Structures,Contents,Anti-α-glucosidase And Antioxidant Activities Of The Chemical Constituents Of Bombax Malabaricum Flower And The Whole Plant Of Viola Yedoensis

α-Glucosidase degrades oligosaccharides to generate glucose and raise postprandial glucose levels in diabetic patients.Elevation of postprandial glucose level in type II diabetes patients can be effectively controlled by inhibiting α-glucosidase.In addition to α-glucosidase inhibitors,antioxidants are known to benefit diabetic patients by enhancing antioxidant defense mechanisms and improving β cell function.The present research was carried out to study the structures,contents,anti-α-glucosidase and antioxidant activities of the chemical constituents in Bombax malabaricum flower and the whole plant of Viola yedoensis.Through ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS)analysis and by comparison with standard compounds,we identified 16 chemical constituents from B.malabaricum flower.Twenty-eight compounds including the presently identified 16 ones and 12 that isolated from B.malabaricum flower in a previously work were studied for their effects on α-glucosidase and rat intestinal disaccharidase,and as antioxidants.As the traditional colorimetric method was of low reproducibility for screening α-glucosidase inhibitors when the samples were yellow or dark colored,an UHPLC-UV method with high reproducibility was established to separate the enzymolysis product from the colored compounds,and the inhibition was evaluated by the relative peak areas of the enzymolysis products.Among the 28 chemical constituents of B.malabaricum flower,muraxanthone(2),isoorientin(5),isoxitexin(7),quercetin(9),isoquercetin(10),chlorogenic acid(17),neochlorogenic acid(18),and1,5-dicaffeoylquinic acid(19)showed anti-α-glucosidase activity;mangiferin(1),2,9-10,protocatechuic acid(13),gallic acid(14),caffeic acid(15),17-19,and 3,5-dicaffeoylquinic acid(20)showed inhibitory activity on rat intestinal disaccharidase;compounds 1-2,5,orientin(6),9-10,rutin(11),13-15,17-20 showed DPPH radical scavenging activity;compounds 2,9,13,15,17,citric acid(21),linoleic acid(24),and linolenic acid(25)showed hydroxyl radical scavenging activity.B.malabaricum flower is not only used as a Mongolian medicine but also as a kind of herbal tea material,so it is interesting to compare the bioactivities and chemical compositions with other teas,such as green tea,black tea,Fagopyrum tataricum seeds Jasminum sambac flowers,Ilex latifolia leaves,Ligustrum quihoui leaves,Lonicera japonica flowers,Rosa rugosa flowers,Plumula nelumbinis seed hearts,Ophiopogon japonicus roots,Hordeum vulgare seeds,Semen sterculiae seeds,Coreopsis tinctoria flowers,Dendranthema morifolium flowers,and Semen cassiae seeds).High levels(>8 mg/g)of the bioactive flavonoids(1-2,9)and organic acids(13,21-27)were found in B.malabaricum flower by UHPLC-MS analysis.Among the other 15 teas,Rosa rugosa flower,green tea and black tea showed strong antioxidant,as well asα-glucosidase and rat intestinal disaccharidase inhibitory activities.Quantification analysis revealed that these 3 teas contained high amounts(1.1±0.1~82.4±5.7 mg/g)of bioactive flavonoids(9-11),phenolic acids(13-16),caffeoylquinic acids(17-20),and organic acids(21,24-25).Theα-glucosidase inhibitory capacity of B.malabaricum flower was similar to that of green tea,ranking the fifth among these 16 teas tested;the DPPH radical scavenging capacity of B.malabaricum flower was similar to that of F.tataricum seeds,and ranked the eighth among these teas;the hydroxyl radical scavenging capacity of B.malabaricum flower ranked the second,only next to green tea.Cluster analysis indicated that the composition of B.malabaricum flower was similar to that of H.vulgare seeds.From the whole plant of V.yedoensis,12 compounds were isolated and purified.Their structures were determined as rutin(33),apigenin-6,8-di-C-β-D-glucopyranoside(34),apigenin-6-C-β-Dglucopyranosyl-8-C-α-L-arabinopyranoside(35),apigenin-6,8-di-C-α-L-arabinopyranoside(36),5-hydroxy-4’-methoxyflavone-7-O-rutinoside(37),esculin(38),6,7-di-O-β-D-glucopyranosylesculetin(39),esculetin(40),5,5′-bi(6,7-dihydroxycoumarin)(41),6,6’,7,7’-tetrahydroxy-5,8’-bicoumarin(42),7R,8S-dihydrodehydrodiconiferyl alcohol 4-O-β-D-glucopyranoside(43),and(7R,8S,8′S)-3,3′-dimethoxy-4,4′,9-trihydroxy-7,9′-epoxy-8,8′-lignan 9-O-rutinoside(44).Among these compounds,44 is a new one;compounds 37,39,and 43 are isolated and identified from this genus for the first time.The planar structure of 44 was determined by analysis of its mass spectrometry and 1D and 2D NMR spectra;the stereo-structure of the aglycone was determined by NOESY and CD spectra.To determine the configurations of the sugar part,a method of hydrolyzing the glycoside to release the sugars followed by reaction with L-cysteine methyl ester and UHPLC-MS analysis were established,and the sugars of 44 were identified as D-glucose and L-rhamnose.Of these compounds from V.yedoensis,37 and 44 were potently active against α-glucosidase,with IC50 values being 24.63±2.33 and 1.89±0.09 μg/ml,respectively.The two dimeric coumarins(41-42)potently inhibited HCV protease with IC50 values being 0.41±0.07 and 3.63±0.13 μg/ml,respectively.Esculetin(40)showed inhibition on rat intestinal disaccharidase(IC50: 58.22±2.31 μg/ml).Rutin(33),esculetin(40),and 5,5′-bi(6,7-dihydroxycoumarin)(41)showed DPPH radical scavenging activity,with EC50 being 18.67±0.99,6.03±0.20 and 7.79±0.38 μg/ml,respectively.A simultaneous quantification method for the 12 constituents was established and the results showed that V.yedoensis contained high levels of esculetin(40),5,5′-bi(6,7-dihydroxycoumarin)(41),and6,6’,7,7’-tetrahydroxy-5,8’-bicoumarin(42)(7.0±0.3、1.4±0.1 和 11.2±0.2mg/g,respectively)as its major chemical constituents.The present research established an UHPLC-UV method for assessing α-glucosidase inhibitory activity and generated repeatable results that were not influenced by the color of the test compounds;identified some bioactive flavonoids,caffeoyl quinic acids,lignans,and coumarins from B.malabaricum flower and the whole plant of V.yedoensis as α-glucosidase inhibitory constituents;found that B.malabaricum flower as a tea material had anti-α-glucosidase and antioxidant activities above the average power 16 teas;established a simple method including hydrolysis-one step derivatization-UHPLC-MS analysis to determine D-or L-sugars,and using this method determined the sugar configurations of a new lignan,(7R,8S,8′S)-3,3′-dimethoxy-4,4′,9-trihydroxy-7,9′-epoxy-8,8′-lignan 9-O-rutinoside,from V.yedoensis.