Studies On The Anti-inflammatory And Antioxidant Effects Of Total Saponins From Leaves And Fruits Of Tribulus Terrestris L.

Tribulus terrestris L.as an annual herb plantand good medicine in the herbs,the whole plant,fruits,roots,stems,leaves and flowers of Tribulus terrestris L.all can be used as herb parts,and it exhibits anti-inflammatory,antioxidant,antibacterial and antitumor activities and other pharmacological effects.Tribulus terrestris L.mainly contains saponins,flavonoids and alkaloids compounds,and the major components are saponins compounds.Saponins compounds have antitumor,hypoglycemic effect,anti-inflammatory,and immunity enhancement,protection of cardiovascular system and central nervous system activities.Lipopolysaccharide(LPS)is one endotoxin constituent from the cell walls of gram-negative bacteria,can induce inflammation and activate the congenital immune system,and lead to the production of cytokine tumor necrosis factor-?(TNF-?),which will activate toll-like receptors pathway and NF-?B in downstream pathway,and also can induce inflammatory by the release of NO,and then induce a series of inflammatory response.Inflammation is closely associated with many diseases in livestock and poultry and impacts on the production practice.At present,the steroids and non-steroidal anti-inflammatory drugs have certain side effects in clinic.Therefore,the research and development of a natural and green anti-inflammatory drug has gradually become a research hotspot.This research has investigated the antioxidant and anti-inflammatory effects,extraction process and chemical components of total saponins of tribulus terrestris leaves and fruits as the research object,and,and provided ideas and methods for the research and development of anti-inflammatory drugs in veterinary clinic.In this study,the single factor test and box-behnken response surface test were used to investigate the optimal extraction conditions of total saponins.The main chemical constituents of leaves and fruits from Tribulus terrestris L.were identified by high performance liquid chromatography-mass spectrometry method.Antioxidant activities of total saponins from Tribulus terrestris L.leaves and fruits were determined by ABTS,DPPH free radicals scavenging capacity and FRAP assays.The inflammatory model was induced by LPS in in RAW264.7 cell and mice,and the content of NO in the lung tissue lavage fluid and cell supernatant was measured by Griess method,the TNF-? content was detected by enzyme-linked immunosorbent assay,the TLR4,TRAF6,I?B and P-I?B protein levels were detected with immune protein imprinting method.The anti-inflammatory activity of total saponins from Tribulus terrestris L.leaves was systematically evaluated,and the mechanism of action was further revealed.The optimum extraction technology of total saponins from Tribulus terrestris L.leaves was as follows: ethanol concentration 30%,extraction time 55 min,ratio of solvent to material 35:1,extraction temperature 46?,and the yield was 5.52% under this condition;the optimum extraction technology of total saponins from Tribulus terrestris L.fruits was as follows: ethanol concentration 100%,extraction time 40 min,ratio of solvent to material 25:1,extraction time temperature 70?,and the yield was 2.94% under the condition These results revealed that the leaves and fruits of Tribulus terrestris L.all exhibited ABTS,DPPH free radical scavenging ability and FRAP.In addition,total saponins of Tribulus terrestris L.leaves and fruits can significantly reduce the content of cytokine TNF-? and inflammatory mediator NO.Total saponins of Tribulus terrestris L.leaves could significantly reduce the levels of TLR4 and TRAF6 proteins in vitro and in vivo,and significantly inhibit the phosphorylation of I?B,and it was further revealed that total saponins of Tribulus terrestris L.leaves can inhibit inflammation through TLR4-NF-?B signaling pathway.This study successfully optimized the extraction technology of total saponins from leaves and fruits of Tribulus terrestris L.,and identified their main components,and evaluated comprehensively their antioxidant and anti-inflammatory effects.Through in vivo and in vitro inflammatory models,It was further revealed that total saponins of Tribulus terrestris L.leaves played an anti-inflammatory role by inhibiting the TLR4-NF-?B signaling pathway,which would provide a theoretical basis for the research and development of the anti-inflammatory medicine in veterinary clinic.