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Banaba Leaf Dry Extract
DEFINITION
Banaba Leaf Dry Extract consists of the dried leaves of Lagetroemia speciosa (L.) Pers. (Fam. Lythraceae) by extraction with hydroalcoholic mixtures. It contains NLT 90.0% and NMT 110.0% of the labeled amount of corosolic acid (C30H48O4), calculated on the dried basis.
IDENTIFICATION
•  A. Meets the requirements for Specific Tests, Botanic Characteristics
•  B. Thin-Layer Chromatography
Standard solution A:  0.2 mg/mL of USP Corosolic Acid RS in methanol
Standard solution B:  10 mg/mL of USP Lagerstroemia speciosa Leaf Dry Extract RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.
Sample solution:  Banaba Leaf Dry Extract in methanol at a concentration equivalent to 0.2 mg/mL of corosolic acid according to the label claim. Sonicate if necessary.
Chromatographic system 
(See Chromatography 621, Thin-Layer Chromatography.)
Mode:  HPTLC
Adsorbent:  Chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates)
Application volume:  6 µL as 8-mm bands
Relative humidity:  Condition the plate to a relative humidity of about 33% using a suitable device.
Column temperature:  25
Developing solvent system:  A mixture of toluene, ethyl acetate, and acetic acid (55: 45: 0.5)
Developing distance:  6 cm
Derivatization reagent:  85 mL of ice-cooled methanol mixed with 10 mL of glacial acetic acid, 5 mL of sulfuric acid, and 0.5 mL of p-anisaldehyde
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 an unsaturated chamber, remove the plate from the chamber, and dry. Treat with Derivatization reagent, heat for 3 min at 100, and examine under visible light.
System suitability:  Under visible light, the chromatogram of Standard solution B exhibits the most intense band, a violet or blue band, with similar RF and color as the corosolic acid band in the chromatogram of Standard solution A; a blue band close to the start (about RF 0.1), consistent with asiatic acid; two minor blue bands in between the corosolic and asiatic bands; a minor blue band due to virgatic acid, above the band due to corosolic acid; and just below the asiatic band, a minor brown band. Standard solution B also exhibits two minor violet bands, separated, at about three-fourths of the chromatogram; the band with the lower RF corresponds to oleanolic acid.
Acceptance criteria:  Under visible light, the chromatogram of the Sample solution exhibits the most intense band as a violet band corresponding in color and RF to the band due to corosolic acid in the chromatogram of Standard solution A, as well as the following bands corresponding to similar bands of Standard solution B: a minor blue band close to the start (about RF 0.1), two minor purple bands below the corosolic acid, two minor blue bands above the corosolic acid, and two minor violet bands, which are separated, at about three-fourths of the chromatogram.
•  C. HPLC
Analysis:  Proceed as directed in Content of Corosolic Acid.
Acceptance criteria:  The chromatogram of the Sample solution exhibits a group of three peaks. The one in the center is the most intense of the group and occurs at a retention time corresponding to that of corosolic acid in the chromatogram of Standard solution A. The peak that elutes before corosolic acid has about one-half to one-third of the intensity of that of corosolic acid, and the peak that elutes after corosolic acid has the lesser intensity of the three and is consistent with virgatic acid. A minor peak due to oleanolic acid elutes later in the chromatogram.
COMPOSITION
•  Content of Corosolic Acid
Solution A:  Dilute 0.1% phosphoric acid in water.
Solution B:  Acetonitrile
Mobile phase:  A mixture of Solution A and Solution B (4:6)
Standard solution A:  0.1 mg/mL of USP Corosolic Acid RS in methanol
Standard solution B:  5.0 mg/mL of USP Lagerstroemia speciosa Leaf Dry Extract RS in methanol. Sonicate if necessary. Before injection, pass through a membrane filter of 0.45-µm or finer pore size. Discard the first few mL of the filtrate.
Sample solution:  Banaba Leaf Dry Extract in methanol at a concentration equivalent to 0.1 mg/mL of corosolic acid according to the label claim. Sonicate if necessary. Before injection, pass through a membrane filter of 0.45-µm or finer pore size. Discard the first few mL of the filtrate.
Chromatographic system 
(See Chromatography 621, System Suitability.)
Mode:  LC
Detector:  UV 205 nm
Column:  4.6-mm × 25-cm; 5-µm packing L1
Flow rate:  1.6 mL/min
Injection volume:  20 µL
System suitability 
Samples:  Standard solution A and Standard solution B
[Note—The approximate relative retention times of the individual peaks for corosolic acid, virgatic acid, and oleanolic acid are 1.00, 1.1, and 3.2, respectively. ]
Suitability requirements 
Chromatogram similarity:  The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Lagerstroemia speciosa Leaf Dry Extract RS being used.
Resolution:  NLT 1.5 between the corosolic acid peak and the preceding peak, Standard solution B
Tailing factor:  NMT 2.0 for the corosolic acid peak, Standard solution A
Relative standard deviation:  NMT 2.0%, determined from the corosolic acid peak in repeated injections, Standard solution A
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Identify the relative retention times of the peaks for corosolic acid, virgatic acid, and oleanolic acid of the Sample solution.
Calculate the percentage of the labeled amount of corosolic acid in the portion of Banaba Leaf Dry Extract taken:
Result = (rU/rS) × (CS/CU) × 100

rU== peak area of corosolic acid from the Sample solution
rS== peak area of corosolic acid from Standard solution A
CS== concentration of corosolic acid in Standard solution A (mg/mL)
CU== concentration of Banaba Leaf Dry Extract in the Sample solution (mg/mL)
Calculate the percentage of the labeled amount of corosolic acid in the portion of Extract taken:
Result = (P/L) × 100

P== content of corosolic acid as determined above (%)
L== labeled amount of corosolic acid (%)
Acceptance criteria:  90.0%–110.0% of the labeled amount of corosolic acid 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 µg/g
Mercury:  NMT 0.2 µg/g
•  Microbial Enumeration Tests 2021: The total aerobic microbial count does not exceed 104 cfu/g, and the total combined molds and yeasts count does not exceed 103 cfu/g.
•  Microbial Procedures for Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
SPECIFIC TESTS
•  Loss on Drying 731
Sample:  2.0 g of Banaba Leaf Dry Extract
Analysis:  Dry the Sample at 105 for 2 h.
Acceptance criteria:  NMT 8%
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, following the official name, the parts of the plant from which the article was derived. It meets other labeling requirements in Botanical Extracts 565.
•  USP Reference Standards 11
USP Corosolic Acid RS Click to View Structure
USP Lagerstroemia speciosa Leaf Dry Extract RS
USP38
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Monograph Christopher O Okunji, Ph.D.
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2021 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
(301) 816-8339		 (GCM2010) General Chapters - Microbiology
2022 Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison
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Reference Standards RS Technical Services
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USP38–NF33 Page 5902
Pharmacopeial Forum: Volume No. 39(6)