| Mung bean,as the seed of Vigna radiate L which belongs to a family of Leguminosae,has been cultivated in the north and south China for more than two thousand years.It is well-known that mung bean is commonly used as food and drug,which is rich in nutritions and has antipyretic-detoxicate effect.Mung bean was collected in Chinese pharmacopoeia 2015 as a medicine in many historical preparations,not as Chinese herbal medicines or medicinal slices.Moreover,there are no reports on the pharmacokinetics study of mung bean.Thus,it is difficult to control the quality of medicinal materials and drugs preparations and their efficacy and safety of clinical application.This dissertation takes the mung bean and their stems and leaves as the research object,and the chemical constituents and pharmacological activity of them are further studied.What's more,this dissertation study on the quality control and pharmacokinetics of mung bean.These findings will increase our knowledge about mung bean and their leaves and seeds and help promote their potential application in functional foods.Chemical constituents 26 constituents were isolated by repeated column chromatography from the n-butanol fractions of mung bean.On the basis of physico chemical properties and spectroscopic analysis,20 constituents were identified.They are isovitexin-6"-O-?-L-glucoside(1),vitexin(2),isovitexin(3),dulcinoside(4),(2R,3R)-taxifolin(5),scopolin(6),p-coumaric acid(7),L-tryptophan(8),D-3-O-methyl inositol(9),muconic acid(10),benzoic acid(11),2H-1,3-Thiazine-2,4(3H)-dione(12),nicotinic acid(13),nicotinamide(14),methyl-?-D-glucoside(15),uracil(16),adenine(17),uridine(18),adenosine(19),and sucrose(20).Among them,including five flavonoids and fifteen other compounds,compound 1 is determined as new compound and compounds 4-6,12,and 15 are isolated from genus Vigna for the first time.40 constituents were isolated by repeated column chromatography from the n-butanol fractions of the stems and leaves of mung bean.On the basis of physico chemical properties and spectroscopic analysis,38 constituents were identified.They are muconic acid(10),uracil(16),vigradiatain(21),2'-hydroxydaidzin(22),daidzein(23),3'-methoxydaidzein(24),2'-hydroxydaidzein(25),genistein(26),daidzin(27),genistin(28),7,2'-dihydroxyisoflavone-4'-O-?-D-glucopyranoside(29),lupinalbin A(30),2,6-dihydroxy-2-[(4-hydroxyphenyl)methyl]-3-benzofuranone(31),isoliquiritigenin(32),?,4,2',4'-tetrahydroxydihydrochalcone(33),isovestitol(34),liquiritigenin(35),erythro-buddlenol B(36),spicatolignan B(37),dehydrodiconiferyl alcohol 9'-O-?-D-glucopyranoside(38),dehydrodiconiferyl alcohol 4-O-?-D-glucopyranoside(39),threo-Guaiacylglycerol-?-O-4'-coniferyl alcohol(40),hyuganosides ?a(41),erythro-guaiacylglycerol-?-O-4'-coniferyl alcohol(42),2-[4-(3-hydroxy-1-propenyl)-2-methoxyphenoxy]-1,3-propanediol(43),C-Veratroylglycol(44),trans-dihydrophaseic acid(45),cis-dihydrophaseic acid(46),phaseic acid(47),citroside A(48),coumestrol(49),aurantiamide acetate(50),neoechinulin A(51),benzyl-O-?-D-glucopyranoside(52),isosalicin(53),vanillic acid(54),salicylic acid(55),and rutin(56).Among them,including nine isoflavonoids,seven flavonoids,nine lignans,four terpenoids,and nine other compounds,compounds 21 and 22 are determined as new compounds and compounds 24,25,29,31-45,48,and 50-53 are isolated from genus Vigna for the first time.The HPLC-ESI-TOF-MS method was used to identify the chemical composition of stems and leaves of mung bean during growth periods.HPLC-DAD method was used to quantitative analysis the main chemical compositions of stems and leaves of mung bean during growth periods.The results shown that isoflavone glycosides were the main chemical compositions from week 3 to 6.However,flavone O-glycosides was the main chemical compositions after week 7.According to the data,there maybe a transition between isoflavone and flavone.flavone C-glycosides were the main chemical compositions of mung bean,thus,there maybe a transition between flavone O-glycosides and flavone C-glycosides.Therefore,this dissertation speculated these transitions and pointed out a series of biological synthesis enzyme in order to provide the theory basis for the research on chemical composition transitions.Pharmacological activities 30 animals were grouped into five groups,including a normal control,a isoproterenol(ISO)model control,a vitexin group,an isovitexin group,and a mung bean seeds extract group.All treatments were performed once daily for 7 consecutive days.Then,all of the rats except for the normal control group were injected subcutaneously with ISO at an interval of 24 h for 2 days to induce myocardial infarction on the sixth and seventh day.The serum levels of creatine kinase(CK),lactate dehydrogenase(LDH),aspartate aminotransferase(AST)as well as the cardiac level of superoxide dismutase(SOD)and malondialdehyde(MDA)were measued.Compared with normal conrol group,in model control group,the serum levels of CK,LDH,AST and cardiac level of MDA were significantly increase.Meanwhile,the activity of SOD was decrease(P<0.05).Compared with model control group,vitexin group,isovitexin group,and mung bean seeds extract group treatment significantly decrease the serum levels of CK,LDH,AST and cardiac level of MDA.Meanwhile,the activity of SOD was increased(P<0.05).In addition,after the administration of vitexin or isovitexin or mung bean seeds extract,cardiac interstitial edema diminished,and less infiltration of inflammatory cells was observed.Furthermore,histopathologic changes induced by ISO were markedly improved by vitexin,isovitexin,and mung bean seeds extract.Vitexin and isovitexin maybe the major constituents responsible for the myocardial preservation effect in mung bean seeds extract.The antioxidant and antidiabetic activities of compounds 1-55 were evaluated in this dissertation.The results show that only compounds 5,7,24,and 36 showed DPPH' scavenging activity.In ABTS assay,compounds 1-5,7-10,21-42,and 44 showed moderate to strong activity(EC50=2.21-88.94 ?M)compared with the positive control L-Ascorbic acid(EC50=11.06 ?M).Compounds 1-8,23,26,30-32,34,36-40,42,and 49-51 showed moderate to strong activity(EC50=2.24-49.34 ?M)compared with the positive control acarbose(EC50=2.62 ?M)in ?-glucosidase inhibition activity assay.The results obtained show that flavonoids and lignans maybe the major constituents responsible for the antioxidant and antidiabetic activities in mung bean and their stems and leaves.Quality control 15 different batches of mung bean were collected.After analyzing 15 batches of samples,13 common peaks were achieved.Vitexin was choosed as the reference substance.The similarity of 15 samples was from 0.8798 to 0.9906,indicating that the HPLC fingerprint of mung bean from different regions varied considerably.The method offers guarantee for the overall control over the quality of medicinal materials.A method for the simultaneously determination of vitexin(2),isovitexin(3),L-tryptophan(8),and benzyl-O-?-D-glucopyranoside(52)by HPLC was developed.The method was simple,accurate and reproducible and can provide a quantitative basis for the quality control of mung bean.Pharmacokinetics A rapid,sensitive and high throughput LC-MS/MS method was established and validated to assay the concentration of isovitexin-6"-O-?-L-glucoside(1),vitexin(2),isovitexin(3),dulcinoside(4)in rat plasma,and the method was successfully applied to the pharmacokinetic study of four C-glycosyl flavones in rats.Following intragastric administration of mung bean extract at doses of 2 g·kg-1 to male rats,four C-glycosyl flavones were absorbed quickly and eliminated faster.They could be detected at 5 min after administration of mung bean extract and the blood concentrations reached the peak concentration at 1?2 h.The t1/2 of four C-glycosyl flavones were 1.2,4.1,5.0,and 3.6 h,respectively,which indicates that the 1 eliminates significantly faster than the other C-glycosyl flavones in vivo after i.g.administration of the mung bean extract owing to different types of glycosidic bonds(? or ?).However,the pharmacokinetic behaviors of 2 was markedly different from the other C-glycosyl flavones(the t1/2 of 1,2,3,and 4 were 0.88,0.38,0.89,and 0.81 h,respectively)possibly due to different types of glycosylation sites(C-8 or C-6)of the analytes.The absolute bioavailability(F%)of 1,2,3,and 4 for i.g.were estimated as 1.37%,5.82%,5.53%,1 7.07%,respectively.Although the structures of four C-glycosyl flavones are the same,their absolute bioavailability are different.This difference was likely due to the complexity of herbal medicines or functional foods because the interactions between multitudinous compounds might greatly affect the in vivo processing of medicines.Tissue distribution A rapid,sensitive and high throughput LC-MS/MS method was developed for quantifying four C-glycosyl flavones in rat tissues.The tissue distribution of four C-glycosyl flavones in male rats was investigated.After oral administration of mung bean extract(2 g·kg-1),the drug was fast and extensively distributed.The highest levels of four C-glycosyl flavones in most tissues were observed about 1.5 h after administration,which in accord with Tmax(1-2 h)in pharmacokinetics.The levels of four C-glycosyl flavones in tissues were almost eliminated in 4 h,which indicated four C-glycosyl flavones were rapidly distributed and eliminated,and little accumulated.From the results,we found that the maximum concentration of four C-glycosyl flavones were observed in stomach and intestine which maybe mainly attributed to the oral mode of administration.The concentration of four C-glycosyl flavones exhibited a higher concentration in the heart,liver,spleen,lung and kidney,which indicates that blood flow and perfusion rate of the organ played a key role in the distribution of four C-glycosyl flavones.In addition,four C-glycosyl flavones could be detected in brain,which illustrated that these four compounds could transfer across the blood-brain barrier. |
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