Study On Preparation Process Optimization And Activity Of Female Gonad Saponins From Apostichopus Japonicus

Sea cucumbers are widely found in the coastal areas of China.It’s an important economic product in China.Sea cucumber contain varieties of biological active components,including sea cucumber polypeptides,phospholipids,saponins,etc.Among them,sea cucumber saponins have the activities of anti-biofouling,anti-tumor,relieve atherosclerosis,improve Parkinson’s disease,improve obesity,obesity-induced insulin resistance and so on.Apostichopus japonicus has a large output in the northern coastal areas of China and has high nutritional value.Due to lack of market guidance,as by-product,Apostichopus japonicus gonads are abundant but not fully utilized.In this paper,the Box-Behnken response surface method was used to optimize the ultrasonic-assisted extraction of female gonad saponins from Apostichopus japonicus.The crude saponin was separated and purified by high-speed countercurrent chromatography.The high-speed countercurrent chromatography conditions were optimized.The antioxidant activity of the crude saponin and the components after separation were explored and compared,and the immunity-enhancing activity of the crude extract were determined.The main results are as follows:（1）Ultrasonic-assisted method was used to extract saponin from Apostichopus japonicus female gonad,the optimal process conditions of response surface methodology were as follow:extraction time 7d,ethanol concentration 83%,ultrasonic time 33 min,solid-liquid ratio 1:20.Under this conditions,the extraction rate was（0.66±0.02）%.（2）The crude saponins of sea cucumber were separated by high-speed countercurrent chromatography.The optimized conditions were as follows:the ratio of solvent system was ethyl acetate:n-butanol:methanol:water（2:3:0.2:4.8,v/v/v/v）,the volume flow of mobile phase was 2 m L·min^-1 and the rotating speed was 800 r·min^-1.Both of them have good separation effect and have obvious characteristic peaks in HPLC.The antioxidant activities of crude saponin,component 1 and component 2 were analyzed and compared.It was found that three of them showed certain antioxidant activity,and the ABTS free radical scavenging ability of component 1 was significantly higher than that of crude saponin（P<0.05）;Component 2 showed significantly higher activity in scavenging DPPH free radical and ABTS free radical than crude saponin and component1（P<0.05）.This provides a reference for the further development and utilization and active molecular mechanism of female gonad saponins from Apostichopus japonicus.（3）The in vitro immune-enhancing activity of crude saponin was explored by macrophage RAW264.7 neutral red assay and cytokine content determination.The saponin-treated RAW264.7 group had significantly enhanced ability to phagocytose neutral red;its secretion of TNF-α,IL-6,and IFN-γwas increased,and the middle and high dose groups had obvious effects.The immunocompromised mouse model was established by injecting cyclophosphamide to explore the immune-enhancing activity of crude saponin.The thymus index and spleen index of mice fed with crude saponin were increased;the carbon clearance index and phagocytic index of monocyte-macrophage were increased;the proliferation activity of peritoneal macrophages and spleen cells were increased;the activity of NK cells were increased;CD4⁺T cells in the spleen were increased,and CD8⁺T cells were decreased,thereby the level of CD4⁺/CD8⁺were increased;the secretion of TNF-α,IL-6,and IFN-γwere increased.The crude saponin has the effect of enhancing immunity of mice,and the effect is obvious in the middle and high dose groups.