Extraction And Purification Of Total Saponins From Nigella Glandulifera And Its Hepatoprotective Activity

Nigella glandulifera Freyn is an annual plant in Nigella genus(Raunnculaceae),and its mature seeds is known as“black cumin seed”.Uighur people often used it in the crepe cake as a seasoning,which has a fragrant and a light taste.In this study,based on the single factor experiment,the optimal process of enzyme-assisted ultrasonic extraction of total saponins from N.glandulifera seed was optimized by using the response surface method.The macroporous resin purification process of the main saponin,the main marker component,saponin A from N.glandulifera was established,and its adsorption thermodynamics and adsorption kinetics were analyzed.The hepatoprotective activity of total saponins was examined by LO2 cells.The main findings are shown as follows:(1)Optimized conditions of modified enzyme-assisted ultrasonic extraction:solution temperature at 60°C,ethanol concentration at 54%,ratio of material to liquid at 1:37 g/m L,enzyme addition amount at 0.98%,ultrasonic frequency at 60 k Hz and extract time at 30 min.The yield of total saponins was 14.09±0.12%under this condition,the theoretical prediction value of the model equation was 14.17%,and the relative error was less than 3%.(2)HPLC determination method of saponin A form N.glandulifera was established,and chromagraphic conditions were shown as follows:Eclipsee XDB-C18column(250 mm×4.6 mm,5?m),acetonitrile:water(28:72)in mobile phase,column temperature at 35°C,flow rate at 1.0 m L/min,detection wavelength at 210 nm.(3)Through the adsorption and desorption experiments,D101 resin was selected from HPD-100A,D101,HPD-400,HPD-500,NKA,HPD-750,X-5 and AB-8macroporous resins,and it has a good enrichment and desorption function for saponin A(static adsorption amount 72.25 mg/g resin,desorption rate 94.08%).The optimum purification conditions of saponin A from N.glandulifera with D101 resin was as follows:the maximum loading mass 16.488 mg/g,loading concentration 4.5816mg/m L,loading rate 1.0 m L/min,the impurity removal water 90 m L,90 m L at 50%ethanol and elution flow rate 2.0 m L/min.The content of saponin in this extract is more than 55.0%and the recovery of saponin A is better than 99.0%.The static results show that both Freundlich equation and Langmuir equation can explain the adsorption behavior of D101 resin on the saponin A from N.glandulifera,the equilibrium concentration and the adsorption amount were increasing.The temperature of the reaction system was appropriately increased,which was beneficial to the improvement of the yield of the saponin A from Nigella glandulifera by purification and enrichment.Thermodynamic model studies show that?H>0,?S>0,?G<0,the system was endothermic,and belonged to the category of physical adsorption.The adsorption behavior of resin on the saponin A from Nigella glandulifera accorded with the quasi-secondary equation(4)Hepatoprotective effect of total saponins from N.glandulifera was preliminary studied in the LO2 cell injuryed by H₂O₂ experiment,and the result showed that this extract had a certain protective effect on H₂O₂-induced liver cell damage.