In Vitro Antioxidant Activity Of Natural Products From Microorganism In Special Eco-environment And Their Structure-Activity Relationships

Oxidative stress is a phenomenon that the body produces large number of free radicals after being stimulated by external factors,which leads to cell and tissue damage.Oxidative stress can cause a series of diseases,such as cardiovascular and cerebrovascular diseases,malignant tumors,neurodegenerative diseases,diabetes,and so on.Therefore,it is of great significance to find out antioxidants that can effectively eliminate free radicals for the treatment of diseases related to oxidative stress.Looking for antioxidant lead compounds from natural products and carrying out structural modification and optimization are important ways to develop effective new drugs for treating oxidative stress-related diseases In order to adapt to the special eco-environment,microorganisms living in the extreme environment(such as high temperature,high permeability,and high pressure,etc.),marine environment,animal and plant internal environment and other special environments can form special secondary metabolic pathways and easily produce novel chemical components with significant bioactivities,which are important sources of antioxidant lead compounds.The antioxidant activity of 18 compounds derived from Penicillium janthinellum HK1-6 and Penicillium sp.HK1-22 in the rhizosphere soil of mangrove forests,endophyte Alternaria sp.W-1 associated with Laminaria japonica,and endophyte Streptomyces rochei SR-1102 associated with cucumber,were determined by four in vitro antioxidant assays in this thesis,including oxygen radical absorbance capacity(ORAC),ABTS(2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium)free radical scavenging ability,DPPH(2,2-diphenyl-l-picrylhydrazyl)free radical scavenging ability,and ferric reducing ability of plasma(FRAP).It was found that peninaphone A(3)and peninaphone B(4)produced by Penicillium sp.HK1-22 showed good antioxidant activity in these four assays.Their ORAC values were 1.91 ± 0.14 and 1.71 ± 0.21 μmol Trolox/mg,respectively.The scavenging rate of peninaphone A(3)and peninaphone B(4)on ABTS.+free radicals was(18.63±0.83)%and(18.53±0.95)%at 100μg/mL.The scavenging rate of peninaphone A(3)and peninaphone B(4)on DPPH.free radicals was(47.34±1.68)%and(47.34± 1.85)%at 100 μg/mL.The FRAP value of peninaphone A(3)and peninaphone B(4)were 12.86±0.66 and 13.99 ± 0.83 μmol FeSO4/mg,respectively.In addition,penicilone B(1)from P janthinellum HK1-6 and peninaphone C(5)from Penicillium sp.HK1-22 showed moderate antioxidant activity in the ORAC,ABTS and DPPH assays.Tricycloalternarene 3a(12)from Alternaria sp.W-1 and dihydrodaidzein(15)and daidzein(16)from S.rochei SR-1102 only showed certain antioxidant activity in the ORAC assay.Density functional theory(DFT)is a widely used quantum chemical calculation method.DFT could be used to predict the antioxidant activity of compounds and find out the key antioxidant groups,which provides theoretical basis for structure optimization of compounds and plays an important role in the development of antioxidant drugs.Therefore,DFT was used to discover the structure-antioxidant activity relationship of some compounds in this thesis.The optimized molecular model was established by Gauss view 6.0,and the geometric structure of the molecule was fully optimized at the level of M06-2x/6-31+G**.Then,Gaussian 16 was used to analyze the antioxidant activity of the compounds from the aspects of molecular structural parameters,bond dissociation enthalpy(BDE),adiabatic ionization potential(IP)and frontier molecular orbital(FMO)at the same level.It could be seen that the hydrogen atom of phenolic hydroxyl group was easy to dissociate,which was the basis of the antioxidant activity for naphthopyranone compounds peninaphones A-C(3-5).The stereoisomerism at C3 position had little effect on their antioxidant activity,while the presence of C2-C3 double bond greatly reduced the antioxidant activity.Furthermore,the activity of phenolic hydroxyl group was closely related to its substitution position.The most active phenolic hydroxyl group at C5 was the most important reaction site of naphthopyranones followed by C6,while the phenolic hydroxyl group at C8 was the most inactive.The DFT calculation of azaphilone compound penicilline B(1)showed that phenolic hydroxyl group at C15 was easy to occur hydrogen-abstraction reaction.Penicilone B also might bind to active free radicals directly to terminate the chain reaction of oxidation,and ring A and ring B were the primary active sites.The DFT calculation of diacylated putrescine alkaloids N,N’-diferuloylputrescine(DFP,13)and N-p-coumaroyl-N’-feruloylputrescine(CFP,14)showed that DFP and CFP could not form intramolecular hydrogen bond and more stable semiquinone free radical structure,and showed very weak antioxidant activity.The DFT calculation results of the above-mentioned compounds were consistent with the experimental results in vitro.The relationships between antioxidant activity and structures of naphthopyranone and azaphilone compounds were studied by DFT method for the first time in this thesis.It provides a theoretical basis for further research and development of naphthopyranones and azaphilones,which is of a broad application prospect.