| The growing attention has been paid to the health benefits of whole grain foods, and the recommendedintake of standards are improved continuously in recent years.In the past five years,the new types of foodswhich are developed for the whole grain foods are three times of the previous. Polyphenols in whole grainfoods have significant antioxidant activity, they can remove the free radicals, and they also haveproliferation inhibition effects on some tumor cells. However, the studies on the polyphenols in whole grainfoods always only focus on the content of its free types of polyphenols, while ignore the conjugatedpolyphenols that also occupy a certain proportion in whole grain foods,which lead to a seriouslyunderestimation of the content of polyphenols in whole grain foods. Adlay, as a kind of medicinal andedible efficacy of whole grain foods, is widely grown in the Asian region,and it has good antioxidantactivity. But few studies and evaluations about the antioxidant activity and the inhibition of proliferation onsome tumor cells of our existing varieties of adlay have been reported at present.So,in this paper,we put thedomestic common varieties of adlay as our research samples, to explore the functional components and thecontents of the polyphenol extract in adlay,then determine the extracellular and intracellular antioxidantactivity of the extract, to study their inhibtion of cytotoxicity and proliferation of different tumor and itsmechanism of action.Besides these,we take the animal experiments to evaluate the lipid-lowering effects ofadlay extract objects on the high-cholesterol diet rats.In this study,different varieties of adlay are the objects, we take different extraction methods to extractthe free and bound polyphenols in the adlay samples,then measure the contents of polyphenols, flavonoids,vitamin E respectively.At the same time,we pre-test the chemical composition of the adlay extracts,thenmeasure and analysis the active ingredient in the adlay extracts by HPLC. Which,the conjugatedpolyphenols are extracted by using NaOH that digest and combine with ethyl acetate to extract, it canextract more than90%of the conjugated polyphenols in the adlay samples, which lead to a morecomprehensive evaluation of the contents of the polyphenols and flavonoids in the adlay samples. Usingboron sodium hydroxide-chloranil colorimetric to determine the total contents of flavonoids can overwritealmost all flavonoids species,and the data is more accurate in comparison with the current domestic AlCl3colorimetric method and high performance liquid chromatographic method. From the results,we find thatthe content of the conjugated polyphenols is close to which of the free polyphenols in the adlaysamples.The conjugated polyphenols contributed45.28%of the total polyphenols,and the conjugatedflavonoids contributed71.08%of the total flavonoids. Polyphenol contents in different varieties of adlayare obviously different. Overall, the adlay named Liaoning5and Long Yi1are significantly better than theblack Guizhou Valley adlay. The contents of the tocopherol in the adlay samples and the other cereal cropsare in the same order of magnitude,they are also close to that of wheat.The content of total tocopherol in theblack Guizhou Valley adlayis the highest one which reachs14.75±0.62μg/mL.There are significantdifferents among the contents of tocopherol in different cultivars（P＜0.05）,and δ-tocopherol can not bechecked out.In the first time,we take the preliminary high-performance liquid chromatography scan andanalysis of different varieties of adlay samples, the results show that HPLC spectras of adlay extracts aresimilar, they contain substantially the same characteristic components. The peak time of the freepolyphenol extract is earlier than that of the conjugated ones,and the peak time is about6min. In the HPLCspectras of the adlay extracts,the peaks’ shapes are all complex and the absorption peaks are more.All theseindirectly proof that there are rich varieties of botanic active ingredients in the extracts of the experimentalvarieties of adlay of the experimental varieties of barley.Use ORAC and PSC as evaluation of vitro antioxidant activity，and CAA as evaluation of intracellularantioxidant activity，to determine the antioxidant capacity of different varieties of adlay extracts. Among them,compare psc method with ORAC method, not only the antioxidant activity for a water-soluble partbut also the lipophilic portion of the sample can be measured, and the result is more rapid andcomprehensive; Intracellular antioxidant activity test(CAA test), however, compared with the commonchemical antioxidant capacity evaluation, it is more practical for the establishment of a cell culture model atthe biological level to analysis of the antioxidant capacity of the material. The results found that: the boundpart (ORAC method and PSC method) contributed48.08%and43.64%to the total antioxidant capacityrespectively, which proved that in the cereal crop adlay samples,although the conjugated polyphenols arenot easy to separated and extracted, and are often neglected, antioxidant capacity is still able to demonstrateclose to the free polyphenols,thus it can enables the antioxdant capacity of adlay doubled.The intracellularantioxidant activity assay results show that the CAA in bound Long Yi1adlay is the highest,which is1.37±0.48μmol QE/100g adlay samples(PBS wash) and9.87±1.53μmol QE/100g adlay samples(no PBSwash). Long Yi1adlay also has the highest total antioxidant capacity,which is the same with chemicalevaluation method in vitro. The total antioxidant capacity is closely related to the total polyphenol content(r2=0.935) in the sample. But the respective antioxidant capacity specific to the free-type and combinationtype, cell evaluation methods compared with external chemical evaluation methods are not exactly thesame.Research adlay extracts’cytotoxic and anti-proliferative effect to MCF-10a, MDA-MB-231, MCF-7and HepG2cells,we found: there are significant differences in varieties of the experimental cells of adlaypolyphenol extracts.They have more significant inhibition of proliferation and toxicity tests proved thetoxicity of the sample itself.Above all,there was no obvious toxicity and inhibition of proliferation toMCF-10a cells; To MDA-MB-231cells,inhibit the proliferation of cell lines in the varieties adlay havelarger differences.Free Liaoning5adlay, however, has the strongest ability of anti-proliferative effect(EC50=124.52±10.17mg/ml) which is significantly higher than the Guizhou Black Valley and Long Yi1adlay (P＜0.05), at the same time, the toxicity of MDA-MB-231cells was not detected (CC50＞320mg/ml);To the MCF-7cells, the differences and anti-proliferation among varieties of adlay are basicallythe same as MDA-MB-231cells; To HepG2cells, however, adlay varieties have significant proliferationinhibition effect.The effects to the cycle of HepG2cell by PCNA/p21and CDK4protein have been researched.We findthat when the concentration of the extracts in adlay was150mg/mL,the expression of p21protein rised;andwhen the concentration of the extracts in adlay was180mg/mL, compared with the control,the amount ofthe expression of p21protein increased100%.In this experiment, CDK4protein was not significantlydetected.The study confirmed that the adlay extracts inhibit the proliferation of human hepatoma HepG2cells by DNA polymerase regulating factor PCNA and p21-mediated cell cycle regulation.The study used high-cholesterol diet rat model to evaluate the hypolipidemic and antioxidant effects ofthe adlay extracts. The results showed that:after we feed Wistar rats with high-cholesterol diets for4weeks,the contents of TC and LDL-C in the serum increased significantly compared with the normalgroup,while there were no obvious changes in body weight gain,feed efficiency,and the index of liver.Adlayextracts can obviously reduce the contents of TC and LDL-C in serum of high cholesterol diet rats.Besidesthis,the high-dose group of adlay extracts are able to put it back to the normal level.It illustrated that adlayextracts have hypolipidemic effect on high-cholesterol diet rats,which has clinical significance.In addition,compared with the model group,adlay extracts can significantly improve the CAT and GSH-PX activity andantioxidant capacity in liver,also can obviously reduce the content of MDA in the serum of high cholesteroldiet rats.In adlay extracts, the antioxidant capacity of dose group, high dose group, free type polyphenolgroup and conjugated polyphenol were significantly increased, and the rising amplitudes are82.89%（P＜0.05）,107.52%（P＜0.01）,129.25%（P＜0.01）,113.64%（P＜0.01）respectively.It proved that adlayextracts can significantly improve the antioxidant capacity in serum of high-cholesterol diet rats. |
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