Study On The Mechanisms Of Phenonic Antioxidants From Chinese Herbal Medicine In Reaction With DPPH· Radical

ObjectivePhenonic antioxidants from Chinese herbal medicine have been proved to exert excellent antioxidant ability by many researchers. To provide a direct evidence to settle the controversy concerning the mechanisms of those phenonic antioxidants in interaction with DPPH· radical (DPPH·), the present study focused on illustrating the pathways these reactions undergo, and further deduced the structure of the reaction products based on the MS spectrograms files. Since most of the current researches analyze the mechanisms from the perspective of thermodynamics, kinetics or functional model, this study tried to explain the antioxidant mechanisms with total different methods, such as GC-MS and HPLC-MS.MethodsPresent study proceeded from the classical DPPH· radical scavenging reaction. Six different phenonic antioxidants from Chinese herbal medicine and other two synthetic antioxidants were chosen as testing reactants with DPPH·radical. Then the reaction products were analyzed using GC-MS or HPLC-MS method. Based on the results of mass spectrograms, the mechanisms of the above reactions were discussed respectively, and the possible solvent effect was further explored. To determine whether reaction conditions had an essential effect on the mechanisms of the reactions or the final product type, BHA was chosen as a representative, and reaction time, reaction temperature, and reactants mole ratio were tested as impact factors, respectively.ResultsThe data indicated that most of the tested antioxidants reacted with DPPH· radical, to yield a dimmer as reaction product. For BHA, as analyzed by GC-MS, the product molecular ion peak was at m/z 358.2, while BHA itself at m/z 180.1; For both eugenol and isoeugenol, the products molecular ion peak were at 326.2 m/z, while eugeno and isoeugenol themselves at 164.1 m/z; For both carvacrol and its isomeride thymol, the products molecular ion peak were at m/z 298.1, while carvacrol and thymol themselves at m/z 150.2; For scopoletin, as analyzed by HPLC-ESI-MS, the product peak gave a clear molecular ion peak at m/z 381.0 [M-H]-, which inferred the gerneration of a dimmer as well.Besides dimmers, another different product type occurred as adduct of antioxidants and DPPH· radical. For the reaction of BHT scravenging DPPH· radical, a molecular ion peak at 430.4 m/z [M-H]-, compared to BHT itself m/z 220, which indicated the formation of an adduct of BHT and DPPH· radical; Moreover, The data showed a similar result when tested with sinapine thiocyanate, HPLC-ESI-MS chromatogram gave a molecular ion peak at m/z 703.6, which also suggested an affixture as product.In addition, the maximum absorption wavelengths of scopoletin and the reaction product were respectively 351 nm and 382 nm. And for sinapine thiocyanate, it was from 344nm to 475nm, the great bathochromic shifts (351 nm→382 nm,344 nm→475 nm) inferred that the reaction products may contain a larger conjugative system than scopoletin or sinapine thiocyanate itself.At last, the research on impact factors of BHA scavenging DPPH* radical reaction suggested that the product peak area maintained a slowly rising trend from 0.5hr to 12 hrs for reaction time; When reaction temperature ranged from 20 ℃ to 80 ℃, the product peak area showed a declining trend instead of a rising trend as usual; The reaction mole ratio test indicated that when BHA reacted with DPPH· at 1:1, the product peak area reached the highest level.ConclusionOn this basis, it can be concluded that it is the phenolic hydroxyl group of the antioxidants that exerts the antioxidant ability when scavenging DPPH* radical; Eight chosen antioxidants convert into radicals at the first step, during which, both HAT and SET have been regarded as two main possible mechanisms, but further exploration is still necessary to confirm which one specifically.According to the present study, RAF mechanism has been proved to take place positively for all of the eight antixoidants. And RAF process includes three forms:firstly, dimerization reaction, such as BHA, eugenol, isoeugenol, carvacrol, thymol and scopoletin; Secondly, the adduct reaction of semiquinone radical and DPPH· radical, such as BHT and sinapine thiocyanate. Thirdly, the adduct reaction of semiquinone radical and solvent radical, such as BHT and isoeugenol. During this process, both p-π conjugative effect and σ-p super conjugative effect shuld be taken into consideration comprehensively, as well as the steric-hinerance effect of the final reaction products. This paper first provides direct evidence for the above three forms of RAF mechanism, and proposes related theory analysis.In addition, the data of the impact factors test indicate that reaction time, reaction temperature and reactant mole ratio all have an effect on the product generation.