Introduction: Milk thistle contains flavonolignan silymarin in its fruits, making it one of the most important medicinal plants for protecting the liver and treating a variety of liver diseases, such as hepatitis and liver diseases containing toxins. The drought resistance mechanisms of medicinal plants, such as milk thistle, are of critical importance. The investigation of the effect of drought stress on the activity of antioxidant enzymes will aid in the identification of these mechanisms. Water stress modifies plant behavior via its effect on plant metabolism and consequently has a substantial effect on plant production. The biosynthesis pathway of secondary metabolites is altered by environmental stresses, altering the production and concentration of active substances in plants. In addition to playing a crucial role in defense mechanisms, secondary metabolites are an important source of bioactive compounds. However, flavonoids not only have antioxidant activity via the release of free radicals, but they also regulate cellular pathways in the defense of plants against environmental stresses, alter plant metabolism, and have a significant impact on plant production.
Materials and method: In order to determine the effect of drought stress on the physiological and biochemical characteristics of milk thistle, treatments of 100%, 75%, 50%, and 25% water requirement were applied in a greenhouse in the village of Shandel, city of Hirmand. There were multiple harvesting stages, including 6 and 13 weeks after planting. Measurements included dry weight, fresh weight, chlorophyll a, chlorophyll b, carotenoids, proteins, proline levels, and antioxidant enzyme activity. First, the data were examined for normality in Minitab software version 18, using the Smearnof Columnograph method to determine data normality, and then the errors were examined for normality to confirm data normality. Then, analysis of variance of all traits and comparison test of Duncan were performed at 5% level with SAS software version 9.2 (SAS 2010).
Results and discussion:  The analysis of variance revealed significant differences in harvest time, irrigation level, and the interaction between harvest time and irrigation level. In both harvest stages, the concentration of carotenoids began to decrease as the level of stress increased. The amount of chlorophyll a at the stage 6 weeks after planting was less than at the stage 13 weeks after planting, but its amount decreased with increasing stress at both stages. The levels of protein and proline also increased as the level of stress increased. The activity of antioxidant enzymes also increased as the intensity of stress increased, to the point where 100% of water requirements reached the minimum value and 25% reached the maximum value. Due to the fact that most oxidative enzymes in this study increased in response to stress, it can be concluded that catalase, superoxide dismutase, guaiacol peroxidase, polyphenol oxidase, and ascorbate peroxidase can eliminate ROS in this plant. Therefore, milk thistle has a good adaptation to drought stress, which is likely due to the reduction of oxidative damage caused by the activation of the antioxidant system and the accumulation of osmolytes such as proline and protein. These modifications are a form of adaptation to stress conditions.
Conclusion: The majority of traits were affected by growth stage and stress level. The levels of protein and proline increased with increasing stress intensity. Most antioxidant enzymes, including catalase, ascorbate peroxidase, and superoxide dismutase, increased in activity under the influence of stress. According to these findings, milk thistle responds to drought stress by increasing proteins and amino acids, decreasing photosynthetic pigments, and increasing the activity of antioxidant enzymes, among other mechanisms. Also, because the amount of metabolites in this plant increases with increasing drought stress, it is possible to determine the stress tolerance threshold of this plant, cultivate it on a large scale, and apply stress to increase secondary metabolites.