CELL SUSPENSION CULTURE ESTABLISHMENT OF
THYMUS PERSICUS FOR THE PRODUCTION OF MEDICINALLY
IMPORTANT TRITERPENOIDS

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Mohammad Hossein Mirjalili1,*, Ziba Bakhtiar1, Ali Sonboli2, Mahdi Moridi Farimani3 Mahdi Ayyari3

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۱Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Tehran, Iran
۲Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Tehran, Iran
۳Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Tehran, Iran E-mail: m-mirjalili@sbu.ac.ir

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In the present study, an efficient protocol has been performed for establishment of Thymus persicus (Lamiaceae) cell suspension culture in shake flasks, as a strategy to obtain an in vitro triterpenoids producing cell line. Betulinic acid (BA), oleanolic acid (OA) and ursolic acid (UA) are highly valuable pentacyclic triterpenoids because of their wide spectrum of biological activities such as anti-inflammatory, hepatoprotective, antitumor, anti-HIV, antimicrobial, antifungal, anti-ulcer, gastroprotective, hypoglycemic, and antihyperlipidemic [1, 2]. Callus culture was initiated from nodal segments and leaf explants on MS [3] medium containing basic salts and 30 g/L sucrose supplemented with different concentrations of auxins 2,4-D and NAA (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L) solely or in combination with cytokinins BAP and Kin (0.1, 0.5 and 1.0 mg/L). High percentage of friable callus was obtained in nodal segments cultured on MS medium supplemented with 1 mg/L 2, 4-D and 0.5 mg/L BAP. Cell suspension culture was established on MS medium supplemented with NAA (0.5 and 2.0 mg/L), BAP (2.0 and 2.5 mg/L) and Kin (0.4 mg/L). The highest growth index obtained from the culture medium containing 2.5 mg/L BAP and 0.5 mg L−۱NAA. The growth kinetics of the cell suspension culture followed a general growth pattern of sigmoid curve and has shown a maximum specific cell growth rate of 0.76g day-1, doubling time of 36 h and cell viability of 86.4 %, respectively. BA, OA and UA content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The maximum BA, OA and UA production was observed in the stationary growth phase, reaching 54.3±۱.۵۸, ۳.۷۸±۰.۳۷, ۶.۲۱±۰.۸۱ mg g−۱ dry weight (DW) at day 25 of the culture, respectively. The results of present study are useful to scale-up process and augment the T. persicus biological research.

References
[۱]    Liu, M. Yang, S. Jin, L. Hu, D. Wu, Z. Yang, S., Molecules, 2012, 17: 6156-6169.
[۲]    Cichewicz, R.H.; Kouzi, S.A., Med Res Rev, 2004, 24: 90–۱۱۴.
[۳]    Murashige ,T. Skoog, F., Physiologia Plantarum, 1962, 15, 473-497.


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