| In broad sense, Leech usually refers to all the animals from Hirudinea of Annelida. It has been more than 3000 years that leech was used as Traditional Chinese Medicine(TCM), starting from being recorded in 《Sheng Nong’s Herbal Classic》. According to the basic theory of TCM, blood stasis is the pathologic substances of blood sediment. The cause of formation was regarded as kinds of factor in vivo or in vitro. Leech has got the tropism of taste: xian, ping, got the function of breaking blood stasis syndrome, stimulate the menstrual flow, treating blood or abdominal mass amassment, amenorrhea, blood tuberculsis, drop damage, and so on. Thrombin is a vital physiological defense process. However the pathological thrombin, thrombogenesis, plays a pivotal role to the deteriorate of cardiovascular and cerebrovascular disease due to its thrombosis formulation. Therefore, antithrombus and anticoagulation are the significance ways to treat the cardiovascular and cerebrovascular disease. At present, Hirudin is the most effective anticoagulant medicine developed from leeches.As a TCM, leech has confirmed therapeutic effect on anticoagulation in the clinical medicine. However, lots of medicinal theoretical problems have not been solved yet. As follow: 1. Medicinal varieties. Only three species of leeches exist in the Chinese Pharmacopoeia, and they refers to those from Hirudinea in the broad sense which includes a large variety of sub species. What is the best way to distinguish its species to maintain best clinical effects? 2.Material basis of effect. Hirudin is only found in Japanese bloodsucking leeches while it is not found in the non-bloodsucking, Whitmania pigra Whitman. In the domestic medicine market, the mainstream medicine are from non bloodsucking W. Whitman which don’t include Hirudin. Does W. Whitman have active substance of anti coagulation? What are the ingredients of active substance? What are the factors related to anti coagulation mechanism? Hirudin can only be exacted from the secration of living Japanese leeches, and it is easy to be damaged in the process of drying and heating. Is it effective in blood circulation and anti coagulation after being dried and boiled? 3. Resource tension. The demands for medical leeches continue to rise, and it becomes precious resources. What is the differences between ancient medical leeches and modern medical leeches? Which species of leeches can be determined as new research ones? 4. Quality assessment. Anticoagulation is the pharmacological foundation of blood circulation from TCM leeches. However, the measurement of anti coagulation from leeches is not stable and accurate in Chinese Pharmacopoeia. A new measurement method needs to be proposed to ensure its clinical effect. All these issues related to TCM basic theories need to be solved in the research.To solve the above topic, this paper researched on the following wide range, including identification with the molecular biology of different varieties of leeches, separation and identification the active anticoagulant ingredients in Whitmania pigra Whitman, analyzation the active anticoagulant ingredients Hirudo nipponia Whitman, Poecilobdella manillensis, the study of in vitro anticoagulant mechanism of Whitmania pigra Whitman, the method of APTT to assay the qualitity of leech, etc. Especially the research of Whitmania pigra Whitman, has got greater progress than predecessor. We hope that these researchs will be the key to answer the question and the basic to leech’s more further researches.Molecular biology technology was used to research the DNA barcoding candidate sequences. Four sequences were compared, ITS2, COⅠ,12 Sr RNA, 16 Sr RNA. Eight species were used as sample, Poecilobdella manillensis Lesson, Poecilobdella javanica Wahlberg, Whitmania laevis Baird, Erpobdella octoculata Linnaeus, Whitmania pigra Whitman, Whitmania acranulate Whitman, Hirudo nipponia Whitman and Haemadipsa japonica. Wilcoxon Two Sample Tests was used to examine the candidate sequences of interspecific and intraspecific variation barcoding gap. Acorrding to the result of the tests, 12 Sr RNA was regarded as the suitable sequence of DNA barcoding for medical leeches. We suppose that to design a specific primer which is produced from position of the COⅠ and 12 Sr RNA, that shows the intra conservatives and the inter differences, is an ideal method to accurately identify medicinal leeches.Basis on the applied characteristics of TCM, preparation of high performance liquid chromatography and gel column chromatography method were both used to separate the heat-resistant, anticoagulant activity of polypeptide composition, named Whitmania pigra Whitman peptide I, which belongs to sugar peptide compounds. Accroding to the analysis of LC-ESI-TOF and MALDI-TOF, the molecular weight was 9374. After enzymolysis of trypsin, the molecular weight of 1832 sugar chains was obtained. A general interpret was given to the material basis of effect.It is reported that the peptides were obtained from anticoagulant active extracted parts of dried bodies of the two leeches and their molecular weights were analyzed by ultra performance liquid chromatography(UPLC) coupled with ESI-Q-TOF mass spectrometric detector. Three peptide components were identified from H. nipponica with their molecular weight separately 14998 、 15988 and 15956, six peptide components were identified from P. manillensis with molecular weight 9590, 13642, 14998,17631,15988 and 16567. So the composition of the extracted active part of the two leeches are very close, difference is in that the extract of P. manillensis has more small peptide peaks but the extract of H. nipponica has not. Furthermore, the tryptic digestion hydrolysates of the extracted active part of each sample were analyzed and the results showed that there were four peaks which only exist in P. manillensis but not in H. nipponia. They may be the identified peak between the two leeches.Combined the in vitro incubation with UPLC-MS, to analysis the reaction of the Whitmania pigra Whitman extraction with the blood coagulation factor Ⅶ,Ⅸ and thrombin. Compared the effect of thrombin with Poecilobdella manillensis Lesson, Poecilobdella javanica Wahlberg, Hirudo nipponia Whitman extraction. Drew a speculation that Whitmania pigra Whitman has the function position on the blood coagulation factor Ⅶ in the channel of blood coagulation. The other three species leech have the function position on thrombin in the channel of blood coagulation. The Whitmania pigra Whitman has got the anticoagulant targets in thrombin and coagulation factor Ⅶ,which can be regarded as multiple targets in integrity, in according to the holistic concept of TCM.Importing the bioassay method and the principles, to evaluate the quantity of Traditional Chinese Medicine(TCM). To assay the anticoagulant activity of leeches by using biological standardization method with the aim to explore the quality control method which can reflect the biological activity. Heparin was used as the reference substance, and APTT value was used as the index of anticoagulant activity evaluation. Four species of leeches were determined, including Hirudo nipponia Whitman, Poecilobdella manillensis Lesson, Poecilobdella javanica Wahlberg, and Whitmania pigra Whitman. The determination results were calculated with standard curve and bioassay statistics. The concentration-response curves of APTT of the four species of leeches were similar to that of heparin, and their variation ranges were parallel. APTT values can reflect the comprehensive anticoagulant activity of different species of leeches, which may have more clinical significance. Biological standardization, which conforms to holistic and synergetic effect of the TCM, is a good supplement to the current quality control methods, also a proper technology for the quality control of TCM.After the paper was completed, the following conclusion can be highlighted. Much advanced analysis technology was used in the research, such as DNA molecular marker technology, LC- MS chromatographic detection, biological standardization and a variety of computer software system for data analysis and processing, which can be the basic of an advanced technology platform of identification, separation, analysis and quality evaluation of animal active peptides. The biological detection technology for quality evaluation and control the development direction of TCM should be pay more attention to. Poecilobdella manillensis Lesson can be used as new medical leeches resources, which can reduce the shortage of the leech medicine source. A series of significant achievements will be both the foundation for further study of leech and the TCM state for identification, substitution, material of leech. |
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