Preparation And Purification Of Zeaxanthin From Corn Husks And Its Mechanism Of Inhibiting The Growth Of Gastric Cancer Cells

Zeaxanthin is a carotenoid that is widely found in foods such as corn,egg yolks,and green vegetables.It is widely used as a pigment in the food industry.At the same time,zeaxanthin also has a potential health regulation effect,and it is widely known in the early stage because of its good effects on the treatment of eye diseases.Recent studies have shown that zeaxanthin has a good biological function in the prevention or treatment of digestive system diseases such as esophageal cancer and colon cancer.In related studies,it has been found that it also has a certain regulatory effect on gastric diseases,but at present,there are few reports on this aspect of research.Zeaxanthin cannot be formed by the body itself,so it needs to be ingested through food.Corn is one of the effective sources of zeaxanthin from food.Corn husk contains part of zeaxanthin,which is a by-product of wet corn starch production with an annual output of more than 2 million tons.It is usually used as feed,causing serious waste of resources.In this study,corn husk was used as raw material to optimize the extraction and separation of zeaxanthin by enzymatic hydrolysis and ultrasonic-assisted solvent method,and then used macroporous adsorption resin and semi-preparative liquid chromatography to purify it;and through in vitro experiments To explore the inhibitory effect and mechanism of zeaxanthin on gastric cancer cells.This research can provide theoretical support for the deep processing and comprehensive utilization of corn husks,and provide new ideas for the application of zeaxanthin in regulating digestive system diseases.The experimental results of each part of this research are as follows:(1)The study optimized the extraction process of zeaxanthin by response surface methodology based on single factor results.With 95%ethanol as the extractant and zeaxanthin concentration as the index,the best extraction process is neutral protease,enzymatic hydrolysis temperature 29?,enzymatic hydrolysis material-liquid ratio of 1:20(m/v),and enzyme activity When 20.9 U/mg,enzymatic hydrolysis for 5 h,extraction time of 1.65 h,extraction temperature of 39?,liquid-to-material ratio of 1:16.2,and ultrasonic power of 400 W,the content of zeaxanthin is 208.36?g/g.(2)Separation and purification of zeaxanthin by macroporous adsorption resin and semi-preparative liquid chromatography and determination of its antioxidant capacity.First,AB-8macroporous resin was used to process the crude zeaxanthin extract,using 70%ethanol as the eluent,and the elution flow rate was 1 m L/min.When eluting 3 BV,the concentration of zeaxanthin was 9.68?g/m L;Secondly,the zeaxanthin concentration obtained was 12.93?g/m L by saponification with 40%KOH and the sample solution at a ratio of 1:1;finally,the saponified extract was purified by semi-preparative liquid chromatography,the purity of zeaxanthin obtained is 73.41%.The scavenging ability of ABTS free radicals was measured to be 95.27%,and the total antioxidant capacity of purified zeaxanthin was 0.235 Fe SO₄(m M),which has good antioxidant capacity.(3)Use flow cytometry and western blotting to explore the inhibitory effect and mechanism of zeaxanthin on human gastric cancer cells.First,the CCK-8 experiment confirmed that zeaxanthin has a significant killing effect on human gastric cancer cells but has low toxicity on human normal cells;secondly,the apoptotic rate of AGS cells under the action of zeaxanthin was tested,and the results showed that With the prolongation of the action time of zeaxanthin,the apoptotic rate of AGS cells increased from 1.71%to 56.36%,while the intracellular mitochondrial membrane potential decreased from 90.35%to 76.86%.Its related anti-apoptotic proteins Bcl-2and pro-caspase-3 and pro-PARP expression levels were inhibited,while the protein expression levels of apoptotic proteins Bax,cytochrome C,cleave(cle)-caspase-3 and cle-PARP increased;the protein expression levels of p-ERK,p-STAT3 and NF-?B were inhibited,while the protein expression levels of p-p38,p-JNK,and I-?B increased,indicating that zeaxanthin can regulate the MAPK pathway;after adding p-ERK inhibitors,p-The protein expression levels of STAT3 and NF-?B were suppressed,and the protein expression levels of p-STAT3 and NF-?B increased after the addition of p38 or JNK inhibitors,thus indicating that MAPK can regulate STAT3 and NF-?B;When the concentration of zeaxanthin increases,the level of reactive oxygen species(ROS)in normal human gastric cells GES-1 decreases,and with the increase of the concentration or time of zeaxanthin,the level of ROS in AGS cells increases;similar to the zeaxanthin group Compared with the apoptotic rate of AGS cells in the ROS scavenger NAC and zeaxanthin group added at the same time,it can indicate that increasing the ROS level can induce apoptosis;when the ROS scavenger NAC is added,p-ERK,The protein expression levels of p-STAT3 and NF-?B increased,while the protein expressions of p-p38,p-JNK,and I-?B were inhibited,indicating that ROS can regulate the MAPK signaling pathway;the action time of zeaxanthin is prolonged,and AGS cells are The arrest rate of G2/M phase increased from 23.99%to 36.73%.At the same time,the protein expression of p-AKT,CDK1/2,Cyclin A and Cyclin B1 was suppressed,while the protein expression levels of p21 and p27 increased.After administration,the protein expression levels of p-AKT,CDK1/2,Cyclin A and Cyclin B1 increased,while the protein expressions of p21 and p27were inhibited.Finally,the mechanism of action of zeaxanthin was discussed,and it was found that zeaxanthin can regulate the MAPK signaling pathway to induce apoptosis by up-regulating the level of ROS in cells;MAPK can also induce apoptosis by regulating the mitochondrial pathway;in addition,zeaxanthin Quality can also increase the level of ROS in cells to arrest cells in the G2/M cycle,leading to apoptosis of gastric cancer cells.In summary,this study optimized the extraction process of zeaxanthin from corn husk and tested the antioxidant capacity after separation and purification.To investigate the inhibitory effect of zeaxanthin on human gastric cancer cells and the mechanism of action,it was found that the optimized method was suitable for the extraction of zeaxanthin from corn husk,and the higher purity of zeaxanthin could be obtained.Its antioxidant capacity is closely related to its inhibitory effect on gastric cancer cells.This study provides a new idea for good source of zeaxanthin and provides a reference for resource integration of maize.