超声萃取对茯苓菌核多糖提取率及结构影响的研究
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摘要
茯苓是(Poria cocos wolf)一种附着在松树根上生长的真菌,为多孔菌科植物,是我国传统的中药材。茯苓多糖是茯苓中重要的生物活性物质,具有利水渗湿、健脾宁心、抑菌杀菌、降血糖、抗病毒、调节免疫力、诱生与促干扰素和白细胞调节素等作用,对人体有较高的保健功能和药用价值。目前关于超声波对茯苓多糖提取率影响的研究工作已有报道,但对超声波强化提取对水溶性茯苓菌核多糖和碱溶性茯苓菌核多糖提取率及结构影响的系统研究相对较少,尚未发现用原子力显微镜对它们的微结构进行直接观测的报道。因此,本项目以茯苓菌核为原料,利用超声波对其中的水溶性多糖和碱溶性多糖分别进行提取,并将提取效果与传统提取方法进行比较,揭示超声萃取茯苓菌核多糖(水溶性多糖、碱溶性多糖)对其提取率、单糖组成种类和各组分间比例及结构的影响规律;通过对超声波强化提取茯苓菌核多糖的内在机理进行初步研究,为人们对超声萃取中草药多糖对其结构及生物活性影响的研究提供必要的基础数据和实验依据。论文的主要研究内容及相应的实验结果可归纳为以下几个方面:
(1)确定超声波辅助热水浸提水溶性茯苓菌核多糖的最佳提取工艺,以多糖得率为指标,在单因素试验的基础上采用正交试验得出了超声波提取水溶性茯苓菌核多糖的最佳工艺条件,即超声时间25min、提取温度80℃、液围比60,并与传统热水浸提法进行比较;同时,用环境扫描电子显微镜对超声波处理前后茯苓菌核细胞结构的变化情况进行研究,结果发现未经超声处理的茯苓菌核细胞整体比较完整,表面几乎没有裂纹,不同细胞间通过胞间层连接在一起:经25min超声处理后,多数茯苓菌核细胞上都出现不同程度的裂痕,有的细胞甚至已经完全崩溃,大多数胞间层也已经被破坏。
(2)采用传统热水浸提法和超声波辅助热水浸提法,按照优化后的工艺组合分别对脱脂茯苓菌核粉末进行提取,得多糖PPTH和PPUH。用傅立叶变换红外光谱(FTIR)分析糖类官能团,气相色谱法测定单糖组成,原子力显微镜观察多糖结构,并将二者的测定结果进行对比,结果表明:采用传统热水浸提得茯苓菌核多糖(PPTH)与超声波辅助热水浸提得茯苓菌核多糖(PPUH)具有相同的单糖组成,都包含核糖、木糖、甘露糖、果糖、半乳糖和葡萄糖,二者的红外吸收谱也基本相同,原子力显微镜扫描分析显示,PPTH整体呈现网状结构,而PPUH主要以长短不一的近棒状结构存在,推测可能是由于超声辐射对水溶性茯苓菌核多糖的网状结构造成破坏,促使其大分子链降解为较小的分子片断。由于这些小分子片段间发生聚集,因而最终形成较大“团簇棒状”结构。
(3)采用传统碱提法和超声波辅助碱提法分别对超声辅助热水浸提所得茯苓残渣进行提取,得碱溶性茯苓菌核多糖,记为PPTA、PPUA,得率76.47%、85.4%。与潘琦、黄才欢报道的方法相比,超声辐射强化浸提缩短了提取时间,提高了碱溶性茯苓菌核多糖的提取效率,同时,在提取过程我们发现,超声辐射强化提取碱溶性茯苓菌核多糖的提取效果主要体现在第一次浸提上,可使绝大部分多糖转移至碱溶液中。用傅立叶变换红外光谱仪、原子力显微镜等仪器分析手段分别对PPTA、PPUA的糖类官能团及分子形貌进行研究,并将二者测定结果进行比较发现:二者的红外吸收光谱基本相同,表明超声波强化提取不会改变碱溶性茯苓菌核多糖的基本结构;原子力显微镜观测表明,云母表面的PPTA整体呈现网状结构,而PPUA则以长短不一,且平均链高度较小的近棒状结构存在,说明超声辐射强化提取不仅会破坏碱溶性茯苓菌核多糖的网状结构,而且对多糖不同分子间的相互作用也有一定影响。
(4)对PPUA进行羧甲基修饰,Sevag法反复除蛋白(重复8次),经紫外光谱检测未观测到色素、蛋白质、核酸之类物质的特征吸收后透析,冷冻干燥备用。用高效液相色谱法对羧甲基茯苓多糖(Carboxymethylpachymaran,CMP)的分子量进行测定,用傅立叶变换红外光谱对CMP的结构进行表征,用原子力显微镜(Atomic force microscopy,AFM)对不同溶液环境下CMP的形态变化进行观测,实验结果表明:经过羧甲基修饰,碱溶性茯苓菌核多糖在水中的溶解性显著增加,890cm~(-1)处的β-D-葡聚糖特征吸收峰明显减弱,1333cm~(-1)处出现次甲基振动吸收峰,1606cm~(-1)处出现C=O非对称伸缩振动吸收峰,表明羧甲基化成功;高效液相色谱法测得CMP的分子量大小为1.45×10~5;原子力显微镜分析表明:在不同溶液条件下,CMP分子以不同形态存在,多糖溶液的浓度、离子强度及溶剂的物化特性均能对CMP的分子链构象及链间相互作用形式产生影响,推测可能与CMP分子内、分子间的氢键缔合及静电作用有关,CMP分子与云母基底间的吸附及静电作用也会对CMP的分子链构象及图像质量产生影响。
Poria cocos wolf, one of the traditional Chinese medicinal materials in china, is a kind of edible fungus attaching to the pine tree root which belongs to the polyporaceae plant. Polysaccharide from the poria cocos wolf is one of the most important biological active substance in the poria cocos wolf which has the activity of inducing diuresis and excreting dampness, strengthening spleen and being easy in mind, restraining and sterilizing bacteria, hypoglycemic effect, antivirus, modulateing immune function, inducing interferon and leukoregulin (LR) and so on. It is of high healthy function and medicinal value on human beings. At present, there have been papers about the effect of ultrasonication on extraction rate of polysaccharide from the poria cocos wolf, but there have been few papers about the systematic research of effect of ultrasonic enhanced extraction on extraction rate and structure of water-soluble polysaccharide and alkali-soluble polysaccharide from the sclerotium of poria cocos. Direct observation of microstructure of polysaccharides from the sclerotium of poria cocos has not been found by atomic force microscopy. Accordingly, water-soluble polysaccharide and alkali-soluble polysaccharide were extracted from the sclerotium of poria cocos by ultrasonic wave respectively in this study. Meanwhile, the extraction effect of ultrasonic wave was compare with that of traditional extraction in order to reveal the effect laws of ultrasonic extraction on extraction rate, monosaccharide composition and proportion among different components, structure of polysaccharide (water-soluble polysaccharide and alkali-soluble polysaccharide) from the sclerotium of poria cocos. Internal mechanism of extracting polysaccharide from the sclerotium of poria cocos by ultrasonic wave was preliminarily studied, which provided necessary basic data and experimental basis for the study of influence of ultrasonic extraction on structure and biological activity of polysaccharide from Chinese herbal medicine. The main research contents and corresponding results reported in the papers were summarized as follow:
(1)The optimal extraction technology of ultrasound-assisted hot water extraction of water-soluble polysaccharide from the sclerotium of poria cocos was determined. Taking polysaccharide extraction rate as the index, the optimal extraction conditions of ultrasound-assisted hot water extraction of water-soluble polysaccharide from the sclerotium of poria cocos was determined with the orthogonal design on the basis of single factor experiment, namely ultrasonic function time: twenty five minutes, extraction temperature: 80℃, the ratio of liquid to solid: 60:1, which were compared with those of traditional hot water extracting method. At the same time, structural changes of poria cocos wolf cells were investigated before and after ultrasonic extraction by environmental scanning electron microscope. The results showed that poria cocos wolf cells without ultrasonic processing were intact and there were few cracks on their surfaces. Different cells were connected together by middle lamella. After twenty five minutes ultrasonic processing, there were craws in varying degrees on the surfaces of most of poria cocos wolf cells, some of which had been completely collapsed. Most of middle lamellas had also been destroyed.
(2)Two kinds of water-soluble polysaccharides, which were named as PPTH and PPUH, were extracted from the sclerotium of poria cocos according to optimal extraction conditions by traditional hot water extracting method and ultrasound-assisted hot water extraction respectively. The polysaccharides functional groups were investigated by fourier transform infrared spectroscopy(FTIR). The monosaccharide compositions of polysaccharides were determined by gas chromatography. The structure of polysaccharides was analyzed by atomic force microscopy. Determination results of PPTH and PPUH were compared. The experimental results indicated that the components of PPUH were identified by GC as: ribose, xylose, mannose, fructose, galactose, glucose, which were the same as those of PPTH. Meanwhile, the infrared absorption spectras of PPUH and PPTH were basically the same. We found that PPTH on mica formed network-like structure by atomic force microscopy while PPUH existed in the form of different rod-like structure. It is presumed that ultrasonic radiation destructed network structure of water-soluble polysaccharide from the sclerotium of poria cocos, and made its macrochains degraded to small molecular fragments. Because of the aggregation of different small molecular fragments, they finally formed large cluster rod-like structure.
(3)Two kinds of alkali-soluble polysaccharides, which were named as PPTA and PPUA, were extracted from the residue of sclerotium of poria cocos after ultrasound-assisted hot water extraction by traditional alkaline extracting method and ultrasound-assisted alkaline extraction respectively. The yields of PPTA and PPUA were 76.47% and 85.4%. Compared with using extraction methods Pan Qi, Huang Caihuan reported, using ultrasonic enhanced extraction shortened the extraction time and improved extraction efficiency. Meanwhile, it was found in the process of extraction that the extraction effects of extracting alkali-soluble polysaccharide by ultrasonic wave embodied in the first extraction, when most of polysaccharide transferred to alkaline solution. Functional groups and molecular morphologies of PPTA and PPUA were investigated by fourier transform infrared spectrometer and atomic force microscopy respectively. It was found by comparing with both of determination results that the infrared absorption spectras of PPTA and PPUA were basically the same, which basic structure of alkali-soluble polysaccharide from the residue of sclerotium of poria cocos was not changed. The observation by atomic force microscopy showed that PPTA on mica formed network-like structure while PPUA existed in the form of different rod-like structure whose average height of chain was low. It was indicated that not only was network-like structure of alkali-soluble polysaccharide from the residue of sclerotium of poria cocos destroyed, but also different intermolecular interactions of polysaccharide were influenced by ultrasonic enhanced extraction.
(4)The structure of PPUA was modified by caboxymethyl group. The obtained polysaccharide, namely carboxymethylpachyman, was deproteinized by using Sevage method (repeated times 8). When there were no characteristic absorption peaks of pigments, protein and nucleic acid in its UV-Vis Absorption spectra, the modified PPUA was lyophilized and was reserved. The molecular weight of carboxymethylpachyman (CMP) was determined by high performance liquid chromatography. The structure of Carboxymethylpachymaran was characterized using fourier transform infrared spectroscopy. Morpological changes of carboxymethylpachymaran (CMP) were observed under different solution conditions by atomic force microscopy (AFM). After the carboxymethylation, the solubility of alkali-soluble polysaccharide from the sclerotium of poria cocos in the water was significantly increased, itsβ-D- glucan characteristic absorption peak at 890cm~(-1) became weak obviously, and its methylene vibration absorption peak and C=O antisymmetrical stretch vibration absorption peak appeared at 1333cm~(-1) and 1606cm~(-1) respectively, which indicated the carboxymethylation succeeded. The molecular weight of carboxymethylpachyman was 1.45×10~5 by high performance liquid chromatography. The analysis of AFM results showed that CMP molecules existed in different morphology under different solution conditions, and that the concentration, ionic strength and solvent physical chemistry characterisitics of polysaccharide solution had effects on the CMP chains conformation and the action mode between different molecular chains. The phenomena were considered to be related to hydrogen bond association and intramolecular and intermolecular electrostatic interactions of CMP. Meanwhile, the affinity and electrostatic interaction between CMP molecules and the mica substrate also had an influence on the CMP chains conformation and the image quality.
(1)确定超声波辅助热水浸提水溶性茯苓菌核多糖的最佳提取工艺,以多糖得率为指标,在单因素试验的基础上采用正交试验得出了超声波提取水溶性茯苓菌核多糖的最佳工艺条件,即超声时间25min、提取温度80℃、液围比60,并与传统热水浸提法进行比较;同时,用环境扫描电子显微镜对超声波处理前后茯苓菌核细胞结构的变化情况进行研究,结果发现未经超声处理的茯苓菌核细胞整体比较完整,表面几乎没有裂纹,不同细胞间通过胞间层连接在一起:经25min超声处理后,多数茯苓菌核细胞上都出现不同程度的裂痕,有的细胞甚至已经完全崩溃,大多数胞间层也已经被破坏。
(2)采用传统热水浸提法和超声波辅助热水浸提法,按照优化后的工艺组合分别对脱脂茯苓菌核粉末进行提取,得多糖PPTH和PPUH。用傅立叶变换红外光谱(FTIR)分析糖类官能团,气相色谱法测定单糖组成,原子力显微镜观察多糖结构,并将二者的测定结果进行对比,结果表明:采用传统热水浸提得茯苓菌核多糖(PPTH)与超声波辅助热水浸提得茯苓菌核多糖(PPUH)具有相同的单糖组成,都包含核糖、木糖、甘露糖、果糖、半乳糖和葡萄糖,二者的红外吸收谱也基本相同,原子力显微镜扫描分析显示,PPTH整体呈现网状结构,而PPUH主要以长短不一的近棒状结构存在,推测可能是由于超声辐射对水溶性茯苓菌核多糖的网状结构造成破坏,促使其大分子链降解为较小的分子片断。由于这些小分子片段间发生聚集,因而最终形成较大“团簇棒状”结构。
(3)采用传统碱提法和超声波辅助碱提法分别对超声辅助热水浸提所得茯苓残渣进行提取,得碱溶性茯苓菌核多糖,记为PPTA、PPUA,得率76.47%、85.4%。与潘琦、黄才欢报道的方法相比,超声辐射强化浸提缩短了提取时间,提高了碱溶性茯苓菌核多糖的提取效率,同时,在提取过程我们发现,超声辐射强化提取碱溶性茯苓菌核多糖的提取效果主要体现在第一次浸提上,可使绝大部分多糖转移至碱溶液中。用傅立叶变换红外光谱仪、原子力显微镜等仪器分析手段分别对PPTA、PPUA的糖类官能团及分子形貌进行研究,并将二者测定结果进行比较发现:二者的红外吸收光谱基本相同,表明超声波强化提取不会改变碱溶性茯苓菌核多糖的基本结构;原子力显微镜观测表明,云母表面的PPTA整体呈现网状结构,而PPUA则以长短不一,且平均链高度较小的近棒状结构存在,说明超声辐射强化提取不仅会破坏碱溶性茯苓菌核多糖的网状结构,而且对多糖不同分子间的相互作用也有一定影响。
(4)对PPUA进行羧甲基修饰,Sevag法反复除蛋白(重复8次),经紫外光谱检测未观测到色素、蛋白质、核酸之类物质的特征吸收后透析,冷冻干燥备用。用高效液相色谱法对羧甲基茯苓多糖(Carboxymethylpachymaran,CMP)的分子量进行测定,用傅立叶变换红外光谱对CMP的结构进行表征,用原子力显微镜(Atomic force microscopy,AFM)对不同溶液环境下CMP的形态变化进行观测,实验结果表明:经过羧甲基修饰,碱溶性茯苓菌核多糖在水中的溶解性显著增加,890cm~(-1)处的β-D-葡聚糖特征吸收峰明显减弱,1333cm~(-1)处出现次甲基振动吸收峰,1606cm~(-1)处出现C=O非对称伸缩振动吸收峰,表明羧甲基化成功;高效液相色谱法测得CMP的分子量大小为1.45×10~5;原子力显微镜分析表明:在不同溶液条件下,CMP分子以不同形态存在,多糖溶液的浓度、离子强度及溶剂的物化特性均能对CMP的分子链构象及链间相互作用形式产生影响,推测可能与CMP分子内、分子间的氢键缔合及静电作用有关,CMP分子与云母基底间的吸附及静电作用也会对CMP的分子链构象及图像质量产生影响。
Poria cocos wolf, one of the traditional Chinese medicinal materials in china, is a kind of edible fungus attaching to the pine tree root which belongs to the polyporaceae plant. Polysaccharide from the poria cocos wolf is one of the most important biological active substance in the poria cocos wolf which has the activity of inducing diuresis and excreting dampness, strengthening spleen and being easy in mind, restraining and sterilizing bacteria, hypoglycemic effect, antivirus, modulateing immune function, inducing interferon and leukoregulin (LR) and so on. It is of high healthy function and medicinal value on human beings. At present, there have been papers about the effect of ultrasonication on extraction rate of polysaccharide from the poria cocos wolf, but there have been few papers about the systematic research of effect of ultrasonic enhanced extraction on extraction rate and structure of water-soluble polysaccharide and alkali-soluble polysaccharide from the sclerotium of poria cocos. Direct observation of microstructure of polysaccharides from the sclerotium of poria cocos has not been found by atomic force microscopy. Accordingly, water-soluble polysaccharide and alkali-soluble polysaccharide were extracted from the sclerotium of poria cocos by ultrasonic wave respectively in this study. Meanwhile, the extraction effect of ultrasonic wave was compare with that of traditional extraction in order to reveal the effect laws of ultrasonic extraction on extraction rate, monosaccharide composition and proportion among different components, structure of polysaccharide (water-soluble polysaccharide and alkali-soluble polysaccharide) from the sclerotium of poria cocos. Internal mechanism of extracting polysaccharide from the sclerotium of poria cocos by ultrasonic wave was preliminarily studied, which provided necessary basic data and experimental basis for the study of influence of ultrasonic extraction on structure and biological activity of polysaccharide from Chinese herbal medicine. The main research contents and corresponding results reported in the papers were summarized as follow:
(1)The optimal extraction technology of ultrasound-assisted hot water extraction of water-soluble polysaccharide from the sclerotium of poria cocos was determined. Taking polysaccharide extraction rate as the index, the optimal extraction conditions of ultrasound-assisted hot water extraction of water-soluble polysaccharide from the sclerotium of poria cocos was determined with the orthogonal design on the basis of single factor experiment, namely ultrasonic function time: twenty five minutes, extraction temperature: 80℃, the ratio of liquid to solid: 60:1, which were compared with those of traditional hot water extracting method. At the same time, structural changes of poria cocos wolf cells were investigated before and after ultrasonic extraction by environmental scanning electron microscope. The results showed that poria cocos wolf cells without ultrasonic processing were intact and there were few cracks on their surfaces. Different cells were connected together by middle lamella. After twenty five minutes ultrasonic processing, there were craws in varying degrees on the surfaces of most of poria cocos wolf cells, some of which had been completely collapsed. Most of middle lamellas had also been destroyed.
(2)Two kinds of water-soluble polysaccharides, which were named as PPTH and PPUH, were extracted from the sclerotium of poria cocos according to optimal extraction conditions by traditional hot water extracting method and ultrasound-assisted hot water extraction respectively. The polysaccharides functional groups were investigated by fourier transform infrared spectroscopy(FTIR). The monosaccharide compositions of polysaccharides were determined by gas chromatography. The structure of polysaccharides was analyzed by atomic force microscopy. Determination results of PPTH and PPUH were compared. The experimental results indicated that the components of PPUH were identified by GC as: ribose, xylose, mannose, fructose, galactose, glucose, which were the same as those of PPTH. Meanwhile, the infrared absorption spectras of PPUH and PPTH were basically the same. We found that PPTH on mica formed network-like structure by atomic force microscopy while PPUH existed in the form of different rod-like structure. It is presumed that ultrasonic radiation destructed network structure of water-soluble polysaccharide from the sclerotium of poria cocos, and made its macrochains degraded to small molecular fragments. Because of the aggregation of different small molecular fragments, they finally formed large cluster rod-like structure.
(3)Two kinds of alkali-soluble polysaccharides, which were named as PPTA and PPUA, were extracted from the residue of sclerotium of poria cocos after ultrasound-assisted hot water extraction by traditional alkaline extracting method and ultrasound-assisted alkaline extraction respectively. The yields of PPTA and PPUA were 76.47% and 85.4%. Compared with using extraction methods Pan Qi, Huang Caihuan reported, using ultrasonic enhanced extraction shortened the extraction time and improved extraction efficiency. Meanwhile, it was found in the process of extraction that the extraction effects of extracting alkali-soluble polysaccharide by ultrasonic wave embodied in the first extraction, when most of polysaccharide transferred to alkaline solution. Functional groups and molecular morphologies of PPTA and PPUA were investigated by fourier transform infrared spectrometer and atomic force microscopy respectively. It was found by comparing with both of determination results that the infrared absorption spectras of PPTA and PPUA were basically the same, which basic structure of alkali-soluble polysaccharide from the residue of sclerotium of poria cocos was not changed. The observation by atomic force microscopy showed that PPTA on mica formed network-like structure while PPUA existed in the form of different rod-like structure whose average height of chain was low. It was indicated that not only was network-like structure of alkali-soluble polysaccharide from the residue of sclerotium of poria cocos destroyed, but also different intermolecular interactions of polysaccharide were influenced by ultrasonic enhanced extraction.
(4)The structure of PPUA was modified by caboxymethyl group. The obtained polysaccharide, namely carboxymethylpachyman, was deproteinized by using Sevage method (repeated times 8). When there were no characteristic absorption peaks of pigments, protein and nucleic acid in its UV-Vis Absorption spectra, the modified PPUA was lyophilized and was reserved. The molecular weight of carboxymethylpachyman (CMP) was determined by high performance liquid chromatography. The structure of Carboxymethylpachymaran was characterized using fourier transform infrared spectroscopy. Morpological changes of carboxymethylpachymaran (CMP) were observed under different solution conditions by atomic force microscopy (AFM). After the carboxymethylation, the solubility of alkali-soluble polysaccharide from the sclerotium of poria cocos in the water was significantly increased, itsβ-D- glucan characteristic absorption peak at 890cm~(-1) became weak obviously, and its methylene vibration absorption peak and C=O antisymmetrical stretch vibration absorption peak appeared at 1333cm~(-1) and 1606cm~(-1) respectively, which indicated the carboxymethylation succeeded. The molecular weight of carboxymethylpachyman was 1.45×10~5 by high performance liquid chromatography. The analysis of AFM results showed that CMP molecules existed in different morphology under different solution conditions, and that the concentration, ionic strength and solvent physical chemistry characterisitics of polysaccharide solution had effects on the CMP chains conformation and the action mode between different molecular chains. The phenomena were considered to be related to hydrogen bond association and intramolecular and intermolecular electrostatic interactions of CMP. Meanwhile, the affinity and electrostatic interaction between CMP molecules and the mica substrate also had an influence on the CMP chains conformation and the image quality.
引文
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