Study On Extraction Process Optimization And Anti-tumor Activity Of Saponins From Sapindus Mukorossi

Sapindus mukorossi is a woody plant of the Sapindus genus in the Sapindaceae family.It is a multifunctional and comprehensive application woody plant resource integrating biomass energy,biochemicals,ecological restoration and landscaping.The active ingredient saponins,rich in its peel,has biological activities such as anti-tumor,anti-inflammatory and antibacterial,and has been widely used in the fields of medicine,daily chemicals,agriculture and forestry.The research on the efficient extraction process and biological activity of Sapindus saponins has been a hot topic in this field.In order to efficiently develop and utilize Sapindus saponins resources,this study firstly screened and determined water and 60%ethanol as extraction solvents using the yield of saponins as the standard.Then this study optimized the water compound enzyme-assisted extraction process and 60%ethanol compound enzyme-ultrasonic assisted extraction process by single factor experiment,combined Plackett-Burman screening experiment and response surface methodology.In this study,the anti-melanoma(skin malignancy)and anti-ovarian cancer activities of Sapindus saponins were analyzed.The main findings are as follows:1.In this study,water and 60%ethanol were screened as the yield of saponins from 60%ethanol,water,ethanol absolute,methanol absolute,n-butanol among five solvents based on the yield of saponins,product safety,ease of solvent recovery and extraction cost.In the study,two extraction methods,water compound enzyme-assisted extraction and 60%ethanol compound enzyme-ultrasonic assisted extraction,were identified for extraction process optimization.2.In the water compound enzyme-assisted extraction process,the effect of each factor on the yield of saponins from largest to smallest was:enzymatic temperature>amount of compound enzyme>enzymatic time>compound enzyme ratio>pH value>solid-liquid ratio.In this study,three factors and three levels of Box-Benhnken optimization and validation experiments were conducted to select the enzymatic temperature,amount of compound enzyme and enzymatic time.The optimal process was finally determined as follows:the solid-liquid ratio of 1:10,pH value of 5,amount of compound enzyme of 7%,pectinase:cellulase=2:1,enzymatic temperature of 56℃ and enzymatic time of 127 min.The average yield of saponins from the Sapindus pericarp was 33.42%,with a relative error of 0.21%from the theoretical prediction,which was 1.41 times higher than the yield of water extraction.3.In the 60%ethanol compound enzyme-ultrasonic assisted extraction process,the effect of each factor on the yield of saponins from the largest to the smallest was:ultrasound time>amount of compound enzyme>ethanol volume fraction>enzymatic time>compound enzyme ratio>enzymatic temperature>ultrasound power>solid-liquid ratio.Ultrasound time,amount of compound enzyme,and ethanol volume fraction were selected for three factors and three levels of Box-Benhnken optimization and validation experiments.The optimal process was finally determined as follows:solid-liquid ratio 1:10,ethanol volume fraction 61%,amount of compound enzyme 7.5%,pectinase:cellulase=1:1,enzymatic temperature 60℃,enzymatic time 90 min,ultrasonic time 40 min,and ultrasonic power 350 W.The average yield of saponins from the Sapindus pericarp was 33.91%,with a relative error of 0.21%from the theoretical prediction,which was 1.41 times higher than the yield of 60%ethanol extraction.4.Saponins from 60%ethanol compound enzyme-ultrasonic assisted extraction and saponins from water compound enzyme-assisted extraction both caused morphological changes in melanoma A375 cells and ovarian cancer OVCAR8 cells,decreased cell proliferation viability,blocked the cell cycle of A3 75 cells and OVCAR8 cells,and promoted cell apoptosis of A375 cells and OVCAR8 cells.In summary,two saponins extraction processes were optimized by single factor experiment,Plackett-Burman factor screening experiments,and Box-Benhnken experiments.The yield of saponins could be increased to 1.41 times of that extracted by single solvent extraction method.Meanwhile,the saponins by both processes showed strong inhibitory activity on the growth and proliferation of human melanoma A375 cells and human ovarian cancer OVCAR8 cells,which provided technical and theoretical basis for improving the resource utilization efficiency and further using Sapindus saponins to develop antitumor drugs.