Expression Analysis of Genes Responsible for Rosmarinic acid Biosynthesis and HPLC Quantification Method Development and Validation of Rosmarinic acid from Isodon rugosis

Abstract

Abstract Natural products from plants, either as pure compounds or as standardized extracts, offer unlimited prospects for new pesticide discovery. In screening programs, because of increased chemical diversity demand, in search of pesticides from natural products, interest mainly in harmless plants has developed all over the world. Botanicals comprise of several types of bioactive compounds. In our previous publications, bioactive pesticidal compound; rosmarinic acid (RA) was isolated from the plant, Isodon rugosus and was identified by using various analytical techniques. In this study two key genes, hydroxyphenylpyruvate reductase (HPPR) and rosmarinic acid synthase (RAS), known to involve in biosynthesis of RA were targeted to clone from Isodon rugosus. Only one of these genes, HPPR was successfully cloned in I. rugosus and its cDNA was fully sequenced through RACE (Rapid Amplified cDNA ends) PCR, which consequently will open the way to explore all other genes responsible for biosynthesis of rosmarinic acid. The expression of HPPR was analyzed in different parts of plant and it was found that RA was expressed in all parts of the plant. Further, RA quantification was performed on RP-HPLC using C18 column, giving a maximum absorbance at 310 nm in isocratic conditions. The methodology was found selective and robust to quantify 1.60+0.14gm/kg RA in I. rugosus with sensitivity of LOD 1.32 µg/ml, and LOQ 4.41 µg/ml. The molecular knowledge regarding biosynthetic pathway and significant quantity of RA in this plant will help in biotechnological production of RA and to produce insect resistant plants through genetic engineering approaches.