| Thrombotic diseases normally occur when blood vessels are partially or completely occluded by excessive thrombosis.There has been an increased morbidity and mortality of cardiovascular and peripheral vascular diseases caused by the formation of thrombosis in various parts of the body.Therefore,prevention of thrombosis formation has been an important strategy to treat thrombotic diseases.A wide range of naturally produced substances such as herbal plants used in traditional Chinese medicines and functional foods have shown significant antithrombotic activity.They can be classified into three major categories according to their main mechanisms of action:（1）anticoagulants which inhibit the clotting cascade process and interfere with thrombus expansion;（2）platelet inhibitors which inhibit platelet aggregation;（3）thrombolytic agents which are capable of directly dissolving thrombosis.Previous studies from our group have evaluated the antiplatelet aggregation activity of crude extracts of 31HuoXueHuaYu traditional Chinese medicines and 14 functional foods.It has been found that some of the investigated natural products profoundly inhibited in vitro rabbit platelet aggregation,indicating there were active ingredients in these natural products.Therefore,this study aims to screen the active antithrombosis compounds from six natural products through establishing a series of novel methods based on offline LC monitoring technology and further evaluate corresponding pharmacological effects on thrombosis inhibition.These natural products include five HuoXueHuaYu traditional Chinese medicines[Corydalis yanhusuo W.T.Wang,Ligusticum chuanxiong Hort.,Salvia miltiorrhiza Bunge,Carthamus tinctorius L.,Selaginella tamariscina（P.Beauv.）Spring]and one herbal food（Citrus limon L.）.Additionally,a rapid extraction and separation method was established to accumulate the polar active ingredients from a HuoXueHuaYu traditional Chinese medicine Salvia miltiorrhiza Bunge.This study provides the basis for further development and utilization of these investigated traditional Chinese medicines and food as drug entities.The thesis mainly consists of the following eight chapters:In the first part,the mechanisms of thrombosis formation and the pharmacological effects of natural products against thrombosis were briefly introduced.The importance of active ingredients in natural products for antithrombotic therapy was highlighted.Then,the necessity of applying pharmacodynamic mechanism to drug screening and development was explored.The development and application of chromatographic technology as a modern and efficient separation tool in the field of natural product research was introduced.Furthermore,methods for screening active ingredients of natural products based on offline LC analysis were reviewed in details.The theoretical basis,technical background and purpose of this research were clarified b y describing the basic ideas and progress of related research.In the second chapter,the direct platelet biospecific affinity extraction and LC-DAD analysis were used to screen the compounds with antiplatelet aggregation activity in Rhizoma Corydalis and Chuanxiong.5 compounds in total alkaloid extract（TAE）were found to bind to platelets,which were identified as glaucine,dehydrocorydaline,canadine,tetrahydrocoptisine and corydaline.In vitro antiplatelet aggregation activity test showed that all these 5 compounds had strong inhibitory effects on THR-induced platelet aggregation.To our knowledge,this is the first time that glaucine,canadine,tetrahydrocoptisine and corydaline were reported to have inhibitory effect on platelet aggregation in vitro.Quantitative determination showed that total contents of the 5 compounds accounted for about 21%of the TAE,which functioned as the main antiplatelet aggregation active ingredients in the TAE of Rhizoma Corydalis.Similar study on Rhizome Chuanxiong found that 5 compounds were attached to platelet in vitro and further identified as caffeic acid,ferulic acid,senkyunolide I,senkyunolide A and（Z）-ligustilide by HPLC-DAD/LC-MS method.Among them,senkyunolide A and（Z）-ligustilide showed significant inhibitory effect on THR-induced washed platelet aggregation in vitro,whereas the other 3 components had weak or no inhibition activity.2 components with the highest contents in ethanol extract of Rhizoma Chuanxiong displayed the most profound antiplatelet aggregation activity.Although the proposed method with a combination of direct platelet biospecific extraction and HPLC is a simple,rapid,and straightforward strategy in the field of discovering potential bioactive components from natural products,it has shown limitations in identifying compounds with specific activity due to the weakness of affinity-intrinsic activity.In the third chapter,a method named platelet adsorbed hollow fiber-based biological fingerprinting was developed to screen potential platelet aggregation inhibitors from Danshen-Honghua（Dan-Hong）decoction.12 components from Dan-Hong decoction were found to be trapped in platelet adsorbed hollow fiber compared with blank fibers.The chemical structures of 9 compounds were identified by LC-MS/MS analysis,among which 3 compounds including lithospermic acid,salvianolic acid A and salvianolic acid B showed inhibitory effects on platelet aggregation induced by THR,adenosine diphosphate（ADP）and U4661,respectively.Another two identified compounds salvianolic acid H（salvianolic acid I）and salvianolic acid were found to have the identical structural units that Danshensu and para-hydroxyl group presented on the same benzene ring structural unit as lithospermic acid,salvianolic acid A,and salvianolic acid B.The similarities on structures indicated the potential of using salvianolic acid H（salvianolic acid I）and salvianolic acid as antiplatelet compounds.The introduction of adsorbed hollow fiber,to some extents,made a progression on the direct platelet affinity extraction method,which not only reduced the platelet damage caused by the continuous centrifugation of platelet extract,but also shortened the screening time.The fourth chapter described the screening of potential thrombin（THR）inhibitor in Rhizoma Chuanxiong by a THR in-solution based bipospecific extraction combined with ultrafiltration and LC-DAD analysis method.It was found that 8 components were attached to active THR when comparing the differences between activated and inactivated THR by ultrafiltration analysis.The chemical structures of 3 components were identified.In vitro activity test showed that there was a strong THR inhibitory activity of isochlorogenic acid C and senkyunolide I with IC50 of 206.48 and 197.23μM,respectively.Further molecular docking analysis showed that both compounds could bind to the key amino acid residues in THR catalytic pocket with low binding energy.In addition,a group of 4 compounds including isochlorogenic acids A and B,senkyunolide J and N with similar structures as the 2 identified active compounds also showed similar binding energy and sites to THR,suggesting the promising role of these compounds as THR inhibitors.Affinity ultrafiltration extraction,which could increase the reliability of screening results through comparing the screening differences between activated and inactivated enzymes,provides an efficient strategy to rapidly screen and separate active small molecules targeting to specific proteins.The fifth chapter established a method of LC-MS analysis combined with chemometrics to assist the phytochemical separation method to screen antiplatelet compounds in extracts of Citrus limon L.（lemon）.7 major compounds were found to be strongly associated with the antiplatelet aggregation activity of lemon.Among them,5compounds were identified by UPLC-IT-TOF-MS,including 2 coumarins,1 flavonoid,1 tricarboxylic acid,and 1 phthalide.4 compounds including oxypeucedanin hydrate,citric acid,diosmin,and limetin showed antiplatelet aggregation effects with concentrations less than 300μM.Moreover,differential proteomic analysis was carried out to study the possible mechanism responsible for the inhibitory effect of screened components on platelet aggregation.It was shown that limetin might inhibit platelet aggregation via interaction with the TPβreceptor of thromboxane A2,which triggers PI3K/Rap-1b signaling pathway throughβγsubunit of GPCR.Additionally,the effect of limetin could rely on inhibition of COX-1 activity which catalyzes the conversion of AA to TXA2.The efficiency of screening and separation of bioactive components was greatly simplified by combination the traditional photochemical separation method with a chemometric multivariate statistical data processing method.Generally,different components and potential active substances could be evaluated by analyzing the components in different fractions obtained from the primary separation stage,resulting in a time-saving and cost-saving process in phytochemical separation.In the sixth chapter,the concept of spectral effect relationship was introduced to study the possible compositional basis for the different coagulation activity measured before and after the stir-frying process of Selaginella tamariscina（P.Beauv.）Spring（ST）.Pharmacological experiments have shown that the extract of S.tamariscina carbonisatus（STC）had strong hemostatic activity on bleeding rat through regulating the properties involved in haemorheology and plasma coagulation system,such as the shortened tail bleeding time,increased whole blood viscosity（WBV）and plasma viscosity（PV）,andreduced erythrocyte sedimentation tate（ESR）,shortened activated partial thromboplastin time（APTT）and increased plasma fibrinogen（FIB）content.In contrast,ST extracts did not lead to significant effect on bleeding time,WBV,PV,ESR and packcd cell volume（PCV）.HPLC-DAD and LC-MS were then used to identify the compositional differences of the extracts of ST and STC.There were 11 components identified as different before and after processing,two of which were further tested for their effects on coagulation activity.Dihydrocaffeic acid as one of the compounds with significantly increased levels in STC had haemostatic activity as shown by enhanced platelet aggregation and reduced APTT and PT.However,amentoflavone which showed a decreased level in STC inhibited ADP-and AA-induced platelet aggregation.The contrary effect of ST and STC on coagulation system could result from their compositional variances before and after stir-frying process.In the seventh chapter,a novel amino-terminated cucurbit[6]uril（CB[6]）pseudorotaxane motif coated magnetic nanoparticles（MNPs）was successfully fabricated through cucurbit[6]uril promoting azide-alkyne cycloaddition.The obtained functional MNPs were used to extract polar salvianolic acids（SAs）in Salvia miltiorrhiza Bge.By investigating the adsorption capacity of materials under different extraction conditions,the possible anion exchange,hydrophobic interaction and hydrogen bonding adsorption mechanisms of the materials were speculated.It was shown that the adsorption behavior of the material belonged to the Freundlich adsorption model as measured by static adsorption behavior experiments.The investigated material exhibited superior adsorption properties with high capacity（15-80mg/g）and fast adsorption speed（10 min）for multiple SAs including rosmarinic acid,lithosperic acid,salvianolic acid B and salvianolic acid A.In addition,the developed pseudorotaxane motif MNPs offered a good recyclability and stability.Satisfied recovery（95.1%-106.5%）was obtained when applying to extract polar SAs from complex spiked Danshen samples.The eighth chapter is the summary of this study and offers suggestions for future research in relevant filed.Generally,offline LC monitoring technology was applied to screen antiplatelet aggregation compounds from 4 HuoXueHuaYu traditional Chinese medicines and a functional food.The same strategy was used to study the compositional basis responsible for bioactivity transformation before and after stir-frying process of ST.The screened active compounds of ST were further evaluated for their pharmacological activities related to thrombosis formation including hemorheology system,coagulation system and platelet aggregation system.The antiplatelet aggregation mechanism of active compound limetin was investigated by differential proteomics analysis.Additionally,magnetic solid phase extraction materials were successfully prepared for separation and enrichment of active SAs in Salvia miltiorrhiza Bge.Taken together,this study focuses on development of methods with high sensitivity and reliable accuracy to separate and analyze active compounds in natural products based on the combination of LC analysis and biological activity measurement.There were a series of methods established including direct platelet biospecific extraction,ultrafiltration screening,adsorbed hollow fiber immobilization,chemometrics assisted the phytochemical separation and spectral effect relationship method,which provides a comprehensive analysis routine for separation and analysis of active compounds in HuoXueHuaYu natural products as well as evaluation of pharmacological effect and corresponding mechanisms.The methods developed in this study therefore benefit not only the quality screening,exploration of material foundation,but also discovering more innovation solutions in the field of natural product research.Moreover,the present study provides the basis for screening of active compounds from a wide range of natural products and the potential application of them as drug entities. |
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