In collaboration with Scientific Association of Iranian Medicinal Plants

Document Type : Research Paper

Authors

1 Faculty of Forest Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

2 Department of Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran

Abstract

This study aimed to enhance the production of betulin and betulinic acid using suspension cultures of birch (Betula pendula Roth) and elicitation of the cell cultures by methyl jasmonate (MeJA) and salicylic acid (SA). To do this, at the first step, the cell growth curve was investigated in a 16-day period. Then, two elicitors, namely, MeJA (at final concentration of 0, 50, 100, 150 and 200 µM) and SA (at final concentration of 0, 100, 200, 300 and 400 µM) were separately supplemented to 8-day-old cell cultures and the cells were harvested 1, 2, 3, 5 and 7 days after elicitations. Fresh weight (FW), dry weight (DW) and cell viability were measured. In addition, betulin and betulinic acid content were analyzed using HPLC. The results showed the significant effects of different concentrations of SA and MeJA on metabolites content and FW and DW. Maximum amount of betulin was observed about 4-fold (2.5 mg g-1 DW) higher than the control treatment by addition of 100 µM SA, two days after elicitation. Moreover, betulinic acid content was enhanced about 5 mg g-1 DW, 4.5-fold compared to control, one day after addition of 200 µM SA. Furthermore, the high accumulation of betulin (2.3 mg g-1 DW) was obtained in the elicited cell by 50 µM MeJA, seven days after elicitation. Also, the maximum amount of betulinic acid, about 3 mg g-1 DW, was observed in the cells elicited by 100 µM MeJA, two days after elicitation. Overall, the effect of SA on the production of betulin and betulinic acid was significantly more than the effect of MeJA.

Keywords

- Ali, M.B., Hahn, E.J. and Paek, K.Y., 2007. Methyl jasmonate and salicylic acid induced oxidative stress and accumulation of phenolics in Panax ginseng bioreactor root suspension cultures. Molecules, 12: 607-621.
- Fan, G.Z., Li, X.C., Wang, X.D. and Zhan, Y.G., 2010. Chitosan activates defense responses and triterpenoid production in cell suspension cultures of Betula platyphylla Suk. African Journal of Biotechnology, 9(19): 2816-2820.
- Fan, G., Zhai, Q., Li, X. and Zhan, Y., 2013. Compound of Betula platyphylla cell suspension cultures in response to fungal elicitor. Biotechnology & Biotechnological Equipment, 27(1): 3569-3572.
- I.U.C.N., 2001. IUCN Red List Categories and Criteria. IUCN, Gland, Switzerland.
- Liu, J., Fu, M.L. and Chen, Q.H., 2011. Biotransformation optimization of betulin into betulinic acid production catalysed by cultured Armillaria luteo-virens Sacc ZJUQH100-6 cells. Journal of Applied Microbiology, 110:90-97.
- Mehrabani, B., Nazeri, S. and Piri, K., 2012. Evaluation of total produced phenol in ChaeiKoohi (Stachys lavandulifolia Vahi) callus culture and possibility of its enhancement using Elicitors. Journal of Agricultural Biotechnology, 4(2): 77-88.
- Mehri Rad, N., 2014. Possibility to Increase Betulin Extract of betula litwinowii Callus in vitro Condition. M.Sc Thesis, Faculty of Forest Sciences, Gorgan University of Agriculture Science and Natural Resources, Iran, 74p.
- Mirjalili, N. and Linden, J.C., 1995. Gas phase composition effects on suspension cultures of Taxus cuspidate. Biotechnology and Bioengineering, 48: 123-132.
- Namadeo, A.G., 2007. Plant cell Elicitation for production of secondary metabolites-A review. Pharmacogenocy Review, 1: 154-160.
- Nazari, J., 2012. Optimization of culture medium and sterilization treatments for Betula litwinowii micropropagatin. M.Sc Thesis, Faculty of Forest Sciences, Gorgan University of Agriculture Science and Natural Resources, Iran, 67p.
- Neumann, K.H., Kumar, A. and Imani, J., 2009. Plant Cell and Tissue Culture: A Tool in Biotechnology. Springer-Verlag Berlin Heidelberg, 333p.
- Payamnoor, V., Jafari Hajati, R. and Nazari, J., 2015. Callogenesis of two species of birch (B. pendula and B. litwinowii) using the bark explant and evaluation of inducted Betulin. Research Project of Gorgan University of Agricultural Sciences and Natural Resources, Iran, 21p.
- Sato, F., Hashimoto, T. and Hachiya, A., 2001. Metabolic engineering of plant alkaloid biosynthesis. Proceedings of the National Academy of Sciences of the United States of America, 98: 367-372.
- Shabani, L. and Ehsanpour, A.A., 2009. Induction of antioxidant enzymes, phenolic and flavonoid compounds in in vitro culture of licorice (Glycyrrhiza glabra L.) using methyl jasmonate and salicylic acid. Iranian Journal of Plant Biology, 21(3): 421-432.
- Smith, P.F., Oundele, A., Forrest, A., Wilton, J., Salzwedel, K., Doto, J., Allaway, G.P. and Martin, D.E., 2007. Phase I and II study of the safety, virologic effect, and pharmacokinetics/pharmacodynamics of single-dose 3-O-(3′,3′-dimethylsuccinyl) betulinic acid (bevirimat) against human immunodeficiency virus infection. Antimicrob Agents Chemother, 51: 3574-3581.
- Wang, J.W., Xia, Z.H., Chu, J.H. and Tan, R.X., 2004. Simultaneous production of anthocyanin and triterpenoids in suspension cultures of Perilla frutescens. Enzyme and Microbial Technology, 34: 651-656.
- Yin, J., Ma, H., Gong, Y., Xiao, J., Jiang, L., Zhan, Y., Li. C., Ren, C. and Yang, Y., 2013. Effect of MeJA and light on the accumulation of betulinand oleanolic acid in the saplings of white birch (Betula platyphylla Suk). American Journal of Plant Sciences, 4: 7-15.
- Yin, J., Li, C.X., Hong-Ran Sun, H.R.,Wang, Z.H., Xiao, J.L., Ya-Guang Zhan, Y.G. and Zhang, M.Y., 2014. The Physiological characteristics, expression of oxidosqualenecyclase genes and accumulation of triterpenoids in white birch (Betula platyphylla Suk) saplings by SA and MeJa treatment. Journal of Plant Biochemistry & Physiology, 2: 2-6.
- Yoon, H.J., Kim, H.K., Ma, C.J. and Huh, H., 2000. Induced accumulation of triterpenoids in Scutellaria baicalensis suspension cultures using a yeast elicitor. Biotechnology Letters, 22: 1071-1075.
- Yu, L.J., Lan, W.Z., Qin, W.M. and Xu, H.B., 2001. Effects of salicylic acid on fungal-elicitor induced membrane-lipid peroxidation and taxol production in cell suspension cultures of Taxus chinensis. Process Biochemistry, 37: 477-482.
- Zhao, G., Yan, W.D. and Cao, D., 2007. Simultaneous determination of betulin and betulinic acid in white birch bark using RP-HPLC. Journal of Pharmaceutical and Biomedical Analysis, 43: 959-962.