| Obesity has become one of the greatest threats to human life and health.The existing weight reducing medicines all have the defect of more side effects.Therefore,the development of more safe and effective weight loss drugs has become a hot topic of current research.Pancreatic lipase（PL）inhibitor extracted from natural plants can not decompose fat in food into free fatty acids by inhibiting PL of gastrointestinal tract to inhibit the utilization and absorption of fat,which can achieve good weight loss effect.At the same time,it has not been found any side effects.Therefore,it has been rapidly developed as a new weight loss drug.Dioscorea nipponica Makino（D.nipponica）is the rhizome of Dioscorea nipponica,which contains steroidal saponins as its main active component.Traditional Chinese medicine believes that it has many functions,such as dispelling wind,removing dampness,relieving pain,relieving cough and asthma,etc.In recent years,steroidal saponins contained in it have also been reported to have the functions of reducing blood lipid and regulating lipid metabolism,suggesting it was a potential weight loss drug.Therefore,in this paper,taking the steroidal saponins from D.nipponica as the research object,the inhibitory effect and mechanism of total saponins and aglycone on PL activity were explored by UV spectrophotometry,LC-MS,macroporous resin separation and purification technology and microwave-assisted biphasic acid hydrolysis method,and the kinds of saponins with inhibitory effect were screened from D.nipponica and the structure-activity relationship between inhibitory effect and structure of saponins and aglycones was clarified.To develop the medicinal value of D.nipponica and develop new plant-derived PL inhibitors,it provides more research ideas and research basis.The main contents and results were as follows:1.The extraction and purification methods of total saponins from D.nipponica were determined.And based on the influencing factors of enzymatic reaction kinetics,the UV spectrophotometry was established to determine the inhibition of total saponins of D.nipponica on Porcine Pancreas Lipase（PPL）.The final reaction conditions were as follows:the concentrations of PPL and p-NPP were 4.00 mg/m L and 0.320 mmol/L respectively,the reaction temperature and p H were 37℃and 7.5,the best storage time of PPL was less than 15 min,the extract of D.nipponica was incubated with p-NPP for 5 min and then added with PPL to react for 5 min,and the influence of DMSO,the background solvent,on the inhibition of PPL activity was deducted when calculating the inhibition rate.Under the optimum reaction conditions,the inhibitory rate of the extract on PPL activity was 68.34±1.47%,and IC50 was107.05μg/m L.The Lineweaver-Burk plot showed that the extract of D.nipponica was the noncompetitive inhibition of PPL.At the same time,the inhibitory ability of the extractive solution to PPL activity was enhanced after it was placed for a long time.2.Qualitative analysis of steroidal saponins in the extract of D.nipponica by HPLC-Q-TOF-MS/MS was carried out,and six known steroidal saponins were identified:protodioscin,protogracillin,pseudoprotodioscin,pseudoprotogracillin,dioscin and gracillin.A HPLC method was established for the determination of steroidal saponins in D.nipponica.The system adaptability test results were good;The linear relationship of dioscin was good in the concentration range of 7.812-500.0μg/m L（R2>0.9999）.The RSD of precision,stability and repeatability of 6 saponins was≤2.84%.The average recovery rate of spiked recovery was 96.72-101.6%,RSD≤1.87%.The established method was applied to the content determination of 4 samples of D.nipponica with different storage time.The determination results showed that the contents of protodioscin and protogracillin decreased from 1450μg/m L and458.8μg/m L to 927.3μg/m L and 313.9μg/m L,respectively,however,the contents of dioscin and gracillin increased from 215.2μg/m L and 60.49μg/m L to 756.8μg/m L and 180.2μg/m L,respectively,when the extraction solution was left for 0 day to 3 months.Finally,the kinds of saponins that have effects on PPL were screened by HPLC.The contents of 4 saponins after incubation with PPL decreased from 0.3148,0.1010,0.2137 and 0.0596 mg/m L to 0.2872,0.0782,0.1350 and 0.0282 mg/m L,respectively.At this time,the kinds of 4saponins and the order of their inhibitory effects on PPL were as follows:dioscin>gracillin>protodioscin>protogracillin.3.By comparing the adsorption and desorption effects of different resins on D.nipponica extract,D101 macroporous resin was finally selected for the separation and purification of the saponins of D.nipponica.The static adsorption curve of the resin showed that the best adsorption time was 60 min.The results of adsorption kinetics showed that the adsorption of D101 resin on D.nipponica extract conformed to the pseudo-second-order kinetic model,and based on the particle diffusion kinetic model,the film diffusion was determined as the main speed control step.The separation and purification process of saponin by D101 macroporous resin was successfully established.The technological conditions were as follows:when the total saponin concentration of D.nipponica was 1.816 mg/m L,the p H of the sample was 7.0,the volume was 40 m L,the flow rate was 30 m L/h,and the amount of water-washed polysaccharide was 20 m L.Using 360±10 m L 40%（v/v）ethanol-water 190±15m L 50%（v/v）ethanol-water and 230±10 m L 90%（v/v）ethanol-water as eluants,we got 0.8508 g mixture of protodioscin and protogracillin,0.3176 g mixture of pseudoprotodioscin and pseudoprotogracillin and 0.6125 g mixture of dioscin and gracillin.The experiment of inhibition of PPL activity proved that 40%（v/v）ethanol eluent and 90%（v/v）ethanol eluent could inhibit PPL activity,and the inhibition rates were 10.05±1.21%and 63.21±1.41%,respectively.Through comparison,it was found that the inhibitory rate of 90%（v/v）ethanol eluate after separation and purification with D101 resin on PPL was 2.51 times that of D.nipponica extract stored for three months before separation and purification.4.The microwave-assisted biphasic acid hydrolysis method was established to obtain diosgenin.Through single factor investigation and BBD,and then adjusted according to the actual conditions:acid concentration of 1.5mol/L,liquid-solid ratio of 21 m L/g,extraction temperature of 91℃and extraction time of 34 min.The yield of diosgenin was 17.23±0.32 mg/g,which was close to the predicted value of the model of 17.16 mg/g,indicating that the designed model was effective and reliable.Comparing the yield of diosgenin by four methods,microwave-assisted biphasic acid hydrolysis,microwave-assisted single-phase acid hydrolysis method,water bath reflux biphasic acid hydrolysis and water bath reflux single-phase acid hydrolysis,it was found that the yield of diosgenin obtained by microwave-assisted biphasic acid hydrolysis was the highest,compared with other methods,and the extraction time was shortened by nearly 90 min by water bath reflux method.A HPLC method was established to determine the content of diosgenin.The system adaptability test results showed that this method meets the analysis requirements.The linear relationship of diosgenin was good in the concentration range of 0.0100-0.640mg/m L（R2>0.9999）.The RSD of precision,stability and repeatability were1.21%,2.23%and 1.86%respectively.The maximum inhibitory rate of diosgenin on PPL activity was 56.18±1.38%,IC50 was 191.42μg/m L,and the Lineweaver-Burk plot showed that diosgenin was the noncompetitive inhibition of PPL.At last,the inhibitory effects of different hydrolysis products containing the same concentration of diosgenin obtained when methanol and ethanol extraction systems respectively on PPL activity were compared.The results showed that there was no obvious difference in the degree of inhibition,which further clarified the inhibitory effect of diosgenin on PPL activity.Diosgenin and PL molecular docking results showed that diosgenin and Leu2,Leu19 and Phe5 amino acid residues on PL formed hydrophobic binding pi-alkyl bond. |
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