| A novel mode for antioxidant compounds screening was developed by this thesis: somenature small-molecular compounds（from a variety of edible Chinese herbal）were used fordocking with some enzymes which are related to biological oxidation and radical reaction.According to the virtual screening results，some small-molecular compounds with potentialantioxidant activity can be validated by determination of antioxidant activity based on HPLCin vitro. Then it was quick and easy to find real antioxidant components. This study includesthe following components:1. A molecule libraries including376nature small-molecular compounds wasestablished，five enzymes (LOX-5, LOX-3, Cu/Zn SOD, GPx-1and XOD) which are relatedto biological oxidation and radical reaction were used to docking with the small-molecularcompounds. According to statistical analysis of virtual screening results, the score of94compounds are ranking top50, some of them are reported to own antioxidant activity byreferences.17compounds of them have strong theoretical antioxidant activity to each target,they are salvianolic acid B, chebulinic acid, sennoside B, isoginkgetin, forsythoside A,echinacoside, cistanoside A, bilobetin, isoacteoside, lobetyolin, benzoylpaeoniflorin,polydatin, sesamolin, neohesperidin, astragaloside, kaempferol-3-O-rutinoside, sesamin,respectively.2. Compared to the spectrophotometric method, high performance liquidchromatography has high accuracy and sensitivity. So this paper established three newdetermination of antioxidant activity based on HPLC in vitro.(1) Determination on inhibitory rate of antioxidants to soybean lipoxygenase by HPLCWith enzymatic reactions, linoleic acid can be catalyzed by soybean lipoxygenasespecificly to form linoleic acid hydroperoxide (LA-OOH). The inhibitory rate was calculatedby the HPLC peak areas of LA-OOH in the above reaction system with or without samples.Then, antioxidant activity of samples can be evaluated. The chromatographic conditionsincluded a SinoChrom300A C8column，a mobile phase consisting of methanol-water (55:45,V/V), ultraviolet detection at234nm.(2) Determination on scavenging rate of antioxidants to hydroxyl radicals by HPLCThe proposed method employed the reaction between hydroxyl radicals generated by theFenton system and dimethyl sulfoxid (DMSO) to form formaldehyde, which then reacted with2,4-dinitrophenylhydrazine (DNPH) to produce the corresponding hydrazone (HCHO-DNPH).The hydroxyl radicals scavenging rate was calculated by the HPLC peak areas ofHCHO-DNPH in the above reaction system with or without samples. Then, antioxidantactivity of samples can be evaluated. The chromatographic conditions included a Diamonsil C18column, a mobile phase consisting of acetonitrile-water (65:35, v/v), ultraviolet detectionat365nm.(3) Determination on inhibitory rate of antioxidants to lipid peroxidation by HPLCThe proposed method employed the reaction between malondialdehyde (MDA)generated by Fe2+induced yolk lipid and2-thiobarbituric acid (TBA) to form2-thiobarbituricacid reactive substances(MDA-TBA).The inhibitory rate was calculated by the HPLC peakareas of MDA-TBA in the above reaction system with or without samples. Then, antioxidantactivity of samples can be evaluated. The chromatographic conditions included a KromasilC18column, a mobile phase consisting of acetonitrile-water-glacial acetic acid (24:76:0.5,v/v/v), ultraviolet detection at365nm.3. According to the actual situation of the compound obtained and the result of virtualscreening, three compounds, including polydatin, resveratrol, and chlorogenic acid, had beenverified by determination of antioxidant activity in vitro. The results as followed:(1) Inhibition of these compounds to lipoxygenase-3. Compared to resveratrol andchlorogenic acid, Polydatin is strongest inhibitor to LOX-3. The half-inhibition rate(IC50) ofthe polydatin was41.28μg/ml. The IC50of resveratrol was＞80μg/ml, the IC50of chlorogenicacid was149.6μg/ml. This result is consistent with the results of virtual screening, it wasshowed that the virtual screening was more reliable.(2) Scavenging activity of these compounds to hydroxyl radicals. It is showed thatresveratrol was strongest among these compounds to hydroxyl radicals scavenging activity,IC50was4.53μg/ml. The IC50of polydatin was19.21μg/ml, the IC50of chlorogenic acid was27.59μg/ml.(3) The inhibition of these compounds to lipid peroxidation. It is showed that resveratrolwas strongest among these compounds to inhibit lipid peroxidation, IC50was6.83μg/ml, TheIC50of polydatin was21.53μg/ml, the IC50of chlorogenic acid was＞100μg/ml. |
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