Forskohlii consists of the dried roots of Plectranthus barbatus Andrews, also known as Coleus barbatus (Andrews) Benth. and Coleus forskohlii Briq. (Fam. Lamiaceae). It contains NLT 0.4% of forskolin, calculated on the dried basis.
• A. Thin-Layer Chromatographic Identification Test 201
Standard solution A: 50 µg/mL of USP Forskolin RS in acetonitrile. Sonicate for about 10 min.
Standard solution B: 5 mg/mL of USP Powdered Forskohlii Extract RS in acetonitrile. Sonicate for about 15 min, centrifuge, and use the supernatant.
Sample stock solution: Use the Sample solution, prepared as directed in the test for Content of Forskolin.
Sample solution: Dilute 10 mL of the Sample stock solution with acetonitrile to 25 mL.
Adsorbent: Chromatographic silica gel with an average particle size of 1015 µm (TLC plates)
Application volume: 10 µL as 4-mm bands
Developing solvent system: Toluene and ethyl acetate (85:15)
Spray reagent: 5% vanillin in glacial acetic acid and 10% sulfuric acid in water (1:1)
Samples: Standard solution A, Standard solution B, and Sample solution
Apply the Samples as bands to a suitable thin-layer chromatographic plate (see Chromatography 621). Use a saturated chamber. Develop the chromatograms until the solvent front has moved up about 90% of the plate. Remove the plate from the chamber, dry, spray with the Spray reagent, heat for 510 min at 105, and examine under visible light.
Acceptance criteria: The chromatogram of the Sample solution exhibits the following: a violet zone due to forskolin at an RF value of approximately 0.3, corresponding in color and RF to that in the chromatogram of Standard solution A; and a minor violet zone, a pink zone, and a brick red zone at RF values of approximately 0.1, 0.62, and 0.69, due to isoforskolin, 1,9-dideoxyforskolin and crocetindialdehyde, respectively. Zones detected in the Sample solution chromatogram correspond in position and color to zones in the chromatogram of Standard solution B. Other minor zones may be observed in the Sample solution and Standard solution B chromatograms.
• B. The chromatogram of the Sample solution obtained in the test for Content of Forskolin shows a main peak at a retention time corresponding to that of forskolin in the chromatogram of Standard solution A. Identify other diterpene peaks in the Sample solution chromatogram by comparison with the chromatogram of Standard solution B and the reference chromatogram provided with the lot of USP Powdered Forskohlii Extract RS. The Sample solution chromatogram shows an additional peak corresponding to isoforskolin.
• Content of Forskolin
Solution A: Use filtered and degassed acetonitrile.
Solution B: Use filtered and degassed water.
Standard solution A: Sonicate a quantity of USP Forskolin RS in acetonitrile to obtain a solution having a known concentration of about 1.0 mg/mL.
Standard solution B: 5 mg/mL of USP Powdered Forskohlii Extract RS in acetonitrile, sonicate for about 15 min, centrifuge, and use the supernatant. Before injection, pass through a membrane filter of 0.45-µm or finer pore size.
Sample solution: Transfer about 3.0 g of Forskohlii, finely powdered, to a 100-mL round-bottom flask fitted with a reflux condenser. Add 50 mL of acetonitrile, reflux on a water bath for 20 min, cool to room temperature, and decant the supernatant. Repeat until the last extract is colorless. Combine the extracts, filter, concentrate under vacuum, and adjust the volume with acetonitrile to 100 mL. Before injection, pass through a membrane filter of 0.45-µm or finer pore size, discarding the first 5 mL of the filtrate.
System suitability solution: Standard solution A and 0.01% toluene in acetonitrile (1:1)
Mobile phase: See the gradient table below.
Detector: UV 220 nm
Column: 4.6-mm × 25-cm; 5-µm, 100
Column temperature: 30 ± 2
Flow rate: 1.8 mL/min
Injection size: 20 µL
Samples: Standard solution A, Standard solution B, and System suitability solution
[NoteThe approximate relative retention times for isoforskolin and forskolin are 0.51 and 1.00, respectively. ]
Suitability requirements: The chromatogram from Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Forskohlii Extract RS being used.
Resolution: NLT 1.5 between the forskolin and toluene peaks, System suitability solution
Relative standard deviation: NMT 2% determined from the forskolin peak for replicate injections, Standard solution A
Samples: Standard solution A, Standard solution B, and Sample solution
Using the chromatogram of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Powdered Forskohlii Extract RS, identify the retention times of the peaks corresponding to isoforskolin and forskolin.
Calculate the percentage of forskolin in the portion of Forskohlii taken:
Result = (rU/rS) × (CS/CU) × 100
Acceptance criteria: NLT 0.4% on the dried basis
• Heavy Metals, Method III 231: NMT 20 ppm
• Procedure 1: Articles of Botanical Origin, Foreign Organic Matters 561: NMT 2.0%
• Procedure 2: Articles of Botanical Origin, General Method for Pesticide Residues Analysis 561: Meets the requirements
• Botanic Characteristics
Macroscopic: Fresh root, pale pinkish yellow, cylindrical to subcylindrical, with tapering ends, 512 in length, 12 cm in diameter; surface rough, shows lateral rootlets or scars of rootlets and transversely running lenticels. Pharmacopeial article, dark brown; surface rough, irregularly cylindrical, longitudinally wrinkled, showing irregular grooves and prominent ridges; fracture short; cut surface is yellowish white; characteristic and pleasant aromatic odor, and slightly bitter to pungent taste.
Transverse section of roots: Irregular circular in outline; showing narrow cork, 1015 rows of tangentially elongated radially arranged cork cells; cortex composed of 1015 rows of thin-wall parenchyma cells showing sclereids and crystals of calcium oxalate; vascular cambium in the form of a continuous ring; xylem showing narrow rays of vessels, few lignified fibers are present in older roots, 38 cell-wide medullary rays, and parenchyma showing few sclereids, oleoresin canals and simple starch grains; pith composed of parenchyma cells in young roots and is replaced by compactly arranged vessels, fibers, and tracheids in mature roots.
• Loss on Drying 731: Dry 1.0 g of finely powdered Forskohlii at 105 for 3 h: it loses NMT 12.0% of its weight.
• Articles of Botanical Origin, Total Ash 561: NMT 6%, determined on 1.0 g of finely powdered Forskohlii
• 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 does not exceed 103 cfu/g.
• Microbiological Procedures for Absence of Specified Microorganisms 2022: Meets the requirements of the tests for absence of Salmonella species and Escherichia coli
• Articles of Botanical Origin, Test for Aflatoxins 561: Meets the 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 contained in the article.
• USP Reference Standards 11
USP Powdered Forskohlii Extract RS
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USP35NF30 Page 1298Pharmacopeial Forum: Volume No. 36(3) Page 695