Terminalia chebula Fruit Dry Extract

Terminalia chebula Fruit Dry Extract

Proposed For Comment Version 0.3

Terminalia chebula Fruit Dry Extract

 


DEFINITION

The extract is prepared from the pericarp of mature dried fruit of Terminalia chebula Retz. (Family Combretaceae) by extraction with hydroalcoholic mixtures. It contains NLT 90% and NMT 110% of the labeled amount of hydrolyzable tannins calculated as the sum of chebulagic acid and chebulinic acid, on the dried basis.

 

POTENTIAL CONFOUNDING MATERIALS

None known.

 

CONSTITUENTS OF INTEREST

Hydrolyzable tannins: Chebulagic acid and chebulinic acid

Triterpene: Arjungenin

 

IDENTIFICATION

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

Standard solution A: 0.1 mg/mL each of USP Chebulagic 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 or filter, and use the supernatant or filtrate.

Sample solution: Sonicate about 1.0 g of Terminalia chebula Fruit Dry Extract in 10 mL of methanol for 10 min, centrifuge or filter, and use the supernatant or filtrate.

Chromatographic system

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

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 an intense band in the lower half similar in RF and color to the chebulagic acid band in the chromatogram of Standard solution A. Another intense band above chebulagic acid band is the chebulinic acid band. Two additional bands appear above the chebulinic acid band, one near RF 0.4 and another near RF 0.7. Two additional bands appear below the chebulagic acid band. Under UV 365 nm after derivatization, about six intense blue fluorescence bands appear in the chromatogram. A pale blue band similar in RF and color to the chebulagic acid band also appears.

Acceptance criteria: Under UV 254 nm prior to derivatization, the chromatogram of Sample solution exhibits two intense bands in the lower half of the chromatogram with the lower band corresponding in RF and color to the chebulagic acid in the chromatogram of Standard solution A. The following bands correspond to similar bands in the chromatogram of Standard solution B: two bands below chebulagic acid, a band above chebulinic acid, and one intense band with an RF of approximately 0.7. Under 365 nm after derivatization, about four intense blue fluorescence bands appear in the lower half, and two blue fluorescence bands appear between RF 0.6 and 0.7. A greenish white band, characteristic to Terminalia bellirica should not be observed near RF 0.5.

• B. 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 those for gallic acid, chebulagic acid, ellagic acid, and chebulinic acid in Standard solution B. The most prominent peak in the chromatogram corresponds to ellagic acid, followed in response by gallic acid with an RT of around 6 min. Chebulagic acid appears before ellagic acid and chebulinic acid appears after ellagic acid, with almost equal responses.

 

ASSAY

• Content of Constituents of Hydrolyzable Tannins

Solution A: Dissolve 140 mg 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

100

0

7

92

8

12

85

15

22

80

20

25

50

50

28

100

0

32

100

0

 

Standard solution A: 0.3 mg/mL each of USP Chebulagic Acid RS in methanol

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 or finer pore size.

Sample solution: Transfer 0.1 g of Terminalia chebula Fruit Dry Extract to a 100-mL volumetric flask. Add 70 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 254 nm

Column: 4.6-mm x 25-cm; 5-µm packing L1 (similar to Phenomenex Luna C18)

Column temperature: 25°

Flow rate: 1.5 mL/min

Injection volume: 10 µL

System suitability

Samples: Standard solution A and Standard solution B

Suitability requirements

Resolution: NLT 2.0 between chebulagic acid and the peak after, Standard solution B

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

Relative standard deviation: NMT 2.0% for the chebulagic acid 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 Terminalia chebula 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 Terminalia chebula Fruit Dry Extract RS being used, identify the retention times of the peaks corresponding to chebulagic acid and chebulinic acid in the Sample solution. The approximate relative retention times of the peaks for chebulagic acid and chebulinic acid are 1.0 and 1.21, respectively.

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

 

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

 

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

rS     = peak area of the chebulagic acid from Standard solution A

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

V      = volume of the Sample solution (mg/mL)

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

F      = conversion factor for the analytes (1 for chebulagic acid and 1.14 for chebulinic acid)

 

Add the percentages of the labeled amounts of chebulagic acid and chebulinic acid.

 

Calculate the percentages of the labeled amounts of hydrolyzable tannins in the portion of Terminalia chebula Fruit Dry Extract taken:

 

Result = (P/L) x 100

 

P     = content of hydrolyzable tannins as determined above (%)

L     = labeled amount of hydrolyzable tannins (%)

 

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 0.2 µg/g

• Articles of Botanical Origin <561>, Pesticide Residues 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 10cfu/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: 1 g of Terminalia chebula Fruit Dry Extract

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

Acceptance criteria: NMT 5%

• Articles of Botanical Origin <561>, Methods of Analysis, Total AshNMT 15%

• Articles of Botanical Origin <561>, Methods of Analysis, Acid-Insoluble Ash: 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 Chebulagic Acid RS

USP Terminalia chebula Fruit Dry Extract RS

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Other Versions

Proposed For Comment Version 0.2
Proposed For Development Version 0.1