Quantitative determination of four constituents of Tinospora sps. by a reversed-phase HPLC-UV-DAD method.: Broad-based studies revealing variation in content of four secondary metabolites in the plant from different eco-geographical regions of India

This paper describes the separation and quantitation of important markers, such as 20β-hydroxyecdysone, tinosporaside, cordioside, and columbin, present in three species of Tinospora viz, T. cordifolia, T. malabrica, and T. crispa. A reverse-phase (RP) high-performance liquid chromatography (HPLC)-UV-diode array detection (DAD) method employing gradient elution is thus developed. The marker compounds isolated from 70% ethanolic extract of T. cordfolia by repeated column chromatography are identified on the basis of 1H NMR, 13C NMR, and mass spectral data. The compounds are separated on a RP (RP-18, 5 μm, 250 x 4.6-mm i.d.) column using water-acetonitrile gradient and are detected by the HPLC-UV-DAD method. The calibration curves that result from marker compounds in the concentration range of 100-2000 ng on column exhibit a good correlation (r2 ≥ 0.99978). The method is successfully applied to separate and study the content of four marker compounds in 40 different accessions of three Tinospora species collected from different regions of India. The studies reveal that the maximum amount of the marker compounds is present in Tinospora cordifoliaspecies, especially from accessions collected from higher altitudes of the Jammu province (North India).