Effect of Drought Stress on Physiological and Phytochemical Traits of Silybum marianum L.

 Plant growth and yield are affected often by stress conditions, especially drought, which is the most important factor in reducing crop production worldwide. Silybum marianum is an important pharmaceutical crop with great potential as a multipurpose plant for low-input cropping systems of the arid and semiarid regions.

 In this experiment, the effect of drought stress (full irrigation at field capacity; no stress, irrigation at 70% of field capacity; mild stress and irrigation at 40% of field capacity; severe stress) on physiologic traits, the amount of silybin a and b and 1000 seed weight in milk thistle was studied in the research field of Shahid Beheshti University in 2017-2018. The experimental site is located in Shahid Beheshti University, Tehran, Iran (51.23°N, 35.48°E, and 1769 m above mean sea level). It has a moderate and mountainous climate with a mean annual rainfall of 145.2 mm and a mean temperature of 22ºC. This experiment was performed in a completely randomized block design with 3 replications. Milk thistle seeds were prepared from Isfahan Pakanbazr Company. The soil composition consisted of 1/3 clay, 1/3 sand, and 1/3 leaf composts. The field area was 150.0 m2. Furrows were created to implement this study. The space of plants on rows was 0.5 m and between rows was 1 m. In general, 15 furrows were created and 15 plants were cultured on each furrow, so the total number of plants cultivated on the field was 225. Drought stress was applied at flowering stage. Soil moisture was measured by the weighing method. The soil samples were taken from various areas (randomly) of the field, three samples each day. After measuring the water content at F.C, based on that, the amount of 70%F.C and 40%F.C was also calculated. At the time of stress, F.C irrigation was performed every two days, 70% F.C irrigation every 4 days, and 40% F.C irrigation every 6 days. After 8 days, leaf sampling was performed to measure catalase, ascorbate peroxidase, proline and malondialdehyde content and seed sampling was performed to 1000 seed weight and extract analysis. For physiologic measurements, 3 plants were randomly assigned to each stack and their leaves were separated and transferred to the laboratory. Then, in the next step, their physiologic parameters (include Catalase and ascorbate peroxidase, proline and malondialdehyde content) were measured according to the relevant protocol. In order to measure silybin, 4 plants were randomly considered to each stack and their seeds were harvested and combined and dried in shade condition in the laboratory for one month. The dried seed samples were completely powdered using a mill, then 10 g of the powders weighed and the oil extract of them was isolated by Soxhlet using n-hexane solvent. The extraction temperature was 70 °C and the extraction time was 6 h. After the extraction was completed, the extract was poured into dark glass. Next, the oil-free powder was dried in an incubator at 37 ° C. Methanol extract of oil-free powder was extracted using methanol solvent. For this purpose, 2 g of samples powder was weighed and 200 ml of 80% methanol was added to each of them and the mixture stirred for two days by Shaker. The mixture was then passed from a filter paper and after that, 200 ml of 80% methanol was added to the sample precipitate (on filter paper) and placed again on the shaker. After 24 h, extraction was performed by the same method and were added to the previous extract. The extracts were then concentrated in the environment temperature for two weeks. Concentrated extracts (powder) as well as standard silymarin with certain concentrations were dissolved in methanol solvent and used for injection into HPLC (Model: Infinity1260, Manufacturer: Agilent) using syringe filters with a diameter of 0.2 μm. After receiving the HPLC results, the data and peaks were analyzed and the amounts of silybin a and b were determined and compared at different levels of water stress. Statistical analysis of data was performed using R 3.6.1 and RStudio 1.1.463 software. Mean data were compared using Duncan's test with agricolae package at a significance level of 0.05.

 The results showed that with increasing water stress intensity, the amount of silybin a and silybin b increased by 24% and 26%, respectively. The amount of these compounds in 40% were significantly higher than other treatments, so that its amount compared to F.C treatment (26.07 mg/g Grain DW in silybin a and 40.74 mg/g Grain DW in silybin b) and compared to the 70%F.C (25.32 mg/g Grain DW in silybin a and 34.64 mg/g Grain DW in silybin b) was higher. This indicates carbon assimilation from photosynthesis to produce secondary metabolites in this treatment. Also, the amount of silybin b compared to silybin a in all treatments was (0.8: 1.2), in which 1.2 is related to silybin b and 0.8 is related to silybin a. In severe stress treatment (40% of field capacity), the amount of silybin a and b (67.30 and 98.92 mg/g, respectively) increased significantly compared to other treatments. According to the mean comparison results, the highest activity of catalase (5.16 U/ml) and ascorbate peroxidase (2.26 U/ml) was observed in mild stress treatment. Proline content gradually increased with increasing stress intensity and reached its peak in severe stress (3.36 µM/gr). Lipid peroxidation also had their maximum in severe stress (8.35 nmol/grFW). The 1000 seeds weight was reduced by 6.8 g in severe stress treatment (40%F.C) compared to the control (F.C). 

 According to the results, the amount of milk thistle flavonoids can be increased for medicinal purposes including the treatment of liver disease and hepatitis by applying dehydration stress.