| Crotalaria sessiliflora L. is a Chinese herbal medicine, widely used in the treatment ofvarious types of cancer, such as esophageal cancer, cervical cancer and skin cancer and soon. Flavonoids and alkaloids are the main constituents in it. Conventional extractionmethods of the effective constituents such as heat-reflux extraction, cold soaking extraction,etc, are using lot of organic slovent, not environmentally friendly and time-consuming,have poor selectivity and productivity. In this paper, the multi-phase extraction was used toextract the effective constituents from Crotalaria sessiliflora L., The formation, propertiesand extraction characteristics of multi-phase system were studied. Multi-phase extractionwas combined with microwave-assisted extraction to extract the effective constituents, theextraction conditions were studied by sigle factor test under the action of microwave. Andthe influencing factors were further optimized. Based on the principle of the dissolution inthe similar material structure, the alkaloids and flavonoids were separated to some extent.The main researches and results were as following:1、 The methods to determination of flavonoids and alkaloids from Crotalariasessiliflora L. were developed by reversed high performance liquid chromatography. Themobile phases were consisted by acetonitrile-water containing0.5%phosphoric acid (14：86), the detection wavelength at340nm for vitexin and isovitexin, the retention time of thetwo constituents were30.66min and34.77min, respectively. Their linear range were from0.2080~20.08mg L1. Recoveries were between98~105.5%. The mobile phaseds wereconsisted by methonal-water containing0.1%ammonia (30：70), the detection wavelengthat220nm for monocrotaline, the retention time was11.13min. Their linear range werefrom1.092~109.2mg L1. Recoveries were between97.55~101.54%. These methods hadbeen already applied to determination of flavonoids and alkaloid from Crotalaria sessiliflora L.，and it turned to be a reliable method.2、 Using ethanol/(NH4)2SO4aqueous two-phase system as extraction agent，microwave-assisted aqueous two-phase extraction of the effective constituents fromCrotalaria sessiliflora L. was investigated. A response surface methodology, with a threelevels, three variable designs was applied based on the single factor optimizationexperiment. The optimum extraction conditions were: the concentration of alcohol was32%, the concentration of ammonium sulfate was22%, ratio of solvent to material50：1,extraction time8min, extraction temperature80℃. An experimental extraction yield ofvitexin243.39μg/g and isovitexin162.09μg/g were achieved under the optimal extractionwhich were very close to the optimal prediction extraction yield of vitexin242.08μg/g andisovitexin164.5μg/g, demonstrated the validation of the RSM model, indicating that themodel was adequate for the extraction process.3、Different kinds of non-ionic surfactant were choosed to form cloud point system,and the cloud point temperature, properties have been studied. The results showed that: thecloud point system was formed stable by Triton X-100and Tween80, but the cloud pointsystem which formed by Triton X-100had the lower cloud point temperature and higherextraction rate, the best extraction conditions were:2%of Triton X-100,63℃oftemperature,30min of time and0.05mol/L of HCl for vitexin and isovitexin.2%of TritonX-100,68℃of temperature,30min of time and0.1mol/L of HCl for monocrotaline. Theextraction rate of the vitexin, isovitexin and monocrotaline were respectively77.61%,75.36%and79.6%. Using Triton X-100solution as the extraction solvent, combinedmicrowave-assisted extraction and cloud point extraction to extract the effectiveconstituents from Crotalaria sessiliflora L.. Based on the single factor experiment, theextraction conditions were optimized by orthogonal experiment. The optimum extractionconditions were:5%of Triton X-100,90℃of microwave temperature, ratio of solvent tomaterial100：1,10min of microwave time for vitexin and isovitexin.3%of Triton X-100,80℃of microwave temperature, ratio of solvent to material100：1,10min of microwavetime for monocrotaline. And the yield of vitexin, isovitexin and monocrotaline were601.37μg/g,386.7μg/g,105.48μg/g respectively on their best extraction conditions. 4、Based on the aqueous two-phase system, we choosed a suitable organic solvent toform the three-phase system, the phase properties of ethanol/(NH4)2SO4/chloroform(petroleum ether, ethyl acetate) and PEG/(NH4)2SO4/chloroform (ethyl acetata) systemswere investigated, and also discussed the properties and extraction rate of the three-phasesystems which formed by different molecular weight of PEG. Finally, we usedPEG2000/(NH4)2SO4/ethyl acetate to extract the effective constituents from Crotalariasessiliflora L.. Using orthogonal experiment to optimize the microwave-assistedthree-phase extraction，results showed that the optimum extraction conditions were:14%of PEG,14%of (NH4)2SO4,90℃of temperature, ratio of solvent to material90：1,15minof time for vitexin and isovitexin.14%of PEG,16%of (NH4)2SO4,80℃of temperature,ratio of solvent to material90：1,20min of time for monocrotaline. Compared differentmethods to extract the vitexin, isovitexin and monocrotaline from Crotalaria sessiliflora L.,the microwave-assisted three-phased extraction had the highest yield.5、 The thermodynamics process of aqueous two-phase extraction, cloud pointextraction and three-phase extraction were probed, and the data was linear fitted. Theresults showed that all of the ΔS0and ΔH0in aqueous two-phase extraction, cloud pointextraction and three-phase extraction were bigger than zero, ΔG0were smaller than zero.From these results, it could be found that the aqueous two-phase extraction, cloud pointextraction and three-phase extraction were both endothermic and entropy increase, it can bespontaneous in certain temperature. Based on the characteristics and properties ofmicrowave-assisted extraction, aqueous two-phase extraction, cloud point extraction andthree-phase extraction, the microwave-assisted extraction technique coupled with aqueoustwo-phase extraction, cloud point extraction and three-phase extraction to extract theeffective constituents were compared. |
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