Study On Aqueous Two-phase Extraction Coupled With Reverse Micelles System For Separation Of Alkaloids From Sophora Flavescens Ait

Modern pharmacological studies had demonstrated that alkaloids in Sophoraflavescens Ait. prossess anticancer, antitumor and antivirus and so on. So, they havewidely used in the clinical treatment and get more and more attention as a greenpesticide. In order to obtaien zhese alkaloids, the extraction and separation of theChinese medicinal herbs such as Sophora flacescens Ait.. Aqueous two-phaseextraction (ATPE) and reverse micelles extraction (RME) are two new separationtechniques developed in recent years. The two techniques have many advantagessuch as less separation procedure, milder operating condition, and easy to enlargemanufacture and less residue of organic solvents in the product etc. Thus, they havebeen extensively used to purify different proteins, enzymes, amino acids and activeconstituents in natural products and traditional Chinese medicines.In this paper, the ATPE and RME techniques with the system of EtOH/(NH4)2SO4and sodium dodecyl benzene sulfonate (SDBS)/isooctant/n-octanolwere used to extract the alkaloids from Sophora flavescens Ait. to get more purifiedalkaloids. The formation, properties and characteristics of extaction systems wereinvestigated systematically. The main researches and results were as following:1. A new method determination of alkaloids in Sophora flavescens Ait. wasdeveloped by reversed haigh performance liquid chromatography (R-HPLC) coupled with Pressurized Microwave-assisted extraction (PMAE) technique. Usingmethanol-acetonitrile-0.1%ammonia solution as mobile phase, the alkaloidsextracted by PMAE were separated on the C18column with gradient elution. Settingdetection wavelength at220nm, the retention times of main alkaloids separatedsuch as oxymatrine, N-oxysophocarpine, sophoridine, matrine and sophocarpinewaere7.31,8.25,26.11,40.37and42.48min, respectively. Their linear range warefrom2.5mg/L to200mg/L. Recoveries were between93.87%and100.9%. TheirRSDsranged from0.53%to2.53%. The method had been applied to determinationof alkaloid in Sophora flavescens Ait. successfully. It was simple, fast with goodprecision and repeatability.2. Several aqueous two-phase systems such as PEG/(NH4)2SO4, EtOH/saltsand Triton X-100/salts ware formed using the PEG, ethanol and Triton X-100andas salts phase substances. The formation, properties and extraction characteristicswere studied and compared in detail for several systems mentioned. EtOH/(NH4)2SO4could form fastly to stable in the wide phase range, and extract alkaloidsto the top phase containing ethanol with higher extraction rate. The experiments ofoptimum extraction conditions were perfomed. The results showed that optimumextraction conditions were38%of ethanol,18%of ammonium in the system, pH=7,30℃of temperature,15min of extraction time. The extraction rate of theoxymatrine and matrine were respectively96.17%and95.74%. Using theEtOH/(NH4)2SO4system to extract alkaloids from the water extract from Sophoraflavescens Ait. The results indicated that the yield of the alkaloids was20.54mg/g,the extraction rate was92.54%and the purity can increase from34.04%to71.01%.The results showed the ΔH0and ΔS0in the ATPE process were greater than zero,the extraction of alkaloids was endothermic and entropy increase, it can bespontaneous essentially due to ΔG0less than zero.3. PMAE was combined with ATPE firstly to extract alkaloids directly fromSophora flavescens Ait.. The method named Microwave-assisted aqueoustwo-phase extraction (MAATPE) was developed. The aqueous two-phase system was not only an extacting solvent to extract the alkaloids from Sophora flavescensAit. but also the extractant to prutiy the alkaloids at the same time. The extractionconditions were investigated by sigle factor test. Based on their results, theinfluencing factors were further optimized with central composite design andresponse surface method (CCD-RSM). With the helpof software Design expert8.0,the optimal extraction conditions were28%of ethanol,18%of ammonium sulphatein ATPE system,60:1of ratio solvent to material and90℃of extractiontemperature. Under this optimal conditions, the yield and extraction rate ofalkaloids were up to66.66mg/g and92.02%respectively, the purity were up to69.64%. This method could set extraction and purification of alkaloids for oneprocedure, and make it easy to complete more fastly and simply.4. Comparative study had been studied systematically for the formation andproperties of several reverse micelles systems, using different anionic surfacetants.The result showed that reverse micelles system of sodium dodecyl benzene sulfonate(SDBS)/isooctant/n-octanol had higher selectivity to extract alkaloids under pH was5.The influencing factors including pH, water content W0, extraction time andtemperature, the concentration of the surfactant and salts were investigated. Theconcentration of SDBS and the water content W0can cause the change of surfacetention and the viscosity, results in effcting on the mass transfer of the alkaloids. Theoptimal extraction conditions were that pH was5.0, water content W0was25, theconcentration of SDBS and KCl were0.05mol/L, the forward-extraction time andbackward-extraction time were5min and20min respectively, the temperature wasroom temperature. Under the optimal condition, there was about84.90%of alkaloidscould be solubilized in the reverse micelles system, and the purity of alkaloids were upto85.43%. The extraction mechanism of RME was discussed thermodynamically. TheΔH0and ΔS0were greater than zero in the lower range of temperature, the extractionprocess was endothermic and entropy increase and the process can be spontaneous.However, At more than40℃of temperature, it would lead to damage the reversemicelle, its physicochemical change depended on the dissimilar mechanism for extraction of alkaloids.5. According to distinguishing features of ATPE and MRE, a new method ofextracting alkaloids from Sophora flavescens Ait. was put forward with microwave-assisted aqueous two-phase extraction coupled with the reverse micellesextraction(MAATPE-RME). Using EtOH/(NH4)2SO4system as solvent to extractmore alkaloids from Sophora flavescens Ait. with microwave assisted extraction(MAE), they can be purified efficiently by RME with SDBS/isooctant/n-octanolsystem. The yield and the purity of alkaloids were up to55.38mg/g and90.92%respectively, being2.5times higher than that of RME and increasing about20%inpurity than that of ATPE. The method could be used to extract alkaloids from not onlySophora flavescens Ait. but also Radix Sophorae Tonkinensis. Similarly, using thefoucus microwave-assisted extraction(FMAE) also combined with ATPE and RME toextract the alkaloids in larger scale, the results showed that the yield of alkaloids wasincreased significantly. Throgh purification of the extract on the silica gel column, theproducts of oxymatrine, sophoridine and matrine monpmer could be obtained in thepurity with98.07%、96.62%and94.37%respectively. The technique can be regulateand enlarge easily. it has a very good application prospects.