| A novel polyphenolic compound,named Oxovitisin,with unique lactone pyranone ring and non-oxonium structures was prepared.In this paper,Oxovitisin A was synthesized by two-step reactions in micro-oxygenation system using carboxypyranoanthocyanins(Vitisin A)formed from pyruvic acid and anthocyanins.The preparation conditions of Oxovitisin A were optimized using combination of single factor and orthogonal array experiments.HPLC-DAD was used to monitor the reaction process and the main adduct was analyzed by HPLC-DAD-EI-MS/MS qualitatively and quantitatively,then the fraction of Oxovitisin A was isolated by a combination of several chromatographic techniques.Oxovitisin A was investigated in terms of characterization of chromatic stability,storage stability and spectra characteristic under different conditions,as well as scavenging free radical including ROS and reducing power.The antiproliferation effects of Oxovitisin A in MCF-7 estrogen dependent cancer cell line,as well as the apoptotic properties were assayed,with addition of a putative structure-activity relationship was discussed.The biologically transport efficiency of Oxovitisin A through gastric epithelial cell models(MKN-28 cells monolayer)was evaluated,compared with Precursors anthocyanins and Methylpyrano-Mv.The main conclusions are as follows:1.The optimal preparation conditions were found to be reaction at 45 ? for 21 d with 20% ethanol acidified to pH 3.6 using 1.0 mg/ml of Vitisin A,the main reaction product was confirmed by HPLC-MS/MS to be Oxovitisin A with the yield of 26.59% after 21 d.The results showed that the purity of Oxovitisin A could reach more than 99% after isolated and purified by hyphenated techniques of polyamide resin,TSK Toyoperal gel column chromatography and preparative HPLC.2.Oxovitisin A demonstrate an obvious stability than precursor anthocyanins(Vitisin A and Mv-3-O-gluc),the half-life of Oxovitisin A at 50? and 90? was 693.14 h and 67.30 h.The stability of three pigments was as follows: Oxovitisin A > Vitisin A > Mv-3-O-gluc,after 7 h of storage without light.Under 7 h of sunlight treatment,the retention rate of Oxovitisin A was 92.49%,but Vitisin A and Mv-3-O-gluc were 63.30% and 73.53 respectively.Which indicated Oxovitisin A had a higher storage and light resistance stability than the precursor anthocyanins.Moreover,Oxovitisin A showed a great resistance to discoloration by 200 ppm of SO2,with lowest absorbance changes than Vitisin A and Mv-3-O-gluc.3.Analysis with UV spectroscopy and color parameter variations method indicated that Oxovitisin A had a higher structural and color stability.The UV-vis and color features study showed that the ultraviolet spectrum and color parameters of Oxovitisin A changed very little in acidic or neutral conditions.While pH > 9,?max of Oxovitisin A UV spectrum moved to the long-wave,with smooth waveform and absorption peak enhancement.Meanwhile,Oxovitisin A displayed a more full-bodied yellow with increasing saturation.All above indicated that Oxovitisin A had a higher structure and color stability than precursor anthocyanins,and less susceptible to influence from pH changes.4.The molar extinction coefficient value(?)of Oxovitisin A in methanol was higher than vitisin A and Mv-3-O-gluc.Moreover,Oxovitisin A could maintain the structure stability and exhibit a higher ? values under alkaline conditions.The fluorescence spectra results revealed that Oxovitisin A had an obvious fluorescence peaks under the excitation wavelength of 440 nm(Em = 490 nm),but no intensity of florescence was detected on Vitisin A and Mv-3-O-gluc,this properties may because of the special structure similar to flavonoids.5.The DPPH,ABTS+ radical scavenging ability and FRAP assay showed that both Oxovitisin A and precursor anthocyanins had strong antioxidant activity.The ROS scavenging activity of Oxovitisin A were evaluated by using a BPCL Ultra-weak luminescence analyzer in vitro,all four compounds(Oxovitisin A,Vitisin A,Mv-3-O-gluc,Vitamin C)showed strong antioxidant activity in scavenging O2-·(IC50 of 71.4,30.7,19,28 ?g/m L,respectively),·OH(1.68,3.524,2.854,8.441 ?g/m L,respectively)and H2O2(1.311,0.4098,0.288,3.265 ?g/mL,respectively).However,Oxovitisin A showed the highest antioxidant ability in scavenging ROS radical compared with Vitisin A,Mv-3-O-gluc and Vitamin C,the result indicated that Oxovitisin A had a stronger ability to dissociate the hydrogen protons,this would be conducive to ultimately a more stable free radical intermediates structure after supplied hydrogen.6.The antiproliferation assay showed that Oxovitisin A displayed obvious inhibitory effect on breast cancer cells(MCF-7),the IC50 value of was 0.083 ±0.011 mg/m L,revealed a obviously higher anti-tumor effect than Vitisin A and Methyl-pyranoanthocyanin.At the same time,this results also showed a correlation between the chemical structure of the compounds and the biological properties.Among the derivatives oxovitisin A seemed to be the most effective compound inhibiting the proliferation and inducing the apoptosis of MCF-7 cells,which detected a highest level of apoptosis promoting factor(Caspase3,9 and TNF-?)than Mv-3-O-gluc,Vitisin A and Methylpyrano-Mv.Futhermore,a kinetic incorporation of Oxovitisin A was assayed and revealed that this pigment was fully incorporated into cells after only 4 minutes of exposure,reacted the cell nucleus and surrounding mitochondria directly,and retain the role for a long time.7.The transport of Mv-3-O-gluc and derivatives across MKN-28 monolayer was time dependent.However,for the higher incubation times,the transport was dependent on the type of anthocyanin derivatives(substitution in ring-D and neutral pyranone structure),as well as the B ring or glucose has been reported,being Oxovitisin A the most absorbed. |
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