0.25-mm layer of chromatographic silica gel
Application volume: 25 µL
Developing solvent system: A mixture of ethyl acetate, toluene, and acetic acid (45:55:3)
Spray reagent: Mix 0.5 mL of anisaldehyde, 10 mL of glacial acetic acid, 85 mL of methanol, and 5 mL of concentrated sulfuric acid in the order given.
Samples: Standard solution and Sample solution
Apply the Samples as bands to a suitable plate (see Chromatography 621). Use a saturated chamber. Develop until the solvent front has moved up about 90% of the length of the plate. Dry the plate, spray with Spray reagent, heat for 510 min at 100, and examine under visible light.
Acceptance criteria: The Sample solution exhibits five main grayish-blue bands with RF values of approximately 0.12, 0.29, 0.47, 0.67, and 0.73 that are similar in position and color to the main bands from the Standard solution. Other less intense bands are observed for the Sample solution and the Standard solution.
• B. The Sample solution in the test for Content of Withanolides shows main peaks at retention times corresponding to those of withanolide A and withanoside IV in Standard solution A and Standard solution B, respectively. Identify other withanolide peaks in the Sample solution by comparison with Standard solution C and the reference chromatogram provided with the lot of USP Powdered Ashwagandha Root Extract RS being used. The Sample solution shows additional peaks corresponding to some of the following withanolides: physagulin D, 27-hydroxywithanone, withanoside V, withanoside VI, withaferin A, withastramonolide, withanone, and withanolide B.
• Content of Withanolides
Solution A: Dissolve 0.14 g of potassium dihydrogen phosphate in 900 mL of water, add 0.5 mL of phosphoric acid, dilute with water to 1000 mL, and mix.
Solution B: Filtered and degassed acetonitrile
Standard solution A: Dissolve, using gentle heat, a quantity of USP Withanolide A RS in methanol to obtain a solution having a known concentration of about 0.1 mg/mL.
Standard solution B: Dissolve, using gentle heat, a quantity of USP Withanoside IV RS in methanol to obtain a solution having a known concentration of about 0.1 mg/mL.
Standard solution C: Dilute 5 mL of the Standard solution prepared in Identification test A with methanol to 10 mL, and mix. Before injection, pass through a membrane filter of 0.45-µm or finer pore size.
Sample solution: Use the Sample solution prepared in Identification test A. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first few mL of the filtrate.
Mobile phase: See the gradient table below.
Detector: UV 227 nm
Column: 4.6-mm × 25-cm, end-capped; packing L1
Temperature: 27 ± 1
Flow rate: 1.5 mL/min
Injection size: 20 µL
Samples: Standard solution A and Standard solution C
Using the chromatogram of Standard solution C and the reference chromatogram provided with the lot of USP Powdered Ashwagandha Root Extract RS being used, identify the retention times of the peaks corresponding to the various withanolide aglycones and glycosides. The approximate relative retention times of the withanolide aglycones and glycosides are provided in the following table.
The chromatogram for Standard solution C is similar to the reference chromatogram provided with the lot of USP Powdered Ashwagandha Root Extract RS being used.
Resolution: NLT 1.0 for the withanolide A and withanone peaks, Standard solution C; NLT 3.0 between the withaferin A and coeluting withanoside V and withanoside VI peaks, Standard solution C
Tailing factor: NMT 1.5 for the withanolide A peak, Standard solution A
Relative standard deviation: NMT 2.0% for replicate injections, withanolide A peak, Standard solution A
Samples: Standard solution A, Standard solution B, Standard solution C, and Sample solution
Calculate the percentage of withanolide aglycones in the portion of Ashwagandha Root taken:
Result = 5(rT/rS)(CS/W)
Calculate the percentage of withanolide glycosides in the portion of Ashwagandha Root taken:
Result = 5(rT/rS)(CS/W)
Acceptance criteria: The sum of the percentages of withanolide aglycones and withanolide glycosides is NLT 0.3%, calculated on the dried basis. [NoteBecause of inherent variations, some of the withanolides mentioned in this test may be present in minor quantities or may be totally absent. The sample will be deemed compliant if the sum of the total withanolides is NLT 0.3%. ]
• Heavy Metals, Method II 231: NMT 20 ppm
• Procedure 1: Articles of Botanical Origin, Foreign Organic Matter 561: NMT 2.0%
• Procedure 2: Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561: Meets the requirements
• Botanic Characteristics
Macroscopic: Primary roots are not branched and are straight, conical, or fingerlike in shape and variable in thickness with age; some carry a crown, consisting of a number of remains of stem base; the outer surface is buff to grayish-yellow with longitudinal wrinkles; facture is short and uneven; secondary roots are thin and fibrous.
Transverse section of roots: It shows a narrow band of yellowish crumpled cork, moderate-size cortex and a wide wood. The cork cells are rectangular, radially flattened, nonlignified, and filled with starch grains and reddish brown content; cork cambium is 24 diffused rows of cells; secondary cortex is formed of 2025 rows of thin-wall parenchymatous cells, filled with starch grains, and shows occasional microsphenoidal crystals of calcium oxalate; phloem consists of sieve tubes, companion cells, and phloem parenchyma; vascular cambium consists of tangentially elongated parenchymatous cells; vessels and tracheids are in radial rows toward the periphery of the wood; medullary rays are uniseriate to 2- to 3-seriate, and are filled with starch grains; scattered vessels in groups are embedded in the parenchyma; vessels have pitted and scalariform thickening, and generally the end walls are perforated; and a few fibers with thick lignified walls are also found scattered in the wood.
• Loss on Drying 731: Dry 1.0 g of finely powdered Ashwagandha Root at 105 for 3 h: it loses NMT 12.0% of its weight.
• Articles of Botanical Origin, Total Ash 561: NMT 7.0%, determined on 1.0 g of finely powdered Ashwagandha Root
• 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 count does not exceed 103 cfu/g.
• Microbiological Procedures for Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
• Articles of Botanical Origin, Aflatoxins 561: Meets the 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, following the official name, the part of the plant contained in the article.
• USP Reference Standards 11
USP Powdered Ashwagandha Root Extract RS
USP Withanolide A RS
USP Withanoside IV RS
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USP35NF30 Page 1193Pharmacopeial Forum: Volume No. 35(4) Page 885