| Oxidation in human body involves in causes of some human diseases, and one of the causes of food deterioration is a radical initiated reaction of oxygen with one or more of the ingredients. Commercial antioxidant supplements or natural antioxidants have been used in order to reduce oxidative damage in the human body and prolong the shelf-life of food. However, the possible toxicity of synthetic antioxidants has been questioned. In recent years the natural antioxidants become a global trend. There are many factors such as collecting time, grown area effecting the antioxidants and antioxidant activity of plants. And to detect antioxidants in mixture such as plant extract is a tough work. To resolve these two questions better, the antioxidant capacity of Eucommia ulmoides Oliv.-a traditional Chinese medicine was studied. And three kinds of methods of rapid detection of antioxidants in plants extracts were set up. After that, several plants extracts were analyzed and their main antioxidants were identified by the new methods.Firstly, the on-line HPLC-RSD (High Performance Liquid Chromatography-Radical Scavnger Detection) system was setup to detect antioxidants in plants extracts. The free radical detection part was optimized as follows:free radical concentration 0.32 mmol/L; free radical solution flow rate 0.5 mL/min and reaction coil length 10.0 m. A mixed juice and its organic solvent extracts were analyzed by the system. About 20 antioxidants were found in t-butyl methyl ether extract. The main active compounds were elucidated as chlorogenic acid and a compound with similar structure by HPLC-DAD-MS. Phloridzin and hypercoside were also the active compounds in the extract.The leaves and male flowers of Eucommia ulmoides Oliv. were also analyzed by the on-line HPLC-RSD system. Eighteen kinds of antioxidants were found in the leaves and 19 antioxidants in the male flower sample. The main active component of the leaves and male flowers was chlorogenic acid. Medioresinol di-glucopyranoside was found in the male flowers and quercetin-glycoside was in the leaves of Eucommia ulmoides Oliv.The method can detect antioxidants in polar mixtures rapidly and simply with low minimum detectable concentration and good repeatability.To further increase its sensitivity and decrease the manpower input, an additional automated on-line sample enrichment system was added. With this system injection enrichment and separation is controlled by computer and by the post-column addition of an ABTS radical solution pump, detection and identification of antioxidants in fruit juices, vegetables, and processed foods is possible. Trap breakthrough and reproducibility of peaks area calculated. The maximum injection volume is 200μL. By this method, various apple sample were investigated. Their profiles revealed more radical scavenging compounds than previously found because of the larger injection volume and sample enrichment. The effects on the components of a mixed juice of storage time, storage temperature and producing batch analyzed by this system. It is found that the content of antioxidants in the juice decreased after stored 9 weeks. There is no remarkable different between juices stored less than 4℃or room temperature. And two series of juices were found although they had similar packages.Fresh apple juice, carrot juice and strawberry juice were analyzed by this HPLC-RSD system with on-line enrichment, and the results showed that apple and strawberry had abundant antioxidants. Apple juice treated under different temperature was detected. It was found that apple components were changed and some negative peaks became smaller or even disappeared. On the other hand, apple juice treated under high temperature showed a new antioxidant having not been found in fresh juice.HPLC-RSD system with on-line enrichment can be used as a technology to detect antioxidants in food or plant medicine more sensitive than normal on-line system.To directly analyze non-polar samples, a rapid normal-phase HPLC method for on-line detection of antioxidants in complex non-polar mixtures was developed. The stable radical galvinoxyl·was applied in this system because leads to higher S/N ratios than DPPH·. The use of a superloop in the system for post-column radical addition decreased the noise remarkably. In the optimized instrumental set-up a radical scavenging solution of 2uM galvinoxyl·in hexane is delivered by a pump equipped with a superloop at 1.0 mL/min into a reaction coil of 15m×0.25mm. Heating the reaction coil increases the sensitivity for some compounds.Wheat germ oil and olive oil were studied by this method and found the main antioxidants in these two oils were tocopherols. By HPLC-DAD-MS method and comparison with UV and MS data of standard compounds, the antioxidants were a-tocopherol,β-tocoherol and y-tocopherol in wheat germ oil and only a-tocopherol in olive oil.In oil-soluble rosemary extract about 15 kinds of antioxidants were found, and three of the main antioxidants were elucidated as carnosic acid, carnosol and a-tocopherol.The system can be equipped with a splitter to direct part of the column effluent to an MS. Hence, structural data and antioxidant activity from mixtures can be obtained in one analysis. The method is simple and rugged. It can be applied for the direct screening of non-polar samples like vegetable oils and lipid-soluble plant extracts for relatively non-polar antioxidants like tocopherols without any prior sample preparation.At last, the antioxidant capacity of Eucommia ulmoides Oliv were characterized by DPPH radical scavenging assay, hydroxyl radical scavenging assay, metal ion chelating activity and reducing power assays, and inhibition effect on the peroxidation of linoleic acid assay, and the contents of active compounds like polyphenoles were measured, too. And the results were as follows. (1) The bark, leaf and male flower of the plant showed a remarkable antioxidant activity, and the leaf exhibited the strongest activity. (2) The leaves collected in August presented a higher DPPH radical scavenging activity, hydroxyl radical scavenging activity, metal ion chelating activity and reducing power, and the content of polyphenols among the leaves collected during the period of May to October. But leaves harvested in May showed the strongest inhibition effect on the peroxidation of linoleic acid. (3) Among leaves grown in Xinjiang, Sichuan and Shaanxi province, leaves harvested in Sichuan and Shaanxi showed higher activity of DPPH radical scavenging, hydroxyl radical scavenging activity, metal ion chelating and reducing power, and higher polyphenol content. But they showed a weaker inhibition effect on the peroxidation of linoleic acid than sample of Xinjiang. (4) The ethyl acetate extract and n-butanol extract from the water extract of the leaves of Eucommia ulmoides Oliv. presented a stronger antioxidant activity and higher polyphenol content than other extracts. (5) The antioxidant activity of aqueous extract of leaves is comparable with Vitamin E and C, and its ethyl acetate extract and n-butanol extract showed a significant higher activity than Vitamin E and C, and higher than commercial antioxidant BHT, too. From these results, the extract was suggested having a potential to be a commercial antioxidant. (6) The polyphenol content of the samples showed a correlation with their antioxidant activity but not significant. |
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