| Epidemiologic studies have demonstrated that regular intake of vegetables and fruits abundant in polyphenols can improve the quality of life and reduce the risk of diabetes,cardiovascular,obesity and other chronic diseases.Brassica rapa L.,belonging to the Cruciferae family,is a biennial herb mainly distributed in the Yangtze River basin and northwest of China.As the plant-derived food,its fleshy roots can be stri-fried,cooked and pickled,and also its pollen is an important functional food.Moreover,the roots,seeds and flowers of B.rapa can be used in medicine to treat indigestion and asthenia as well as strengthen the immune system.In this thesis,twelve compounds were isolated from the aqueous extracts of B.rapa through bioactivity-guided fractionation and various open column chromatographies(macroporous resin HPD-100,polyamide,MCI GEL,ODS,Sephadex LH-20)and preparative HPLC.On the basis of physicochemical properties,extensive spectroscopic analyses(1D NMR,2D NMR,UV,HRESIMS and ORD)and comparison with literature data,their structures were identified as:p-hydroxybenzaldehyde(BR-1),protocatechuate(BR-2),caffeic acid(BR-3),2-O-β-D-glucopyranosyl-(1S)-(4-methoxyphenyl)ethylene glycol(BR-4),2-(4-O-β-D-allopyranosyl)phenyl-ethanol(BR-5),1,3-propanediol-2-O-4’-(3’,5’-dimethoxy-1’-hydroxymethyl)phenyl ether(BR-6),2-{4-[(1E)-3-Hydroxyprop-1-en-1-yl]-2,6-dimethoxy-phenoxy}propane-1,3-diol(BR-7),3,5-dimethoxy-4-hydroxybenzyl alcohol-l-O-β-D-glucop yranoside(BR-8);Lignans:vladinol D(BR-9),9α-hydroxypinoresinol(BR-10),(+)-isolariciresinol(BR-11),(+)-cycloolivil(BR-12).Among them,compounds BR-4 and BR-5 were new compounds,and compounds BR-6-12 were isolated from this genus for the first time.Brassica juncea var.multiceps(Cruciferae),the variant of Brassica juncea,is an annual herb mainly distributed in the Yangtze River basin.In addition to being the common vegetable that can be pickled,it is of medicinal value for treating cough and cold.In this thesis,ten compounds were isolated from the aqueous extracts of B.juncea var.multiceps through bioactivity-guided fractionation and various open column chromatographies(macroporous resin HPD-100,polyamide,MCI GEL,ODS,Sephadex LH-20)and recrystallization.Their structures were identified as:apigenin(BJ-1),luteolin(BJ-2),kaempferol(BJ-3),quercetin(BJ-4),kaempferol-7-O-β-D-glucopyranoside(BJ-5),quercetin 3-O-β-D-glucopyranoside(BJ-6),(+)-pinoresinol 4-O-β-D-glucopyranoside(BJ-7),(+)-8’-hydroxypinoresinol-4-O-β-D-glucopyranoside(BJ-8);neoandrographolide(BJ-9),daucosterol(BJ-10).Among them,compounds BJ-7-9 were isolated from this genus for the first time.The ethanolic extracts of B.rapa and B.juncea var.multiceps along with their aqueous fractions were initially determined to possess varying in vitro α-glucosidase,aldose reductase(AR)inhibitory and antioxidant activities.The isolated compounds(BR-1-12,BJ-1-9)were evaluated their α-glucosidase,AR inhibitory activities together with DPPH·and ABTS.+scavenging and FRAP capacities.And the preliminary structure-activity relationships have also been discussed.(1)Compound BR-5(IC50=14.43 μM)as well as BJ-2(IC50=14.81μM),BJ-3(IC50=15.17 μM)and BJ-4(IC50=12.26 μM)exhibited more potent α-glucosidase inhibitory activities than the positive control acarbose with corresponding IC50 values of 15.76μM and16.81μM.while compounds BR-3,BR-4,BR-6,BR-7,BR-9-12,BJ-1 and BJ-6 also showed effective α-glucosidase inhibition.(2)Compounds BR-4,BR-5,BR-10,BJ-2 and BJ-4 displayed significant AR inhibitory effects with IC50 values of 13.51 μM,9.30 μM,15.82μM,13.62 μM and 15.75 μM,which overmatched the positive control quercetin(IC50=17.35,16.23 μM),and compounds BR-9,BJ-1,BJ-3 and BJ-6 performed considerable inhibition against AR.(3)In the antioxidant activity tests,BJ-2(DPPH IC50=32.17 μM,ABTS IC50=24.48 μM,FRAP 10.34 mmol/g)and BJ-4(DPPH IC50=23.25 μM,ABTS IC50=15.17 μM,FRAP 12.11 mmol/g)exhibited the strongest antioxidant activity compared to the positive control Trolox(DPPH IC50=33.21 μM,ABTS IC50=25.87 μM,FRAP IC50=9.17 mmol/g).Compounds BR-4,BR-5,BR-11,BJ-3,BJ-5 and BJ-6 could effectively scavenged DPPH free radicals.Compounds BR-11(IC50=19.25 μM)and BR-12(IC50=20.72 μM)showed more significant ABTS free radical scavenging capability than the positive control Trolox(IC50=25.87 μM).and compounds BR-4,BR-5,BR-9 and BJ-6 exhibited fairly strong ABTS free radical scavenging activities.In the FRAP assay,compounds BR-2(10.66 mmol/g),BR-3(11.21 mmol/g).BR-11(9.78 mmol/g)and BR-12(10.40 mmol/g)possessed potent FRAP capability compared to the positive control Trolox(9.17 mmol/g),and compounds BR-4-6,BR-9.BR-10 and BJ-6 also exhibited considerable FRAP activities.Enzymatic kinetics results indicated that new compounds BR-4 and BR-5 noncompetitively inhibited α-glucosidase activity,while flavonoids BJ-2 and BJ-4 showed mixed type inhibition on α-glucosidase.The molecular docking results indicated that all the four compounds formed stable complexes with maltase-glucoamylase(MGAM),inserting into the narrow-1 substrate binding site of NtMGAM and CtMGAM and occupying the substrate binding site to inhibit enzyme activity.Besides,BR-4 and BR-5 interacted with the+2 substrate binding site of CtMGAM.The results of AR inhibitory kinetics revealed that compound BR-4 competitively inhibited AR activity,while BR-5 and BJ-2 suppressed AR activity in the mixed type manner,and B J-4 was the noncompetitive AR inhibitor.According to the molecular docking analysis,compounds BR-4 and BR-5 not only inserted into the catalytic site of AR,but also interacted with its specific site.It illustrated that both BR-4 and BR-5 could selectively inhibit AR activity which may help decrease the possibility of inhibiting the activity of other enzymes in the aldo-ketoreductase family.These findings indicated that B.rapa and B.juncea var.multiceps with the potential in alleviating acute postprandial hyperglycemia and preventing the chronic complications resulting from sustained hyperglycemia,may be developed as the promising dietary source for multi-targeted prevention and treatment towards diabetes and its chronic complications,and also provided the scientific basis for the further investigation and functional food exploitation of the two plants. |
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