Powdered Garlic Extract
Powdered Garlic Extract is prepared from fresh Garlic bulbs by extraction with alcohol. The ratio of the starting crude plant material to Powdered Extract is 9.5:113.5:1. It contains NLT 4.0% of alliin (C6H11NO3S). It may contain added Powdered Garlic or other suitable substances.
• A. Thin-Layer Chromatographic Identification Test
Standard solution A: 0.5 mg/mL of USP l-Methionine RS in a mixture of methanol and water (1:1)
Standard solution B: 0.5 mg/mL of USP Alliin RS in a mixture of methanol and water (1:1)
Sample solution: Transfer a quantity of Powdered Extract, equivalent to about 5 mg of alliin, to a suitable container. Add 40 mL of a mixture of methanol and water (1:1), and shake until the powder is fully dispersed. Centrifuge, and decant the supernatant into a round-bottomed flask. Concentrate to a small volume (about 5 mL) using a rotary evaporator.
Adsorbent: 0.25-mm layer of chromatographic silica gel, typically 20 cm long (TLC plates).
Application volume: 20 µL, applied separately as 10-mm bands
Developing solvent system: Butyl alcohol, n-propyl alcohol, glacial acetic acid, and water (3:1:1:1)
Spray reagent: 0.2% solution of ninhydrin in a mixture of butyl alcohol and 2 N acetic acid (19:1)
Samples: Standard solutions and Sample solution
Develop the chromatograms until the solvent front has moved up about three-fourths of the plate, in a saturated chamber. Remove the plate, and allow the solvent to evaporate. Spray with the Spray reagent, heat at 100105 for 10 min, and immediately examine the plate.
Acceptance criteria: The chromatogram of the Sample solution shows the following orange and pinkish violet zones: a violet zone having an RF value of about 0.89; a pink zone having an RF value of about 0.5 and corresponding in color and RF value to that of the chromatogram of Standard solution A; a pinkish zone having an RF value of about 0.43; a strong orange zone having an RF value of about 0.38; a pinkish violet zone having an RF value of about 0.3 and corresponding in color and RF value to that of the chromatogram of Standard solution B; and additional pinkish orange zones situated very close to each other just below the zone attributed to alliin in the chromatogram of Standard solution B.
• B. The retention time of the alliin peak of the Sample solution corresponds to that of the Standard solution, as obtained in the test for Content of Alliin.
• Content of Alliin
Allinase inhibitor solution: 1.09 mg/mL of carboxymethoxylamine hemihydrochloride
Solution A: Monobasic sodium phosphate 0.045 M in water adjusted with 0.2 M sodium hydroxide to a pH of 7.1
Buffer: Monobasic sodium phosphate 0.05 M in water adjusted with 0.2 M sodium hydroxide to a pH of 9.5
Derivatization reagent: Dissolve 140 mg of o-phthaldialdehyde in 5 mL of methanol. Add 100 µL of t-butylthiol, dilute with Buffer to 50 mL, and mix. [NoteThis reagent may occasionally become opaque during preparation. Store at room temperature, and use within 1 week. ]
Mobile phase: Acetonitrile, 1,4-dioxane, tetrahydrofuran, and Solution A (25: 2.9: 2.2: 69.9)
Standard solution: 0.05 mg/mL of USP Alliin RS in a mixture of methanol and water (1:1)
Sample solution: Transfer about 0.10 g of Powdered Extract, accurately weighed, to a 50-mL volumetric flask. Add 30 mL of Allinase inhibitor solution, and shake until the Powdered Extract is fully dispersed. Dilute with Allinase inhibitor solution to volume. Centrifuge, transfer 5 mL of the clear supernatant to a 10-mL volumetric flask, and dilute with Allinase inhibitor solution to volume.
Detector: UV 337 nm
Column: 4-mm × 10-cm; packing L1
Flow rate: 1 mL/min
Injection size: 10 µL
Sample: Standard solution
[NoteAlliin exhibits two major peaks representing its diastereomers. ]
Relative standard deviation: NMT 2.0% for each of the major peaks
Samples: Standard solution and Sample solution
Using a volumetric syringe, transfer 0.1 mL of the Sample solution or the Standard solution to separate septum-capped vials, and add 0.5 mL of the Derivatization reagent to each vial. Allow a reaction time of NLT 2 min before injection into the chromatograph. Record the chromatograms, and measure the areas of the alliin diastereomer peaks.
Calculate the percentage of alliin in the portion of Powdered Extract taken:
Result = (rU/rS) × CS × (V/W) × 100
Acceptance criteria: NLT 4.0% on the dried basis
• Heavy Metals, Method I 231: NMT 10 ppm
• Microbial Enumeration Tests 2021: The total aerobic bacterial count does not exceed 104 cfu/g, and the total combined molds and yeasts count 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
• Alliinase Activity
Allinase inhibitor solution, Solution A, Buffer, Derivatization reagent, Mobile phase, Standard solution, Chromatographic system, and Analysis: Proceed as directed in the test for Content of Alliin.
Sample solution: Incubate 200 mg of Powdered Extract with 20 mL of water at room temperature for 5 min. Immediately after incubation, add 80.0 mL of Allinase inhibitor solution, mix, and centrifuge.
Acceptance criteria: The area of the alliin peak of the Sample solution is NMT 1% of the area of the alliin peak of the Standard solution.
• Alcohol Determination, Method II 611: NMT 0.5%
• Articles of Botanical Origin, Water Content 561: NMT 5.0%
• Other Requirements: Meets the requirements under Botanical Extracts 565, Packaging and Storage and Pesticide Residues
• Packaging and Storage: Preserve in tight containers, in a cool place, protected from light.
• Labeling: The label states the Latin binomial and, following the official name, the part of the plant from which the article was prepared. The label also indicates the content of alliin, the extracting solvent or solvent mixture used for preparation, and the ratio of the starting crude plant material to Powdered Extract. It meets the requirements under Botanical Extracts 565, Labeling.
• USP Reference Standards 11
USP Alliin RS
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USP35NF30 Page 1313