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Standards and chemicals:

HPLC grade methanol, other chemicals
(acetone, ethanol, benzene, petroleum ether) and Authentic polyphenol standards
were purchased from Himedia Laboratories (Mumbai, India)

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Sample preparation and HPLC Analysis:

Powdered leaf was used for HPLC analysis. The
samples were then heated under reflux for 1 hour with 6 ml of 25% hydrochloric
acid and 20 ml MeOH, the hydrolysate was diluted with methanol to 100ml and
filtered. (PTFE Syringe filter, Whatman, UK). 1 ml of this solution was
injected for HPLC analysis. Analysis was performed after three separate
extraction of each sample and each extract was diluted and injected in
triplicate. Polyphenols in the samples was identified by comparison of their
retention times with the standard compounds.

Chromatographic equipment and condition:

The chromatographic analyses were performed
on a 250 mm × 4.6 mm i.d., C18 (ODS), Shimadzu, Japan with 0.5% aqueous
solution of Orthophosphoric acid and Methanol (HPLC Grade) as mobile phase at a
flow rate of 1 mL min-1. The HPLC equipment comprised Hewlett-Packard (HP) 1050
ChemStation Software, an HP model35900 interface unit, an HP 9000 Series 300
computer, and an HP DeskJet 500 Printer. A Waters 486 tunable absorbance
detector was operated at 254 nm; detector sensitivity was 0.05 AUFS and the
column oven temperature was 30°C. Determinations were performed after three
separate extractions of each sample, and each extract was injected in
triplicate.

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