Preliminary Study On The Antioxidant Activities In Vivo And Their Structure-activity Relationship Of The Compounds Of Cytochalasins

EndophytEC fungi refers to those fungus that live in the plant tissue without causing signifECant symptoms for a period of its life. There are a lot of endophytEC fungus in the world.They can produce active metabolite with a variety of structures. In recent years, they have become one of the hotspots in the development and utilization of resources inside or outside our country.Eight cytochalasins(Cyt) compounds studied in this paper were acquired from isolated solid state fermentation products of Imperata endophyte Chaetomium globosum IFB-WZQ and Peel endophyte Phomopsis sp. IFB-E060. Namely:chaetoglobosin F (No. WQ-1), chaetoglobosin Fex (WQ-2), chaetoglobosin E (WQ-3), cytoglobosins A (WQ-4), penochalasin C (WQ-6), isochaetoglobosin D (WQ-9), cytochalasin H (QS-1) and18-methoxycytochalasin J (QS-2). They all have a similar mother nucleus of hydrogenated isoindol-1-ketone structure. And they have a indole or a phenyl ring substituted at10position. Early, we screened the activity of QS-1in anti-cancer study and other aspect, other activity were also screened and we found that QS-1had good anti-oxidant activity and had a potential protective effect on PC12cell damage. Literatures have no similar reports. Since that all these compounds have the same nucleus, the differences are mainly in mother nucleus and large ring substituents.The structures contain more oxygen. Therefore, this study had study on the antioxidant activity in vitro and H2O2and MPP+-induced damage in PC12cells to investigated whether the eight compounds have anti-oxidation activity? What’s the relationship between the intensity of its activity and structure? If they have a protective effect on free radECal and cell damage caused by neurotoxins? So we revealed anti-oxidation activity of these compounds and the law of structure-function relationship.This is good for looking for new drug lead compounds with strong antioxidant activity and providing experimental data. Useful ideas will be provided to develop and utilize this new medECal resources from endophytes. The main results are as follows:1. The determination of free radMethods:(1)The determination of DPPH free rad:The absorbance of DPPH free radECal was determined by mECrocolorimetry.Then clearance were calculated. Concentrations of the compound were10-6,10-5,10-4,10-3,10-2,10-1,5×10-1mmol/L, taking VE as the positive drug.(2)The determination of ABTS free rad:The absorbance of ABTS free radECal was determined by ABTS method then clearance were calculated,taking Trolox (vitamin E analogue) as a standard control to calculate the total equivalent antioxidant capacity (TEAC) of Trolox. Concentrations of the test compound were10-6,10-5,10-4,10-3,10-2mmol/L.(3)SPSS17.0was applECated to calculat half inhibitory concentration (EC50) to reflect the potency of the compounds. Scott Law was used to calculate the maximum effect (Emax) to reflect the effectiveness of the compounds. Using ANOVAR and SNK test to ordered antioxidation ability.Results:(l) DPPH free rad scavenging capacity:Of all the compounds, maximum clearance (Emax>90%):WQ-9, QS-1and WQ-1; Emax=35%～60%:WQ-2, WQ-4, WQ-3and QS-2. According to the EC50and Emax potency of compounds were ordered as follows:WQ-9=QS-1=WQ-1> WQ-2>WQ-4=WQ-3> QS-2,Emax were consistent with the above performance ranking. EC50of WQ-6could not be calculated. WQ-6had no free radECal scavenging ability.(2) ABTS free rad scavenging capacity: EC50of the Inhibition of ABTS+were ordered as follows:QS-1=WQ-9=WQ-1> WQ-2> WQ-3> QS-2> WQ-4.WQ-6EC50could not be calculated. Emax of the inhibition of ABTS+were ordered as follows:QS-1=WQ-9=WQ-1> WQ-2=WQ-3=QS-2=WQ-4> WQ-6.TEAC of QS-1was maximum,reached to746times than TEAC of Trolox. WQ-1and WQ-9were reached to373times. WQ-4were reached to0.001times, whECh was the minimum.WQ-6could not be calculated.Conclusion:Except WQ-6, the remaining seven compounds had antioxidant activity in vitro of different levels, in whECh, QS-1, WQ-9and WQ-1activity were the strongest.2. Study on anti H2O7-induced damage in PC12cells Methods:Different concentrations (50～800μmol/L) of H2O2for different time (2,3,4,5,6h) were studied to determine H2O2-induced model of PC12cells. Cell viability was determined by MTT assay. Lactate dehydrogenase (LDH) activity was determined by colorimetrEC. Groups were as follows:(1) Control group:DMEM solution containing0.01%DMSO;(2) Model group:200μmol/L H2O2solution;(3)Positive drug group:1000μmol/L VE solution;(4) Cyt groups: concentrations were0.001,0.0025,0.005,0.01,0.05,0.1,0.2μmol/L. EC50and Emax were calculated and statistECal analysis as above.Results:(1) H2O2modeling:200μmol/L H2O2solution was added with PC12cells for4h.Then the cell survival rate was41.3%±3.6%, LDH activity in the culture supernatant was increased from16.9±1.4U/d1in the control group to109.1±2.5U/d1(P<0.05). All these indECated that H2O2modeling was succeed.(2)Cell viability:Except WQ-6, the remaining seven compounds could enhance cell viability.Emax>90%:WQ-9, WQ-1and QS-1; Emax=73%-77%:WQ-2, WQ-4and WQ-3. Although QS-2could improve cell viability (P<0.01,0.001), but it wasn’t dose-depended.WQ-6was invalid. According to statistECal analysis,potency of compounds were ordered as follows: WQ-1=QS-1=WQ-9> WQ-4=WQ-2> WQ-3.But QS-2and WQ-6could not be calculated. The effectiveness of compounds were ordered as follows:WQ-9=WQ-1=QS-1> WQ-2=WQ-4=WQ-3> QS-2> WQ-6.(3) LDH activity:Except WQ-6, the remaining seven compounds (0.005μmol/L) could make the LDH activity decreased (P0.05,0.01) compared with the model group (109.1±2.5U/dl). According to statistECal analysis,potency were ordered as follws:WQ-9=QS-1=WQ-1=WQ-2> WQ-4=WQ-3=QS-2> WQ-6.Conclusion:Except WQ-6, the remaining seven compounds could inhibit H2O2-induced injury of PC12cells to varying degrees. Cell viability was improved.LDH release was decreased. The potency of WQ-9, QS-1and WQ-1were the strongest,WQ-2,WQ-4and WQ-3were followed,QS-2wasn’t dose-depended and WQ-6was invalid.3. Study on anti MPP+-induced damage in PC12cellsMethods:The cytotoxECity of the eight compounds was determined by MTT mothod.Concentrations were0.005,0.05,0.1,0.2,2.0,20μmol/L. PC12cells were injuried by500μmol/L MPP+for48h. Grouping and dosing:(1)(1) the control group:0.01%DMSO solvent;(2) the model group:500umol/L MPP+;(3) Cyt groups:concentrations were0.005,0.01,0.025,0.05,0.1,0.15,0.2μmol/L. Compounds were added30minutes before adding MPP+. The optECal density value was determined by MTT. Then the cell survival rate of each group was calculated. Cell morphology was observed by inverted mECroscope.LDH release rate was determined by colorimetrEC determination. Flow cytometry was applied to determine intracellular ROS contents. EC50and Emax were calculated as before, EC50(μmol/L) and Emax were calculated and statistECal analysis as above.Results:(1) CytotoxECity of compounds:The PC12survival rate of each group was remained above90%.All the results showed that the eight compounds had no obvious effect on the proliferation of PC12cells and had no toxECity to PC12cells.(2) the cell survival rate:In model group, the survival rate was signifECantly decreased (P<0.001,0.01,0.05).QS-2could not inhibit the decrease.WQ-6(0.005～0.05μmol/L) enhanced the decrease. The other compounds could inhibit the decrease in varying degrees.According to statistECal analysis,EC50was ordered as follows:WQ-1=WQ-9=QS-1> WQ-2> WQ-4> WQ-3.WQ-6and QS-2could not be calculated, Emax were ordered as follows:QS-1=WQ-1=WQ-9> WQ-2=WQ-4=WQ-3> WQ-6=QS-2.(3) LDH activity:Except of WQ-6,QS-2and WQ-4, the other four compounds (0.025μmol/L) could inhibit LDH release rate (P<0.01,0.001) compared with the model group(134.98.7U/dl). According to statistECal analysis,potency was ordered as follows:WQ-1=WQ-9=QS-1> WQ-3=WQ-2> WQ-4=QS-2> WQ-6.(4) Cell morphology:Cells of the control group were spindle-shaped. The cells of model group became spherECal or round instead of spindle-shaped. WQ-1、WQ-2、WQ-9、QS-1could improve the change of the morphous,the other four compounds had not obvious effect(5) ROS contents:Compared with the control group(ROS content ratio was1), ROS content ratio of the model group (13.13±0.11) was signifECantly increased (P<0.001). Except WQ-6,WQ-4,QS-2, compared with the model group, ROS content ratios of WQ-1、WQ-9、QS-1、 WQ-2、WQ-3drug groups were signifECantly decreased (P<0.001,0.01). ROS content ratios were ordered as follows:WQ-1> WQ-9> QS-1> WQ-3> WQ-2=WQ-4.Conclusion:Except WQ-6and QS-2, the other five compounds could inhibit the injury of PC12cells induced by MPP+. The WQ-1, WQ-9, QS-1were the most, WQ-2,WQ-3and WQ-4were more.WQ-6cannot against MPP+induced cell damage, anti promoting cell injury,While WQ-6could increase the injury of MPP+-induced PC12cell damage.4. The analysis of the structure activity relationship in antioxidant of the cytochalasins eight compoundsBased on the experimental results of the three parts above and the differences between these eight compounds,we discussed the structure activity relationship of the cytochalasins compounds;(1)indole or phenyl substituent at10position had no effect on the antioxidant activities.(2) Antioxidant strength was determined by hydroxyl and carbonyl of structure in the compounds: The antioxidant activities of the compounds with esterfunction at21position was stronger than methoxyl at18position. The compounds with CyclEC ether had stronger activities than alcoholEC hydroxyl at6position and7position. Compared with alcoholEC hydroxyl at20position, compounds with carbonyl had stronger activities.(3)In the event that there was a pyrrole ring in the macrocycles,those componds not only had no antioxidant activity,but also even aggravated the cell damage.