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Banaba Leaf
DEFINITION
Banaba Leaf consists of the dried leaves of Lagerstroemia speciosa (L.) Pers. (Fam. Lythraceae). It contains NLT 0.2% 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 RS in methanol. Sonicate for 10 min, centrifuge, and use the supernatant.
Sample solution:  About 0.2 g of Banaba Leaf, finely powdered, in 10 mL of methanol. Sonicate for 15 min, centrifuge, and use supernatant.
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 each of Standard solution A and Standard solution B and 8 µL of the 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:  A mixture of toluene, ethyl acetate, and acetic acid (55: 45: 0.5)
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), a minor brownish band above the corosolic acid, and a minor violet band 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:  Transfer about 5.0 g of Banaba Leaf, finely powdered and accurately weighed, to a round-bottom flask. Add 75 mL of methanol, reflux for 15 min, set aside to settle, and decant the supernatant. Repeat the extraction three more times, then combine the extracts. Filter, and concentrate under reduced pressure. Transfer to a 100-mL volumetric flask, adjust with methanol to volume, and mix. 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
Column temperature:  25
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 1.0, 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 in the Sample solution.
Calculate the percentage of corosolic acid in the portion of Banaba Leaf taken:
Result = (rU/rS) × CS × (V/W) × 100

rU== peak area of corosolic acid from Sample solution
rS== peak area of corosolic acid from Standard solution A
CS== concentration of corosolic acid in Standard solution A (mg/mL)
V== volume of Sample solution (mL)
W== weight of Banaba Leaf taken to prepare the Sample solution (mg)
Acceptance criteria:  NLT 0.2% 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 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 count does not exceed 103 cfu/g.
•  Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
SPECIFIC TESTS
•  Botanic Characteristics
Macroscopic:  Banaba leaves vary in shape, including lanceolate, oblong-lanceolate, oblong, and elliptic ovate. They are up to 34 cm long and 11 cm wide; olive green to yellowish brown; entirely or slightly wavy at the margins; base acute; apex acute to acuminate; leathery in texture; and petiolate with petioles up to 1 cm long.
Microscopic 
Transverse section of the midrib:  A layer of upper epidermis composed of rectangular to round cells covered with thin cuticle; a few layers of collenchyma cells; numerous layers of parenchyma cells, some containing cluster crystals of calcium oxalate, with large intercellular spaces; groups of lignified fiber bundles; and secretory canals scattered in the parenchyma zone. Bicollateral vascular bundle encircled by a continuous sheath of fibers accompanied by sclerenchyma and cells containing cluster crystals of calcium oxalate; secretory canals between the vascular bundles; numerous layers of parenchyma cells, some containing cluster crystals of calcium oxalate, with large intercellular spaces; a few layers of collenchyma; a layer of lower epidermal cells.
Transverse section of the lamina:  A layer of upper epidermis composed of rectangular cells about twice as large as those of the lower epidermis. Some cells are secretory cells, which tend to protrude into the mesophyll and sometimes appear to be below the upper epidermis. Two layers of rectangular palisade cells; 4–6 layers of parenchyma cells, some containing prisms of calcium oxalate and others containing clusters crystals of calcium oxalate, with large intercellular spaces; groups of vascular bundles scattered in the parenchyma zone; lower epidermis showing stomata.
•  Loss on Drying 731
Analysis:  Dry 2.0 g of Banaba Leaf, finely powdered, at 105 for 2 h.
Acceptance criteria:  NMT 10%
•  Articles of Botanical Origin, Total Ash 561
Analysis:  2.0 g of Banaba Leaf, finely powdered
Acceptance criteria:  NMT 7.0%
•  Articles of Botanical Origin, Acid-Insoluble Ash 561
Analysis:  4.0 g of Banaba Leaf, finely powdered
Acceptance criteria:  NMT 2%
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 part(s) of the plant contained in the article.
•  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
(301) 816-8339		 (GCM2010) General Chapters - Microbiology
Reference Standards RS Technical Services
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Pharmacopeial Forum: Volume No. 39(6)