Terminalia chebula Fruit

Terminalia chebula Fruit

Proposed For Comment Version 0.2

Terminalia chebula Fruit

 


DEFINITION

The article consists of the dried fruit of Terminalia chebula Retz. (Family Combretaceae). It contains NLT 15% of hydrolyzable tannins calculated as the sum of chebulagic acid and chebulinic acid, on the dried basis.

 

SYNONYMS

Myrobalanifera citrina Houtt.

Myrobalanus chebula (Retz.) Gaertn.

Myrobalanus gangetica (Roxb.) Kostel.

Terminalia gangetica Roxb.

Terminalia reticulata Roth.

 

POTENTIAL CONFOUNDING MATERIALS

Terminalia citrine

 

SELECTED COMMON NAMES

Chinese: 诃子, 訶梨勒

English: Myrobalan, black myrobalan, chebulic myrobalan, gali nut, Indian gall-nut, ink nut, yellow myrobalan

French: Badamier chébule, Myrobalan chébule

German: Chebulische Myrobalane, Rispiger Myrobalanenbaum

Hindi: हर्रा,  हरीतक

Japanese: ハリタキ, イ エロ-ターミナリア, ミ ロバランノキ

Russian: Харитаки

Spanish: Mirobalanos índicos

Thai: สมอ ไทย

 

CONSTITUENTS OF INTEREST

Hydrolyzable tannins: Chebulagic acid and chebulinic acid

Triterpene: Arjungenin

 

IDENTIFICATION

• A. Botanical Characteristics

Macroscopic

Fresh fruit: A glabrous, shining ellipsoidal, or broadly ovoid or obovoid; obscurely or faintly five-angled and shallowly furrowed; greenish-yellow drupe from 25–50 mm long and about 25 mm or less wide. The stone cells are present in the mesocarpium and these are of two types: the horizontally elongated ones are present in the peripheral region and the round ones are present in the interior part.

Dry fruit: The surface of the dry fruit is somewhat wrinkled and shows five slightly thick but well-defined longitudinal ridges that are 2–3 mm wide and 2 mm thick. The surface color varies from light yellowish-brown to a nearly uniform brown with yellowish markings or patches. In some fruits the basal portion is narrower and somewhat elongated or tapering.

Microscopic

Fresh fruit: The epidermis consists of a single layer, the cells are rectangular and tangentially elongated, and covered with a thick cuticle. Below the epidermis, the outer mesocarpic region consists of 6–10 layers of cells filled with abundant starch grains and tannin. In the mesocarpic region, sclereids are present, about 4–6 cells thick and arranged tangentially. The sclerified cells are thick-walled, lignified, and elongated. Below the sclerenchymatous layer occurs a layer of 20–25 mesocarpic cells which are parenchymatous in nature, oval to round in shape, and filled with starch grains and tannin. In the mesocarpic region, vascular strands occur at a certain place and consist only of spiral elements. The mesocarpic cells contain abundant rosettes of calcium oxalate. Starch grains present in cells of the outer and inner mesocarpic regions are simple or compound, and oval to round.

• B. Thin-Layer Chromatography

Standard solution A: 0.1 mg/mL each of USP Chebulagic Acid RS and USP Chebulinic Acid RS in methanol

Standard solution B: 100 mg/mL of USP Terminalia chebula Fruit Dry Extract RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.

Sample solution: Sonicate about 1.0 g of Terminalia chebula Fruit, finely powdered, in 100 mL of methanol for 10 min, centrifuge, and use the supernatant.

Chromatographic system

(See Chromatography <621>, Thin-Layer Chromatography.)

Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)

Application volume: 5 µL of Standard solution A, and 1 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.

Temperature: 25°

Developing solvent system: Ethyl formate, toluene, formic acid, and water (30: 1.5: 4: 3)

Developing distance: 7 cm

Derivatization reagent: Natural products reagent (NP reagent): 1 g of diphenylboinic acid aminoethylester dissolved in 200 mL of ethyl acetate

Analysis

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

Apply the Samples as bands to a suitable HPTLC plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry. Examine under UV 254 nm before derivatization. Heat the plate at 100° for 3 min, then treat with Derivatization reagent. Examine under UV 366 nm.

System suitability: Under UV 254 nm prior to derivatization, the chromatogram of Standard solution B exhibits two intense bands in the lower half similar in RF and color to the chebulagic acid and chebulinic bands in the chromatogram of Standard solution A, with chebulagic acid having a lower RF. About four or five additional bands appear with increasing RF: two bands below chebulagic acid, a band above chebulinic acid, and one intense band at an RF of approximately 0.7. Under UV 366 nm after derivatization, about four intense blue fluorescence bands appear in the lower half and two blue fluorescence bands appear between RF of 0.6 and 0.7.

Acceptance criteria: Under UV 254 nm prior to derivatization, the chromatogram of Sample solution exhibits two intense bands in the lower half corresponding in RF and color to the chebulagic acid and chebulinic bands in the chromatogram of Standard solution A, with chebulagic acid having a lower RF. Two additional bands appear, one near the origin and another near an RF of approximately 0.7. Under UV 366 nm after derivatization, about four pale blue fluorescence bands appear in the lower half, and one blue fluorescence band appears with an RF of approximately 0.7.

• C. HPLC

Analysis: Proceed as directed in the Assay for Content of Constituents of Hydrolyzable Tannins.

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

 

ASSAY

• Content of Constituents of Hydrolyzable Tannins

Solution A: Dissolve 0.136 g of monobasic potassium phosphate in 900 mL of water, add 0.5 mL of o-phosphoric acid, dilute with water to 1 L, and mix.

Solution B: Acetonitrile

Mobile phase: See Table 1.

Table 1

Time
(min)

Solution A
(%)

Solution B
(%)

0

95

5

18

65

35

25

45

55

28

45

55

35

65

35

40

95

5

45

95

5

 

Standard solution A: 1.0 mg/mL of USP Chebulagic Acid RS in boiling water

Standard solution B: Dissolve 0.1 g of USP Terminalia chebula Fruit Dry Extract RS in 100 mL of boiling water, sonicate, and pass through a membrane filter of 0.45-μm pore size.

Sample solution: Transfer 0.1 g of Terminalia chebula Fruit, finely powdered and accurately weighed, to a 100-mL volumetric flask. Add 50 mL of boiling water and sonicate for 10 min. Dilute with water to 100 mL, mix well, and pass through a membrane filter of 0.45-μm pore size.

Chromatographic system

(See Chromatography <621>, System Suitability.)

Detector: UV 270 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1 (similar to Merck kGaA Purospher Star LP HPLC Column, RP-18)

Flow rate: 1.5 mL/min

Injection volume: 20 µL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

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

Resolution: NLT 11.4 between chebulagic acid and chebulinic acid peaks, Standard solution B

Tailing factor: NMT 1.5, Standard solution A

Relative standard deviation: NMT 2.5%, Standard solution A

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 Terminalia chebula Fruit Dry Extract RS being used, identify the retention times of the peaks corresponding to chebulagic acid and chebulinic acid. The approximate relative retention times of the peaks for chebulagic acid and chebulinic acid are 1.0 and 1.15, respectively.

Separately calculate the percentages of chebulagic acid and chebulinic acid in the portion of Terminalia chebula Fruit taken:

 

Result = (rU/rS) × CS × (V/W) × F × 100

 

rU     = peak area of the relevant analyte from the Sample solution

rS     = peak area of the relevant analyte from Standard solution A

CS    = concentration of the relevant analyte in Standard solution A (mg/mL)

V      = volume of the Sample solution (mL)

W     = weight of Terminalia chebula Fruit taken to prepare the Sample solution (mg)

F      = conversion factor for the analytes (1 for chebulagic acid and 0.79 for chebulinic acid)

 

Add the percentages of chebulagic acid and chebulinic acid.

Acceptance criteria: NLT 15% on the dried basis

 

CONTAMINANTS

• Elemental Impurities—Procedures <233>

Acceptance criteria

Arsenic: NMT 2.0 µg/g

Cadmium: NMT 1.0 µg/g

Lead: NMT 5.0 µg/g

Mercury: NMT 0.2 µg/g

• Articles of Botanical Origin, General Method for Pesticide Residues Analysis <561>: 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

• Identification of Arjungenin

Standard solution: 0.2 mg/mL of USP Arjungenin RS in methanol

Sample solution: Sonicate about 10 g of Terminalia chebula Fruit, finely powdered, in 10 mL of dichloromethane for 10 min, centrifuge, and use the supernatant.

Chromatographic system

(See Chromatography <621>, Thin-Layer Chromatography.)

Adsorbent: Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)

Application volume: 15 µL each of Standard solution A, 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.

Temperature: 25°

Developing solvent system: Ethyl acetate, toluene, and glacial acetic acid (55:45:2)

Developing distance: 6 cm

Derivatization reagent: Anisaldehyde-sulfuric acid reagent created by combining 0.5 mL of anisaldehyde with 10 mL of glacial-acetic acid, 85 mL of methanol, and 5 mL of sulfuric acid.

Analysis

Samples: Standard solution and Sample solution

Apply the Samples as bands to a suitable HPTLC plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry. Treat with Derivatization reagent. Examine under visible light.

Acceptance criteria: Under visible light, the Sample solution exhibits the most intense band in the middle, as a dark blue band corresponding in color and RFto the band due to arjungenin in the Standard solution.

• Articles of Botanical Origin, Foreign Organic Matter <561>: NMT 1.0%

• Articles of Botanical Origin, Alcohol-Soluble Extractives, Method 1 <561>: NLT 40.0%

• Articles of Botanical Origin, Water-Soluble Extractives, Method 2 <561>: NLT 60.0%

• Loss on Drying <731>

Sample: 1 g of Terminalia chebula Fruit, finely powdered

Analysis: Dry the Sample at 105° for 2 h.

Acceptance criteria: NMT 8%

• Articles of Botanical Origin, Total Ash <561>: NMT 15%

• Articles of Botanical Origin, Acid-Insoluble Ash <561>: NMT 5%

 

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 Arjungenin RS

USP Chebulagic Acid RS

USP Chebulinic Acid RS

USP Terminalia chebula Fruit Dry Extract RS

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Proposed For Development Version 0.1