| Thrombotic diseases are one kind of diseases that severely threaten our health, and their fatality and disability are on the top of all other diseases worldwide. Various reasons which cause the whole or local blood to slow down are the factors of thromboembolism, such as hyperfibrinogenemia, hyperlipemia, and circulatory disturbance. Thrombotic diseases can bring severe injury if they take place. Therefore, it is a necessary to maintain the normal condition of blood in long term for prophylaxis. As a result, it becomes a trend to develop antithrombotics with mild efficacy, no bleeding, and long term safety.Heparinoid is a sort of natural, semisynthetic, or synthetic substances with bioactivities similar to heparin. Porcine intestinal mucosal heparinoid (HPND), with its mild and definite efficacy, small side effect, low cost, proper molecular weight, and proper chemical characteristics, are suitable for oral taking, and hence can be developed to convenient oral taken antithrombotics. Currently, porcine intestinal mucosal heparinoid antithrombotics, such as Danaparoid, Lomoparan, Sulodexide, Mesoglycan have been on oversea market for several years. The byproduct of heparin manufacturing, the mentioned heparinoid, is of large quantity in China and sold in low price. However, there was still no such antithrombotics on domestic market. With large quantity and low cost, HPND has immeasurable potential and bright prospect to be developed into a new safe and effective antithrombotic.HPND consists of heparan sulfate (HS), dermatan sulfate (DS), heparin (HP), and small quantity of chondroitin sulfate (CS).It was demonstrated that the major components of HPND possess antithrombotic bioactivity. HS/HP accelerate the inhibition of thrombin and other clotting factors by binding to antithrombinâ…¢(ATâ…¢). Whereas DS accelerate the inhibition of thrombin via binding to another serine proteinase, heparin cofactorâ…¡(HCâ…¡), to decrease platelet aggregation, exhibiting antithrombotic effects. In blood coagulation,1/3 of the total thrombin is inhibited by its binding to HCâ…¡, while the other 2/3 to ATâ…¢. The two ways of thrombin inhibition proceed independently in vivo, and there is synergism when DS and HS exist together. However, the biological value of this kind drugs such as Sulodexide is measured by lipoprotein lipase release unit (LSU). But there were few reports on their preparation, details of their components, and the effects of each component on its biological value.This study started from crude HPND to obtain pure HPND via deproteinization, decolouration, separation, and purification procedures. Benedict precipitation and strong anion exchange chromatography were applied to separate and purify each component of HPND and its positive control SLDX. Overall structural certification and physicochemical property analysis were carried out for each component, including disaccharide analysis, NMR analysis, molecular weight determination, specific rotation deternimation, cellulose acetate membrane electrophoresis, SO42-/COO- ratio determination. The in vitro anti-Fâ…©a and anti-Fâ…¡a activities of each component, and LPL and t-PA release activities of HPND in vivo were tested. In addition, the synergism of HS and DS on LPL and t-PA release activities was investigated.The results and conclusions of our research were as follows. 1. Preparation of HPND and purification of each componentCrude HPND was taken as the raw material. Trypsin, papain or pronase E was used to hydrolyze protein impurities, the protein impurities were precipitated with CaCl2, and the purified HPND was obtained after H2O2 oxidation, desalting, and lyophilization. UV full wavelength scan method was applied to detect protein and nucleotide impurities in HPND, and the result showed that HPND was in accordance with USP standard.HPND and SLDX were separated into 2 parts, DS and HS, by Benedict precipitation method. Flame atomic absorption spectrophotometry method was applied to test the content of Cu HPND, DS and HS, with the results of 2.83 ppm 35.1 ppm and 18.4 ppm Cu, respectively.HS was applied to QFF strong anion exchange chromatography for a second separation, and three fractions, HSO, HS1 and HS2, were obtained. According to the chromatography, the corresponding peaks of HPND and SLDX overlayed very well.2. Structure certification and physicochemical property analysis of HPND and SLDX(1) HPND-DS:Mw was 22,070 Da (0.70%); major disaccharideα-â–³UA-[1â†'3]-GaINAc; specific rotation-56; SO42-/COO- ratio 1.14±0.03.1H and 13C NMR demonstrated that it was dermatan sulfate.(2) SLDX-DS:Mw was 18,860 Da (0.25%); major disaccharideα-â–³UA-[1â†'3]-GaINAc; specific rotation-51°; SO42-/COO- ratio 1.45±0.09.1H and 13C NMR demonstrated that it was dermatan sulfate.(3) HPND-HSO:Mw was less than 1,000 Da; major disaccharide a-â–³UA-[1â†'4]-GIcNAc; nalmost no rotation value. Saccharide content of HPND-HSO determined by anthracenone colorimetry method was (15.31±0.96)%. (4) SLDX-HSO:Mw was less than 1,000 Da; major disaccharideα-â–³UA-[1â†'4]-GIcNAc:almost no rotation value was detected; saccharide content of SLDX-HSO determined with anthracenone colorimetry method was (3.60±0.38)%.(5) HPND-HS1:Mw was 14,070 Da (0.70%); major disaccharidesα-â–³UA-[1â†'4]-GIcNAc andα-AUA-2S-[1â†'4]-GIcN-6S; specific rotation 57°; SO42-/COO- ratio 1.06±0.06.1H and 13C NMR demonstrated that it was heparan sulfate.(6)SLDX-HS1:Mw was 11,150 Da (0.80%); major disaccharides areα-â–³UA-[1â†'4]-GIcNAc andα-â–³UA-2S-[1â†'4]-GIcN-6S; specific rotation 50°; SO42-/COO-ratio 1.50±0.08; 1H and 13C NMR demonstrated that it was heparan sulfate.(7) HPND-HS2:Mw was 14,300 Da (2.50%); major disaccharides a-AUA-[1â†'4]-GIcNAc and a-AUA-2S-[1â†'4]-GIcNS-6S; specific rotation 40°; SO42-/COO- ratio 1.72±0.11; 1H and 13C NMR demonstrated that it was heparin.(8) SLDX-HS2:Mw was 10,370 Da (1.30%); major disaccharides are a-AUA-[1â†'4]-GIcNAc and a-AUA-2S-[1â†'4]-GIcNS-6S; specific rotation 36°; SO42-/COO- ratio 1.85±0.13; 1H and 13C NMR demonstrated that it was heparin.3. Anti-F Xa and anti-Fâ…¡a activities of each component purified from HPND and SLDXThe anti-F Xa and anti-Fâ…¡a value of each component of HPND and SLDX was determined by US pharmacopeia method. The results showed that the anti-F Xa and anti-Fâ…¡a value of HPND-DS, SLDX-DS, HPND-HSO and SLDX-HSO were relatively small, ranging from 2-8 IU/mg, and with a same anti-F Xa/anti-Fâ…¡a ratio of 1.2. The anti-F Xa and anti-Fâ…¡a value of HPND and SLDX attributed to their HS component. The detailed results are shown as follow.(1) HPND-HS:Anti-F Xa value 122.6 IU/mg, anti-F II a value 97.7 IU/mg, and anti-F Xa/anti-Fâ…¡a ratio 1.3;(2) SLDX-HS:Anti-F Xa value 64.6 IU/mg, anti-Fâ…¡a value 46.9 IU/mg, and anti-F Xa/anti-Fâ…¡a ratio 1.4;(3) HPND-HS1:Anti-F Xa value 19.6 IU/mg, anti-F II a value 11.3 IU/mg, and anti-F Xa/anti-Fâ…¡a ratio 1.7;(4) SLDX-HS1:Anti-F Xa value 8.6 IU/mg, anti-Fâ…¡a value 2.4 IU/mg, and anti-F Xa/anti-Fâ…¡a ratio 3.6;(5) HPND-HS2:Anti-F Xa value 173.6 IU/mg, anti-Fâ…¡a value 148.5 lU/mg, and anti-F Xa/anti-Fâ…¡a ratio 1.2;(6)SLDX-HS2:Anti-F Xa value 118.4 IU/mg, anti-F II a value 79.5 IU/mg, and anti-F X a/anti-F II a ratio 1.5.4. LPL and t-PA release bioactivity of HPNDHPND was given to healthy wistar rats via intravenous injection. The quantity of LPL and t-PA in plasma was measured by corresponding ELISA kit. HPND was injected at doses of 2.5 mg/kg,5.0 mg/kg,10.0 mg/kg for experiemntal groups respectively, SLDX was given at a dose of 5.0 mg/kg for positive control group, and saline was given for negative control group.The results demonstrated that HPND evidently promoted vascular endothelial cells to release LPL with a dose dependence manner, and was equivalent to SLDX at the same dosage. The detailed LPL contents were: saline group, (0.0893±1.7069) ng/mL; low dose HPND, (3.926±0.984) ng/mL; medium dose HPND, (9.502±1.532) ng/mL, high dose HPND, (10.481±4.681) ng/mL; SLDX group, (10.368±4.453) ng/mL HPND did not increase t-PA content in plasma very significantly, though the content of t-PA increased slightly with HPND dose increasing. The detailed t-PA contents were: saline group, (0.5336±0.1564) pg/mL; low dose HPND, (0.5336±0.1564) pg/mL; medium dose HPND, (0.7051±0.1538) pg/mL; high dose HPND, (0.7771±0.1091) pg/mL; SLDX group, (0.5259±0.0595) pg/mL.5. Synergism of HS and DS on LPL and t-PA releaseIn order to determine the main component in HPND in promoting LPL and t-PA release, HS and DS were set into low dose group, medium dose group, and high dose group, according to their percentage in HPND. The results demonstrated that DS had stronger potential to promote LPL release, while neither DS nor HS could significantly promote t-PA release..LPL contents of DS groups:low dose group, (1.5355±0.8644) ng/mL; medium dose group, (2.0118±0.9767)ng/mL, and high dose group, (3.7870±2.2535) ng/mL. LPL contents of HS groups:low dose group, ((1.6134±0.4838) ng/mL; medium dose group, (2.5573±0.6038) ng/mL; and high dose group, (2.8604±0.8253) ng/mL.t-PA contents of DS groups:low dose group, (0.6507±0.2116) pg/mL; medium dose group, (0.7429±0.2547)pg/mL, and high dose group, (0.7401±0.1543) pg/mL. t-PA contents of HS groups:low dose group, (0.5044±0.1049) pg/mL; medium dose group, (0.6297±0.2148) pg/mL; and high dose group, (0.5883±0.1786) pg/mL.6. Main achievements of this study(1) Established a set of procedures to prepare HPND, DS, and HS from the by product of heparin production in laboratory, and HPND, DS, and HS of high purity were obtained.(2) Anti-F Xa and anti-Fâ…¡a values of each component purified from HPND and SLDX were evaluated.(3) LPL and t-PA release activities of HPND were determined.(4) It is the first time to systematically evaluate synergistic effects of HS and DS on LPL and t-PA release from vascular endothelial cells. |
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