| This work was part of the project "Exploration, preservation and utilization of germ plasm resources on several important officinal marine organisms" supported by Marine Industry Research Special Funds for Public Welfare Projects (201205024-1).This dissertation was divided into three chapters, and the major results were summarized as follows:In Chapter 1, many aspects including the origin, classification and physiological activities of Sipuncula are summarized by the researches of scholars at home and abroad from the past to the present. Moreover, the utilization of the resources is analyzed, and the achievements of artificial culture, new drug development and pollutant monitoring of Sipuncula are confirmed. The nutritional substance, physiological activators and pharmacologic active substance as well as pharmacological activities of Sipuncula are focused in the research, and many shortcomings in the present research and expectation of the direction of the research in the future are discussed. At last, the emphases and differences of domestic and foreign researches are compared for connection and complementation of each other to make better protection and researches on Sipuncula and more contribution to human beingsIn Chapter 2, the resource chemistry evaluation on S. nudus is performed, and the results are summarized in three sections.In Section 1, to evaluate the resource chemistry of nucleosides, nucleobases and free amino acids in S. nudus, a rapid, simple and sensitive analytical method was developed by using ultra-high performance liquid chromatography coupled with a triple quadrupole mass detection in multiple-reaction monitoring mode for the simultaneous quantitative determination of 16 nucleosides, nucleobases and 25 free amino acids in S. nudus, which was confirmed to be reproducible and accurate. The limits of detection (LODs) and quantification (LOQs) are between 0.003-0.229μg/mL and 0.008-0.763μg/mL for the 41 analytes, respectively. The intraday precisions and interday precisions are< 3.72% and< 3.42%, respectively. The RSDs of the repeatability are< 4.48%. And the RSDs of the storage stability are< 4.92%. The Recoveries are between 94.03% and 106.33% with RSDs in the range of 0.64%-3.76% for all target compounds. The established method was applied to analyze 19 batches of S. nudus samples from four habitats with two different processing methods. The results showed that S. nudus contained a variety of free amino acids, nucleosides and nucleobases in sufficient quantity and reasonable proportion, and the total contents of these investigated compounds varied from 111.70 mg/g to 268.55 mg/g. They also demonstrated that the contents of these compounds in different parts of S. nudus were significantly discriminating, which were in the order:(highest) coelomic fluid> body wall> intestine (lowest). The results of PC A performed on the basis of the contents of 41 tested compounds further show the difference of the parts in S. nudus samples. As the contents of xanthine,2’-deoxyguanosine, taurine and glycine were the highest in each category, all of them could be proposed as markers for quality control of S. nudus. Moreover, the research results also provide a firm basis for further exploitation and development of S. nudus.In Section 2, to compare the difference of fatty acids in S. nudus,26 fatty acids in 12 batches of S. nudus samples are determined by gas chromatography coupled with mass spectrometer (GC-MS). The limits of detection (LODs) and quantification (LOQs) are between 0.028-0.359 μg/mL and 0.093-1.197 μg/mL for the 26 analytes, respectively. The RSDs of precisions and repeatability are< 3.35% and< 3.84%, respectively. And the RSDs of the stability are< 3.73%. The Recoveries are between 93.91% and 106.36% with RSDs in the range of 1.13%-4.40% for the investigated compounds. The results of the analytes in S. nudus show that the total contents of 26 fatty acids in the samples varies from 198.20μg/g (S9) to 578.05 μg/g (S12), and the total contents of 26 fatty acids in coelomic fluid are higher than those in body wall and intestine in S. nudus collected from all the regions. The total contents of unsaturated fatty acid are higher than saturated fatty acid. Among the polyunsaturated fatty acids, the contents of linoleic acid between 1.54% and 5.65% and a-linolenic acid between 1.61% and 4.94% are higher than others. The contents of cis-sinapic acid are the highest in monounsaturated fatty acid, which varies from 14.66% to 46.41%. Moreover, the results of PC A performed on the basis of the contents of 26 tested compounds show the difference of the parts in S. nudus samples.In Section 3, to investigate the difference of mineral elements in S1. nudus, a method established by inductively coupled plasma mass spectrometry (ICP-MS) is used to determine 27 mineral elements in 12 batches of of S. nudus collected from 7 regions. The limits of detection (LODs) and quantification (LOQs) are between 0.018-2.386μg/mL and 0.060-7.953μg/mL for the 27 analytes, respectively. The RSDs of precisions and repeatability are< 2.91% and< 3.66%, respectively. And the RSDs of the stability are< 3.56%. The Recoveries are between 96.17% and 103.30% with RSDs in the range of 1.01%-4.63% for the investigated compounds. The results of the analytes in S. nudus show that the total contents of 27 mineral elements in the samples varies from 149.01μg/g (S4) to 330.55μg/g (S12), and the total contents of 27 mineral elements in coelomic fluid are higher than those in body wall and intestine in S. nudus collected from all the regions. The total contents of macroelements including Na, K, Ca and Mg are between 13.14μg/g and 133.24μg/g. The samples contain all the 15 essential microelements including Fe, Cu, Zn, Co, Mn, Cr, Mo, Ni, V, Sn, Se, Sr, B, As and I, and the total contents varies from 94.97 μg/g to 170.76μg/g, which account for 45.08% to 64.58% of the total essential mineral elements. The essential mineral elements of Sn and Sr have the first and second highest percentage of the total contents of 15 essential mineral elements, which varies from 25.63% to 58.39% and 8.32% to 19.70%, respectively. Moreover, the results of PCA performed on the basis of the contents of 27 investigated components show the difference of the regions in S. nudus samples.In Chapter 3, three sections about the extraction, separation and purification technology of SK and its acting mechanism, enzymatic properties and thrombolytic activities in vitro and in vivo are studied.In Section 1, SK, an active enzyme with thrombolytic activity is obtained by tracking fibrinolytic activity. The fresh Sipunculus nudus worms are processed in the procedure of homogenate, freeze, unfreeze and centrifugation for two times after cleaned up and dissection. And then the supernatant is mixed and separated by the process of ammonium sulfate precipitation with the concentration of 30%-60% and the deposit is dialyzed by 3500 Da dialysis bag for the purpose of removing sulfate and other low molecular protein and polypeptide. After that, the crude protein is further purified by the combination of hydrophobic interaction chromatography of Phenyl Sepharose High Performanc, anion exchange chromatography of Q Sepharose High Performance and gel filtration chromatography of Superdex prep grade G-75 and dialysis and ultrafiltration. A method combines electrophoresis with SDS-PAGE and Native-Page and gel filtration chromatography equipped with a HPLC and a TSK gel G4000PWXL column, a method equipped the AB Sciex 5800 MALDI-TOF/TOF and a method of Edman degradation are applied for purity identification, determination of molecular weight and determination of N-terminal amino acid sequence of SK, respectively. And the results show SK is electrophoretic pure and chromatographically pure, and the molecular weight and N-terminal amino acid sequence are 28003.67 Da and PFPVPDPFVWDTSFQ, respectively.In Section 2, the effect of different temperature (10,20,30,40,50 and 60℃) on SK is studied by determining the residual activities of SK by fibrin plate method after SK pre-incubated at different temperature. And the effect of pH on SK is studied by determining the residual activities by fibrin plate method after SK pre-incubated dissolved in different buffer solution with a series pH (pH=4.0,5.0,6.0,7.0,8.0,9.0,10.0 and 11.0). In order to explore the effect of metal ions (K+, Na+, Mg2+, Ca2+, Cu2+, Fe2+, Zn2+, Al3+, Fe3+ and Mn2+) and different inhibitors (EDTA, PMSF, Aprotinin, Leupeptin and Pepstatin A) on SK, the residual activities is determined by fibrin plate method after SK pre-incubated with different metal ions and different inhibitors. The results show that the enzymatic properties of SK are specific. SK treated at 30 ℃-40℃ shows stronger remaining activities, and SK treated at 60℃ is almost inactivated. SK treated at pH 8-9 shows stronger remaining activities, and SK treated at pH 11 is almost inactivated. The metal ions do not show significant effects on SK. The PMSF and Aprotinin show strong inhibiting effect on SK, which indicates SK is a serine protease.In Section 3, the researches of thrombolytic activities of SK in vitro by fibrin plate method show that SK has obvious effect in dissolving fibrin, and there is dose-effect relation. And compared to urokinase, SK not only has direct effects on fibrinogen but also has indirect effects on fibrinogen by activating plasminogen determined in both plasminogen-rich and plasminogen-free fibrin plates. The mechanism of fibrinogenolytic activity of SK is studied by gel electrophoresis analysis of fibrinogen degraded by SK for a series times (5 min,10 min,30 min,1 h,2 h,4 h,6 h,8 h and 16 h). And the results show the fibrinogen is degraded by its chain a, chain β and chain y are degrade by SK in order.In Section 4, the FeCl3-induced arterial thrombosis model in rat is used to investigate the effect of SK in thrombosis in vivo, and results show that SK significantly inhibits thrombosis and improves the examination of artery. Furthermore, SK shows satisfactory effect in improving a series of indexes including four blood coagulation indexes, platelet aggregation rate, TXB2, PAI-1, PLG, tPA, CGRP, ET-1, FDP,6-keto-PGF1α and PGI2, and there is also dose-effect relation.In section 5, the metabolomics strategy is used to study thrombolysis mechanism of SK in arterial thrombosis rat. The urine and plasma samples are analyzed by UHPLC-Q-TOF/MS. The PCA, PLS-DA and PLS-DA are performed for multiple classification analysis of samples in control groups, model groups and SK groups. The results show that the samples are significantly separated in three groups, and 9 potential biomarkers including leukotriene C4, thromboxane A2, dihydroceramide, tryptamine, sphinganine, arachidonic acid, niacinamide, phytosphingosine and LysoPC(18:1(9Z)) are identified and the relative intensity of the biomarkers are investigated to evaluate the model and effect of SK on the model. Those endogenous metabolites were chiefly involved in sphingolipid metabolism, arachidonic acid metabolism, nicotinate and nicotinamide metabolism, glycerophospholipid metabolism, tryptophan metabolism and biosynthesis of unsaturated fatty acids. The metabolic deviations could be regulated closer to normal level after SK intervention. The results above indicate that the metabonomics method is a promising tool in the efficacy and mechanism research of SK on thrombogenesis. |
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