Aegle marmelos Fruit Dry Extract

DEFINITION

The article is prepared from the dried ripe or unripe fruit of Aegle marmelos (L.) Corréa (Family Rutaceae) by extraction with a hydroalcoholic mixture. It contains NLT 90.0% and NMT 110.0% of the labeled amount of marmelosin on the dried basis.

POTENTIAL CONFOUNDING MATERIALS

Limonia acidissima Groff

CONSTITUENTS OF INTEREST

Furocoumarin: Marmelosin

IDENTIFICATION

• A. HPTLC for Articles of Botanical Origin <203>

Standard solution A: 0.1 mg/mL of USP Marmelosin RS in methanol

Standard solution B: 10 mg/mL of USP Aegle marmelos Fruit Dry Extract RS in methanol. Sonicate for 5 min, centrifuge or filter, and use the supernatant or the filtrate.

Sample solution: Sonicate about 100 mg of Aegla marmelos Fruit Dry Extract in 10 mL of methanol for 10 min, centrifuge or filter, and use the supernatant or the filtrate.

Chromatographic system

Adsorbent: Chromatographic silica gel F₂₅₄ mixture with an average particle size of 5 µm

Application volume: 10 µL of Standard solution A and 5 µL each of 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: Toluene, ethyl acetate, methanol, and acetic acid (40: 10: 1.25: 1)

Developing distance: 6 cm

Analysis

Samples: Standard solution A, Standard solution B, and Sample solution

Apply the Samples as bands and dry in air. Develop in a saturated chamber (20 min with filter paper), remove the plate from the chamber, and dry in air. Examine under UV light at 254 and 365 nm.

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements: Under UV 254 nm, the chromatogram of Standard solution B exhibits about 7 black bands with the most intense and uppermost band similar in R_F and color to the marmelosin band in the chromatogram of Standard solution A. Under UV 365 nm, a pale greenish-blue band appears near the R_F of 0.6, similar to the color of the marmelosin band in the chromatogram of Standard solution A. One pale blue and one pale green band appear above the marmelosin band. Below the marmelosin band, one blue band appears right below the marmelosin band and several bands, with decreasing R_F, one pale green band, two pale blue bands, one bright blue band, two pale blue bands, one green band, one pale green band, and one light blue band.

Acceptance criteria: Under UV 254 nm, the chromatogram of the Sample solution exhibits the most intense and uppermost band similar in R_F and color to the marmelosin band in the chromatogram of Standard solution A. Six minor black bands appear below the marmolesin band. Under UV 365 nm, a pale greenish blue band appears near the R_F of 0.6 in the Sample solution similar to the color of the marmelosin band in the chromatogram of Standard solution A. One pale blue and one pale green band appears above the marmelosin band. Below the marmelosin band, one blue band appears  right below the marmelosin band and several bands, with decreasing R_F, one pale green band, two pale blue bands, one bright blue band, two pale blue bands, one green band, one pale green band, and one light blue band.

• B. HPLC

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

Acceptance criteria: The chromatogram of the Sample solution exhibits peaks at retention times corresponding to the peaks due to marmelosin in Standard solution B.

ASSAY

• Content of Marmelosin

Solution A: 0.2% acetic acid in water

Solution B: Acetonitrile

Mobile phase: See Table 1.

Table 1

Standard solution A: 0.01 mg/mL of USP Marmelosin RS in methanol

Standard solution B: Dissolve 0.1 g of USP Aegle marmelos Fruit Dry Extract RS in 100 mL of methanol, sonicate, and pass through a membrane filter of 0.45-µm or finer pore size.

Sample solution: Transfer 0.1 g of Aegle marmelos Fruit Dry Extract to a 100-mL beaker and add 50 mL of methanol. Sonicate for 30 min and transfer the solution to a 100-mL volumetric flask. Rinse the beaker with 20 mL of methanol and add the extract to the volumetric flask. Dilute with methanol to volume. Pass through a membrane filter of 0.45-µm pore size.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Detector: UV 300 nm

Column: 4.6-mm × 10-cm; 2.7-μm packing L1

Flow rate: 0.6 mL/min

Injection volume: 20 µL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Tailing factor: NMT 2.0 for the marmelosin peak, Standard solution A

Relative standard deviation: NMT 2.0% for the marmelosin peak in repeated injections, Standard solution A

Chromatogram similarity: The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Aegle marmelos Fruit Dry Extract RS being used.

Analysis

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 Aegle marmelos Fruit Dry Extract RS being used, identify the retention times of the peaks corresponding to marmelosin in the Sample solution.

Calculate the percentages of marmelosin in the portion of Aegle marmelos Fruit Dry Extract taken:

Result = (r_U/r_S) x (C_S/C_U) x 100

r_U   = peak area of marmelosin from the Sample solution

r_S   = peak area of marmelosin from Standard solution A

C_S  = concentration of USP Marmelosin RS in Standard solution A (mg/mL)

C_U  = concentration of Aegle marmelos Fruit Dry Extract in the Sample solution (mg/mL)

Calculate the percentage of the labeled amount of marmelosin in the portion of Dry Extract taken:

Result = (P/L) x 100

P   = content of marmelosin as determined above (%)

L   = labeled amount of marmelosin (%)

Acceptance criteria: 90.0%–110.0% on the dried basis

CONTAMINANTS

• Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 0.5 µg/g

Lead: NMT 5.0 µg/g

Mercury: NMT 1.0 µg/g

• Articles of Botanical Origin <561>, Pesticide Residue Analysis: 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

SPECIFIC TESTS

• Loss on Drying <731>

Sample: 2 g of Aegle marmelos Fruit Dry Extract 

Analysis: Dry the Sample at 105° for 5 h.

Acceptance criteria: NMT 6%

ADDITIONAL 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 the part(s) of the plant contained in the article.

• USP Reference Standards <11>

USP Aegle marmelos Fruit Dry Extract RS

USP Marmelosin RS