| This dissertation includes four parts:Part One literature review.Research on Status of the chemical constituents and pharmacological effect of stevia,and to explore its medicinal value.The general study situations of caffeoylquinic acids which are the main chemical components of stevia.The research progresses on correlation of pharmacokinetics and pharmacodynamics of Chinese prescription in recent years were reviewed.129 references were cited.Part Two Study on the in vivo and in vitro chemical constituents of phenolic effective fraction of the steviaChapter 1 to Chapter2This paper,based on the early research of the stevia phenolic effective fraction preparation technology,the preparation of the phenolic effective fraction of the stevia,total phenol content is 53.78%,fla is 16.75%,compliance with preparation and quality standards.And using a variety separation technology,to research chemical constituents of the phenolic effective fraction of the stevia.Finally,the use of1H-NMR,13C-NMR,2D-NMR,ESI-MS,HRFAB-MS and UV spectra to identify 11 compounds,two new compounds and six compounds first isolated from the plant,and clarifies the chemicals foundation of the phenolic effective fraction of the stevia.Summarizes some of the fragmentation patterns of known composition,and provides the basis of the stevia phenolic effective fraction for latter HPLC-MS analysis.Use pre-trial reaction to analysis the components type of the stevia phenolic effective fraction,indicating the stevia phenolic effective fraction in addition to containing phenolic constituents and tannin components,may also contain sugar,flavonoids,coumarins,lignans,quinones ingredients.Established the HPLC-PDA,HPLC-PDA-MS spectrum analysis features of the stevia phenolic effective fraction,the characteristic absorption peak of 51 chemical types were confirmed,and by adding a reference to identify the 16 chemical composition.And analysis each secondary characteristic peaks of mass spectrometry,suggesting the 19 possible structures.Using UPLC-PDA method for determining the 31 ingredients into the blood of the stevia phenolic effective fraction,including 11 components which natural ingredients directly into the blood(prototype into the blood),there are 40 vitro components not found in vivo,in addition to 9 components into the blood are not found invitro,presumably newborn metabolite(metabolites into the blood);Besides 11 components which blanks and drug-containing serum contain,may be endogenous component.Part Three Study on the in vivo and in vitro determination method of the antidiabetic drug-like componentsChapter 3Through the study of the chemical composition of the body of stevia,F1,F2,and F3 in Stevia was found as its main drug-like components of antidiabetic.The main drug-like ingredients include:F1,characteristic index components was f1a;F2,characteristic index components was f2a;F3,characteristic index components was f3a.To consider the dynamic changes of Stevia antidiabetic drug-like ingredient content in the herbs,the phenolic effective fraction and serum(the ratio was also congsidered),also to provide a means for further determination of pharmacokinetic characterization of stevia.The trial was the first to establish stevia quality control methods by UPLC-based stevia major antidiabetic drug-like ingredients f1a,f2a and f3a,considerations the content of the main drug-like ingredients of Stevia herbs and phenolic effective fraction.f1a,f2a,f3a,respectively,within the range of 0.0401~1.202 μg,0.0084~0.2526 μg,0.0088~0.2652 μg mass and peak area showed a good linear relationship,the recoveries were 98.18%,98.75%and 97.47%.6 samples assay results show that the method is simple,accurate,reliable,and can be used for the main content of Stevia drug-like ingredient determination,and as one of quality methods of stevia and its major antidiabetic drug-like components.Use this method to determine the content of three types of antidiabetic drug-like component in the herbs and the phenolic effective fraction of Stevia.Further,carry on the correlation analysis of the 11 prototyp composition of the herbs and the phenolic effective fraction of Stevia,the results confirm that the content of prototype in the phenolic effective fraction of Stevia are higher than the herbs.In addition,we have found that the change of relative proportion of the prototype component is a little change,it is confirmed that during the preparation process,the natural proportions of herbs in phenolic effective fraction of Stevia are preserved very well.UPLC method for measuring the concentration of plasma has been established of a rat serum of Stevia major antidiabetic drug-like ingredients fla,f2a,f3a.It was turned out that plasma concentration of fla,f2a,f3a were in 0.0517~46.6 μg/mL,0.731-658 μg/mL,0.0491~44.2 μg/mL,with good linear relationship,extraction recoveries were more than 75%,recoveries between 90%to 110%,intra-day precision and stability of RSD were less than 10%.The measurement results showed that established method is accurate and reliable,high sensitivity,and it can be better used in stevia pharmacokinetic studies.Part Four The drug system characterization of stevia in antidiabetic system model.Chapter 4 to Chapter 61 Study of PK-DI characterization in different parts of the state for the stevia phenoliceffective fraction.Based on f1a,f2a and f3a,the characteristics drug-like ingredients of stevia,the multifaceted holistic medicine effective part of the stevia phenolic effective fraction pharmacokinetic characterization were discussed.Based on the plasma concentration of stevia characteristics drug-like ingredients,the plasma concentration of drug-related component of characteristics drug-like ingredients expression,the PK research methods of the drug plasma concentration ratio comprehensive characterization of prototype and metabolic components of characteristics drug-like ingredients expression.Obtained multifaceted PK features and parameters for the PK-PD-DI association studies,and provided information support for drug system.Established UPLC measuring method of index drug-like ingredients to measure the drug concentration of Stevia components into the blood and calculate the pharmacokinetic parameters,and dynamic characterization the plasma concentration and the relative composition proportions.Use ALX-induced and STZ-induced diabetic model to characterize the multi-level parts of PK-DI of the stevia phenolic effective fraction.(1)According to the results of the Tmax of the stevia phenolic effective fraction,the normal state,the two coordinated absorption peak time were 0.167 and 4 h.The ALX(hypoglycemic)state,four coordinated absorption peak time were 0.083,0.5,1 and 4 h.The ALX(sugar tolerance)state,3 coordinated absorption peak time were 0.25,1.5 and 6 h.The STZ(hypoglycemic)state,5 coordinated absorption peak time were 0.167,0.5,1.5,2 and 6 h.The STZ(sugar tolerance)state,5 coordinated absorption peak time were 0.167,0.5,0.75,2 and 6 h.Results show that the same type or different types of drug-like ingredients at the same time to peak,illustrate the collaborative class of drug-like ingredients,embodies the medicinal properties of the more ingredients coordination absorption of the Chinese traditional medicine.Through the comparative analysis under different pathological state of the collaborative point,found the each ingredients into the blood of the stevia phenolic effective fraction play a synergistic effect of three time points as follows:0.083~0.5 h,1-2 h and 4-6 h.(2)The results of the Cmax of the stevia phenolic effective fraction showed that it was compared the prototype and metabolic components in Cmax of different physiological states and found that except the peaks 15 and 28,the other prototype components Cmax in pathological states were higher than normal,except peaks 17,20 and 24,the other metabolic components in Cmax pathological conditions were higher than normal,indicating that the extent of absorption of these prototype components(peaks 8,12,14,18,19,22,23,27,29)and these metabolic components(peaks 10,13,16,21,30,31)in pathological states is higher than the normal state,play the role of the strength efficacy.Comparing Cmax of different components of the prototype in the same physiological state,it was found that Cmax of the five ingredients of peaks 14,18,19,22,28 and 4 ingredients of peaks 10,16,24,31 were higher than the other prototype and metabolic components in different pathological state,indicating that the five prototype ingredients and 4 metabolic ingredients play the role of the strength efficacy.(3)The results of area under the curve(AUC)of the stevia phenolic effective fraction showed that by comparing AUC of prototype and metabolic components in different physiological state,except peaks 8,15,28 and 29,the AUC values of other prototype components in the pathological state were higher than normal group,except peaks 17,30 and 31,the AUC values of other metabolic components in a pathological state were higher than normal,indicating that these prototype components(peaks 12,14,18,19,22,23,27)and metabolic components(peaks 10,13,16,20,21,24)affinity for different pathological states is higher than the normal state.In the same physiological state,after comparing AUC value of the different prototype components of peaks 18,19,22,23,28 and metabolic components of peaks 10,16,24,30,31,these ingredients AUC values in different pathological states are higher than other prototype and metabolic ingredients,indicating that these components in different pathological states have a high affinity.(4)The half-life sites(T1/2)and mean residence time(MRT)of drug-like ingredients of stevia phenolic effective fraction showed that different physiological states T1/2 and MRT comparison and found that the T1/2 and MRT of prototype F1(peaks 8,12,14,28,29)in pathological states has been shortened to more normal state,indicating FI have a shorter residence time in the body from the pathological state,and a faster metabolize;the T1/2 and MRT metabolism F1 peaks 13,20,21 in pathological states has been shortened to more normal state,indicating that the peaks 13,20,21 is likely the metabolites of prototype F1 components.Also found that the T1/2 and MRT of prototype T1 peak 19 and prototype F3 peaks 23,27 in pathological states has been extended to more normal state,and the T1/2 and MRT of metabolic F2 peaks 10,16,24 and F4 peaks 30,31 in pathological states has been extended to more normal state,indicating that the peaks 10,16,24 and 30,31 is probably the metabolites of prototype F3,F2 and F4components.This shows that the metabolic drug-like ingredients from prototype drug-like ingredients,prototype drug-like ingredients derived from natural stevia phenolic effective fraction,reflecting the nature of the drug-like ingredients.2 The PD-DI characterization studies of the stevia phenolic effective fraction in different states,to provide the PD data for PK-PD-D1 association studies.Using ALX-induced and STZ-induced diabetic model,according to the results of pharmacokinetic curve choose the same point in time as the efficacy time of the experiment,hypoglycemic and glucose tolerance are used as indicators of the efficacy of the stevia phenolic effective fraction in antidiabetic pharmacodynamic study.Using SAS software analysis ANOVA statistical between groups,whether there was a significant difference based on P value judgment.Determined ALX(hypoglycemic)experimental for hypoglycemic efficacy time points according to the order:1.5,4,0.25 and 0.75 h;ALX(glucose tolerance)test for glucose tolerance efficacy time points according to the order:0.5,0.167 and 1.5 h;STZ(hypoglycemic)experimental for hypoglycemic efficacy time points according to the order:6,1.5,2.5 and 0.25 h;STZ(glucose tolerance)test for glucose tolerance efficacy time pointsaccording to the order:1.5,0.75 and 0.167 h.Under ALX-induced and STZ-induced diabetic model,the effects time of the hypoglycemic experiment is slower than the efficacy time of the glucose tolerance test.Efficacy time points is also divided into three sections:0.167-0.75 h,1.5~2.5h and 4~6 h.The drug-like ingredients peak contribution analysis under different pathology based on the efficacy time point.The ALX(hypoglycemic)state,peaks 23>24>22>10>12>13>21>16>20>15>8>19>31>30;The ALX(sugar tolerance)state,peaks 23>19>22>27>31>10>30>16>21>20>12>28>15>17>24>13>8>14>18;The STZ(hypoglycemic)state,peaks 23>13>15>12>21>20>30>31>28>14>10>16>18>8;The STZ(sugar tolerance)state,peaks 18>24>21>28>29>22>15>19>13>30>20>12>17.Through the different pathological condition kinds of medicine composition of the efficacy of time point of peak contribution analysis,found that different pathological model of common components:F1 peaks 12,13,15,20,21,F3 peaks 22,27,F2 peaks 10,18,20,T1 peak 19 and F4 peaks 30,31,shows these ingredients are a particular focus on hypoglycemic directly effect.3.Under the high blood sugar model,summary the blood drug concentration data and efficacy data of the index drug-like ingredients of the stevia phenolic effective fraction,carried PK-PD-DI association analysis.Screening antidiabetic drug system of the stevia phenolic effective fraction,and to determine the effects associated composition of the stevia phenolic effective fraction.(1)Screening hypoglycemic drug system of the stevia phenolic effective fraction under ALX-induced diabetic model:F1(peaks 8,12,13,14,20,21,28,29),T1(peak 19),F2(peaks 10,16,18,24),F3(peaks 22,23,27).Contribution analysis for these efficacy components:peaks 23>22>24>28>12>19>13>16>10>14>29>21>8>20>18>27.Peak 22(F3,f3c),the largest contribution and as the prototype components,can be used as characteristic indexes composition of ALX hypoglycemic state.The blood concentration of peak 28 is reference 1,other components are the ratio,get the following combination between prototype and metabolite effective components.The best ratio of prototype effective components:peaks 8:12:14:18:19:22:23:27:28:29=0.111:0.573:0.586:16.34:0.954:2.540:1.723:0.088:1:0.473,metabolic effective components:peaks 10:13:16:20:21:24=33.01:1.465:10.56:0.433:0.293:31.91.(2)Screening sugar tolerance drug system of the stevia phenolic effective fraction under ALX-induced diabetic model:F1(peaks 8,12,13,14,20,21,28,29),T1(peak 19),F2(peaks 10,16,18),F3(peaks 22,27).Contribution analysis for these efficacy components:peaks 16>10>13>22>14>28>19>12>29>21>8>27>18>20.Peak 14(F1,f1g),the largest contribution and as the prototype components,can be used as characteristic indexes composition of ALX sugar tolerance state.The blood concentration of peak 28 is reference 1,other components are the ratio,get the following combination between prototype and metabolite effective components.The best ratio of prototype effective components:peaks 8:12:14:18:19:22:27:28:29=0.174:0.547:0.728:1.419:0.803:0.567:0.077:1:0.399,metabolic effective components:peaks 10:13:16:20:21=15.39:3.519:3.463:0.308:0.215.(3)Screening hypoglycemic drug system of the stevia phenolic effective fraction under STZ-induced diabetic model:F1(peaks 8,12,13,14,15,20,21,28,29),F2(peaks 10,24),F3(peaks 22,23),F4(peaks 17,30,31).Contribution analysis for these efficacy components:peaks 24>17>23>22>28>14>12>15>13>21>20>29>31>30>10>8.Peak 23(F3,f3b),the largest contribution and as the prototype components,can be used as characteristic indexes composition of ALX hypoglycemic state.The blood concentration of peak 28 is reference 1,other components are the ratio,get the following combination between prototype and metabolite effective components.The best ratio of prototype effective components:peaks 8:12:14:15:22:23:28:29=0.139:0.529:1.876:0.765:1.341:0.794:1:0.929,metabolic effective components:peaks 10:13:17:20:21:24:30:31=34.54:2.441:0.297:0.513:0.354:27.25:8.533:7.176.(4)Screening sugar tolerance drug system of the stevia phenolic effective fraction under STZ-induced diabetic model:F1(peaks 12,13,14,15,20,21,28,29),T1(peak 19),F2(peaks 10,18),F3(peak 22),F4(peak 30).Contribution analysis for these efficacy components:peaks 28>29>30>13>12>19>20>18>14>22>15>10>21.Peak 28(F1,f1a),the largest contribution and as the prototype components,can be used as characteristic indexes composition of STZ sugar tolerance state.The blood concentration of peak 28 is reference 1,other components are the ratio,get the following combination between prototype and metabolite effective components.The best ratio of prototype effective components:peaks 12:14:15:18:19:22:28:29=0.462:0.631:1.082:6.559:0.526:0.139:1:0.641,metabolic effective components:peaks 10:13:20:21:30=8.146:3.603:0.421:0.290:8.136.(5)Screening the common antidiabetic drug system of the stevia phenolic effective fraction F1(peaks 12,13,14,20,21,28,29),F2(peak 10),F3(peak 22).Which phenolic acids(peaks 12-f1d,14-f1g,28-f1a,29-f1f)and flavonoids(peak 22-f3c)for the total of the prototype effective ingredients,and can be used as the index composition of stevia herbs and phenolic effective fraction for quality control.(6)According to PK-PD-DI associate analysis,summed up the three major property of drug system characterization:nature,collaborative,and affinity.Part Five Summary and discussionChapter 7Analysis of the experimental results of the various parts of the discussion of this paper is to clarify the main innovations:1 The chemical constituents of stevia phenolic effective fraction,11 compounds were identified,TYJ-10 and TYJ-12 are 2 new compounds,and found a new kind ingredients of stevia--phenylethanoidics.Chemical type of 51 characteristic absorption peak confirmed,identified 16 chemical composition,and infer 19 possible structures.2 It is the first time to establish the UPLC-PDA method to determine f1a,f2a and f3a,three different types of drug-like ingredients of antidiabetic.And determine the content of three different types of drug-like ingredients of antidiabetic,and carry on associate analysis between stevia herbs and phenolic effective fraction based on the 11 components of prototype into the blood.3 A Comparative Study of stevia phenolic effective fraction PK-DI behavior was carried out firstly under the normal group,ALX hypoglycemic model group,ALX glucose tolerance model group,STZ hypoglycemic model group,STZ glucose tolerance model group,and carry on associate analysis of PK-DI characterization,and show the property of nature,collaborative,and affinity of drug-like ingredients.4 A Comparative Study of stevia phenolic effective fraction PD-DI behavior was carried out firstly under the ALX hypoglycemic model group,ALX glucose tolerance model group,STZ hypoglycemic model group,STZ glucose tolerance model group.and carry on associate analysis of PD-DI characterization,and show the collaborative property of drug-like ingredients.5 Screening out stevia phenolic effective fraction antidiabetic drug system for the first time according to PK-PD-DI associate analysis.Filter prototype efficacy ingredients,which can be used for the index compositions on stevia herbs and phenol effective parts quality control.Provide pharmaceutical chemical basis for developing the stevia phenolic effective fraction to the 5 kinds of new antidiabetic drug. |
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