Optimization of ultrasound-assisted extraction based on response surface methodology using HPLC-DAD for the analysis of red clover (Trifolium pretense L.) isoflavones and its anti-inflammatory activities on LPS-induced 3D4/2 cell

Introduction: Trifolium pratense L. has anti-inflammatory, antioxidant, cardiovascular disease prevention, and estrogen-like effects. The existing method for the assay of effective components is commonly based on a spectrophotometer, which could not meet the requirement of quality control. Furthermore, although there have been many studies on the anti-inflammation effect of red clover, a few have been reported on the regulatory effect of red clover isoflavones (RCI) on lipopolysaccharide (LPS)-induced inflammatory response in porcine alveolar macrophages (3D4/2 cells), and its mechanism of action is still unclear.Methods :The main components of RCI including daidzein, genistein, and biochanin A were accurately quantified by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) after optimizing the extraction process through response surface methodology. The anti-inflammatory potential of RCI was carried out by detecting the level of inflammatory cytokines and mRNA expression of related genes. Furthermore, its anti-inflammatory mechanism was explored by investigating two signaling pathways (NF-kappa B and MAPK).Results :The optimal extraction conditions of RCI were as follows: the concentration of ethanol is 86% and the solid-liquid ratio is 1:29, with the herb particle size of 40 mesh sieve. Under the optimal conditions, the total extraction of target components of RCI was 2,641.469 mu g/g. The RCI could significantly suppress the production and expression of many pro-inflammatory cytokines. The results of the Western blot revealed that RCI dramatically reduced the expression of p65, p-p65, I kappa B-alpha, p38, and p-p38. These results are associated with the suppression of the signal pathway of p38 MAPK, and on the contrary, activating the NF-kappa B pathway. Collectively, our data demonstrated that RCI reversed the transcription of inflammatory factors and inhibited the expression of p65, p-p65, I kappa B-alpha, and p38, indicating that RCI had excellent anti-inflammatory properties through disturbing the activation of p38 MAPK and NF-kappa B pathways.Conclusion :The extraction conditions of RCI were optimized by HPLC-DAD combined with response surface methodology, which will contribute to the quality control of RCI. RCI had anti-inflammatory effects on the LPS-induced 3D4/2 cells. Its mechanism is to control the activation of NF-kappa B and p38 MAPK pathways, thereby reducing the expression of inflammatory-related genes and suppressing the release of cytokines.