Study On Extraction, Purification And Separation Of Alkaloids From Common Fenugreek Seeds

Fenugreek (Trigonella foenum-grae-curn L.) belongs to the Leguminosae Papilionoideae. is an annual herb. Alkaloids of fenugreek have effect on hypoglycemic, adjusting lipids, anti-cancer, anti-tumor, anti-ulcer effect, keeping from inJury cerebral ischemic and kidney impotence, losing weight and else. As far as we know, fenugreek seeds contain Gentianine, Carpaine, Choline, Ttrigonelline. In this study, raw material is powder of mature seeds of fenugreek. The heating reflux method and response surface methodology to be used to optimize in the extraction process of the alkaloids. AB-8macroporous resin and HSCCC were applied to purify fenugreek alkaloids.The study will provide a theoretical basis for alkaloid extraction, purification and separation of further study.The main results of the test were which as follows:The first electing three factors by solvent extraction method with the single factor experiment. There is a peak at18-19min of the standard sample of Trigonelline by HPLC. The standard curve is:y=75970494x+77968, the equation shows a good linear relationship in the16～96μg·mL-1(r=0.9999, n=6). Then, the response of alkaloids yield was investigated by ethanol concentration, extraction time, pH with response surface experimental design method. The results showed that: influencing factors impact from strong to weak of fenugreek alkaloid yield are:ethanol concentration, extraction time, pH value. The most significant factor is ethanol concentration which performance surface is relatively steep. By the Design Expert software for each regression fitting, the final regression equation is: Y=0.86+0.068×A+0.070×B-0.025×C+0.023×A×B+0.010×A×C-0.021×B×C-0.044×A2-0.073×B2-0.066×C2.Useing the software to optimize the yield of alkaloids,the analysis of the results of the response surface are:the concentration is83.8%, time is5.32h, pH is2.64. According to the actual situation, the correct process parameters are:the concentration is85%test time is5.5h, pH is2.6. Confirmatory test, the average yield of fenugreek alkaloids is0.904%(n=3, RSD=1.17%).Using AB-8macro porous resin to purify fenugreek alkaloids, alkaloids were identified by ultraviolet spectrophotometer, characteristic precipitation reaction, the thin-layer chromatography. Come to the Conclusion is:to determine the elution conditions of the AB-8macro porous resin purification of fenugreek alkaloids. The specific steps are as follow:AB-8macro porous resin which pretreatment is wet packed to the column, washed to alcohol. Stand for2h to absorb the liquid water,50%ethanol,70%ethanol elution are used with three times of the column volume. Collect the finally target liquid. Water elution can wash out a large number of water-soluble impurities, and50%ethanol elution can wash out the impurities of the Flavonoids, and after the elution of70%ethanol washing, the desired target solution is collected. Separate alkaloids by HSCCC and the solvent system of the test is N-hexane-ethyl acetate-methanol-water (2:5:2:5).There needs1000mL solvent system volume. The Optimization values by solvent system selection software, n-hexane-ethyl acetate-methanol-water as131.69,423.96,212.77,330.97mL. In the procession for separating sample liquid, it needs:the weight of sample is20mg, each of2mL of up and down relation, the speed of machine is800r/min, the wavelength of detection is33nm, the time of detection is150min, then injection once. There were four peaks, according to the identification of thin-layer chromatography (TLC) with iodine vapor color, four peaks be inferred that are alkaloids. Four components were measured by high performance liquid chromatography (HPLC), the peak time of the component C was similar as Trigonelline, that can inferred that group C is Trigonelline. The other three components can not be determined.