Silybin, the Main Active Component of Silybum marianum, Affects Blood Coagulation: An In Vitro Pilot Study

The health-promoting properties of Silybum marianum have been acknowledged since antiquity. This plant is credited with substantial hepatoprotective properties and is also protective in cardiovascular diseases, diabetes mellitus, and neurodegeneration, mainly for its anti-inflammatory and antioxidant effects. Only a few experimental studies have described the impact of Silybum marianum extract on the blood coagulation process; furthermore, these data are unsatisfactorily fragmented and need to be supplemented to understand the plant’s properties better. The predominant biologically active flavonolignan extracted from Silybum marianum is silybin, a mixture of two diastereomers, silybin A and silybin B, in approximately equimolar ratio. This study investigated the effect of silybin on the fundamental laboratory parameter for blood coagulation, namely prothrombin time (PT), an assay used to assess the extrinsic and common coagulation pathways. To evaluate the effect of silybin on PT, we prepared three solutions of silybin (Silybin (A + B mixture), PhytoLab GmbH & Co. KG, Vestenbergsgreuth, Germany) in 0.1% dimethylsulfoxide (DMSO, Sigma-Aldrich, Co., St. Louis, MO, USA): 10 μM, 50 μM, and 100 μM. PT was measured on a Coag 4D coagulometer (DIAGON Kft., Budapest, Hungary) using rabbit calcium thromboplastin (Dia-PT, DIAGON Kft., Budapest, Hungary) and control plasma, which is pooled plasma obtained from healthy donors (Dia-CONT, DIAGON Kft., Budapest, Hungary). A total of 10 µL of silybin solution was added to 40 µL of plasma; the sample was incubated for two minutes at 37 °C, and then 100 µL of thromboplastin, pre-warmed to 37 °C, and was added to the mixture. The coagulometer automatically gives the PT result in seconds (s). At the same time, PT was measured in the control plasma both without additional solutions and with the addition of tris-buffered saline (TBS) and 0.1% DMSO (10 µL of TBS or DMSO + 40 µL of plasma). Each measurement was performed eight times. Student’s t-test and the Friedman test with post-hoc analysis were used in the statistical analysis (Statistica 13, TIBCO Software Inc., Palo Alto, CA, USA). In the first step of our study, we tested how the dilution of the plasma sample affected PT. We did not observe statistically significant differences in PT between the control plasma and the control plasma supplemented with TBS (mean ± standard deviation 14.00 ± 0.77 s vs. 13.88 ± 0.38 s, p = 0.606). We also found no statistically significant differences in PT between the control plasma and the control plasma with the addition of 0.1% DMSO (mean ± standard deviation 14.00 ± 0.77 s vs. 14.10 ± 0.26 s, p = 0.728); therefore, we further analyzed the effect of silybin on PT using DMSO at this level (0.1%). The addition of silybin solutions to the control plasma resulted in a statistically significant PT-shortener (p < 0.001). Post-hoc analysis revealed a substantial shortening of PT under the influence of 50 μM (median 13.55 s) and 100 μM solution (median 13.40 s) of silybin, compared to plasma with the addition of 0.1% DMSO alone (median 14.10 s) and plasma with the addition of the lowest, 10 μM, level of silybin (median 14.20 s). At the same time, PT in the plasma with the addition of a 50 μM and 100 μM solution of silybin did not significantly differ statistically. Our in vitro analysis characterized the possible effect of Silybum marianum on the blood coagulation process. These results require further investigation to validate their validity and clinical utility.