Increasing Biological Activities Of Saponins From Aralia Elata (Mig.) Seem By Hydrolyzing With β-Glucosidase

In this study the material is Aralia elata(Miq) Seem planted in Benxi county of Liaoning province.The method and parameters of extracting and purifying saponins from Aralia elata(Miq) Seem,the basic property of saponins,the hydrolyzing influence ofβ-Glucosidase on saponins and the boosting degree of biological activities hydrolyzed byβ-Glucosidase are studied by the numbers in this paper.The technics of extracting and purifying saponins from Aralia elata(Miq) Seem and the method of reducing toxicity and boosting activity are confirmed by this study.The applied extension is expanded and the economic value of saponins from Aralia elata(Miq) Seem is enhanced.The important conclusions gained in this study are as follows:The ultraviolet method of measuring saponins is established,the standard sample is the oleanolic acid,and the metrical wavelength is 291nm.It proves:the Aralia elata(Miq) Seem contains abundant oleanolic acid and homologous saponins,the saponins concentration can be calculated briefly by the oleanolic acid content.The orthogonal test analysed by difference demonstrates:the sequence of the ponderance affecting extracting saponins from high to low is the extracting temperature,the extracting time,the solvent ratio and the concentration of alcohol.The best extracting parameters are as follows:the extracting temperature is 70℃;the extracting time is 3h;the solvent ratio is 1:20;the concentration of alcohol is 90%.At these conditions the maximum extraction rate are 8.1%.The orthogonal test of ultrasonic-assisting treatment demonstrates:the sequence of the ponderance affecting extracting saponin from high to low is the solvent ratio,the extracting temperature,the ultrasonic-assisting time and the concentration of alcohol.The best extracting parameters are as follows:the solvent ratio is 1:20;the extracting temperature is 65℃;the ultrasonic-assisting time is 20min;the concentration of alcohol is 100%.At these conditions the maximum extraction rate are 8.7%.81.9%protein in the crude saponins of Aralia elata(Miq) Seem is removed by treating four times with chloroform.The orthogonal test of removing protein with pawpaw proteinase analysed by difference demonstrates:the sequence of the ponderance affecting doffing protein from high to low is the treating temperature,enzyme quantity,the treating time and the pH value.The best treating parameters are as follows:the treating temperature is 50℃;the enzyme dosage is 2%;the treating time is 2h;the pH value is 6.0.At these conditions 80.2% protein can be removed again,the protein concentration falls to 0.4mg/mL in the saponins solution.The adsorptive capacity of AB-8 resin reachs to 63.9mg/g,and desorption rate is 96.5%.The best purification condition of AB-8 resin is 400mL eluting solution,70%alchohol, 0.6mL/min velocity of flow and pH8.0.Six samples can be separated by eluting through silica gel column,the quantitive proportion is 34.3%,21.1%,16.9%,15.1%,7.9%and 4.7%.Foam test,Liebermann reaction,stibium chloride-saturated chloroform reaction and TCL test prove:there are abundant saponins component in extraction of Aralia elara(Miq) Seem.The monosaccharide are made up of rhamnose,glucose,mannitose,galactose and xylose.It offers foundation for boosting biological activities by hydrolyzing saponins.The examining condition of saponins components by HPLC is established,the inspecting wavelength is 215nm,acetonitrile and water are mobile phase eluting by gradient.There are four monomers in sample 1,2 and 3,five monomers in sample 4 and 5,six monomers in sample 6.The six samples all contain the oleanolic acid which retaining time is 14.7min.The examining method of the oleanolic acid content by HPLC is established,the inspecting wavelength is 215nm,acetonitrile and water are mobile phase.The oleanolic acid retaining time is 7.0min at these conditions.The oleanolic acid content is 45.6%,36.1%,31.4%,10.7%, 26.6%and 25.5%differently in the six samples,and 33.5%in the general saponins.The ultraviolet test indicates:there are six obvious absorbing peaks in the general saponins,the biggest absorbing peak is at 299nm.It shows:the general saponins contain multiplicate monomers,its molecule is big comparatively.All the six saponins samples contain several obvious absorbing peaks,the main absorbing peak closes with long wavelength gradually.It shows:the saponins monomers molecule become bigger,its polarity become lower.The infrared test indicates:all the saponins absorbing waves are similar,they contain functional groups such as aliphatic hydrocarbons,primary aliphatic alcohols and secondary aliphatic alcohols(Aliphatic carboxylic acids in sample 1 and 2) estimated by basic analyzed chart. They may contain long carbon chain with hydroxy,isomaltose and galactose estimated by particular analyzed chart.The orthogonal test ofβ-Glucosidase hydrolyzation analysed by difference indicates: the sequence of the ponderance affecting hydrolyzing saponins from high to low is the hydrolyzing temperature,the enzyme dosage and the hydrolyzing time.The best hydrolyzing parameters are as follows:the hydrolyzing temperature is 40℃;the enzyme dosage is 3.0mg; the hydrolyzing time is 120min.Six samples can be separated by eluting saponins hydrolyzed byβ-Glucosidase through silica gel column with methanol,the quantitive proportion is 34.8%, 24%,11.8%,11.3%,8.1%and 10.0%.The examining condition of hydrolyzed saponins components by HPLC is established,the injecting dosage is 10μL,the mobile phases are methanol and water with 70:30 proportion,the velocity of flow is 1.0mL/min,the inspecting wavelength is 215nm,the column temperature is 25℃.At these inspecting conditions sample E1,E2 and E3 contain four monomers,sample EA,E5and E6 contain five monomers.The six samples all contain the oleanolic acid which retaining time is 5.1min.The ultraviolet test of hydrolyed saponins indicates:All the six hydrolyzed saponins samples contain several obvious absorbing peaks,the main absorbing peak closes with short wavelength gradually,the biggest absorbing peak is at 236nm in most hydrolyzed samples.It shows:the hydrolyed saponins monomers molecule become smaller,its amount decreases.The infrared test indicates:all the hydrolyed saponins absorbing waves are similar,they contain functional groups such as aliphatic hydrocarbons,primary aliphatic alcohols,secondary aliphatic alcohols,aliphatic Primary amines and aliphatic carboxylic acids estimated by basic analyzed chart.They may contain long carbon chain with hydroxy or amino,unsaturated fatty acid, phenyl glucoside,mannitol,benzoic acid,isomaltose and galactose estimated by particular analyzed chart.Low concentration saponins solution can eliminate hydroxy and oxygen free radicel, the effect of eliminating free radicel submits positive pertinence with saponins concentration. The result of dispelling alcohol and defending ebriety test indicates:saponins of Aralia elata (Miq) Seem can restrain alcohol absorbed,extend alcohol bearing time,reduce ebriety maintaining time remarkably,and have a function of dispelling alcohol,defending ebriety and protecting liver.The result of antioxidating effect in vivo test indicates:saponins of Aralia elata(Miq) Seem can boost SOD activity,reduce the MDA concentration of organic tissue. Saponins of Aralia elata(Miq) Seem can increase spleen exponent and thymus exponent, enhance rat immunity;it also debases totall grease and totall cholesterin content,boosts high density lipoprotein content of blood serum.Saponins of Aralia elata(Miq) Seem can mediate blood sugar and insulin level of normal rat at low degree,can debase blood sugar and enhance insulin level of restraining model rat remarkably,can cure high blood sugar.All the activities become prominent after saponins hydrolyzed byβ-Glucosidase,it indicateβ-Glucosidase can enhances biological activities of saponins of Aralia elata(Miq) Seem.