Study On The Anti-platelet Aggregation Activity Induced By PAF Of Ginkgolide Monomers,Compounds And GB Derivatives

Ginkgolide is the main active component of Ginkgo biloba leaves,including more than ten kinds of sesquiterpenoids and diterpenoid lactones,such as Ginkgolide A（GA）,Ginkgolide B（GB）,Bilobalide（BB）.Ginkgolide can prevent thrombosis,improve brain blood circulation,improve blood oxygen carrying level,enhance myocardial contractility and obvious central nervous system protection.U.S.Pharmacopoeia USP 44 and European Pharmacopoeia EP 10.5 put forward the content range requirements for the composition of ginkgo lactone monomer in Ginkgo biloba extract（GBE）.Optimizing the proportion of different lactone monomers in total lactones in order to achieve activity synergy and achieve the optimal pharmacological effect is the core and hotspot of Ginkgo biloba extract and traditional Chinese medicine preparation research.GB is one of the most intensively studied and active compounds.Our research group continues to carry out structural modification and optimization of ginkgolide to improve its solubility and anti platelet aggregation activities.In this paper,a new green process for the separation and purification of Ginkgolides by semi preparative supercritical fluid chromatography（SFC）was developed.A series of GB benzyl ether derivatives with anti platelet aggregation were synthesized by chemical modification with GB as the mother nucleus.The activity of blood platelet aggregation induced by platelet activating factor（PAF）was evaluated and analyzed for the prepared series of ginkgolide monomers,compositions and GB derivatives.The research contents and results of this paper are as follows:（1）The optimum semi preparative SFC separation conditions were optimized by using crude Ginkgolides as raw materials:nucifera SI column（10 mm×250 mm,10μm）The mobile phase is CO₂,the entrainer is 7%CH₃OH,the flow rate is 10 m L/min,and the injection volume is 100μL.The column temperature is 40,the back pressure is 12 MPa and the separation time is 25 min.the five constant ginkgolide monomer compounds have good separation on semi preparative SFC chromatography,followed by BB,GB,GA,GC and GJ.The corresponding monomers with purity>98.0%can be prepared at one time.The maximum sample loading is 2 mg at a time,and the yields are 21.8%,14.8%,20.3%,5.3%and 3.8%respectively.It has the advantages of rapid separation and green environmental protection,which lays a foundation for the amplification of the preparation of ginkgolide by SFC and the continuation of laboratory projects in the future.（2）The activity of seven lactone monomers against PAF induced platelet aggregation was evaluated by rabbit platelet aggregation experiment in vitro.The results showed that different concentrations of ginkgolide monomers GA,GB,GC,GL,GJ,GK and BB could inhibit rabbit platelet aggregation,and the concentration was 156.25μg/m L The inhibition rate was the highest.The half inhibition rate concentration,that IC₅₀ values were 153.22μg/m L、102.62μg/m L、173.58μg/m L、291.86μg/m L、139.11μg/m L、206.67μg/m L、201.80μg/m L respectively。The activity of GB and GK against PAF induced rabbit platelet aggregation is better than that of other compounds,in which GB has the strongest activity and BB has the weakest activity.（3）According to the quality standard requirements of Ginkgo biloba extract in USP 44:Based on the content requirements of terpene lactones in GBE（total lactones 5.4%～12%,BB:2.6%～5.8%;GA+GB+GC:2.8%～6.2%）,analyze the correlation between the composition of different monomer proportions of Ginkgo biloba lactone and its anti platelet aggregation activity,and explore the activity of different monomer compositions of Ginkgo biloba lactone against platelet aggregation induced by PAF in rabbits.The results showed that the monomer composition of ginkgolide was BB:（GB+GA+GC）and the ratio was 29:31.The optimal ratio of GB:GA:GC was 2:3:1.The inhibition rate of anti PAF induced platelet aggregation in rabbits was 52.92%±2.19%.（4）Based on the principle of optimal activity,10 benzyl halide side chains were selected with GB as substrate and etherified at the C-10 position of GB.The synthetic products were separated and purified by recrystallization or high-pressure preparation chromatography,and their structures were characterized by ~1H NMR,¹³C NMR and HRMS.The purity of 10 target compounds was more than 90.0%and the yield was 5.46%～17.50%.In vitro platelet aggregation test of rabbits showed that the antiplatelet aggregation activity of compound 1,compound 2,compound 9 and compound 10 was better than that of other derivatives,and at the concentration of was 156.25μg/m L,the inhibition rate of platelet aggregation was the highest,which were 85.28%±4.01%,86.12%±4.54%,75.49%±3.37%,77.09%±1.98%respectively,and the IC₅₀ values were respectively 72.01μg/m L、49.11μg/m L、62.87μg/m L、58.54μg/m L,the anti platelet aggregation activities of other derivatives were weaker than GB in varying degrees.