Published:Journal of Chromatographic Science,
ISSN 0021-9665 Volume 44,
Number 8, September 2006, pp. 504-509
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
S.M. Ahmed, L.R. Manhas, V. Verma, and R.K. Khajuria
Regional Research Laboratory, Canal Road, Jammu-180001, India
This paper describes the separation and quantitation
of important markers, such as 20b-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 ¥ 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 cordifolia species, especially
from accessions collected from higher altitudes of the Jammu province
(North India).
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