EXPRESSION PROFILING OF SELECTED MICRO RNAs COUPLED WITH MOLECULAR AND BIOCHEMICAL ANALYSES OF ASIATICOSIDES IN CENTELLA ASIATICA (L.)URB IN VITRO
Expression profiling of microRNAs in C. asiatica
Objective: Centella asiatica (L.) Urb from Umbelliferae is a potential source of secondary metabolites having immense medicinal value. Asiaticoside is the major therapeutic compound. In the present study, Identification of a possible relationship between concentration/transcript level expression of asiaticoside and concentrations of growth hormones at different growth stages was observed. The current study includes molecular and biochemical evaluation of stress generated in C. asiatica at different time intervals in vitro.
Methods: The enhancement in auxin, cytokinin and final asiaticoside content were determined using immunoassay kits for auxin, cytokinin and HPLC analysis respectively. Transcript level expression at different growth phases was carried out using real-time RT-PCR. For isolation of stress-related miRNAs, reverse transcription of total RNA using miScript II RT Kit PCR System was carried out as per instructions. The differential expression of five selected miRNAs was done by Real-Time RT-PCR. The analysis of stress in vitro was done by quantification of Hydrogen Peroxide (H2O2), total phenolics and total antioxidants by H2O2 assay kit, total antioxidant assay kit and Folin Ciocalteau reagent respectively. The final asiaticoside content was determined by HPLC.
Results: Differential expression of key genes involved in asiaticoside pathway showed significantly higher transcript expression, which is in correlation with the final asiaticoside content. The enhanced expression of miRNAs and the analysis of H2O2, total antioxidant capacity and total phenolics are suggestive of generation of oxidative stress under controlled conditions.
Conclusion: The present study shows a direct correlation between oxidative stress and transcript/phytochemical estimation of asiaticoside content under in vitro conditions.
2. Tuteja N, Gill SS, Trivedi PK, Asif MH, Nath P. Plant growth regulators and their role in stress tolerance. Plant Stress. Globan Science Books; 2010.
3. Shriram V, Kumar V, Devarumath RM, Khare TS, Wani SH. MicroRNAs as potential targets for abiotic stress tolerance in plants. Front Plant Sci 2016;7:817.
4. Ambros V. The functions of animal microRNAs. Nature 2004;431:350–5.
5. Bushati N, Cohen SM. MicroRNA functions. Annu Rev Cell Dev Biol 2007;23:175–205.
6. Anjali N, Nadiya F, Thomas J, Sabu KK. Discovery of MicroRNAs in cardamom (Elettaria cardamomum Maton) under drought stress. Hindawi Dataset Papers Sci 2017. https://doi.org/ 10.1155/2017/9507485.
7. Zheng CJ, Qin LP. Chemical components of Centella asiatica and their bioactivities. Chin J Integr Med 2007;5:348–51.
8. Tao Y, Li JM, Li YC, Pan Y, Qun Xu, Ling-Dong K. Antidepressant-like behavioral and neurochemical effects of the citrus-associated chemical apigenin. J Life Sci 2008;82:741-51.
9. Subathra M, Shila S, Devi MA, Panneerselvam C. Emerging role of Centella asiatica in improving age-related neurological antioxidant status. Exp Gerontol 2005;40:707–15.
10. Rao MKG, Rao MS, Rao G. Centella asiatica (L.) leaf extract treatment during the growth spurt period enhances hippocampal CA3 neuronal dendritic arborization in rats. eCAM 2006;3:349–57.
11. James JT, Dubery IA. Pentacyclic triterpenoids from the medicinal herb Centella asiatica (L) Urban. Molecules 2009;14:3922-41.
12. Inamdar PK, Yeole RD, Ghogare AB, de Souza NJ. Determination of biologically active constituents in Centella asiatica. J Chromatogr 1996;746:127-30.
13. Jisha S, Anith KN, Manjula S. Induction of root colonization by Piriformospora indica leads to enhanced asiaticoside production in Centella asiatica. Mycorrhyza 2012;22:195-202.
14. Haslam E, Haworth RD, Knowles PF, Gallotannins IV. The biosynthesis of gallic acid. J Chem Soc 1961;1854-9.
15. Mc Donald S, Prenzler PD, Antolovich M, Robards K. Phenolic content and antioxidant activity of olive extracts. Food Chem 2001;73:73–84.
16. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9.
17. Pils B, Heyl A. Unraveling the evolution of cytokinin signaling. Plant Physiol 2009;151:782-91.
18. Tognetti VB, Muhlenbock P, Breusegem FV. Stress homeostasis-the redox and auxin perspective. Plant Cell Environ 2012;35:321-33.
19. Bielach A, Hrtyan M, Tognetti VB. Plants under stress: involvement of auxin and cytokinin. Int J Mol Sci 2017;18:E1427.
20. Moller IM. Plant mitochondria and oxidative stress: electron transport, NADPH turnover and metabolism of reactive oxygen species. Annu Rev Plant Physiol Plant Mol Biol 2001;52:561–91.
21. Orozco Cardenas ML, Narvaez Vasquez J, Ryan CA. Hydrogen peroxide acts as a second messenger for the induction of defence genes in tomato plants in response to wounding, system in and methyl jasmonate. Plant Cell Online 2001;13:179-91.
22. Slesak I, Libik M, Karpinska B, Karpinski S, Miszalski Z. The role of hydrogen peroxide in regulation of plant metabolism and cellular signalling in response to environmental stresses. Acta Biochimica Polonica 2007;54:39-50.
23. A-H-Mackerness S, Surplus SL, Blake P, John CF, Buchanan Wollaston V. Ultraviolet–B induced stress and changes in gene expression in Arabidopsis thaliana: Role of signaling pathways controlled by jasmonic acid, ethelene and reactive oxygen species. Plant Cell Environ 1999;22:1413-23.
24. Wahdanimgsin S, Wahyuono S, Riyanto S, Murwanti R. Antioxidant activity of red dragon fruit peel (Hylocereus polyrhizus (F. A. C. Weber) Britton and Rose) isolates using 2,2-diphenyl-1-picryl hydrazyl method. Asian J Pharm Clin Res 2018;11:124.
25. Fidrianny I, Fikayuniar L, Insanu M. Antioxidant activities of various seed extracts from four varieties of rambutan (Nephelium lappaceum) using 2,2-diphenyl-1-picryl hydrazil and 2,2’-azinobis (3-ethyl benzothiazoline-6-sulfonic acid) assays. Asian J Pharm Clin Res 2015;8:227-31.
26. Bhanu DRC, Sabu KK. Fatty acid composition of the fruits of Syzygium zeylanicum (L.) DC. var. zeylanicum. Int J Curr Pharm Res 2017;9:155-7.
27. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci 1993;84:407–12.
28. Kikuzaki H, Nakatani B. Antioxidant effect of some ginger constituent. J Food Sci 1993;58:1407-10.
29. Hao J, Li L, Wolf M, Xu MM, Brinsko B, Yanik M, et al. Antioxidant properties and phenolics components of grape seeds. Funct Plant Sci Biotechnol 2009;1:60-8.
30. Jirovetz L, Buchbauer G, Stoilova I, Stoyanova A, Krastanov A, Schmidt E. Chemical composition and antioxidant properties of clove leaf essential oil. J Agric Food Chem 2006;54:6303-7.
31. Hamid AA, Shah MZ, Muse R, Mohamed S. Characterisation of anti-oxidative activities of various extracts of Centella asiatica (L.) urban. Food Chem 2001;77:465-9.
32. Zainol MK, Abdul-Hamid A, Yusof S, Muse R. Antioxidative activity and total phenolic compounds of leaf, root and petiole of four accessions of Centella asiatica (L.) urban. Food Chem 2003;81:575–81.
33. Chalker Scott LS, Fuchigami. In: Low temperature stress physiology in crops. (Ed) Paul HL, CRC Press; Boca Raton, Florida: 1989.
34. Dixon RA, Paiva N. Stress induced phenyl propanoid metabolism. Plant Cell 1995;7:1085-97.
35. Liu SR, Zhou JJ, Hu CG, Wei CL, Zhang LZ. MicroRNA-mediated gene silencing in plant defense and viral counter-defense. Front Microbiol 2017;8:1801.
36. Sunkar R. MicroRNAs with macro-effects on plant stress responses. Seminars Cell Dev Biol 2010;21:805-11.
37. Verardo LL, Nascimento CS, Silva FF, Gasparino E, Toriyama E, Barbosa AR, et al. Identification and expression levels of pig miRNAs in skeletal muscle. Livest Sci 2013;154:45–54.
38. Izumi H, Kosaka N, Shimizu T, Sekine K, Ochiya T, Takase M. Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey. PLoS One 2014;9:e88843.
39. Srivastava S, Srivastava AK, Suprasanna P, D’Souza SF. Identi?cation and pro?ling of arsenic stress-induced microRNAs in Brassica juncea. J Exp Bot 2013;64:303–15.
40. Akdogan G, Tufekci ED, Uranbey S, Unver. TmiRNA-based drought regulation in wheat. Funct Integr Genomics 2016;16:221-33.
41. Wu L, Zhang Q, Zhou H, Ni F, Wu X, Qi Y. Rice MicroRNA effector complexes and targets. Plant Cell 2009;21:3421-35.
42. Chen X, Xia J, Xia Z, Zhang H, Zeng C, Lu C, et al. Potential functions of microRNAs in starch metabolism and development revealed by miRNA transcriptome profiling of cassava cultivars and their wild progenitor. BMC Plant Biol 2015;15:33.
43. Ferdous J, Hussain SS, Shi B. Role of microRNAs in plant drought tolerance. Plant Biotech J 2015;13:293–305.
44. Kantar M, Lucas SJ, Budak H. miRNA expression patterns of Triticum dicoccoides in response to shock drought stress. Planta 2011;233:471–84.
45. Xu M, Hu T, Zhao J, Park MY, Earley KW, Wu G, et al. Developmental functions of miR156-regulated Squamosa promoter binding protein-like (SPL) genes in Arabidopsis thaliana. Plos Genetics 2016;12:e1006263.
46. Dong CH, Pei H. Over-expression of miR397 improves plant tolerance to cold stress in Arabidopsis thaliana. J Plant Biol 2014;57:209-17.
This work is licensed under a Creative Commons Attribution 4.0 International License.