Quick Screening Of Antioxidant Compounds From Traditional Chinese Medicinal Herbs Using On-line HPLC Analysis

The antioxidants, not only play an important role in the prevention and treatment of human disease, but also are important preservative in food and industrial products. In view of potential toxicities and carcinogenic effects of synthetic antioxidants, researchers have taken more attention to natural antioxidants with high efficiency and low toxicity, such as phenolic acids, flavonoids and polysaccharides from the rich resources of Chinese herbal medicines. There are many methods for antioxidant activity evaluation, and each method has its own advantages, disadvantages and scope of application. At the end of1990s, the combination of chromatographic analysis and antioxidative assay realized simultaneous separation and activity detection of chemical compounds, which was able to rapidly screen antioxidative effects of a variety of substances from mixtures. And the compound structures could be preliminarily identified through the MS analysis. This method solved the problems including low efficiency, heavy workload and the compound variability of traditional methods.In this thesis, based on this method, antioxidants in extracts of some traditional Chinese medicines including Erigeron breviscapus and three kinds of salvia (Salvia yunnanensis.C. H.wrigh (SY), Salvia przewalskii Maxim var. przewalskii (SP) and Salvia castanea f.castanea (SC)) were screened. Also, the advantages and disadvantages of HPLC-ABTS method and HPLC-DPPH method were compared. The main contents and results of the current research are as follows:1.70%methanol extracts of Erigeron breviscapus (EBME) performed different degrees of inhibition on DPPH and ABTS free radicals in off-line assay, and the activities were weaker than the positive control VC in both asays.2. Based on the online HPLC-ABTS method and HPLC-DPPH method,15antioxidants have been found in EBME. The structures of13compounds have been elucidated by means of MS data, including4-CQA,1,4-DCQA,3,5-DCQA,4,5-DCQA,1-Malonyl-3,5-DCQA,9-COA,3-CDOA,4,9-DCDOA,3,9-DCDOA, Erigeside I, Scutellarin, Quercetin-3-O-glucuronide, Eriodictyol-7-O-glucuronide. This is the first time to report the antioxidant activity of9-COA,3-CDOA,4,9-DCDOA,3,9-DCDOA and Eriodictyol-7-O-glucuronide. Isochlorogenic acid A and Scutellarin were the main two antioxidants in EBME. Off-line testing results showed that their free radical scavenging activities were stronger than VC.3. Reverse anti-oxidative activity order of scutellarin and3,5-di-CQA was observed in on-line HPLC-ABTS assay and on-line HPLC-DPPH assay. To validate their anti-oxidative activities, the off-line ABTS and DPPH assays were performed. Given sufficient reaction time,3,5-di-CQA showed higher activity than scutellarin, which was consistent with the order obtained in on-line HPLC-ABTS assay. These results revealed that on-line HPLC-ABTS assay is a more sensitive method for screening and determining free radical scavengers, especially more suitable for those compounds with slower reaction kinetics.4. Antioxidant activities orders of95%ethanol extracts of six samples were measured by off-line ABTS assay, the order is SP1>SC2>SC1≈SY1>SP2>SY2, and the activities were weaker than the positive control VC in both asays.5.13free radical scavengers have been discovered from the underground part of Salvia yunnanensis.C. H.wrigh and the aerial part of Salvia przewalskii Maxim var. przewalskii using on-line ABTS assay. And seven compounds (Yunaneic acid G, Salvianolic acid B,11,12-di-O-methylisorosmanol, Tanshinone Ⅰ, Rosmanol, Tanshinone ⅡA and6-oxo-ferruginol) were identified. And on-line ABTS method has been used to analyze the antioxidant abilities of them and the order was:salvianolic acid B> VC> Tanshinone Ⅰ> Tanshinone ⅡA.The current research revealed that HPLC-ABTS/DPPH is a rapid and efficient method for screening of free radical scavengers from the complex Chinese herbal samples. Comparing with HPLC-DPPH method, HPLC-ABTS assay has a more sensitive response to some of the chemical composition.