Abstract
Withaferin-A (WA) is a major bioactive compound in the roots of Withania somnifera. It is reported to possess inhibitory effects against breast cancer, liver cancer, melanoma cancer and prostate cancer cells. We developed an elicitation based on the hairy root culture (HRC) protocol for the enhancement of the WA production. Agrobacterium rhizogenes strain R1000 was used for the establishment of hairy root culture. PCR analysis confirmed the integration of the root loci C (rol C) gene in transformed roots. Elicitation of the hairy roots with natural polysaccharides of sodium alginate (SA), k-carrageenan (kC) and chitosan (CH) at various concentrations in the most cases resulted in a higher amount of WA compound. Among the various elicitors, 100 mg L−1 CH resulted 4.03-fold increase in WA production than control. This study is the first report in W. somnifera hairy roots, where the natural polysaccharides are used as elicitors for the large-scale production of WA.
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Abbreviations
- CH:
-
Chitosan
- HRC:
-
Hairy root culture
- kC-k:
-
Carrgeenan
- rol C :
-
Root loci C gene
- SA:
-
Sodium alginate
- WA:
-
Withaferin-A
References
Aalinkeel R, Hu Z, Nair BB, Sykes DE, Reynolds JL, Mahajan SD, Schwarts SA (2010) Genomic analysis highlights the role of the JAK-STAT signaling in the anti-proliferative effects of dietary flavonoid-‘Ashwagandha’ in prostate cancer cells. Evidence-Based Compl Altern Med 7(2):177–187. https://doi.org/10.1093/ecam/nem184
Aftab T, Khan MMA, Idrees M, Naeem M, Moinudeen HN, Varshney L (2011) Enhancing the growth, photosynthetic capacity and artemisinin content in Artemisia annua L. by irradiated sodium alginate. Radiat Phys Chem 80(7):833–836. https://doi.org/10.1016/j.radphyschem.2011.03.004
Alfaifi MY, Saleh KA, El-Boushnak MA, Elbehairi SE, Alshehri MA, Shati AA (2016) Antiproliferative activity of the methanolic extract of Withania somnifera leaves from Faifa mountains, Southwest Saudi Arabia, against several human cancer cell lines. Asian Pac J Cancer Prev 17(5):2723–2726. https://doi.org/10.7314/APJCP.2016.17.5.2723
Aslam A, Mohamed Rafi K, Kathiravan K, Shajahan A (2010) Class-based stratification matrix for physical leaf traits in phonetic relations of Withania somnifera (L.) Dunal accessions. Plant Syst Evol 28(1):99–111. https://doi.org/10.1007/s00606-010-0316-z
Chaurasiya ND, Sangwan NS, Sabir F, Misra L, Sangwan RS (2012) Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L. (Dunal). Plant Cell Rep. https://doi.org/10.1007/s00299-012-1302-4
Chen HL, Seguin P, Archambault A, Constan L, Jabaji S (2009) Gene expression and isoflavone concentration in soybean sprouts treated with chitosan. Crop Sci 49(1):224–236. https://doi.org/10.2135/cropsci2007.09.0536
Estruch JJ, Chriqui D, Grossmann K, Schell J, Spena A (1991) The plant oncogene rol-C is responsible for the release of cytokinins from glucoside conjugates. EMBO J 10(10):2889–2895. https://doi.org/10.1002/j.1460-2075.1991.tb07838.x
Ge X, Wu J (2005) Induction and potentiation of diterpenoid tanshinone accumulation in Salvia miltiorrhiza hairy roots by β-aminobutyric acid. Appl Microbiol Biotechnol 68(2):183–188. https://doi.org/10.1007/s00253-004-1873-2
Georgiev MI, Ludwig-Muller J, Alipieva K, Lippert A (2011) Sonication-assisted Agrobacterium rhizogenes-mediated transformation of Verbascum xanthophoeniceum Griseb. for bioactive metabolite accumulation. Plant Cell Rep 30(5):859–866. https://doi.org/10.1007/s00299-010-0981-y
Godoy-Hernandez G, Loyola-Vargas VM (1991) Effect of fungal homogenates, enzyme inhibitors and osmotic stress on alkaloid content of Catharanthus roseus cell suspension cultures. Plant Cell Rep 10(11):537–540. https://doi.org/10.1007/BF00232506
Halder B, Singh S, Thakur SS (2015) Withania somnifera root extract has potent cytotoxic effect against human malignant melanoma cells. PLoS One 10:e0137498. https://doi.org/10.1371/journal.pone.0137498
Hashmi N, Khan MMA, Moinuddin IM, Khan ZK, Ali A, Varshney L (2012) Depolymerised carrageenan ameliorates growth, physiological attributes, essential oil yield and active constituents of Foeniculum vulgare Mill. Carbohydr Polym 90(1):407–412. https://doi.org/10.1016/j.carbpol.2012.05.058
Hu ZB, Du M (2006) Hairy root and its application in plant genetic engineering. J Integr Plant Biol 48(2):121–127. https://doi.org/10.1111/j.1744-7909.2006.00121.x
Jeong GT, Park DK (2005) Enhancement of growth and secondary metabolite biosynthesis: effect of elicitors derived from plants and insects. Biotechnol Bioprocess Eng 10(1):73–77. https://doi.org/10.1007/BF02931186
Katiyar D, Hemantaranjan A, Singh B (2015) Chitosan as a promising natural compound to enhance potential physiological responses in plant: a review. Indian J Plant Physiol 20(1):1–9. https://doi.org/10.1007/s40502-015-0139-6
Kim OT, Bang KH, Shin YS, Lee MJ, Jung SJ, Hyun DY, Kim YC, Senong NS, Cha SW, Hwang B (2007) Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) urban elicited by methyl jasmonate. Plant Cell Rep 26(11):1941–1949. https://doi.org/10.1007/s00299-007-0400-1
Komaraiah P, Naga Amrutha R, KaviKishor PB, Ramakrishna SV (2002) Elicitor enhanced production of plumbagin in suspension cultures of Plumbago rosea L. Enzym Microb Technol 31(5):634–639. https://doi.org/10.1016/S0141-0229(02)00159-x
Kupper FC, Kloareg B, Guern J, Potin P (2001) Oligoguluronates elicit an oxidative burst in the brown algal kelp Laminaria digitata. Plant Physiol 125(1):278–291. https://doi.org/10.1104/pp.125.1.278
Luczkiewicz M, Kokotkiewicz A (2012) Elicitation and permeabilisation affect the accumulation and storage profile of phytoestrogens in high productive suspension cultures of Genista tinctoria. Acta Physiol Plant 34(1):1–16. https://doi.org/10.1007/s11738-011-0799-4
Maliyakkal N, Udupa N, Pai K, Rangarajan A (2013) Cytotoxic and apoptotic activities of extracts of Withania somnifera and Tinospora cordidolia in human breast cancer cells. Int J Appo Res Nat Prod 6(4):1–10
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Naeem M, Idrees M, Aftab T, Khan MMA, Moinuddin VL (2012) Depolymerised carrageenan enhances physiological activities and menthol production in Mentha arvensis L. Carbohydr Polym 87(2):1211–1218. https://doi.org/10.1016/j.carbpol.2011.09.002
Oh JH, Lee TJ, Kim SH, Choi YH, Lee SH, Lee JM, Kim YH, Park JW, Kwon TK (2008) Induction of apoptosis by withaferin-A in human leukemia U937 cells through down-regulation of Akt phosphorylation. Apoptosis 13(12):1494–1504. https://doi.org/10.1007/s10495-008-0273-y
Potin P, Bouarab K, Kupper F, Kloareg B (1999) Oligosaccharide recognition signals and defence reactions in marine plant-microbe interactions. Curr Opin Microbiol 2(3):276–283. https://doi.org/10.1016/S1369-5274(99)80048-4
Potrykus I (1990) Gene transfer to plants: assessment and perspectives. Physiol Plant 79(1):125–134. https://doi.org/10.1111/j.1399-3054.1990.tb05874.x
Ramawat KG, Goyal S (2008) The Indian herbal drugs scenario in glonl perspectives, pp. 325–347. In: Ramawat KG, Merillon JM (eds.), Bioactive molecules and medicinal plants. https://doi.org/10.1007/978-3-540-74603-4_18
Roy RV, Suman S, Das TP, Luevano JE, Damodaran C (2013) Withaferin-A, a steroidal lactone from Withania somnifera, induces mitotic catastrophe and growth arrest in prostate cancer cells. J Nat Prod 76(10):1909–1915. https://doi.org/10.1021/np400441f
Sangwan RS, Chaurasiya ND, Lal P, Misra L, Uniyal GC, Tuli R, Sangwan NS (2007) Withanolide A biogeneration in in vitro shoot culture of Ashwagandha (Withania somnifera), a main medicinal plant in Ayurveda. Chem Pharm Bull 55:1371–1375. https://doi.org/10.1248/cpb.55.1371
Saravanakumar A, Aslam A, Shajahan A (2012) Development and optimization of hairy root culture systems in Withania somnifera (L.) Dunal for withaferin-A production. Afr J Biotechnol 11:16412–16420. https://doi.org/10.5897/AJB11.3867
Saucedo S, Contreras RA, Moenne A (2015) Oligo-carrageenan kappa increases C, N and S assimilation, auxin and gibberellins contents, and growth in Pinus radiata trees. J For Res 26(3):635–640. https://doi.org/10.1007/s11676-015-0061-9
Shilpha J, Satish L, Kavikkuil M, Largia MJV, Ramesh M (2015) Methyl jasmonate elicits the solasodine production and anti-oxidant activity in hairy root cultures of Solanum trilobatum L. Ind Crop Prod 71:54–64. https://doi.org/10.1016/j.indcrop.2015.03.083
Sivanandhan G, Arun M, Mayavan S, Rajesh M, Mariashibu TS, Manickavasagam M, Selvaraj N, Ganapathy A (2012) Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal. Ind Crop Prod 37:124–129. https://doi.org/10.1016/j.indcrop.2011.11.022
Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Arjunan A, Muthuselvam M, Manickavasagam M, Selvaraj N, Ganapathi A (2013) Increased production of withanolide A, withanone, and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell Tissue Organ Cult 114(1):121–129. https://doi.org/10.1007/s11240-013-0297-z
Sivanandhan G, Selvaraj N, Ganapathi A, Manickavasagam M (2014) An efficient hairy root culture system for Withania somnifera. Afr J Biotechnol 13(43):4141–4147. https://doi.org/10.5897/AJB2014.14128
Thilip C, Soundar Raju C, Varutharaju K, Aslam A, Shajahan A (2015) Improved Agrobacterium rhizogenes-mediated hairy root culture system of Withania somnifera (L.) Dunal using sonication and heat treatment. 3 Biotech 5(6):949–956. https://doi.org/10.1007/s13205-015-0297-2
Xu A, Zhan JC, Huang WD (2015) Oligochitosan and sodium alginate enhance stilbene production and induce defense responses in Vitis vinifera cell suspension culture. Acta Physiol Plant 37(8):144. https://doi.org/10.1007/s11738-015-1900-1
Zhao J, Zhu WH, Hu Q (2001) Selection of fungal elicitors to increase indole alkaloid accumulation in Catharanthus roseus suspension cell culture. Enzym Microb Technol 28(7–8):666–672. https://doi.org/10.1016/S0141-0229(01)00309-X
Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23(4):283–333. https://doi.org/10.1016/j.biotechadv.2005.01.003
Acknowledgments
Dr. A. Shajahan is thankful to the University Grants Commissions (UGC), Govt. of India, New Delhi for the financial support through UGC Major Research Project (F. No. 42-946/2013-SR). The authors also thankful to DST, Govt. of India for providing facilities through DST-FIST program.
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Thilip, C., Mehaboob, V.M., Varutharaju, K. et al. Elicitation of withaferin-A in hairy root culture of Withania somnifera (L.) Dunal using natural polysaccharides. Biologia 74, 961–968 (2019). https://doi.org/10.2478/s11756-019-00236-9
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DOI: https://doi.org/10.2478/s11756-019-00236-9