Studies On HEPG2Cell Protective Effects Of Blackberry After In Vitro Digestion

The active ingredients of fruit will be transformed and degraded in the process of human gastrointestinal digestion. Based on the in vitro digestion model and cell model, this paper studied the antioxidants content (polyphenols, flavonoids, anthocyanins and proanthocyanidins) in different stages of blackberry in vitro digestion, the antioxidant activity (oxygen radical scavenging capacity), the protective effects on oxidative damage in cells and in depth inquired its protective mechanism.The study provide a more scientific method to evaluate the nutritional value, antioxidant activity and function of the fruits.Oxidative stress can overproduce free radicals which will disorder the redox balance of human metabolism and resulting in potential damages. The field of functional food that mining safe and effective function factors from the plant and explore its protective function of cell oxidative damage has become a hot spot in recent years. Blackberry can fall blood pressure, hematic fat and anti-arrhythmic, but its digestion protective effect on liver cell injury caused by acrylamide (AA) is not reported so far. In order to explore the cell protection mechanism of blackberry digestions, this paper use AA as oxidative inducer, combined with the in vitro digestion model, HepG2cell model, use molecular probes and fluorescence labeling technologies to study the oxidation resistance, cell vitality, enzyme activity and gene expression. The main results were as follows:(1) The active ingredients content and antioxidant activity of blackberry in vitro digestionAfter the gastric and intestinal digest, the results showed that the content of polyphenols, flavonoids, anthocyanins and procyanidins in blackberry have all been influenced significantly in different degrees. The experiment of oxygen radical scavenging ABTS antioxidant showed that blackberry antioxidant activity of intestinal digestion increased by47.06%compared with original group, IC50of original, gastric digestion and intestinal digestion group were respectively6.12mg/mL,4.5mg/mL,3.24mg/mL. FRAP result showed that the antioxidant capacity of original, gastric digestion and intestinal digestion group were respectively2.46,4.32,2.45μg TEAC/mg blackberry(fresh weight).(2) The protective effect of blackberry in vitro digestion on HepG2cellThe HepG2cell toxic experiment showed that no toxic components in blackberry digestion. The MTT experiment showed that AA can cause oxidative damage to HepG2cell (cell survival rate63.37%), the blackberry intestinal digestion (0.5mg/mL) can significantly inhibit the cell toxicity caused by AA (cell survival rate85.19%). Using fluorescent dynamic visual technology, molecular probes and other modern molecular biology techniques to studied the mechanism of cell protective effect, results showed that the blackberry digestion can effectively restrain reactive oxygen species such as superoxide anion in the cell level, reduce the cell mitochondria oxidative damage, cell nucleus damage, restore the mitochondrial membrane potential and cellular glutathione levels, so as to achieve the purpose of protective cell oxidative damage.(3) The protective mechanism of blackberry in vitro digestion on HepG2cellThe activity of SOD and CAT were studied by kit, the result showed that the blackberry intestinal digestion (0.5mg/mL) can restore the activity of SOD and CAT decreased by AA, so blackberry digestion can improve the activity of intracellular antioxidant enzymes such as SOD, CAT and so on, enhances the body’s antioxidant ability and protect cell from damaging. The qPCR experiment detected the expression of antioxidant gene Nrf2, HO-1, NQO1, GCS, GSTP, SOD and CAT, the results showed that AA can induce cells to produce oxidative stress response, increase the mRNA expression of NRF2, HO-1, NQO1and Y-GCS, however blackberry intestinal digestion can further increase the amount of antioxidant gene expression to enhance cell antioxidation ability, but had no significant effect on GSTP. Thus we concluded that the blackberry digestion can activate HepG2cell two phase detoxifying enzymes, such as HO-1, NQO1and Y-GCS, reinstate the activity of the SOD and CAT, these may be the important reasons to provide protection against oxidative damage in HepG2cells.To sum up, the results of this paper provide a train of thought to evaluate the functional ingredients of fruits and its cell protective mechanism, a certain scientific basis for the development of natural, safe and effective functional products of blackberry.