Study On The Inhibitory Effect Of Ginkgolides And GB Derivatives On Ovarian Cancer SKOV3 Cells

Ginkgolides are the main active substances in Ginkgo biloba extracts.At present,about12 species have been reported,namely macroginkgolide GA,GB,GC,GJ,bilobalide BB,and trace ginkgolide GK,GL,GN,GP,GQ,GM,etc.Ginkgolide has a wide range of physiological effects such as anti-platelet aggregation,anti-inflammatory,and scavenging free radicals,and has significant effects on protecting cerebral ischemic injury,enhancing myocardial contractility and protecting nerve damage.Ginkgolide B（GB）is one of the most actively researched compounds.It also has anti-tumor effects.It can inhibit the growth and proliferation of ovarian cancer,breast cancer and other tumor cells.It has rich anti-tumor targets and pathways.The anti-tumor research of lactone compounds other than GB has not been reported yet.In order to construct a library of natural ginkgolide compounds and study the anti-tumor activity of ginkgolides,develop a purification process for the preparation of lactone monomers by liquid chromatography,realize the rapid preparation of macroginkgolides,and develop synthetic processes for rare lactone compounds GL,GK,and GN,Solve the problem of difficulty and high cost in separating and purifying rare lactones from plant materials.Preliminary study of the inhibitory activity of ginkgolides on SKOV3 ovarian cancer cells,using GB as the mother nucleus to splice small benzoic acid side chains with anti-tumor effects,synthesize a series of GB-benzoic acid derivatives,and preliminarily evaluate the anti-tumor activity of the derivatives.The research content and results are as follows:（1）Using total ginkgo lactones as raw materials,the optimal separation conditions for preparative liquid chromatography were optimized as follows:mobile phase:methanol:water=30:70,flow rate 95 m L/min,column temperature 35°C,injection volume 10.0m L/time,The separation time is 60 min;the detection conditions of the evaporative light detector:the temperature of the spray chamber is 70℃,the carrier gas is N₂,the flow rate is2.5 L/min,and the maximum sample load is 300.0 mg.The five macrolactone monomers that can be prepared at one time are GC,GJ,BB,GA,and GB,with a purity of>97.0%and a yield of>95.0%,which have the advantages of rapid and efficient separation.A simple and convenient method to prepare rare lactones GK,GL,GN with GA,GB,and GC as substrates.The best synthesis process conditions are:100.0 mg of each of GA,GB,and GC are dissolved in dry dichloromethane,stirred,and the temperature is reduced to-25°C under nitrogen protection,and 3.0 m L/g DAST is added dropwise to react for 0.5 h,and stirred at room temperature until the substrate When the reaction is complete,it is quenched with pure water,the organic solvent is removed by rotary evaporation,extracted with ethyl acetate,concentrated,dissolved in methanol,and purified by high-pressure preparative liquid chromatography.Rare lactone GL 22.8 mg,purity 98.3%,yield 22.8%,GK 44.0 mg,purity97.0%,yield 42.7%,GN 7.3 mg,purity 98.6%,yield 9.0%were prepared respectively.（2）In vitro cell experiments with thiazolyl blue（MTT）method have preliminarily studied the growth inhibitory effects of the obtained 8 natural lactones on SKOV3 ovarian cancer cells.The results show that:100μmol/L GA,GB,GC,GJ,GK,GL,GN and The inhibition rates of BB on SKOV3 cells were 28.76%,37.5%,36.79%,22.8%,20.2%,26.74%,11.12%,14.79%,respectively.The IC₅₀values of the concentration of the half inhibition rate were28.22μmol/L,14.93μmol/L,15.33μmol/L,34.21μmol/L,39.36μmol/L,>100μmol/L,>100μmol/L,23.39μmol/L,respectively.Among them,GB has the strongest inhibitory activity on ovarian cancer cell SKOV3.（3）Based on the principle of optimal activity,GB（1）is selected as the raw material,and p-chlorobenzoic acid,p-fluorobenzoic acid,p-methoxybenzoic acid,3-methoxybenzoic acid,p-nitrobenzoic acid,3’5’-Dinitrobenzoic acid undergoes esterification reaction,synthesized under the action of 4-dimethylaminopyridine（DMAP）catalyst and 1-ethyl-carbodiimide hydrochloride（EDC·HCl）condensing agent.After the reaction,the synthetic derivatives 2-12were purified by high-pressure preparative chromatography,and the structure was confirmed by 1H NMR and 13C NMR structural characterization by nuclear magnetic resonance spectroscopy.Among them,the derivatives of GB C-1 position are:1-（4-chlorobenzoyl）GB（2）,1-（4-fluorobenzoyl）GB（4）,1-（4-methoxybenzyl）Acyl)GB（6）,1-（4-nitrobenzoyl）GB（8）,1-（3-methoxybenzoyl）GB（10）,1-（3’5’-dinitro Benzoyl）GB（12）;GB C-10 derivative products include:10-（4-chlorobenzoyl）GB（3）,10-（4-fluorobenzoyl）GB（5）,10-（4-methoxybenzoyl）GB（7）,10-（4-nitrobenzoyl）GB（9）,10-（3-methoxybenzoyl）GB（11）.Sci Finder database search confirmed that these 11 GB derivatives are new compounds that have not been reported in the literature.（4）In vitro cell experiments have preliminarily studied the growth inhibitory effects of GB and 11 GB-benzoic acid derivatives on SKOV3 ovarian cancer cells.In vitro anti-tumor proliferation activity test of thiazole blue（MTT）method showed that compounds 2,3,6,7,10,11have good inhibitory activity on human ovarian cancer cell SKOV3,with IC₅₀values of 16.05μmol/L,13.72μmol,63.30μmol/L,31.79μmol/L,46.93μmol/L,24.65μmol/L.Annexin V/PI double staining test showed that compound 3 induced apoptosis of SKOV3 cells slightly better than 1,6,and10,with an apoptotic rate of 37.10%.Analysis of the structure revealed that the C-10 position esterified derivative product induced SKOV3 cell apoptosis activity higher than the C-1 position esterified derivative product.