| In this paper,the constituents of 12 anthocyanin extracts from different sources were identified and the inhibition mechanisms onα-glucosidase,α-amylase and lipase were studied in vitro.Three samples were screened out for the HepG2 cell in vitro glucose consumption experiment and one sample was selected for the evaluation of lipid-lowering effect in high-fat mice model.The main results of this paper are as follows:1 Component analysis and structure identification of anthocyanin extracts from different sourcesThe contents of total phenolic acids,total flavones and total anthocyanins in 12 samples were analyzed by chemical methods.Differences in varieties and contents among anthocyanins and copigments in the extracts from different sources were compared using High Performance Liquid Chromatography-Photo Diode Array（HPLC-PDA）,and the Ultra Performance Liquid Chromatography-Quadrupole Time-of-flight Mass Spectrometry（UPLC-QTOF-MS/MS）was combined to systematically identify the chemical structures.The results showed that the total phenolic acid contents were higher in samples 1,2,3,9 and 10（>40 mg of GAE/100 mg）.Sample 10 contained the highest total flavonoids content（172.4±2.95 mg of rutin/100 mg）,while the total flavonoids in samples 1-3 were below the average level.Anthocyanin in sample 8 was the richest（30.218±0.72 mg/100 mg）with the lowest level in in sample 10（4.087±0.13mg/100 mg）.Comparing the liquid chromatograms at 520 nm wavelength,we could directly compare the contents and varieties of anthocyanins in 12 samples.The results illustrated that there were as many as 18 kinds of anthocyanins in samples 1-3,while in samples4-12,only one anthocyanin was dominant.Comparing the liquid chromatograms at 280 nm wavelength,we found that samples 10 and 12contained several kinds of copigments with large peak area.Three anthocyanins and 10 phenolic compounds were identified using liquid chromatography-mass spectrometry.The major anthocyanins in samples 4-12werecyanidin-3-O-galactoside/glucoside,whosecopigmentswere caffeoylquinic acid and rutin.In addition,the proanthocyanidin monomer,dimer and triploid were found in sample 10.2 Screening of inhibition of anthocyanin extracts from different sources on key enzymes for glycolipid digestionBy comparing the effects of anthocyanin extracts from different sources on the activities ofα-glucosidase,α-amylase and lipase under the same concentrations,the anthocyanin extracts with the best activity were selected.Moreover,the component and enzymatic inhibition of samples 1-3 with strongα-glucosidase inhibitory effect were systemically analyzed and compared.On this basis,sample 10 was set as representative material to further investigate the inhibitory type and kinetic characteristics ofα-glucosidase.The results suggested that sample 1,2,3,8 and 10 demonstrated better inhibition onα-glucosidase.Compared with the positive group under the same concentration（40.38±0.70%）,the inhibitory rate of sample 10 was reach up to the highest 94.57±1.43%.Inhibitory rates of sample 2 and 3 were both more than 60%.Sample 5（35.90±3.00%）and 10（35.56±6.16%）had significant influence on the activity ofα-amylase,while sample 5 also presented a strong inhibitory effect on lipase at the same time.Based on the results of the composition analysis and the glycolipid digestion enzymes inhibition,sample 1-3 were further divided into copigment fragments and anthocyanin fragments by semi-preparation HPLC technique.18 anthocyanin structures were identificated in the anthocyanin fragments.Sample 1contained 4 acylated anthocyanins,exclusively.Hydroxycinnamic acids belonging to phenolic acids were the main compounds in copigment fragments.Besides,4 kinds of iridoids were dicovered in all three kind of copigment fragments.The results ofα-glucosidase inhibition manifested that the concentrations of anthocyanin extracts in samples 1-3 had a good linear relationship with enzyme inhibition.What’s more,compared with the anthocyanin fragments,copigment fragments displayed the stronger inhibition effects onα-glucosidase with a clear dose-effect relationship.Sample 10 with the highest content of total phenolic acid was used as representative material to evaluate the inhibition type and kinetic characteristics ofα-glucosidase.The results suggested that the IC50 value of sample 10 onα-glucosidase was 11.37μg/mL with a reversible inhibition effect,and Lineweaver-Burk double reciprocal analysis indicated that the inhibition mechanism was anti-competitive effect.3 Regulation the glucose consumption to HepG2 cells by samples 1-3In normal and insulin-resistant HepG2 cells,the effects of sample 1-3 on glucose consumption activity of cells were studied.Firstly,through investigating the concentrations of insulin or glucose used to induce the transformation of HepG2 cells into insulin-resistant HepG2 cells,the best scheme for modeling insulin-resistant HepG2 cell was:incubating 1×10-8mol/L insulin for 24 h.The results of cell viability experiments showed that three kinds of samples could influence the cell proliferation at 200μg/mL,in which samples 2 and 3 had a cell survival rate of less than 50%at the concentration of 800μg/mL.Insulin promoted cell proliferation significantly at the concentration of 1×10-9 mol/L while glucose had no significant effect on the proliferation of HepG2 cells at the concentration of 22-99 mmol/L.In the normal HepG2 cells,sample 1-3 promoted the glucose consumption in varying extent from 7.8125μg/mL to 31.25μg/mL,in which sample 1 increased glucose consumption greatly at 15.625μg/mL,while sample 2 and 3 showed significant differences at 31.25μg/mL.In the insulin-resistant HepG2 cells,all of the three samples promoted cell glucose consumption in varying extent within the concentration ranging from 7.8125 to 31.25μg/mL.Moreover,the promotion was in a dose-dependent manner and achieved maximum with the concentration 31.25μg/mL,which showed an extremely significant difference compared with the model group,indicating that the anthocyanin extracts of samples 1-3 could improve the glucose consumption of insulin-resistant HepG2 cells.4 Evaluation of sample 5 on lipid-lowering effect in high fat diet-induced obese mice modelThe sample 5 was performed as the research material to probe the lipid-lowering effect in high fat diet-induced obese mice model.The results illustrated that samples 5 could effectively reduce the dietary intake and the high dose could reduce the weight of mice and increase the serum HDL-C levels significantly.The organ index displayed that sample 5 could obviously reduce the fat content in liver,epididymis,and kidney.Liver morphology showed that the number of lipid droplets and inflammatory cells in the high and low dose groups of sample 5 were greatly reduced compared with the model group and the cell morphology was relatively regular.In week 6 and week 8,the contents of stool cholesterol and total bile acid in mice feces were measured.The results illustrated that high dose of sample 5 can significantly increase the cholesterol level in the mice feces in week 8.The total bile acid content of the extracted feces experienced a significant increase in the low dose group of sample 5 for two weeks.The fasting blood glucose measurement indicated that the sample 5 could reduce the fasting blood glucose in high fat diet-induced obese mice.In summary,this article initially revealed the inhibitory effects and mechanisms of anthocyanin extracts onα-glucosidase,suggesting that the copigments（phenolic acids）in anthocyanin extracts may provide the main contribution to the inhibition of the enzyme.Anthocyanin extracts could effectively promote the glucose consumption of HepG2 cells and improve the insulin resistance in vitro.Animal experiments showed that anthocyanin extracts had a certain lipid-lowering effect on high fat diet-induced obese mice.This study may lay a preliminary foundation for further study in the mechanism of anthocyanin extracts on glucose and lipid metabolism. |
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