Studies On Chemical Constituents With Antithrombotic Activity From Whitmania Pigra

The leech Whitmania pigra Whitman(Haemopidae)is widely farmed in China as an important medicinal resource.It is traditionally used to promote blood circulation,relieve stasis and activate meridians.Some preparations made by W.pigra,such as ‘Shu-Xue-Tong' injection and ‘Nao-Xue-Kang' tablet,have been clinically used to treat thrombus and paralytic stroke in China.However,the bioactive components of W.pigra and their pharmacological effects are not fully understood.Only several furanones,pteridines and peptides were isolated from this medicinal animal.The medicinal importance and our interest in the animal chemistry promoted us to investigate the chemical constituents of W.pigra.In this thesis,the dried whole-bodies of W.pigra were extracted by freeze-thaw and ultrafiltration methods.The isolation process was guided by the rate of platelet aggregation inhibition and the expression level of endothelial cell coagulation factor.As a result,21 peptides were isolated by Sephadex G25,Sephadex G10,ODS,HPLC and other chromatographic methods.Their structures were identified by spectral techniques,amino acid sequencing and organic synthesis.These peptieds were identified as: whitmantide A(WP-1),whitmantide B(WP-2),whitmantide C(WP-3),whitmantide D(WP-4),whitmantide E(WP-5),whitmantide F(WP-6),whitmantide G(WP-7),whitmantide H(WP-8),whitmantide I(WP-9),whitmantide J(WP-10),whitmantide K(WP-11),whitmantide L(WP-12),whitmantide M(WP-13),whitmantide N(WP-14),whitmantide O(WP-15),whitmantide P(WP-16),whitmantide Q(WP-17),whitmantide R(WP-18),whitmantide S(WP-19),cyclo(L-Pro-L-Leu)(WP-20)and cyclo(L-Pro-L-Ile)(WP-21).Among them,WP-1~WP-9 are new peptides containing D-amino acids(DAACPs),which were discovered from leeches for the first time.WP-10~WP-19 were new linear peptides.WP-21 was isolated from W.pigra for the first time.In order to confirm the structure of the above natural peptides and provide samples for bioactivity testing,we also synthesized and purified a total of 34 related peptides.The result of anti-platelet aggregation test indicated that WP-5,WP-17 and WP-18 have moderate anti-platelet activity,with inhibition rate over 60% at 1.0 mg/m L.Moreover,WP-1,WP-2,WP-19 and WP-21 can significantly reduce the expression of v WF and PAI-1.Thus,the effect of W.pigra may be related to the reduction of the procoagulant factors.In addition,this thesis also investigated the plasma stability of DAACPs WP-1 and WP-2,and the results showed that WP-1and WP-2 were resistant to protease degradation.The above research results enrich the chemical constituents of W.pigra,and providing a scientific basis for further development and utilization.