Study On Protective Effect Of Purple Cabbage On Cell Damage Induced By Sodium Cyclamate Joint Lemon Yellow

Synthetic sweetener and synthetic pigment,as a large category of compounds widely used in daily life,its safety has been a widespread concern.The current evaluation on safety of food additives is based on a single species to formulate the appropriate maximum use,and one kind of food may contain more than a dozen or even dozens of food additives,in real life,the daily intake of food additives via food for an individual may reach dozens or even hundreds of species.Single additive in the provisions of the scope of use are safe,but the combination of several additives may induce addition,synergy,antagonism and other joint effects,this poses new challenges to the risk assessment of food additives.At present,planty of relevant studies have shown that synthetic sweetener and synthetic pigment has a certain degree of joint toxicity at a certain dose.In daily life,sodium cyclamate and lemon yellow are commonly used in many kinds of food,so they are easily ingested at the same time.Currently,there is not much report on the combined effects of these two food additives,so the exploration in this area is necessary.The aim of this article is to investigate the mechanism of sodium cyclamate together with lemon yellow on suppressing proliferation of Caco-2 cells,which is based on high content analysis(HCA),factorial design experiment analysis of variance as the joint toxicity evaluation method,qRT-PCR and Western blot as technical methods.The experiment indexes evaluated mainly include calcium concentration,reactive oxygen species(ROS)accumulation and mitochondrial membrane potential.The active ingredients in fruits and vegetables will degrade and transform during human digestion.In this paper,in order to evaluate the nutritive value and antioxidant activity of purple cabbage scientifically,the changes of the content of antioxidant substances(flavonoids,polyphenols,proanthocyanidins and anthocyanins)and the changes of antioxidant capacity in cell level,before and after simulated gastrointestinal digestion,were all studied.At the same time,relevant studies have found that purple cabbage has anti-inflammatory,anti-oxidation and other functions.In this paper,based on the study of the mechanism of the potential joint toxicity of sodium cyclamate and lemon yellow on intestinal cells,the preventive effect of purple cabbage against sodium cyclamate joint lemon yellow induced intestinal cell injury was studied from the aspects of cell viability,mitochondrial health,intracellular reactive oxygen species,related antioxidant enzyme activity and protein expression.The main results were as follows:(1)The results indicated that sodium cyclamate and lemon yellow dose-dependently decreased the growth of Caco-2 cells with an IC50 of 11.37±0.15 g/L and 1.21±0.12 g/L at 24 h,respectively.Compared with the high dose group,mix group could significantly decrease the cell number and mitochondrial membrane potential,accompanied by the increase of membrane permeability,ROS accumulation and calcium concentration(P<0.5).Both sodium cyclamate and lemon yellow has synergistic effect on calcium concentration,oxidative stress and additive effect on mitochondrial membrane potential.The results obtained by qRT-PCR and Western blot showed that mix group could inhibit Caco-2 cells proliferation by significantly increasing the mRNA levels of Bax,cytochrome c,caspase-3 and protein levels of Bax,cytochrome c,cleaved-caspase-3 and decreasing the protein level of Bcl-2(P<0.5),Which indicated that reactive oxygen species may be involved in mitochondrial pathway,regulating apoptosis.(2)The simulated gastrointestinal digestion in vitro would affect the content of related antioxidant substances in purple cabbage and cell antioxidant capacity.After in vitro simulation digestion,the contents of flavonoids and polyphenols in purple cabbage were significantly increased,and the content of procyanidins and anthocyanins decreased apparently(P<0.5).Meanwhile,after in vitro simulation digestion,cellular antioxidant capacity of purple cabbage was also improved.It was speculated that,may be the significant increase in the content of flavonoids and polyphenols after digestion of purple cabbage that improved the cellular antioxidant capacity.The promotion of cellular antioxidant capacity provided experimental basis for further investigation that the product of purple cabbage after in vitro simulation digestion could remove extra ROS in the cells to prevent the apoptosis of Caco-2 cells induced by sodium cyclamate joint lemon yellow.(3)Purple cabbage had a protective effect on the damage of Caco-2 cells induced by sodium cyclamate joint lemon yellow.Compared with the joint group,after pretreatment with purple cabbage,the cell viability and mitochondrial membrane potential increased significantly and the intracellular reactive oxygen species level decreased apparently(P<0.5),furthermore,the protective effect was also improved after pretreatment with purple cabbage.It was also found that the joint group could induce excessive ROS,inhibite the activity of antioxidant enzymes(SOD and CAT)significantly and reduce the antioxidant capacity of cells(P<0.5),in addition,excessive ROS may stimulated the activation of Nrf2,activated the Nrf2-ARE pathway and initiated the expression of downstream detoxification enzymes genes.After the pretreatment of purple cabbage,cells could significantly reduce the level of ROS,repair mitochondrial damage,restore cell viability,and recover the activity of SOD,CAT and other antioxidant enzyme(P<0.5),meanwhile,purple cabbage could further stimulate the activation of Nrf2,activate the Nrf2-ARE pathway,it had a strong induction effect mostly on the downstream HO-1 detoxification enzyme,thus,purple cabbage played a certain preventive role againsting the damage of Caco-2 cells induced by sodium cyclamate joint lemon yellow.