Study On The Therapeutic Material Bases And Their Mechanisms Of Arundina Graminifolia (D.Don) Hochr.

Arundina graminifolia（D.Don）Hochr.（A.graminifolia）called“wenshanghai”and“baiyangjie”,is widely used antidote in the Dai medicine.It is not only used for the treatment of food intoxication and urinary tract infection and snakebite,but also applied to the Dai medicine“Ya Jie”（antidote）therapeutical principles of“detoxification goes before symptoms”and“detoxification goes before treatment”.Besides,the Bulang and the Va people use it to treat tracheitis,pneumonia and pulmonary abscess.However,the research on its bioactivities and the corresponding mechanisms has still been in a gap.To explore pharmacological activities and the corresponding mechanism and also the structure-activity relationship of the ethnic medicine A.graminifolia,this thesis conducted a bioactivity-oriented study on the pharmacodynamic material basis using chromatographic separation technology.Combined with the clinical application of A.graminifolia,the thesis systematically evaluated the anti-bacterial,AChE inhabitation,anti-aging and anti-tumor activities of the main stilbene derivatives in A.graminifolia,and further elucidated the related pharmacological activity mechanisms.The main contents of this thesis are as follows:1.Pharmacodynamic material basis of A.graminifolia.Oriented by the bioactivities,thirty-two compounds were separated by column chromatography（siligel,RP-ODS,sephadex LH-20,HPLC）.The types of the compounds included stilbenoids,phenanthrenes,flavonoids,esters and quinones.These compounds were identified by a basis of modern spectroscopic methods,including the 1D-NMR(¹H-and ¹³C-NMR spectra),2D-NMR,UV,MS and X-ray diffraction.Besides,seventeen ones were isolated from A.graminifolia for the first time.Two single crystals were cultivated using the solvent evaporation method.Orchinol belonged to monoclinic system with cell parameters:a=8.548（3）（?）,b=13.426（4）（?）,c=11.369（3）（?）,β=103.0934°,V=1270.7（7）（?）³,Z=4,R_gt（F）=0.0421,w R_ref（F²）=0.1243.Shancidin belonged to orthorhombic system with cell parameters:a=11.355（5）（?）,b=15.084（7）（?）,c=20.078（9）（?）,V=3439（3）（?）³,Z=4,R_gt（F）=0.0481,w R_ref（F²）=0.0994.The isolated compounds provided the material basis for the study on activity evaluation and the structure-activity relationship.Moreover,the crystal structures provide the elementary information for not only the precise binding patterns between target and small molecule inhi-bitors but and the relationship between bioactivities and structures.2.Anti-bacterial activity evaluation and mechanism.Blestiarene A and densiflorol B exhibited excellent inhibitory effects on E.coli and S.aurenus with the MIC ranging from25-50μg/m L（positive control amoxicillin 0.5-5μg/m L）.An in-depth anti-bacterial mechanism study was conducted by detecting AKP and ROS levels,the fluorescence intensity of membrane proteins,membrane permeability,adhesion of compounds to bacteria and the fluorescence intensity of PI probe binding to DNA and observing the morphologies of bacteria.The results demonstrated that blestiarene A and densiflorol B were able to induce the generation of active oxygen,change the structure of residues Phe,Trp and Tyr on the membrane,promote the release of AKP,increase the permeability of membrane,decrease the surface adhesion of the bacteria and destroy the cell walls,causing the leakage of DNA and leading to the apoptosis of bacteria.By DFT calculations,we established the relationship between frontier orbitals and electrostatic potential of the compounds and their antibacterial activities and elucidated how the number and position of hydroxyl groups on the aromatic ring affected the charge distribution and the orbital energy.In other words,adding the electron-withdrawing groups on the aromatic ring was beneficial to lowering the E_LUMO,and enhancing the antibacterial efficacies.This study made a theoretical analysis and elaboration on the anti-bacterial mechanism for the anti-infection material bases of A.graminifolia.3.AChE inhibitory activity and molecular dynamics simulation.AChE is on the key targets for the treatment of Alzheimer’s disease,Parkinson’s disease.Given the applications of A.graminifolia on drug poisoning relieving and neuroprotection,the AChE inhibitory effects of compounds isolated from A.graminifolia were evaluated.Blestriarene A,coeloin,ephemeranthoquinone exhibited excellent inhibitory effects on AChE with the IC₅₀ values of0.285,0.402,0.116 mmol/L,respectively.The relationship between structure and activity indicated that the electron withdrawing groups on the 2,7-positions in the phenanthrene structure may have a better interaction with AChE protein,resulting in enhanced inhibitory effect.However,larger steric hinderance groups on the 1-position may reduce the inhibitory effect.What’s more,the structural flexibility was conducive to the binding for compounds and enzyme,which elevated the AChE inhibitory activity of compounds.The enzyme kinetics study suggested that the phenanthrene compounds had mixed inhibition to AChE.The inhibition constants K_iof blestriarene A,coeloin,ephemeranthoquinone against free AChE were 0.542,0.748,0.193 mmol/L,respectively,larger than the inhibition constants K_i’of compounds against enzyme-compound complexes（0.387,0.693,0.157 mmol/L）,which indicated that these compounds inhibited the free AChE dominantly.Molecular dynamics simulation data demonstrated that ephemeranthoquinone was able to insert deeply into the active pocket of AChE by conformation transformation.Further,ephemeranthoquinone interacted with the amino acid residues Trp83,Asn84,Thr154,His480,Tyr370 and Trp83 via hydrogen bonds andπ-πpacking interaction,resulting in a stable complex.The inhibitory effect was mainly attributed to the flexibility of the compounds with a same type of structure and the compounds binding to residues Trp 83 and Tyr 370,which provided a new insight for designing brand-new AChE inhibitors.4.Anti-aging assay and mechanism using C.elegans as a mode.Neurodegenerative diseases are closely related to aging.The specific mechanisms of detoxication and life prolong of are still unclear.C.elegans is an ideal mode creature for the neurobiology study such as cell apoptosis and aging.In this thesis,the effects of different skeletons compounds（bibenzyls,phenanthrenes and quinones）from A.graminifolia on C.elegans’lifespan was explored for the first time.We explained the mechanism that bibenzyls had contribution to C.elegans lifespan prolonging and the MDA level decrease inside C.elegans,from the aspect of molecular biology.The bibenzyl compounds resveratrol and batatasin III enhanced the enzyme activities of SOD and CAT enzymes and decrease the level of8-hydroxydeoxyguanosine（DNA oxidation product）.The changes of the enzyme activities indicated that these compounds may exert anti-oxidative effect in vivo.However,all the compounds elevated the level of MDA,which demonstrated that these compounds exerted anti-oxidative effect while caused the oxidative damage on C.elegans.The opposite results suggested that the compounds had a greater effect on boosting anti-oxidant enzyme activity than causing the damage on C.elegans,as a general result of increasing lifespan of C.elegans.The results above revealed that the mechanism of the active components from A.graminifolia on anti-aging was attributed to many factors.5.Anti-tumor activity evaluation in vitro and mechanism.The stilbene compounds had a potential for anti-tumor.In vitro study demonstrated that shancidin,coelonin and densiflorol B displayed moderate and excellent inhibitory effects on bladder cancer cell 5637,with IC₅₀values ranging from 35 to 80μM.In the bladder cancer cell migration and proliferation experiment,shancidin exhibited the strongest anti-tumor activity,followed by densiflorol B.For the mechanism study on how the compounds affected the migration and proliferation of balder cancer cell,Western Blot was used to detect the expression of EMT biomarkers（E-Cadherin,N-Cadherin,Vimentin）,cell adhesion proteins（DSP,ZO-1）,signal transduction proteins（P-AKT S473,AKT Pan）and cell cycle regulatory proteins（P-ERK,ERK,P27,P-P27）after drugs administration.The active components isolated from A.graminifolia reduced migratory ability of cancer cell via up-regulating the expression of E-Cadherin and down-regulating the expression of Vimentin and N-Cadherin.Meanwhile,these compounds increased the phosphorylation of AKT（a downstream target protein of P27）,which may block the cell cycle in S phase.Nevertheless,the active expression of ZO-1 and overexpression of signal transduction protein ERK suppressed the phosphorylation of P-ERK,which depressed the cell proliferation.In conclusion,combined with the folk applications of the Dai medicine A.graminifolia,this thesis systematically conducted a study on pharmacodynamic material basis of A.graminifolia and deeply evaluated diverse bioactivities of A.graminifolia.The mechanisms that blestiarene A and densiflorol B were helpful to anti-bacterial,resveratrol and batatasin III to anti-aging and shancidin to anti-tumor were revealed.Besides,the theoretical calculation results indicated that the substitution groups,steric hindrance and molecular flexibility of the compounds with a similar structure had a great impact on the bioactivities.These results proved a basic data to profile the complex relationship between efficacies and chemical compounds of A.graminifolia,which will be not only the scientific supports for the clinical use and promotion but also the modern pharmacological theoretical supports for the traditional efficacies of ethnic medicine.