Preparation Of Pyranoanthocyanidins From Blueberry And Its Inhibitory Effects And Mechanisms On HeLa Cells

The research content of this thesis belongs to the 13th Five-Year Project of Jilin Science and Technology Department?JJKH201800 90KJ?.Pyranoanthocyanin is a novel anthocyanin derivative detected in the wine filtrate of the past 20 years.It is widely used due to its high stability.Concerned,it has a good application prospect in food and health products.In this paper,wild blueberry from Changbai Mountain was used as raw material to extract blueberry anthocyanins and anthocyanidins,The pyranoanthocyanidins were synthesized by the reaction of anthocyanidins with acetone,and its composition and structure were analyzed and identified by high performance liquid chromatography and high performance liquid chromatography-MS.Then the stability,antioxidant activity and inhibitory effect of pyranoanthocyanidins on cancer cells were compared and analyzed.The effect of pyranoanthocyanidins on the activity,morphology,cell cycle distribution and apoptosis of human cervical cancer HeLa cell line was investigated.The anthocyanidins and anthocyanins were used as control samples for analysis and evaluation.The changes of related protein expression in HeLa cells were evaluated by blot.The main conclusions are as follows:1.Anthocyanins and anthocyanidins were extracted from wild blueberry fruits by combination column chromatography,and then pyranoanthocyanidins were synthesized by nucleophilic addition reaction from anthocyanidins and acetone.By optimizing the synthesis method,the optimum synthesis method of pyranoanthocyanidins were obtained as follows:first,the anthocyanidins powder were dissolved in 60%ethanol aqueous solution at 3.0 mg/mL concentration,then mixed with acetone with 3times volume ratio,and the pH of the system was adjusted to 3.0 with HCl solution,and the light-avoidance reaction was carried out for 15 days in the water environment at 42?.The purity of blueberry anthocyanin extracts,anthocyanidin extracts and pyranoanthocyanidin extracts were 63.5%,71.8%and 70.2%,respectively.2.After purifying pyranoanthocyanidins by semi-preparative high performance liquid chromatography,the composition and structure of the pyranoanthocyanidins were analyzed and identified by high performance liquid chromatography-mass spectrometry.The mass-to-charge ratio of the five components in the extract of pyranoside anthocyanidins were identified,respectively,in the order of the peaks,which were methyl pyran-delphinidin,methyl pyran-cyanidin,methyl pyran-petunidin,methyl pyran-peonidin and methyl pyran-malvidin.This is in turn corresponding to the five groups of pigments in the anthocyanidins extract,and is consistent with the results of high performance liquid chromatography analysis.3.The stability of pyranoanthocyanidins under different pH,temperature,warming-up time,metal ions and the scavenging ability of DPPH and ABTS⁺free radicals were studied.Stability study results show that:?1?The stability of anthocyanin and anthocyanidins decreased significantly above pH 3.0,while the stability of pyranoanthocyanidins remained almost unchanged within the acidity-basicity range of 3.0⁹.0.?2?The thermal stability of anthocyanin and pyranoanthocyanidins aqueous solution under different heating temperatures and time was significantly higher than that of anthocyanidins.?3?Fe³⁺ions had obvious fading effect on the three blueberry anthocyanin extracts,while Na⁺,Al³⁺,Cu²⁺,Mn²⁺ions had certain color enhancement effect at lower concentration and decreased the absorbance value at higher concentration.Among them,metal ions had the greatest effect on anthocyanins,while pyranoanthocyanidins and anthocyanins showed higher tolerance.?4?By calculating the antioxidant equivalent of Trolox,it is proved that the three anthocyanins have strong scavenging capacity for DPPH and ABTS⁺free radicals,and the order from strong to weak is:anthocyanidins>pyranoanthocyanidins>anthocyanin.4.HeLa cells were treated with pyranoanthocyanidins,anthocyanidins and anthocyanins at different concentrations.The effects of pyranoanthocyanidin,anthocyanin and anthocyanin extracts on cell viability were detected by MTT assay.Cell death/viability was observed by Calcein-AM/PI double staining method.Cell cycle and apoptosis were detected by flow cytometry.It was found that the cell viability was 24 hours after sample treatment.The strength of HeLa cells treated with anthocyanidins decreased in a dose-dependent manner.The lowest IC₅₀ value was found in HeLa cells treated with anthocyanidins?275.33±1.67?g/mL?,followed by pyranoanthocyanidins?536.91±2.03?g/mL?,and the lowest toxicity of anthocyanin was found in HeLa cells treated with anthocyanidins?1095.28±1.72?g/mL?.The results of Calcein-AM/PI double staining were consistent with the toxicity evaluation by MTT method.All the results showed that anthocyanidins had the strongest inhibitory effect on HeLa cells,followed by pyranoanthocyanidins.However,anthocyanin had the weakest effect in the three samples.Flow cytometry results showed that the three anthocyanins could induce cell cycle arrest in G₂/M phase and significantly induce apoptosis in advanced stage to inhibit the proliferation of HeLa cells.5.In order to further elucidate the possible mechanism of pyranoanthocyanidins affecting cell cycle arrest and cell apoptosis,the expression levels of intracellular related proteins?p53,p38-MAPK and Caspase-3?were detected by Western blotting.The expression level of p53 protein is usually used as a marker of cell cycle arrest in G2/M phase.The up-regulation of p53 and p38-MAPK and the activation of Caspase-3 suggest that HeLa cells may induce apoptosis through mitochondrial apoptosis pathway,thus inhibiting the proliferation of HeLa cells.