Study On Chemical Constituents And Anti-platelet Aggregation Activity Of Ilex Pubescens

Objective:As part of a systematic research on seeking the bioactive constituents from Ilex Pubescens,a phytochemical investigation on the roots of Ilex Pubescens was undertaken,and the advancing spectroscopic methods were used for structural elucidation.These compounds were evaluated in vitro for their anti-platelet aggregation effects,as well as preliminary mechanism was explored.Methods:1.Crude power of the roots of Ilex Pubescens was soaked by methanol,and thenextracted by ethyl acetate(EtOAc)and n-butanol according to the polarity of the solvent.The EtOAc fraction was chromatographed on silica gel column,ODS column,Sephadex LH-20 column and semi-preparative HPLC.2.Structures were elucidated on the basis of chemical and spectroscopic methods including 1D(1H-MR,13C-NMR)and 2D-NMR experiments(1H-1H COSY,HSQC,HMBC and NOESY),HR TOF-MS and IR analysis,as well as acid hydrolysis.3.The anti-platelet aggregation activity of isolated compounds was assessed using the optical method in vitro by platelet aggregometer.Different inducers(ADP,AA,Collagen and Thrombin)and blockers(Aspirin,Clopidogrel,Ozagrel and Dipyridamole)were applied to observe the potential targets of active compounds.Results:1.A phytochemical investigation of Ilex Pubescens led to the isolation of eighteen compounds.Among them,including seventeen triterpenoids and one lignan glycoside were identified,and their names were as following:19?-hydroxy-3-oxo-24-norurs-12-en-28-oic acid(1),3?,19?-dihydroxyolea-24-oxo-12-en-28-oic acid(2),19?-hydroxy-3-oxo-24-norurs-12-en-28-O-?-D-glucopyranosyl ester(3),3-O-?-D-xylpyranosyl-3?,19a-dihydroxyolea-24-oxo-12-en-28-oic acid(4),3-0-?-D-xylpyranosyl-3?,19?-dihy droxyurs-24-oxo-12-en-28-oic acid(5),(20S)-3-O-?-D-glucuronopyranosyl-3?,19a,24?-trihydroxyurs-12-en-28-oic acid(6),3-O-?-D-glucuronopyranosyl-3??19?,24?-trihydroxyolea-12-en-28-oic acid(7),rotundanonic acid(8),siaresinolic acid(9),oleanolic acid-3-O-6-O-methyl-?-D-glucuronopyranoside(10),ilexpublesnin S(11),calenduloside E(12),ilexpublesnin M(13),pedunculoside(14),ilexgenin B-3-O-?-D-xylpyranoside(15),ilexosideA(16),tortoside A(17)and ilexsaponin B3(18).2.Compounds 1-18 were evaluated for their anti-platelet activities in vitro.Among these isolates,compounds 1,3,5,6,11,14,15 and 18(40 ?M)showed inhibitory activities on ADP induced platelet aggregation with inhibition rates of 57.3±4.1%,53.5±8.3%,34.1 ±2.4%,65.2±7.5%,54.6±5.8%,50.7±4.6%,62.1 ± 11.3%and 93.2± 10.1%,respectively,and they also showed inhibitory activities on thrombin induced platelet aggregation with inhibition rates of 57.8±6.4%,51.4±5.8%,55.1±12.3%,47.2±5.5%,63.5±10.2%,58.8±9.5%,49.7±7.2%and 86.4±9.3%,respectively.Ilexsaponin B3(18)exhibited an obvious anti-platelet aggregation effect in vitro with IC50 values on different inducers(ADP,AA,Collagen and Thrombin)of 12.1 ±0.7,10.2±0.8,11.3± 1.5,12.4±1.2 ?M,respectively.The cooperative relationships on ADP and FIB-induced aggregation of the ilexsaponin B3 combination with Aspirin,Clopidogrel,Ozagrel and Dipyridamole,respectively,were as follows:Dipyridamole>Clopidogrel>Ozagrel>Aspirin.Conclusion:1.Seven new triterpenoids(1-7),including three oleanane-,four ursane-type triterpenoids,were isolated from roots of Ilex Pubescens.Compounds 1 and 3 are ursane pentacyclic triterpenoids include with a C-3-ketone group and the absence of a C-24-methyl group,and,these structural characteristics are found in the triterpenoids from plants of Ilex family for the first time.Compounds 2,4 and 5 contain a C-24-active aldehyde group,which is rare for triterpene saponins from Ilex family.Compounds 6 and 7 are relatively rarity triterpenoids with C-24-hydroxyl group-substituted.Compounds 8,9 and 10 were obtained from this plant for the first time.2.In our research,eithght compounds(1,3,5,6,11,14,15 and 18)showed potential inhibition effects on platelet aggregation in vitro.compound 18(ilexsaponin B3)showed the most obvious activity among these isolates.3.ilexsaponin B3 showed inhibition effects on a variety of inducers and could cooperate with many blockers in vitro evaluation.The evident inhibition effect on platelet aggregation induced by AA has been proved.In addition,the combination of ilexsaponin B3 with Dipyridamole or Clopidogrel that on platelet aggregation induced by ADP and FIB exhibited more obvious cooperation than other blockers,which indicated that the anti-platelet activity targets of ilexsaponin B3 may have relationship with AA,cAMP and P2Y12 receptor.