Biological Activity Studies Of Phenylpropene And Fakarindiol Natural Products

Research on neuroprotective effects of natural products of styreneObjective:To detect the protective effects of natural products of styrene on the PC12 cell injury induced by NaN₃,found that trans-anethole exhibits the most significant protective effect;Discussing the possible mechanism of trans-anethloe on the PC12 cell injury induced by sodium azide;To provide a theoretical and experimental basis for the research or the treatment of natural products of styrene in brain diseases.Methods:1Cell lines and cell culture:Rat adrenal pheochromocytoma monoclonal lines（PC12）were grown in DMEM culture medium with ten percent heat-inactivated fetal bovine serum（FBS）,one percent penicillin and streptomycin at 37 celsius under an atmosphere of 95%air and 5%CO₂.2Establishment of NaN₃-induced injury model in PC12 cells:The log phase cells were seeded in 5-6×10³ wells per well in a 96-well plate and cultured for 24 hours.NaN₃ at a final concentration of 9mM,18mM,27mM,36mM,45mM,After cultured for 24 hours,the effect of NaN₃on the proliferation of PC12 cells was detected by CCK-8 assay.3Effect of natural products of styrene on cell proliferation in PC12 Cells and NaN₃model cells:The logarithmic phase cells were seeded in 96-well plates at 5-6×10³ per well for 24 hours.The cells were treated with10-200μM natural products of styrene（trans-anethole,trans-asarone,cis-asarone,methyl eugenol,methyl isoeugenol）were cultured in DMEM medium for 48 hours and preincubated for 8hours,16hours and 24hours,later adding NaN₃ with a final concentration of 27mM for 24 hours.The effects of the natural products of styrene on the proliferation of PC12cells and NaN₃model cells were detected by CCK-8 assay.4Possible protection mechanism of trans-anethole on NaN₃model cells:The PC12 cells were preincubated with trans-anethole（10μM and60μM）for 8hours later adding NaN₃（27mM）for 24 hours,the protective effects of trans-anethole against NaN₃-induced PC12 cells injury on the apoptosis of PC12 cells was observed by microscopy and Hoechst 33258staining;The changes of lactate dehydrogenase（LDH）content in the cell supernatant were detected by microplate reader method;The content of malondialdehyde（MDA）in cells was detected by TBA method.WST-1method was used to detect the content of intracellular superoxide dismutase（SOD）;The expression of Cycs,Calm,Fas,Bax and other genes were detected by reverse transcription polymerase chain reaction（RT-PCR）.5Data processing:Statistical software SPSS20.0 for data analysis and processing;Using drawing software Graph Pad prism6.01 data drawing.Results:1As the incubation time,PC12 cells reflect the neuronal-like states,grow rapidly,and can provide sufficient cells for subsequent experiments.2Different concentrations of NaN₃ can cause PC12 cells damage,of which NaN₃（27mmol/l）cell survival rate of about 50%of the control cell,so selecting NaN₃（27mmol/l）induced PC12 cell injury for 24h as the best model concentration.3The PC12 cells were treated with（10-200μM）natural products of styrene（trans-anethole,trans-asarone,cis-asarone,methyleugenol,methyliso eugenol）for 48h separately,compared with the control groupach drug group the cell proliferation rate increased or decreased,but no significant difference（P>0.05）.the rest of each drug con-centration no difference compared with model group;PC12 cells were pretreated with the natural products of styrene for 8h later adding a final concentration of NaN₃（27μM）for 24h,compared with model group,（10-100μM）trans-anethole,（150-200μM）cis-asarone,（30μM）methyl eugenol and（30μM）methyl isoeugenol the difference was significant（P<0.05）,the rest of each drug concentration no difference compared with model group（P>0.05）;PC12cells were pretreated with the natural products of styrene for 16h later adding a final concentration of NaN₃（27μM）for 24h,compared with model cells,（30-60μM）trans-anethole,（200μM）cis-asarone,（30μM）methyl eugenol the difference was significant（P<0.05）,the rest of each drug concentration no difference compared with model group（P>0.05）;PC12cells were pretreated with the natural products of styrene for 24h later adding a final concentration of NaN₃（27μM）for 24h,compared with model group,（60μM）trans-anethole,（200μM）cis-asarone,the difference was significant（P<0.05）,the rest of each drug concentration no difference compared with model group（P>0.05）.Among them,trans-anethole（10-100μM）precultured for 8h and then added to a final concentration of27mM NaN₃cultured for 24 hours,the cell proliferation rate is better than the other four drug concentration of each group,and in a dose-dependent manner in the range of 10-60μM,So select trans-anethole（10μM and60μM）pre-cultured for 8 hours and then added to a final concentration of27mM NaN₃ for 24 hours and then cultured for the subsequent study of the possible mechanism.4After trans-anethole（10μM and 60μM）precultured for 8 hours and NaN₃（27mM）for 24 hours,the trans-anethole group could improve NaN₃-induced cell morphological changes and decrease the number of apoptotic cells;The levels of LDH and MDA in trans-anethole group were lower than those in NaN₃model group,while the content of SOD in trans-anethole group was higher than that in NaN₃ model group（P<0.05）;The expression of Cycs,Calm,Fas,Bax and other genes is under experiment.Conclusions:1The natural products of styrene have protective effects on the PC12 cell injury induced by NaN₃,found that trans-anethole exhibits the most significant protective effect;2Trans-anethole reduced the injury of PC12 cells by improving cell morphology,reducing apoptotic cells,decreasing LDH,MDA content and increasing SOD content.Study on the Antitumor Activity of Structural Analogues of FalcarindiolObjective:Cancer threatens the health of human beings in modern society.Because cancer cells gradually become resistant to various anti-cancer drugs,it is very urgent to find new anti-cancer drugs.At present,the discovery of lead compounds with anti-cancer effects is mainly through the discovery from natural products,from the discovery of existing anticancer drugs through structural modification and from the body’s endogenous substances.In our laboratory,a series of compounds were synthesized by structural modification of the existing anticancer drug Falcarindiol.Fifteen structures of Falcarindiol analogues with high yield and Ee were screened out.Fifteen Falcarindiol structural analogs of the anticancer activity of HeLa cells and HepG2 cells of human cervical cancer cells provide the basis of structure-activity relationship for the subsequent synthesis experiments,so as to find a better anti-cancer lead compounds.Methods: 1Cell lines and cell culture:HepG2 cells and HeLa cells were grown in DMEM culture medium and RPMI1640 culture medium respectively,with ten percent heat-inactivated fetal bovine serum（FBS）,one percent penicillin and streptomycin at 37 celsius under an atmosphere of 95% air and 5% CO₂.2Antitumor activity of Falcarindiol structural analogs and 5-fluorouracil on HeLa cells:The log phase cells were seeded in 5-6×10³ wells per well in a 96-well plate and cultured for 24 hours.After adding Falcarindiol structural analogues and 5-fluorouracil for 48 hours respectively,the effect of each group of drugs on the proliferation inhibition of HeLa cells was tested by CCK-8 assay.3Antitumor activity of Falcarindiol structural analogs and 5-fluorouracil on HepG2 cells:The log phase cells were seeded in 5-6×10³ wells per well in 96-wells plate and cultured for 24 hours.After adding Falcarindiol structural analogues and 5-fluorouracil for 48 hours respectively,the effect of each group of drugs on the proliferation inhibition of HepG2 cells was tested by CCK-8 assay.4Experimental data analysis:The statistical software SPSS20.0 and IC₅₀ calculation software Graph Pad prism6.01 were used for data analysis.The experimental data were expressed as IC₅₀±Std.Results: 1Falcarindiol structural analogs and 5-fluorouracil can affect the proliferation of HepG2 cells and HeLa cells,the structure of phenyl and TIPS,compared with other groups,showed better anti-cancer activity.2Based on the anticancer activity of the Falcarindiol structural analogs,in our laboratory,the racemates of the structural analogues of Falcarindiol were synthesized and their activities were determined,the results showed racemates and 5-fluorouracil also affected the proliferation of HepG2 cells and HeLa cells,among number1-5 and 15 inhibited proliferation significantly better than the isomers,the structure of phenyl and TIPS,compared with other groups,showed better anti-cancer activity.Conclusions:1Both isomers and racemates of the analogues of Falcarindiol can affect the proliferation of HepG2 cells and HeLa cells,and the inhibition ability of racemates is more than that of isomers;2Falcarindiol analogues have phenyl and TIPS structures,exhibiting better anticancer activity than the other groups.