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

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Abstract

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.

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Abbreviations

µg/g f.w.:

Microgram per gram fresh weight

ANOVA:

Analysis of variance

DMRT:

Duncan’s multiple range test

FT-IR:

Fourier transform infra red

HPLC:

High performance liquid chromatography

IBA:

Indole-3-butyric acid

IPTG:

Isopropyl-β-thio galactopyranoside

LB:

Luria Bertani

MEGA:

Molecular evolutionary genetics analysis

MEP:

2-C-methyl-d-erythritol 4-phosphate

MVA:

Mevalonic acid

MS:

Murashige and Skoog

NAA:

α-Naphthalene acetic acid

PDA:

Photo diode array

PDGF:

Platelet derived growth factor

SPSS:

Statistical package for social sciences

TCA:

Tri carboxylic acid

X-gal:

5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside

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Acknowledgments

V. Agrawal is grateful to the University Grants Commission, New Delhi for providing financial assistance in the form of a UGC’s Major Research Project (F. 41-499/2012) “In vitro evaluation, isolation and up regulation of anticancerous bioactive compounds from C. angustifolia (Senna) through elicitors and their bioefficacy against human cancer cell lines”. The authors extend thanks to the University of Delhi, Delhi for sanctioning funds for Research and Development. SKC is indebted to Indian Council of Medical Research, New Delhi for the award of SRF and HK is grateful to the University Grants Commission, New Delhi for the award of UGC-JRF & SRF.

Author contributions

This paper pertains to elicitation of sennoside A and B in Cassia angustifolia callus cultures exposed to various elicitors and cloning and characterization of isochorismate synthase gene involved in sennoside biosynthesis. All the authors have equal contribution for the research paper.

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  1. Department of Botany, University of Delhi, Delhi, 110007, India

    Siva K. Chetri, Himanshi Kapoor & Veena Agrawal

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  1. Siva K. Chetri
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  2. Himanshi Kapoor
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  3. Veena Agrawal
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Correspondence to Veena Agrawal.

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Chetri, S.K., Kapoor, H. & Agrawal, V. Marked enhancement of sennoside bioactive compounds through precursor feeding in Cassia angustifolia Vahl and cloning of isochorismate synthase gene involved in its biosynthesis. Plant Cell Tiss Organ Cult 124, 431–446 (2016). https://doi.org/10.1007/s11240-015-0905-1

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