The Anti-inflammatory Activity Evaluation And Action Mechanism Of Effective Constituents Isolated From Salvia Miltiorrhiza Bunge Var. Alba Wu And Li, Mss

Salvia miltiorrhiza Bunge var. alba C.Y. Wu et H.W. Li, mss., the variant of Salvia miltiorrhiza Bunge, is native to Laiwu, Zhangqiu and Taian in Shandong province. Because of the quantity of wild S. miltiorrhiza Bge. var. alba is very few, it was listed as one of Taishan rare and endangered species and was mainly produced by artificial cultivation. The main chemical constituents of S. miltiorrhiza Bge. var. alba are tanshinones and phenolic acids, roughly same as S. miltiorrhiza Bge.. In clinical, the root of S. miltiorrhiza Bge. var. alba is often used for the treatment of cardiovascular diseases instead of S. miltiorrhiza Bge.. In addition, it has a unique curative effect for the treatment of thromboangiitis obliterans (TAO). And these vascular diseases have a close relationship with inflammation. Our research on the chemical constituents of S. miltiorrhiza Bge. var. alba roots is sufficient, but the biological activity study of the effective constituents is less. So this study mainly studied the anti-inflammatory activity of effective components of S. miltiorrhiza Bge. var. alba.In this study, we determined the immune anti-inflammatory activities of eleven tanshinones isolated from S. miltiorrhiza var. alba roots using lipopolysaccharide (LPS)-induced THP-1 macrophages, with the tanshinone IIA as positive control. MTT assay was used to test the cell viability of THP-1; Production of tumor necrosis factor (TNF)-a, interleukin (IL)-1β, and interleukin (IL)-8 were measured by enzyme-linked immunosorbent assay (ELISA); Quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the mRNA expression of TNF-a, IL-1β, and IL-8. The results showed that all the tanshinones, except for compound 5, significantly inhibited the mRNA and protein expression of TNF-a, IL-1β, and IL-8, and their anti-inflammatory activities were stronger than that of tanshinone IIA with the same concentration. Compound 9 showed the highest inhibitory effects. The anti-inflammatory mechanism may be associated with the NF-κB signaling pathway, it also need to be further investigated.We also determined the anti-inflammatory activities of nine phenolic acids isolated from S. miltioirhiza var. alba roots in LPS-induced THP-1 macrophages, with the salvianolic acid B as positive control. MTT assay was used to test the cell viability of THP-1; Production of TNF-a, IL-1β, and IL-6 were measured by ELISA; qRT-PCR was used to determine the mRNA expression of TNF-a, IL-1β, and IL-6; Protein level of TLR4, p65, p-p65, IκB, p-IκB were measured with Western Blot. The results show that all the phenolic acids significantly inhibited the mRNA and protein expression of TNF-a, IL-1β, and IL-6, and compound 8 has the best inhibitory effects. In addition, the compound 8 can significantly inhibited the high expression of p-p65, p-IκB and TLR4. The results show that the phenolic acids have anti-inflammatory activity, and its anti-inflammatory effects are achieved by inhibiting TLR4/NF-κB signaling pathway.S. milliorrhiza var. alba root has been used for treatment of TAO for a one hundred years history, and the curative effect is unique. To investigate the effect of tanshinone IIA on the rat models with TAO, we prepared TAO rat models by injecting lauric acid into the femoral artery of rats, and observed the changes of physical signs, pathologic grading of thrombus and the contents of TXB2, ET-1 and 6-K-PGF1α in serum of these rat models. Significantly improved lesion was obseved in the rats of treating group, with reduced number of thrombus formation, decreased content of TXB2 and ET-1, the ratio of TXB2/6-K-PGF1α, and increased content of 6-K-PGF1α. Tanshinone IIA hold significant therapeutic effect on TAO rats. The possible mechanisms may be achieved by improving anti-thrombus thrombolysis, antioxidant function of vessel endothelia and inhibiting inflammatory reaction in vascular wall.In conclusion, this thesis reveals the anti-inflammatory activity of part trace constituents of S. miltiorrhiza Bge. var. alba is better than that of tanshinone IIA or salvianolic acid B by studying on their anti-inflammatory activity. It provides the theory basis for the clinical application of S. miltiorrhiza Bge. var. alba for the treatment of vascular diseases such as AS or TAO from the perspective of anti-inflammatory. The pharmacodynamics research of tanshinone IIA to the rat model with TAO proves that tanshinone IIA is one of the effective constituents of S. miltiorrhiza Bge. var. alba for the treatment of TAO, and provides a reference for new clinical application of tanshinone IIA in practice.