Studies On The Chemical Constituents Of Callicarpa Peii And Interaction Between Its Caffeoyl Derivatives And Biomacromolecules

Callicarpa peii H.T.Chang(Verbenaceae),a deciduous liane of genus Callicarpa,is mainly distributed in southern China.C.peii spend cold in nature and bitter taste,which has been used for the treatment of bleeding,the common cold,cough,arthritis,and diarrhoea in traditional Chinese medicine.In this thesis,the systematic phytochemical study on Callicarpa Peii was carried out.On the basis of the isolated chemical constituents,the interaction between the three caffeoyl derivatives and biomacromolecule was studied firstly in this field.1.Studies on chemical constituents of Callicarpa peii H.T.ChangSeventeen compounds were isolated from the aerial parts of C.peii by isolated measure s of normal phase,reversed phase and molecular sieve column chromatograpies.On the basis of spectroscopic data(IR,UV,1H-NMR,13C-NMR,HMQC,HMBC,’H-’H COSY),Mass spectrum interpretation and chemical analysis,the compounds were identified and their structures were elucidated as peiioside A(1),peiioside B(2),forsythoside B(3),acteoside(4),cistanoside F(5),decaffeoylacteoside(6),3,4-dihydroxyphenylethyl-8-O-β-D-glucopyranoside(7),kelampayoside A(8).lyoniresinol 3a-O-β-D-glucopyranoside(9),kaempferol-3-O-glucuronide(10),hydnocarpin(11).5,7,3’-trimethoxy,4’-hydroxylflavone (12),velutin(13),5,7,4’-trihydroxy-3’-methoxyflavone(14),luteolin(15),oleanic acid(16),2β,3β,19α-trihydroxy-ursolic acid(17).All these compounds were obtained from the species of this plant first time,and compounds 5,6,7,8,10,11,and 12 were isolated from genus Callicarpa first time.Compounds isolated from C,peii include two pairs of epimers(peiioside A1/A2 and cistanoside F1/F2),which is rarely reported from genus Callicarpa.Spectral characters of glucose epimers were demonstrated on the basis of our work and the references.The distribution and type of chemical constituents about C.peii have shown that phenylethanoid glycosides are the main components in the n-BuOH soluable extraction and flavones are the main components of EtOAc soluable extraction in this plant.Forsythoside B and acteoside can be regarded as the two representative secondary metabolites of C.peii.And five presumably biogenetic related glycoside intermediates were isolated from C.peii,which provided theoretical evidence for the presumable biogenetic pathway of the biologically interesting phenylethanoid glycosides about forsythoside B and acteoside.The research has fulfilled the clarification of chemical structures,the main types they belong to,and distribution characteristic of chemical constituents in C.peii and has enriched the types of chemical constituents in genus Callicarpa.2.Studies on the interactions between three caffeoyl derivatives in C.peii and bovine serum albuminThe interaction between three caffeoyl derivatives(cistanoside F,acteoside,and forsythoside B)isolated from C.peii and bovine serum albumin(BSA)in physiological condition was investigated by means of fluorescence,UV-vis absorbance,circular dichroism(CD)and molecule modeling techniques.It was suggested that the fluorescence quenching of BSA under physiological condition by the compounds resulted mainly from the formation of compound-BSA complexes.The binding constants Ka and the number of binding sites n of the caffeoyl compounds with BSA at different temperatures were determined.At 25 ℃,Ka was found as 4.36×104,6.83×105,and 2.20×105 L·mol-1,and n was 1.04,1.22 and 1.08 for cistanoside F-BSA,acteoside-BSA and forsythoside B-BSA,respectively.The results of thermodynamic parameters ΔG,AH and AS indicated that the hydrogen bond played a major role in the interaction of cistanoside F-BSA,while the hydrophobic force played a major role in the interactions of acteoside-BSA and forsythoside B-BSA.According to the Forster’s theory of non-radiative energy transfer,the donor-to-acceptor distance r between tryptophan residues Trp212 of BSA and the compounds was determined as 3.09 nm for cistanoside F,3.13 nm for acteoside,and 2.97 nm for forsythoside B,respectively.The results showed that the fluorescence quenching mechanism in the compound-BSA binary systems was a combination of static quenching and non-radiative energy transfer.The UV-vis,CD and synchronous fluorescence spectra showed that the binding of the three caffeoyl compounds to BSA induced conformational changes in BSA,but with little effect.In addition,the binding constants of the caffeoyl compounds with BSA were calculated after the addition of the some common metal ions Mg2+,Cu2+,Zn2+ and Fe3+.The displacement experiments confirmed that the caffeoyl compounds could bind to site I which located in subdomain IIA of BSA with different affinity.And the relevant interaction models of the compounds with serum albumin were obtained by the molecular modeling study,which were in accordance with spectral results.The research results have not only provided interaction information between the caffeoyl compounds and BSA at the molecular level,but also given a better theoretical reference for future studies of pharmacological mechanism of the caffeoyl derivarives.3.Studies on the interactions between three caffeoyl derivatives in C.peii and calf thymus DNAThe interaction of three caffeoyl derivatives(cistanoside F,acteoside,and forsythoside B)with calf thymus DNA(ctDNA)in physiological condition was investigated using neutral red as fluorescence probe by means of fluorescence,UV-visible spectrophotometry,melting experiment,viscometric assays,and molecule modeling techniques,respectively.According to the experimental results,it can be concluded that the three caffeoyl compounds could bind to ctDNA by forming compound-DNA complexes.The binding interaction of three caffeoyl compounds to ctDNA was not caused by intercalation,but mainly by groove binding.Furthermore,the results of molecular modeling study showed the major mode of recognition between the compounds and ctDNA is groove binding by strong hydrogen bonds,which confirmed the conclusions obtained from spectroscopic and viscosimetric investigations.The binding intensity between compounds and ctDNA was acteoside>forsythoside B>cistanoside F.The research results have provided molecular biological information for studying the anti-tumor and antiviral mechanism of the caffeoyl derivarives.