Validated RP-HPLC-UV method for the determination of betulin in Asteracantha longifolia (L.) Nees. extract
Keywords:
Asteracantha longifolia, betulin, RP-HPLCAbstract
Asteracantha longifolia (L.) Nees. (Acanthaceae) is a well known medicinal plant of Indian traditional medicines. The aim of this work was to develop a validated reversed phase-high performance liquid chromatography (RP-HPLC) method for the quantitative determination of betulin in A. longifolia extract. The analysis was performed by RP-HPLC on Luna C18 (2) 100 Å, 250 x 4.6 mm column under isocratic elution of acetonitrile and water (80:20, v/v) with a flow rate of 1.0 ml/min and the total run time was 20 min. The column temperature was adjusted at 25°C and the detection wavelength was set at 210 nm. The method was validated for suitability, specificity, accuracy, precision, limits of detection and quantification (LOD and LOQ), robustness and ruggedness. The betulin content in A. longifolia extract was found to be 15.96 ± 0.34% (w/w). The calibration curve was linear over a concentration range of 10-125 μg/ml (r2 = 0.997) and the recovery range was 98.29-99.59%. The LOD and LOQ were 1.11 and 7.35 μg/ml, respectively. The intra- and inter-day assay precisions were satisfactory and the relative standard deviations were found to be always less than 2%. The developed method was found to be simple, sensitive, accurate, robust and rugged for the quantification of betulin. This validated method can be useful for the routine quality control analysis of betulin content in A. longifolia extract and its formulations.
References
Chauhan NS, Dixit VK. Asteracantha longifolia (L.) Nees, Acanthaceae: chemistry, traditional, medicinal uses and its pharmacological activities - a review. Braz J Pharmacog. 2010;20(5):812-817.
Kshirsagar AD, Ingale KG, Vyawahare NS, Thorve VS. Hygrophila spinosa: A comprehensive review. Pharmacogn Rev. 2010; 4(8): 167-171.
Khdair A, Mohammad MK, Tawaha K, Al-Hamarsheh E, AlKhatib HS, Al-khalidi B, Bustanji Y, Najjar S, Hudaib M. A validated RP-HPLC-PAD method for the determination of hederacoside C in Ivy-Thyme cough syrup. Int J Anal Chem. 2010; 2010:1-5.
Li Y, He K, Huang Y, Zheng D, Gao C, Cui L, Jin YH. Betulin induces mitochondrial cytochrome c release associated apoptosis in human cancer cells. Mol Carcinog. 2010;49(7):630-640.
Pavlova NI, Savinova OV, Nikolaeva SN, Boreko EI, Flekhter OB. Antiviral activity of betulin, betulinic and betulonic acids against some enveloped and non-enveloped viruses. Fitoterapia. 2003;74(5):489-492.
Lin YC, Cheng HY, Huang TH, Huang HW, Lee YH, Peng WH. Analgesic and anti-inflammatory activities of Torenia concolor Lindley var. formosana Yamazaki and betulin in mice. Am J Chin Med. 2009;37(1):97-111.
Badami S, Gupta MK, Ramaswamy S, Rai SR, Nanjaian M, Bendell DJ, Subban R, Bhojaraj S. Determination of betulin in Grewia tiliaefolia by HPTLC. J Sep Sci. 2004;27(1-2):129-131.
Zhao G, Yan W, Cao D. Simultaneous determination of betulin and betulinic acid in white birch bark using RP-HPLC. J Pharm Biomed Anal. 2007;43(3):959-962.
Dehelean CA, Peev C, Cinta-Pinzaru S, Şoica C, Kaicsa A. FT-IR, FT-Raman and TLC densitometry analysis of some plane tree extracts concerning their content in betulinic acid and betulin. Revista de Chimie, 2006;57(11):1105-1107.
ICH-Q2 (R1). Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization: 2005, Geneva.
