| In this study, the isolation, purification, characterization and sulfated modification of an exopolysaccharide (LEP-1) from a strain of Lachnum were performed. The anti-hypoxia activity of LEP-1 was evaluated, and the antioxidant activity and anticoagulant activity in vitro of sulfated LEP-1 (SLEP-1) were detected as well. Additionally, the anticoagulant activity of SLEP-1 in hypercoagulable model mice and its possible mechanisms were also investigated.The physicochemical properties of LEP-1 were determined by high performance gel permeation chromatography (HPGPC), fourier transform infrared spectrometry (FT-IR), gas chromatography-mass spectrometry (GC-MS) and 1D-and 2D-nuclear magnetic resonance spectroscopy (NMR) including’HNMR,13CNMR, HSQC and HMBC. LEP-1 was a heteropolysaccharide with an average molecular weight of about 473kDa, which consisted of glucose (Glc) and mannose (Man) in a molar ratio of 1.3:1.0. The backbone of LEP-1 was composed of '1)-a-Manp-(3,6'、 '2)-a-Manp-(6'、'1)-a-Glcp-(6'and'1)-β-Glcp-(6', and its branches consisted of '1)-β-Glcp,'1)-a-Manp-(3,6' and'2)-a-Manp-(6'. Meanwhile, LEP-1 had no triple helical structure. The study found that LEP-1 could significantly prolonged the survival time of the mice of the normobarie hypoxia test and the sodium nitrite toxicosis test, and the RBC amounts and HGB concentrations of the experimental mice were increased under the treatment of LEP-1, and the dose-effect relationship of them were revealed.SO3-pyridine method was used for sulfated modification of LEP-1 and SLEP-1 was obtained, the substitution degree (DS) of sulfate of SLEP-1 was 1.97. SEM image showed that the surface morphology of SLEP-1 had web-like structure which was different from that of LEP-1. The characteristic absorption peak of sulfate groups of SLEP-1 was appeared at 264nm in the UV spectrum. FT-IR spectrum showed that characteristic absorption peaks of sulfate groups of SLEP-1 were appeared at 1220cm-1 and 818cm-1. According to the 13CNMR spectrum of LEP-1 and SLEP-1,-SO3H were attached at C-6 of'1)-β-D-Glcp, C-6 of'2)-a-D-Manp-(6', C-3 of '2)-a-D-Manp-(6' and C-3 of'1)-β-D-Glcp-(6' of SLEP-1, of which C-6 of '1)-β-D-Glcp was almost replaced by-SO3H. The result of the antioxidant assay in vitro showed that LEP-1 and SLEP-1 had strong scavenging effects on hydroxyl radical (·OH), diphenyl picryl hydrazinyl radical (DPPH·) and superoxide radical (02-·), of which the scavenging effects of SLEP-1 were higher than those of LEP-1, and dose-effect relationships were existed. The result of the anticoagulant activity in vitro showed that both of LEP-1 and SLEP-1 could effectively prolong APTT and TT of the normal mice plasma, of which SLEP-1 could more effectively prolong APTT and TT of the normal mice plasma than those of LEP-1, and dose-effect relationships were also found.Hypercoagulable mice model which was induced by adrenaline was established, and then the mice of the hypercoagulable mice model were peritoneal injection with the same anount of normal saline, 10mg·kg-1 heparin, 30mg·kg-1 LEP-1,90mg-kg-1 LEP-1, 30mg-kg-1 SLEP-1 and 90mg-kg-1 SLEP-1, respectively. And indicators, including bleeding time (BT), whole blood clotting time (CT), partial thrombin time (APTT), prothrombin time (PT), thrombin time (TT), the concentration of fibrinogen (FIB), the activity of antithrombin Ⅲ(AT-Ⅲ) and the inhibition of the generation of factor Xa (FXa), were determinated, respectively. Compared with the model group (hypercoagulable model mice), the BT and CT of the low-dose group of LEP-1, the high-dose group of LEP-1, the low-dose group of SLEP-1 and the high-dose group of SLEP-1 were obviously prolonged. The APTT of the low-dose group of SLEP-1 and the high-dose group of SLEP-1 were significantly prolonged. The TT of the high-dose group of LEP-1, the low-dose group of SLEP-1 and the high-dose group of SLEP-1 were significantly prolonged. The concentrations of FIB of the low-dose group of LEP-1, the high-dose group of LEP-1, the low-dose group of SLEP-1 and the high-dose group of SLEP-1 were significantly decreased. The activity of AT-III of the high-dose group of LEP-1, the low-dose group of SLEP-1 and the high-dose group of SLEP-1 were all above 70%, and the concentrations of FXa were significantly decreased. Therefore, the low-dose SLEP-1 (30mg·kg-1) and high-dose SLEP-1 (90 mg-kg-1) had strong inhibitory effect on endogenous coagulation pathway, and could enhance the activity of fibrinolytic system. |
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