The effect of temperature and pH on biomass and bioactive compound production in Silybum marianum hairy root cultures

Document Type: Original paper


1 Department of Molecular Physiology, Agricultural Biotechnology Research Institute of Iran, Mahdasht Road, Karaj, Iran. School of Biotechnology and Ginseng Bank, College of Life Sciences, Kyung Hee University, Yongin, Republic of Korea.

2 Department of Molecular Physiology, Agricultural Biotechnology Research Institute of Iran, Mahdasht Road, Karaj, Iran.


Background and objectives: The seed extract of Silybum marianum contains seven flavonolignans known collectively as silymarin. These metabolites can be produced in hairy root cultures of S. marianum. The effect of different physical factors can change root biomass and silymarin production which has been investigated in the present study.
Methods: The effect of different physical factors of temperature (30 ºC/25 ºC, 25 ºC/25 ºC and 15 ºC/20 ºC in 16 h/8 h cycle) and pH (5, 5.7, 6 and 7) were evaluated with respect to the root biomass and silymarin production in hairy root cultures of the plant.
Results: Incubation temperature, 25 ºC /25 ºC promoted the silymarin production in 4-week old hairy roots (0.18 mg/g DW) as compared with the cultures treated with 15 ºC/20 ºC and 30 ºC/25 ºC (0.13 and 0.12 mg/g DW, respectively). Maximal increases in biomass and silymarin accumulation occurred in the root cultures grown in pH 5 and 25 ºC/25 ºC (0.45 g and 0.26 mg/g DW). The content of silybin, isosilybin, silychristin, silydianin were 0.025, 0.024, 0.061 and 0.095 mg/g DW, respectively which were higher than those grown in higher pH.
Conclusion: The results of the present study suggest that 25 ºC/25 ºC and acidic environment of medium are beneficial for silymarin production using hairy root cultures. Furthermore, lipoxygenase (LOX) activity was strongly affected by pH which suggested that acidic environment may act as inducing signal for LOX activity and subsequently greater silymarin production.  


[1] Davis-Searles PR, Nakanishi Y, Kim NC, Graf TN, Oberlies NH, Wani MC, Wall ME, Agarwal R, Kroll DJ. Milk thistle and prostate cancer: differential effects of pure flavonolignans from Silybum marianum on antiproliferative end points in human prostate carcinoma cells. Cancer Res. 2005; 65[MH1] : 4448-4457.

[2] Kim NC, Graf TN, Sparacino CM, Wani MC, Wall ME. Complete isolation and characterization of silybins and isosilybins from milk thistle (Silybum marianum). Org Biomol Chem. 2003; 1: 1684-1689.

[3] Flora K, Hahn M, Rosen H, Benner K. Milk thistle (Silybum marianum) for the therapy of liver disease. Am J Gastroenterol. 1998; 93: 139-143.

[4] Tyagi A, Bhatia N, Condon M, et al[MH2] . Antiproliferative and apoptotic effects of silibinin in rat prostate cancer cells. Prostate. 2002; 53: 211-217.

[5] Rhodes MJC, Robins RJ, Hamill JD, Parr AJ, Hilton MH, Walton NJ. Properties of transformed root culture. In: Charlwood BV, Rhodes MJC, editors. Proceedings of the Phytochemical Society of Europe Secondary Product from Plant Tissue Culture. Clarendon Press, Oxford, 1990;  pp. 201-225.[MH3] 

[6] Merkli A, Christen P, Kapetanidis I. Production of diosgenin by hairy root cultures of Trigonella foenum-graecum L. Plant Cell Rep. 1997; 16: 632-636.

[7] Kittipongpatana N, Hock RS, Porter JR. Production of solasodine by hairy root, callus, and cell suspension cultures of Solanum aviculare Forst. Plant Cell Tiss Org. 1998; 52: 133-143.

[8] Jeong CS, Chakrabarty D, Hahn EJ, Lee HL, Paek KY. Effects of oxygen, carbon dioxide and ethylene on growth and bioactive compound production in bioreactor culture of ginseng adventitious roots. Biochem Eng J. 2006; 27: 252-263.

[9] Zhong JJ, Yoshida T. Effects of temperature on cell growth and anthocyanin production by suspension cultures of Perilla frutescens cells. J Ferment Bioeng. 1993; 76: 530-531.

[10] Sivakumar G, Yu KW, Hahn EJ, Paek KY. Optimization of organic nutrients for ginseng hairy roots production in large-scale bioreactors. Curr Sci. 2005; 89: 641-649.

[11] Saenz-Carbonell LA, Maldonado-Mendoza IE, Moreno-Valenzula O, Ciau-Uitz R, Lopez-Meyer M, Loyola-Vargas VM. Effect of the medium pH on the release of secondary metabolites from roots of Datura stramonium, Catharanthus roseus, and Tagete spatula cultured in vitro. Appl Biochem Biotech. 1993; 38: 257-267.

[12] Rahnama H, Hasanloo T, Shams MR, Sepehrifar R. Silymarin production in hairy root culture of Silybum marianum (L.) Gaertn. Iran J Biotechnol. 2008; 6: 113-118.

[13] Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962; 15: 473-497.

[14] Cacho M, Moran M, Corchete P, Fernandez-Tarrago J. Influence of medium composition on the accumulation of flavonolignans in cultured cells of Silybum marianum (L.) Gaertn. Plant Sci. 1999; 144: 63-68.

[15] Hasanloo T, Khavari-Nejad RA, Majidi E, Shams-Ardakani MR. Analysis of flavonolignans in dried fruits of Silybum marianum(L.) Gaertn from Iran. Pak J Biol Sci. 2005; 8: 1778-1782.

[16] Hasanloo T, Khavari-Nejad RA, Majidi E, Ziai SA, Shams-Ardakani MR. Determination of flavonolignan of dried fruits of Silybum marianum L. Gaertn collected from different areas of Iran by spectrophotometer, TLC and HPLC. Res J Med Plant. 2005; 4: 25-32.

[17] Chance B, Maehly AC. Assay of catalases and peroxidises. In: Colowick SP, Kaplan NO, editors. Methods in enzymology. New York: Academic Press, 1955.

[18] Nakano Y, Asada K. Purification of ascorbate peroxidase in spinach chloroplasts; its inactivation in ascorbate depleted medium and reactivation by monodehydroascorbate radical. Plant Cell Physiol. 1987; 28: 131-140.

[19] Axelroad B, Cheesbrough TM, Laakso S. Lipoxygenase from soybeans. Method Enzymol. 1981; 17: 441-451.

[20] Bradford MA. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248-254.

[21] Ten Hoopen HJG, Vinke JL, Moreno PRH, Verpoorte R, Heijnen JJ. Influence of temperature on growth and ajmalicine production by Catharanthus roseus suspension cultures. Enzyme Mirob Tech. 2002; 30: 56-65.

[22] Yu KW, Murthy HN, Hahn EJ, Paek KY. Ginsenoside production by hairy root cultures of Panax ginseng: influence of temperature and light quality. Biochem Eng J. 2005; 23: 53-56.

[23] Wu J, Zhong JJ. Production of ginseng and its bioactive components in plant cell culture: current technological and applied aspects. J Biotechnol. 1999; 68: 89-99.

[24] Gangopadhyay M, Sircar D, Mitra A, Bhattacharya S. Hairy root culture of Plumbago indica as a potential source for plumbagin. Biologia Plantarum. 2008; 52: 533-537.

[25] Taya M, Mine K, Kino-Oka M, Tone S, Ichi T. Production and release of pigments by culture of transformed hairy root of red beet. J Ferment Technol. 1992; 73: 31-36.

[26] Mukundan U, Bhide V, Singh G, Curtis WR. PH-mediated release of betalains from transformed root cultures of Beta vulgaris L. Appl Microbiol Biot. 1998; 50: 241-245.

[27] Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2000; 9: 405-410.

Mittler R, Vanderauwera S, Gollery M, Breusegem FV. Abiotic stress series. Reactive oxygen gene network of plants. Trends Plant Sci. 2004; 9: 490-498.