Document Type : Research Paper
Author
Assistant Professor, Research Division of Natural Resources , East Azarbaijan Agricultural and Natural Resources Research and Education Center , Agricultural Research, Education and Extention Organization (AREEO), Tabriz, Iran
Abstract
Background and objectives: Milk thistle (Silybum marianum L.) is a significant medicinal plant that has gained a prominent place in the pharmaceutical industry. This annual or biennial plant contains bioactive compounds, particularly flavonoids, which are primarily concentrated in its seeds. The collective active components of this plant are referred to as silymarin, a compound recognized for its anti-cancer properties. Cell culture systems provide a promising method for large-scale cultivation of plant cells to produce secondary metabolites. This research aimed to investigate the effects of genotype, micro-sample type, phytohormone treatments, and callus type on the synthesis of bioactive compounds in S. marianum calluses using high-performance liquid chromatography (HPLC).
Methodology: In this research, a combination of Tween-20 solution, 70% ethanol, hydrogen peroxide, and sterile distilled water was used to disinfect Silybum marianum seeds to produce sterile seedlings. The sterilized seeds were then transferred to sterilized water and an agar culture medium (12 g/L) and kept in dark conditions at a temperature of 25°C for 15 days. After germination and initial growth, the seedlings were moved to light conditions. An experiment was conducted using two genotypes, Hungary and Borazjan, with cotyledon and hypocotyl micro-samples. The experiment utilized Murashige and Skoog (MS) medium and hormonal treatments of 2,4-Dichlorophenoxyacetic acid (2,4-D) at concentrations of 1, 2.5, and 5 mg/L, along with Benzyl Amino Purine (BAP) at 0.25 and 0.5 mg/L. This setup was maintained in dark conditions and organized as a factorial design based on a completely randomized block structure. After 30 days, the calluses were transferred to MS culture medium with half the concentration of hormones. The effective compounds were extracted from the callus through defatting with petroleum ether, followed by extraction with methanol solvents. The components of silymarin were then separated into taxifolin, silychristine, silydianin, silybin, and isosilybin using high-performance liquid chromatography (HPLC). Statistical analysis of the data was performed using SPSS software.
Results: The test results demonstrated the effects of different micro-sample treatments, ecotypes, and hormone concentrations on the levels of silymarin flavonoid compounds (taxifolin, silychristin, silydianin, silybin, and isosilybin) in Silybum marianum extracts. Analysis of variance and comparison of the mean values of flavonolignans in the callus samples revealed that the highest amount of taxifolin was obtained from the hypocotyl micro-sample. The highest silychristin content was associated with the Hungarian genotype and a treatment of 5 mg/L 2,4-D. The highest silydianin concentration was observed in the Hungarian genotype treated with 1 mg/L 2,4-D and 0.25 mg/L BAP. The maximum silybin content was found in the Hungarian cultivar using the hypocotyl micro-sample, with 1 mg/L 2,4-D and 0.25 mg/L BAP. The highest isosilybin levels were achieved in the Hungarian genotype, hypocotyl micro-sample, and 1 mg/L 2,4-D. The overall highest silymarin concentration was recorded in the Hungarian genotype treated with 1 mg/L 2,4-D and 0.5 mg/L BAP.
Conclusion: The results showed that improved varieties of Silybum marianum, such as the Hungarian genotype, produced higher levels of bioactive compounds compared to native ecotypes. In the production of these compounds, the type of micro-sample played a more critical role than the size and type of callus derived from it. Specifically, the hypocotyl micro-sample, which produced small, non-embryogenic calluses, yielded a higher percentage of effective substances compared to the cotyledon, which produced larger, embryogenic calluses. Additionally, lower concentrations of auxin and cytokinin hormones were more effective than higher concentrations in promoting the production of bioactive compounds.
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