Marked enhancement of sennoside bioactive compounds through precursor feeding in Cassia angustifolia Vahl and cloning of isochorismate synthase gene involved in its biosynthesis

Cassia angustifolia Vahl, a chief source of anthraquinone glycosides (sennosides), extensively employed as a laxative is also reported to possess significant anticancerous activity against various cancer cell lines. HPLC analysis of different in vivo plant parts viz., leaves, nodes, roots and seeds revealed that the maximum content of both sennoside A (3816.10 µg/g fresh wt.) and sennoside B (646.74 µg/g fresh wt.) occur in leaf. Elicitation in sennoside content in leaf callus therefore, was achieved employing organic elicitors (glycine, myo-inositol, glutamine, proline, yeast extract, casein hydrolysate and sucrose) as well as precursors (α-keto glutaric acid and pyruvic acid) of anthraquinone pathway. Though there was enhancement at all levels of stress, the optimum elicitation in sennoside A and B was seen at 0.1 % pyruvic acid, their respective percentages being 16 and 32 %. Overall improvement in sennoside A and B content was seen in the order: pyruvic acid > α-keto-glutaric acid > sucrose > yeast extract > glycine > myo-inositol > proline > casein hydrolysate > glutamine. Most importantly, isochorismate synthase (ics), the key enzyme gene involved in the anthraquinone biosynthetic pathway has also been cloned from leaf and sequenced which comprised of 1377 bp. Neighbor joining tree generated through MEGA6 analysis of the nucleotide sequences revealed varied degree of homology with the ics gene sequences of Morus notabilis, Medicago truncatula, Solanum lycopersicum, Cicer arietinum, Glycine max, etc. This is the first report of elicitation of sennoside A and B from leaf callus cultures and cloning of ics gene in C. angustifolia.