CHEMOMETRICS ASSISTED VALIDATED HPLC METHOD DETECTING SEASONAL VARIANCES OF SECONDARY METABOLITES OF Camellia sinensis CONSIDERING THE ABIOTIC STRESSES

Abstract

Climatic changes have great impact on the crops and agro-eco systems and such changes influences the concentration of secondary metabolites. IIT Kharagpur, India is a non-traditional tea growing zone where Tocklai Vegetative 25 variety was used as the research material. This study reports the development of a chemometrics assisted HPLC method validated as per ICH guidelines to explore the effect of seasonal variations in polyphenolics viz. catechins and methyl xanthenes like caffeine in fresh tea leaves and processed CTC black tea prepared from them. Further study was done on the variances amongst the concentration of secondary metabolites and abiotic stress factors. Good resolutions of secondary metabolites were obtained using 92% of 0.2% acetic acid and 8% of acetonitrile as the mobile phase, with a flow rate of 1 mL/ min, injection volume of 20 µl,  PDA detector was set at 200-600 nm and chromatograms were recorded at 274 nm. Results of quantitative HPLC analysis have clearly shown that highest yield of catechins and caffeine were observed in fresh tea leaves plucked during spring (24.3ºC temperature and average rainfall of 34 mm) and also the processed black tea made from it, followed by tea leaves plucked during monsoon (28.8 ºC temperature and 282 mm rainfall) and processed black tea prepared from it. The lowest concentrations of secondary metabolites were found in leaves plucked during autumn (26.2 °C temperature and 132 mm rainfall) and the processed tea prepared from it. The developed quantitative HPLC method showed an inter day precision of 0.3, intraday precision of 0.2, repeatability value of 0.31, ruggedness value of 0.33 and robustness value of 0.2. Considering temperature and rainfall as abiotic stress factors, highest total polyphenolic content was obtained during spring and lowest in autumn. From our experimental findings, the fresh tea leaves of spring season and also the processed black tea prepared from it showed higher yield of catechins