Aegle marmelos Fruit

Aegle marmelos Fruit

Proposed For Comment Version 0.2

Aegle marmelos Fruit

 


 

DEFINITION

The article consists of the dried ripe or unripe fruit of Aegle marmelos (L.) Corréa (Family Rutaceae). The fruits are harvested and processed during summer. It contains NLT 0.2% of marmelosin on the dried basis and no aegeline is present.

 

SYNONYMS

Below marmelos (L.) Lyons

Bilacus marmelos (L.) Kuntze

Crateva marmelos L.

Feronia pellucida Roth

 

POTENTIAL CONFOUNDING MATERIALS

Limonia acidissima Groff

 

SELECTED COMMON NAMES

Chinese: 木橘

English: Bael, bael tree, belfruit tree, Bengal quince, Indian baelfruit

French: Bel indien

German: Belbaum

India: Beli

Spanish: Bela, milva

 

CONSTITUENTS OF INTEREST

Furocoumarin: Marmelosin

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic: Dried fruit pulps rarely negligible quantity of fruits with fruit rind is found in commercial samples. It is de-skinned or de-peeled spheres, or pieces of hemispheres or quadrants of spheres. It is stony, hard, tough, brittle, but not easy to break and generally around 5–6 cm in size. They are pale yellow to golden-yellow or pale brown to reddish or reddish-brown or nearly blackish in color with a pleasant odor and slightly bitter and marmalade in taste. The shapes are variable from perfect or irregular sphere or ellipsoid to somewhat triangle and few of them are amoeboid. Almost all the pieces show deep or shallow longitudinal groves, which are the gaps between the locules. Some of the chopped pieces are circular disc-shaped. When the fruits are cut transversely, they show a central core of hard tissue with a ring of pale-colored vascular traces and another ring of seed cavities with seeds near the periphery. However, vertically cut pieces showed long elongated groves or locules with 5–8 seeds in each. Generally the seed locules are filled with dried, transparent, crystalline, brownish to reddish mucilage. In the cut ends, seeds are visible in the seed cavities. The seeds are hard, buff or white to yellowish in color, oblong, flattened, acuminoid and about 0.75 cm × 0.5 cm in size. The seed surface is rough and covered with trichomes. The seeds are non-endospermic, attached with a delicate funicle. The cut ends of the seeds show the outer seed coat and inner buff white coloured two-seed cotyledons.

Microscopic: Cross section of a portion of the fruit pulp shows loosely arranged, polygonal or circular or elliptical, thick-walled parenchyma cells 50–100 µm in size with large-sized intercellular spaces. Sometimes, the oil from cotyledons get spilled on the parenchyma cells. Regular dicotyledonous vascular bundles are scattered throughout the pulp. Both simple and compound starches are observed in parenchymatous cells. They are spherical or polygonal or angular, 2–5 µm in size with distinct hilum. The cross section of seed shows an outer testa and an inner tegmen which in turn enclose the cotyledons. The testa can be distinguished into outer, middle, and the inner layers. The outermost layer of testa is sclerotic and ornamented with elongated thick-walled, sclerotic epidermal hairs followed by 3–6 layers of parenchymatous cells that usually contain oil globules. The inner testa is made of 2 layers of thick-walled sclerenchymatous cells. Inner to it, the tegmen is distinguished into outer, middle, and the inner layers. The outermost layer of tegmen is thick-walled followed by 6–8 layers of polygonal to irregular shaped parenchymatous cells. The inner layers of tegmen are made of 1–2 layers of circular- to oval-shaped cells containing the large and distinct oil globules. The cotyledon is made of outer single-layered epidermis and the inner parenchymatous tissue. The epidermal cells are relatively smaller and slightly transversely elongated. Inner parenchymatous cells are oval to polygonal in shape. Most of these cells are filled with oil globules. Simple starch grains as same as in pulp are rarely observed.

 

B. 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 1 g of Aegla marmelos Fruit, finely powdered, 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 (HPTLC plates)

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 (30 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 RF 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 RF 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 RF, 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 RF and color to the marmelosin band in the chromatogram of Standard solution A. Two minor black bands appear in the lower half of the chromatogram (between the RF of 0.3 and 0.4). Under UV 365 nm, a pale greenish-blue band appears near the RF 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 band 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 RF, 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.

 

• C. 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

Time
(min)

Solution A
(%)

Solution B
(%)

0

60

40

20

40

60

24

40

60

27

60

40

30

60

40

 

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 0.2 g of Aegle marmelos Fruit, finely powdered and accurately weighed, to a 250-mL flask and add 30 mL of methanol. Reflux at 80° for 30 min and decant the extract into a 100-mL volumetric flask. Repeat the extraction process with another 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 x 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

Resolution: NLT 2.0 between the marmelosin peak and the peak before, and the marmelosin peak and the peak after

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 time of the peak corresponding to marmelosin in the Sample solution.

Calculate the percentage of marmelosin in the portion of Aegle marmelos Fruit taken:

 

Result = (rU/rS) x CS x (V/W) x 100

 

rU   = peak area of marmelosin from the Sample solution

rS   = peak area of marmelosin from Standard solution A

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

V    = volume of the Sample solution (mL)

W   = weight of Aegle marmelos Fruit taken to prepare the Sample solution (mg)

 

Acceptance criteria: NLT 0.2% 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 absence of Salmonella species and Escherichia coli

 

SPECIFIC TESTS

Limit of Aegeline: HPTLC

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

Sample solution, Chromatographic system, and System suitability: See HPTLC for Articles of Botanical Origin <203>.

Analysis

Samples: Standard solution and Sample solution

Apply the Samples as bands and dry in air. Develop in a saturated chamber (30 min with filter paper), remove the plate from the chamber, and dry in air. Examine under UV light at 254 nm.

Acceptance criteria: No dark band observed corresponds with aegeline near the RF of 0.26.

Limit of Aegeline: HPLC

Standard solution: 0.1 mg/mL of aegeline in methanol

Sample solution and Chromatographic system: Proceed as directed in the Assay for Content of Marmelosin, except the Detector.

Detector: UV 274 nm

Analysis

Samples: Standard solution and Sample solution

Using the chromatograms of the Standard solution, identify the retention time of the peak corresponding to aegeline in the Sample solution.

Acceptance criteria: No peak corresponding with aegeline is observed.

• Articles of Botanical Origin <561>, Methods of Analysis, Foreign Organic Matter: NMT 2.0%

• Articles of Botanical Origin <561>, Methods of Analysis, Alcohol-Soluble Extractives, Method 1: NLT 30%

• Articles of Botanical Origin <561>, Methods of Analysis, Water-Soluble Extractives, Method 2: NLT 10%

• Loss on Drying <731>

Sample: 2 g of Aegle marmelos Fruit, finely powdered

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

Acceptance criteria: NMT 10%

• Articles of Botanical Origin <561>, Methods of Analysis, Total Ash: NMT 10%

• Articles of Botanical Origin <561>, Methods of Analysis, Acid-Insoluble Ash: NMT 2.0%

 

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

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Other Versions

Proposed For Development Version 0.1