Iridoid Glycosides And Ester Components Are Based On Biological Activity Studies Against Oxidative Stress

ObjectiveIridoid is a class of secondary metabolites found in a wide variety of plant and some animals.It is widely distributed in nature with varied,potent,and important biological activities including the strong protective effects on the central nervous system.It is applied for the treatment of Alzheimers's disease(AD),depression,Parkinson's disease(PD),cardiovascular disease,diabetes,and various cancers.Studies have demonstrated that the occurrence of these diseases closely related to oxidative stress,due to the excessive generation of excess reactive oxygen species(ROS or RNS).ROS and oxidative stress are associated with intro-cellular injuries:viability,DNA and protein damage.Therefore,this study will be helpful to fill the natural health products and drug's vacancy in alleviating body oxidative stress with low toxicity and high performance.Persent study was focused on the oxidative stress to evaluate the pharmacological activity of iridoids on oxidative stress-induced diseases.Iridoids glycosides and esters,including six kinds of compounds(geniposide,picroside ?,liganin,valtrate,acevaltrate and 1-? acevaltrate),were taken into consideration,mainly due to their similar structures but different signal pathways and bioactivities.The structure identification,radicals scavenging abilities,cytotoxic activities and neuronprotection were investigated to uncover of the activity of iridoid compounds,possible mechanisms and structure-activity relationships.Methods1.Structure identification and antioxidant of iridoids(1)Acevaltrate and 1-? acevaltrate are isomers,therefore their structures have been confused in the literature.Thus,LC-MS,UV and NMR analyses were carried out to estimate their chemical structures.2D-NMR including heteronuclear multiple bond coherence(HMBC)and heteronuclear multiple quantum coherence(HMQC)studies were conducted to confirm the structure elucidation.(2)By assessment of antioxidant capacity in vitro,free radical scavenging activity of different iridoid compounds has been evaluated using FRAP,OH·,O-·,ABTS+· and DPPH·,methods.2.The possible mechanism of tumor cell apoptosis induced by glycoside and ester of iridoid compoundsHuman liver carcinoma cells(HepG2),human cervix carcinoma cells(HeLa),breast cancer cells(MDA-MB-231)cells and human umbilical vein endothelial cells(HUVECs)were obtained for cytotoxicity analysis via MTT assay,Flow cytometry analysis for ROS measurements(DCF assay)and flow cytometry analysis for apoptosis assessment(Annexin V-FITC/PI labelling assay)were performed to evaluate the ROS role during HepG2 apoptosis process.3.The possible mechanism of neuroprotective effect by the iridoid glycoside and ester compoundsHydrogen peroxide(H2O2)-induced apoptosis in PC 12 cells(PC 12 + H2O2)was established.The apoptosis rate was measured by MTT assay and the available concentrations and time were considered.When compared with model control group,the recovery rates were calculated by drug groups.Flow cytometry analysis for ROS measurements(DCF assay)and flow cytometry analysis for apoptosis assessment(Annexin V-FITC/PI labelling assay)were used for PC 12 recovery rate analysis in order to uncover the possible mechanism between the iridoid compounds and intracellular ROS rates.4.Antioxidant activities of Vleriana jatamansi Jones compounds,raw drug and preparations,and the structure-activity relationship analysis.Two batches of raw drug and three batches of preparations were studied.The main polar and nonpolar chemical compositions were isolated by HPLC,and their structures were estimated based on the mass spectra analysis and/or comparison with references.The does-effect relationships among chemical compounds,raw drug and preparation were measured by neuroprotective effect experiments.5.The antioxidative diversities beween valepotriates compounds and their degradations.The stress degradation studies of iridoid valepotriates were performed.The optimized stress conditions were following:acidic hydrolysis(0.01M HCl at 25? for 5 h),alkaline hydrolysis(0.01M NaOH at 25 ? for 10 min),oxidative hydrolysis(3%?6%H2O2 at 25 0C for 24 h),thermal degradation(menthol solution at 60 ? for 0,1,2,4,or 6 h;solids at 4 ?for 7 days;solids at 25 ? for 7 days),and light illumination(intensity of 5000 Ix for 3 h).The TLC-DPPH·-MS and LC-MS assay were conducted to speculate the possible structure.We choose comparative analysis on proliferation,cytotoxicity,ROS and apoptosis in human cancer cell lines.The potential antioxidative and neuroprotective activities on H2O2-induced oxidative injury in PC 12 cells were also evaluated by integrate to analysis the structure and activities relationship.Results1.Acevaltrate and 1-? acevaltrate are isomers with acyl groups connected to different positions of the iridoid rings.Based on the HMBC data,the chemical shifts of the four ester carbonyls were assigned as ?C?C 171.2(C-13),169.8(C-16),170.4(C-22)and 170.8(C-2')for acevaltrate and ?C 171.2(C-13),171.6(C-16),167.6(C-22)and 170.9(C-2')for 1-?acevaltrate.During antioxidant capacity study in vitro,Picroside ? showed the highest effects:it may due to the structure of phenolic hydroxyl and oxygen ring.Valtrate,acevaltrate and 1-?acevaltrate exhibited relatively higher radical scavenging activities in the assays.Comparably,geniposide and liganin exhibted no radical scavenging activities during the experiments,which suggested that they had no obvious antioxidant effects in vitro.2.The anti-proliferative effects of iridoids were investigated on three human cancer cells(HepG 2,HeLa and MDA-MB-231)and one normal cell lines(HUVECs).Iridoid glycosides did not induce cytotoxicity but promotes tumor grouth at high concentration in this study.Our work also indicates that iridoid esters(valtrate,acevaltrate and 1-? acevaltrate)can induce apoptosis in HepG2 cells by alleviating ROS level.3.In order to study the protective effect of iridoids,PC 12 cells were chosen as an oxidative damage model injured by H2O2 solution.Iridoids under certain dose improved the damaged cell morphology significantly in 80 ?M H2O2 damage model.Cell viability assay showed that compared with the normal group,the survival rate of model cells injured by H2O2 was 48.8%(p<0.01);compared with the model group,the cells survival rate of geniposide under the 10 mM dose was increased 17.5%(p<0.01).Picroside II and liganin took second place(16.3%and 12.9%,respectively).The repair rates were relatively low for valtrate,acevaltrate and 1-? acevaltrate(11.5 and 15.5%,respectively)at the concertation of 75 mM.4.Crude drug in Valeriana jatamansi Jones and the fix extract combination of valerian and hop(preparations)were investigated.They presents similar effects as the pure chemical compuounds(valtrate,acevaltrate and 1-? valtrate)did.Valeriana iridoid esters,drug and preparations and recovery the PC 12 cells injured by alleviating high ROS level inducded by H2O2.5.During the acidic and alkaline hydrolysis,the ester linkages of the iridoid valepotriates readily produced baldrinals.The oxirane nucleus was firstly hydrolysezed during thermal degradation.It had been demonstrated as important for exerting anti-proliferative and radicals scavenging activities in neuroblastoma cells.ConclusionAs a result,iridoids glycosides and esters show significant biological activity due to the nuclear parent(hemiacetal-enylcyclopentane structure).Oxidative stress may be a critical mechanism to present bioactivities.Iridoid valepotriates are key component in Valeriana jatamansi Jones and preparations.The results suggested that the oxirane nucleus is important for defining the antioxidant profile of iridoid valepotriates.Our work also indicates that there was a correlation with the protective effect of injured nerve cell.