礁膜（Monostroma nitidum）多糖的提取分离、结构和抗凝血活性研究
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摘要
礁膜(Monostroma nitidum)隶属于绿藻门,绿藻纲,礁膜科,礁膜属。多糖是其主要的生物活性物质。本文以礁膜为原料,对礁膜多糖进行提取、分离纯化,研究其理化性质、结构和抗凝血活性。
礁膜藻体粉末依次经冷水、热水、稀碱和浓碱液提取,得到五种粗多糖CW、HW1、HW2、AM1和AM2,提取得率分别是9.1%、30.6%、5.3%、5.5%和3.7%,总提取率达54.2%。采用苯酚-硫酸法、硫酸钡明胶比浊法、薄层层析、气相色谱、高效液相色谱等方法对其中三种水提取粗多糖CW、HW1和HW2的理化性质和单糖组成进行了比较分析。结果表明:总糖含量HW2最高,CW最低;蛋白含量以CW最高,HW1最低;硫酸基含量以HW1最高,CW最低。三种粗多糖的单糖组成均以鼠李糖为主,含有少量葡萄糖和木糖,CW含有微量甘露糖,HW1含有微量的半乳糖和甘露糖;糖醛酸的类型均为葡萄糖醛酸。
在对粗多糖理化性质和单糖组成测定的基础上,选取HW1作为研究对象,采用离子交换色谱和凝胶排阻色谱相结合对其进行分离纯化得到两种纯化多糖WF1和WF3。采用高效液相色谱法、气相色谱法、红外光谱法对两种多糖的重均分子量、纯度及单糖组成进行研究。结果表明:多糖WF1和WF3经SB-804HQ柱洗脱呈单一对称峰,表明两种多糖的纯度较高,重均分子量分别是870 kD和70 kD,WF1的分子量明显大于WF3;两种多糖的单糖组成均以鼠李糖为主,含有少量木糖和葡萄糖,另外,WF1含有微量半乳糖,WF3含有微量甘露糖;糖醛酸类型是葡萄糖醛酸。红外光谱图在1250 cm-1左右有S=O的伸缩振动吸收峰,表明了两种多糖均是带有硫酸基的鼠李聚糖。
采用脱硫酸基反应、高碘酸氧化、Smith降解、甲基化反应、气相色谱、高效液相色谱、红外光谱、核磁共振波谱和气质联用等方法对多糖WF1和WF3的结构进行研究。结果表明:WF1的糖链中是以1, 2-Rha为主,含有有1, 4和1, 3-Rha,硫酸基的位置以C-3、C-4较多,C-2位相对较少,也可能C-3、C-4和C-2均带有硫酸基。WF3的糖链中是以1, 3-Rha为主,含有1, 2-Rha,存在1→2,3连接的Rha分支点,硫酸基的位置以C-4位较多,C-2、C-3位相对较少,也可能C-3、C-4和C-2均带有硫酸基。脱硫前后的红外谱图对比解析表明两种多糖的构型为α型。核磁共振谱图进一步说明WF3是α构型,C-3位是糖链的连接位点,C-2位、C-4位可能有硫酸基取代。
通过活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)和凝血酶时间(TT)分析,对礁膜多糖WF1和WF3的抗凝血活性进行研究。结果表明:WF1和WF3对APTT和TT均有延长作用,但对PT作用不明显,说明WF1和WF3主要抑制内源性凝血途径和/或共有凝血途径以及抑制凝血酶活性或纤维蛋白原转化为纤维蛋白,而对外源性凝血途径没有影响。而且,WF1抗凝血活性明显高于WF3,这主要是由于它们的分子量、硫酸基含量和结构的差异所引起的。WF1和WF3的抗凝血活性比肝素低。
本研究提取、分离纯化得到两种礁膜多糖WF1和WF3,关于这两种多糖的结构到目前为止国内外尚未见研究报道。
本课题为礁膜多糖的研究提供更多的基础资料,为进一步研究其结构与活性的关系奠定了基础,对促进“海洋糖库”的建设,发展具有我国特色的海洋新药都有重要意义。
Monostroma nitidum is a kind of marine green seaweed. The polysaccharide is its important bioactive substance.
In this paper, Monostroma nitidum was extracted by cool water, boiling water and alkali liquid successively. Five kinds of polysaccharides were obtained and named as CW, HW1, HW2, AM1 and AM2, respectively. The physicochemical properties and monosaccharide compositions of these three polysaccharides CW, HW1 and HW2 were analysed by Phenol-sulphuric acid method, Folin-phenol reagent method, barium sulfate turbidimetry method, GC and HPLC. The results showed that the total yield of the crude polysaccharides was 54.2%. The yield of HW1 was 30.6%, and it was the highest in the three polysaccharides. The contents of the sulfate group were different, they were 21.8%, 29.1% and 26.9% respectively. These three polysaccharides mainly contained rhamnose with a little glucose and xylose. However, the CW also had trace of mannose and HW1 had trace of galactose and mannose. They all contained glucuronic acid.
Based on the physicochemical analysis, HW1 was pured by ion-exchange chromatography and low-pressure gel permeation chromatography. WF1 and WF3 were obtained. The physicochemical properties and monosaccharide compositions of WF1 and WF3 were analysed by Phenol-sulphuric acid method, Folin-phenol reagent method, barium sulfate turbidimetry method, IR, GC and HPLC. The results showed that they were high purity, the molecular weights of them were 870 kD and 70 kD, respectively. The molecular weights of WF1 was higher than that of WF3. They both contained rhamnose, a little glucose and xylose. WF1 also had trace of galactose, WF3 had trace of mannose. In their IR spectrum, 1250 cm-1 showed strong absorption peaks of S=O stretching vibration.
The structures of WF1 and WF3 were studied by desulfate reaction, periodate oxidation, Smith degradation, methylation reaction, HPLC, GC-MS, IR and NMR. The results showed that the main linkage pattern of WF1 was 1, 2-Rha, also had small amount of 1, 4 and 1, 3-Rha. The sulfate group position were mainly at C-3 and C-4, minor at C-2. However, WF3 was mainly consisted by 1, 3-Rha, minor 1, 2-Rha and 1, 2, 3-Rha. The sulfate group was at C-4, minor at C-2, C-3. From the NMR spectrum, the branching point of WF3 was at C-3. The sulfate groups were mainly at C-2 and C-4. The IR spectrum of WF1 and WF3 showed that they were bothαconfiguration.
The anticoagulant activities of WF1 and WF3 were studied by APTT, TT and PT. APTT and TT were effectively prolonged by WF1 and WF3. WF1 and WF3 were lack of prolongation effect on PT. The prolongation of APTT indicates inhibition of the intrinsic and/or common pathway. TT assay is helpful to investigate the sample effect in thrombin accelerated clot formation in platelet poor plasma, whereas prolongation of TT suggests inhibition of thrombin activity or fibrin polymerization. Thus, WF1 and WF3 inhibited both the intrinsic and/or common pathways of coagulation and thrombin activity or conversion of fibrinogen to fibrin. WF1 and WF3 did not inhibit extrinsic pathway of coagulation. The differences between the anticoagulant activities of WF1 and WF3 were pronounced. WF3 had higher anticoagulant activity than WF1 at the same concentration in the APTT assay. The similar result was observed in the TT assay. The differences of anticoagulant activities between WF1 and WF3 may be directly due to their structural features variation. The anticoagulant activities of WF1 and WF3 were weaker than that of heparin.
In this paper two polysaccharides WF1 and WF3 were obtained from Monostrom -a nitidum. So far there is no report about their structures.
This research can supply basic data on polysaccharides from Monostroma nitidum. It is helpful to study structure-activity relationship and enrich‘marine saccharide bank’. It is important to develop marine drug with Chinese features.
礁膜藻体粉末依次经冷水、热水、稀碱和浓碱液提取,得到五种粗多糖CW、HW1、HW2、AM1和AM2,提取得率分别是9.1%、30.6%、5.3%、5.5%和3.7%,总提取率达54.2%。采用苯酚-硫酸法、硫酸钡明胶比浊法、薄层层析、气相色谱、高效液相色谱等方法对其中三种水提取粗多糖CW、HW1和HW2的理化性质和单糖组成进行了比较分析。结果表明:总糖含量HW2最高,CW最低;蛋白含量以CW最高,HW1最低;硫酸基含量以HW1最高,CW最低。三种粗多糖的单糖组成均以鼠李糖为主,含有少量葡萄糖和木糖,CW含有微量甘露糖,HW1含有微量的半乳糖和甘露糖;糖醛酸的类型均为葡萄糖醛酸。
在对粗多糖理化性质和单糖组成测定的基础上,选取HW1作为研究对象,采用离子交换色谱和凝胶排阻色谱相结合对其进行分离纯化得到两种纯化多糖WF1和WF3。采用高效液相色谱法、气相色谱法、红外光谱法对两种多糖的重均分子量、纯度及单糖组成进行研究。结果表明:多糖WF1和WF3经SB-804HQ柱洗脱呈单一对称峰,表明两种多糖的纯度较高,重均分子量分别是870 kD和70 kD,WF1的分子量明显大于WF3;两种多糖的单糖组成均以鼠李糖为主,含有少量木糖和葡萄糖,另外,WF1含有微量半乳糖,WF3含有微量甘露糖;糖醛酸类型是葡萄糖醛酸。红外光谱图在1250 cm-1左右有S=O的伸缩振动吸收峰,表明了两种多糖均是带有硫酸基的鼠李聚糖。
采用脱硫酸基反应、高碘酸氧化、Smith降解、甲基化反应、气相色谱、高效液相色谱、红外光谱、核磁共振波谱和气质联用等方法对多糖WF1和WF3的结构进行研究。结果表明:WF1的糖链中是以1, 2-Rha为主,含有有1, 4和1, 3-Rha,硫酸基的位置以C-3、C-4较多,C-2位相对较少,也可能C-3、C-4和C-2均带有硫酸基。WF3的糖链中是以1, 3-Rha为主,含有1, 2-Rha,存在1→2,3连接的Rha分支点,硫酸基的位置以C-4位较多,C-2、C-3位相对较少,也可能C-3、C-4和C-2均带有硫酸基。脱硫前后的红外谱图对比解析表明两种多糖的构型为α型。核磁共振谱图进一步说明WF3是α构型,C-3位是糖链的连接位点,C-2位、C-4位可能有硫酸基取代。
通过活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)和凝血酶时间(TT)分析,对礁膜多糖WF1和WF3的抗凝血活性进行研究。结果表明:WF1和WF3对APTT和TT均有延长作用,但对PT作用不明显,说明WF1和WF3主要抑制内源性凝血途径和/或共有凝血途径以及抑制凝血酶活性或纤维蛋白原转化为纤维蛋白,而对外源性凝血途径没有影响。而且,WF1抗凝血活性明显高于WF3,这主要是由于它们的分子量、硫酸基含量和结构的差异所引起的。WF1和WF3的抗凝血活性比肝素低。
本研究提取、分离纯化得到两种礁膜多糖WF1和WF3,关于这两种多糖的结构到目前为止国内外尚未见研究报道。
本课题为礁膜多糖的研究提供更多的基础资料,为进一步研究其结构与活性的关系奠定了基础,对促进“海洋糖库”的建设,发展具有我国特色的海洋新药都有重要意义。
Monostroma nitidum is a kind of marine green seaweed. The polysaccharide is its important bioactive substance.
In this paper, Monostroma nitidum was extracted by cool water, boiling water and alkali liquid successively. Five kinds of polysaccharides were obtained and named as CW, HW1, HW2, AM1 and AM2, respectively. The physicochemical properties and monosaccharide compositions of these three polysaccharides CW, HW1 and HW2 were analysed by Phenol-sulphuric acid method, Folin-phenol reagent method, barium sulfate turbidimetry method, GC and HPLC. The results showed that the total yield of the crude polysaccharides was 54.2%. The yield of HW1 was 30.6%, and it was the highest in the three polysaccharides. The contents of the sulfate group were different, they were 21.8%, 29.1% and 26.9% respectively. These three polysaccharides mainly contained rhamnose with a little glucose and xylose. However, the CW also had trace of mannose and HW1 had trace of galactose and mannose. They all contained glucuronic acid.
Based on the physicochemical analysis, HW1 was pured by ion-exchange chromatography and low-pressure gel permeation chromatography. WF1 and WF3 were obtained. The physicochemical properties and monosaccharide compositions of WF1 and WF3 were analysed by Phenol-sulphuric acid method, Folin-phenol reagent method, barium sulfate turbidimetry method, IR, GC and HPLC. The results showed that they were high purity, the molecular weights of them were 870 kD and 70 kD, respectively. The molecular weights of WF1 was higher than that of WF3. They both contained rhamnose, a little glucose and xylose. WF1 also had trace of galactose, WF3 had trace of mannose. In their IR spectrum, 1250 cm-1 showed strong absorption peaks of S=O stretching vibration.
The structures of WF1 and WF3 were studied by desulfate reaction, periodate oxidation, Smith degradation, methylation reaction, HPLC, GC-MS, IR and NMR. The results showed that the main linkage pattern of WF1 was 1, 2-Rha, also had small amount of 1, 4 and 1, 3-Rha. The sulfate group position were mainly at C-3 and C-4, minor at C-2. However, WF3 was mainly consisted by 1, 3-Rha, minor 1, 2-Rha and 1, 2, 3-Rha. The sulfate group was at C-4, minor at C-2, C-3. From the NMR spectrum, the branching point of WF3 was at C-3. The sulfate groups were mainly at C-2 and C-4. The IR spectrum of WF1 and WF3 showed that they were bothαconfiguration.
The anticoagulant activities of WF1 and WF3 were studied by APTT, TT and PT. APTT and TT were effectively prolonged by WF1 and WF3. WF1 and WF3 were lack of prolongation effect on PT. The prolongation of APTT indicates inhibition of the intrinsic and/or common pathway. TT assay is helpful to investigate the sample effect in thrombin accelerated clot formation in platelet poor plasma, whereas prolongation of TT suggests inhibition of thrombin activity or fibrin polymerization. Thus, WF1 and WF3 inhibited both the intrinsic and/or common pathways of coagulation and thrombin activity or conversion of fibrinogen to fibrin. WF1 and WF3 did not inhibit extrinsic pathway of coagulation. The differences between the anticoagulant activities of WF1 and WF3 were pronounced. WF3 had higher anticoagulant activity than WF1 at the same concentration in the APTT assay. The similar result was observed in the TT assay. The differences of anticoagulant activities between WF1 and WF3 may be directly due to their structural features variation. The anticoagulant activities of WF1 and WF3 were weaker than that of heparin.
In this paper two polysaccharides WF1 and WF3 were obtained from Monostrom -a nitidum. So far there is no report about their structures.
This research can supply basic data on polysaccharides from Monostroma nitidum. It is helpful to study structure-activity relationship and enrich‘marine saccharide bank’. It is important to develop marine drug with Chinese features.
引文
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