DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR QUANTITATIVE ESTIMATION OF alpha-MANGOSTIN IN THE RIND EXTRACT AND FRACTIONS OF GARCINIAMANGOSTANA L. AND THEIR CYTOTOXIC ACTIVITY ON T47D BREAST CANCER CELL LINE
Objective: To develop a cheap, accurate, precise, linear and rapid Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method and validate as per ICH guidelines for the quantitative estimation of Î±-mangostin in the rind extract and fraction of mangosteen (Garcinia mangostana L.) as well as to determine their cytotoxic activity against T47D breast cancer cell line.
Methods: The optimized method uses a reverse phase column, Shimadzu Â®Shimp-pack VP â€“ ODS (4.6 x 250 mm; 5Âµ), a mobile phase of 0.1 % v/v H3PO4 in water: acetonitrile (15:85), flow rate of 1 ml/min and a detection wavelength of 243,2 nm using a UV detector. The cytotoxic activity against breast cancer cell line T47D was determined as percentage of cell viability by using MTT (Microculture Tetrazolium Assay) colorimetric assay and IC50(concentration that inhibits cell growth by 50%) were calculated.
Results: The developed method resulted in Î±-mangostin eluting at 8.87 min. Î±-Mangostin exhibited linearity in the range 0.5 â€“ 30 Î¼g mL-1, and precise (intra-day variation â‰¤ 0.10 %, inter-day variation â‰¤ 2.28 %). The average percentage mean recovery was 94.41-102.01 %, during accuracy studies. The limit of detection (LOD) and limit of quantification (LOQ) was found to be 0.2807 and 0.9357 Î¼g mL-1 respectively. The concentration of Î±-mangostin in the 70% ethanol extract, n-hexane fraction, ethyl acetate fraction and n-butanol fraction were 50.73; 11.12; 98.66; 2.29 % w/w, respectively. This extract and fractions had IC50 of 1.375; 5.879; 0.463; and 51.839 Î¼g mL-1 against T47D cell line respectively.
Conclusion: A cheap, accurate, precise, linear and rapid RP-HPLC method was developed and validated for the quantitative estimation of a-mangostin in the rind extract and fraction as per ICH guidelines and hence it can be used for the quality control of crude extract and herbal formulation. The strongest cytotoxic activity was showed by an ethyl acetate fraction of fruit rind of Garcinia mangostana L, against T47D cell line. These results were in agreement with the concentration of Î±-mangostin.
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