金针菇多糖的分离纯化、结构分析及其记忆功能改善作用机制研究
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摘要
金针菇(Flammulina velutipes)是目前国内消费量较大的食用菌类之一,其含有丰富的营养成分和生物活性物质,是一种药食两用菌。据研究和报道显示,金针菇能够增强儿童生长和智力发育,尽管许多报道已经表明金针菇具有改善记忆力功能的作用,然而金针菇中主要的改善记忆功能成分物质仍然不明确,金针菇活性成分的改善记忆功能的作用机制一直未得到很好的阐明。
本文通过Box-Behnken响应面优化金针菇多糖(FVP)的超声波辅助提取工艺,并采用高效凝胶过滤色谱法(HPGFC)、气相色谱法(GC)、傅里叶红外光谱(FT-IR)、紫外扫描法光谱(UV)、核磁共振技术(NMR)等光谱技术和一些化学分析方法对金针菇多糖结构进行分析。进一步利用Morris迷宫试验隐藏平台试验和定位航行试验,研究金针菇多糖对东茛菪碱诱导大鼠记忆损伤的改善作用。并分析脑组织海马和大脑皮质中生理生化指标变化,通过检测超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)等抗氧化酶的活性,乙酰胆碱(ACh)、5-羟色胺(5-HT)、多巴胺(DA)、去甲肾上腺素(NE)等神经递质含量,胆碱乙酰转移酶(ChAT)和乙酰胆碱酯酶(AChE)活性,Ca2+/钙调蛋白依赖的蛋白激酶Ⅱ(CaMKⅡ)、细胞外信号调节激酶(ERK)、以及细胞间隙连接蛋白Connexin36等一些蛋白激酶分子的变化,研究金针菇多糖改善记忆功能作用机制。研究结果表明：
(1)通过研究金针菇四种不同溶剂提取物(水、乙醇、石油醚、乙酸乙酯)抑制AChE活性,水萃取物组分具有最强的AChE抑制活性,抑制率为19.75%。进一步研究水溶液中的多糖成分AChE抑制活性为20.0%(2.0mg/mL).通过Box-Behnken响应面优化金针菇多糖超声波辅助提取工艺条件(料液比(X1)、超声波功率(X2)、超声时间(X3)、超声温度(X4)),建立金针菇多糖提取模型方程为Y=-11.94+0.18·X1+0.03-X2+0.30·X3+0.25·X4+6.88×10-5·X1X2+1.0×10-3·X1X3+1.2×10-3·X1X4-2.59×10-4·X2X3-1.11×10-4·X2X4-1.33×10-3·X3X4-5.99×10-3·X12-1.86×10-5·X22-2.87×10-3·X32-2.06×10-3·X42.确定金针菇多糖超声波辅助提取最佳工艺条件为料液比为25mL/g、超声波功率为620W、超声时间为20min、超声温度为45℃,在此条件下金针菇多糖得率为8.33%,比普通热水浸提法的多糖得率提高了62.7%。
(2)金针菇粗多糖经DEAE纤维素-52离子交换色谱、Sephadex G-100葡聚糖凝胶色谱分离纯化得到FVP-1和FVP-2两种金针菇多糖组分,其总糖含量分别是98.65%和96.24%。经过高效凝胶过滤色谱测得分子量分别为28kD和268kD。FVP-1和FVP-2糖醛酸含量分别为1.56%和3.42%,硫酸基含量分别为0.09%和0.14%。FVP-1由葡萄糖、半乳糖、甘露糖和岩藻糖4种单糖组成,其摩尔比为葡萄糖：半乳糖：甘露糖：岩藻糖=81.3：12.1：3.6：3.0。FVP-2由葡萄糖、甘露糖、半乳糖、木糖和岩藻糖5种单糖组成,其摩尔比为葡萄糖：甘露糖：半乳糖：木糖：岩藻糖=57.9：12.0：15.1：9.5：5.5。FVP-1和FVP-2均是具有三股螺旋结构的多糖分子。紫外光谱图显示FVP-1和FVP-2不含有核酸和蛋白质类物质。红外光谱扫描结果,FVP-1和FVP-2含有多糖类物质的特征吸收峰,FVP-1属于吡喃糖,FVP-2属于呋喃糖。FVP-1和FVP-2均是以β-糖苷键链接的多糖分子。
(3)通过Morris水迷宫试验研究金针菇多糖对东莨菪碱诱导大鼠记忆损伤模型的改善作用。研究结果发现：腹腔注射5mg/kg.bw剂量东莨菪碱能够造成大鼠学习记忆功能损伤,显著延长大鼠在隐藏平台实验中的平均逃避潜伏期,增加大鼠的总游泳路程；并能够降低大鼠在空间探索实验中穿过原平台位置的次数和在原平台象限区域的游泳时间。200mg/kg.bw和400mg/kg.bw金针菇多糖能够显著降低大鼠在隐藏平台实验中的平均逃避潜伏期,减少逃避潜伏期的总路程长度,增加空间探索实验中大鼠穿过原平台位置的次数和在原平台象限区域的游泳时间。表明金针菇多糖对记忆功能损伤大鼠的空间学习和记忆能力有显著的修复作用,为金针菇辅助改善记忆功能的作用提供了科学的证明。
(4)通过分析多糖对大鼠海马和大脑皮质的生理生化指标的变化,研究金针菇多糖改善记忆机制。结果表明,5mg/kg.bw ip的东茛菪碱能够显著抑制海马和大脑皮质中SOD和GSH-Px活性,降低多巴胺、去甲肾上腺素、5-羟色胺、乙酰胆碱等神经递质含量,抑制ChAT活性,促进AChE的活性升高。同时,还能够显著降低海马和大脑皮质中Connexin36、p-ERK和p-CaMK Ⅱ蛋白的表达。最终导致学习记忆能力下降。与损伤模型组相比,金针菇多糖能够显著提高海马和大脑皮质中SOD和GSH-Px活性,修复东茛菪碱引起的海马和大脑皮质中DA、NE、5-HT和ACh等神经递质含量的降低,特别是高浓度的FVP对海马中DA、NE和ACh的含量效果最显著。FVP通过增强ChAT活性和降低AChE的活性以加速ACh的合成和降低ACh的分解速度,有效促进ACh含量的增加。此外,金针菇多糖能够显著增强海马和大脑皮质中Connexin36、p-ERK和p-CaMKⅡ蛋白的表达水平,从而达到改善大鼠学习记忆功能。
Flammulina velutipes is one of the most popular mushrooms in China, which contains plentiful nutrients and active substance and can be used for pharmaceutical and edible functions. A few reports have shown that F. velutipes was benefit for children growth and intellectual development. Although many researches have proved its improvement of learning and memory functions, the functional ingredients and mechanism haven't been elucidated.
In this study, response surface methodology, based on Box-Behnken design was used to optimize the ultrasonic extraction conditions of F. velutipes polysaccharides (FVP), and chromatographic and spectroscopy technologies such as high performance gel filtration chromatography (HPGFC), gas chromatography (GC), Fourier transform infrared spectrum (FT-IR), UV scanning spectrum (UV), and nuclear magnetic resonance (NMR), as well as chemical analysis methods were employed to characterized FVP structure. Hidden platform test and probe test of Morris water maze were applied to evaluate the improvement of scopolamine-induced memory impairments of FVP. Effects of FVP on the biochemical and neurological changes in hippocampus and cerebral cortex of rats were investigated, SOD and GSH-Px activities, content of some neurotransmitters such as acetylcholine (ACh),5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE), choline acetyltransferase (ChAT) and acetylchohnesterase (AChE) activities, phosphorylation of Calcium/calmodulin dependent protein kinase (CaMKⅡ) and extracellular regulated protein kinases (ERK), and expression of gap junction protein connexin36are were analyzed to clarify the mechanism. The following work was performed and the results are summarized:
(1) Four solvent extracts of F. velutipes were prepared for AChE inhibitory activities assay. Results showed that the AChE inhibitory activity of water extract was significantly better than the other solvents extracts (ethanol, petroleum ether, and ethyl acetate), with19.75%inhibition rate. FVP as the main component in water extract was further shown to have20.0%AChE inhibition rate (2.0mg/mL). Box Behnken design was used to optimize the ultrasonic extraction conditions of FVP and four independent variables (ratio of water to raw material, ultrasonic power, ultrasonic time, and ultrasonic temperature) were taken into consideration. A quadratic model was constructed between ultrasonic conditions and yield of FVP, Y=-11.94+0.18·X1+0.03·X2+0.30·X3+0.25X4+6.88×10-5·X1X2+1.0×10-3·X1X3+1.2×10-3·X1X4-2.59×10-4·X2X3-1.11×10-4·X2X4-1.33×10-3·X3X4-5.99×10-3·X12-1.86×10-5·X22-2.87×10-3X32-2.06×10-3·X42。A modified optimization condition was ratio of water to material of25ml/g, ultrasonic power of620W, ultrasonic time of20min, and ultrasonic temperature of45℃, and a yield of FVP of8.33%was obtained under this condition, which is62.7%higher than that obtained with hot water extraction.
(2) FVP was further purified by DEAE cellulose-52chromatography and Sephadex G-100size-exclusion chromatography to afford FVP-1and FVP-2, with total sugar content of98.65%and96.24%, respectively. Molecular weight of FVP-1and FVP-2was28kDa and268kDa, respectively. Uronic acid content was1.56%and3.42%, respectively. Sulfate content was0.09%and0.14%, respectively. FVP-1was composed of glucose, galactose, mannose and fucose in an approximate molar ratio81.3:12.1:3.6:3.0and FVP-2was composed of glucose, mannose, galactose, xylose and fucose in an approximate molar ratio57.9:12.0:15.1:9.5:5.5. Both FVP-1and FVP-2were triple helical structures. Ultraviolet spectrum indicated that FVP-1and FVP-2were without protein and nucleic acid. Characteristic absorptions of polysaccharides were observed in infrared spectra of FVP-1and FVP-2, which indicated the possible presence of pyranose ring in FVP-1and furanose ring in FVP-2, respectively, and glycosyl residues of FVP-1and FVP-2were mainly β-type glycosidic linkages.
(3) Morris water maze was employed to evaluate effect of FVP on scopolamine-induced learning and memory impairments. Results showed that, compared with control,5mg/kg.bw ip scopolamine can injure the learning and memory of rats, presenting an increase in the escape latency and total swimming distance in hidden platform test, and decreased the numbers of crossing the platform and distance of moving around platform of rats in probe test.200mg/kg.bw and400mg/kg.bw FVP significantly decreased the escape latency and total swimming distance of rats in hidden platform test, and increased the numbes of crossing the platform and distance of moving around platform of rats in probe test. All these results indicated that FVP could improve scopolamine-induced learning and memory impairments, which afford scientific proof for learning and memory function of F.velutipe.
(4) To clarify the learning and memory improvement mechanism of FVP, effects of FVP on the biochemical and neurological changes in hippocampus and cerebral cortex of rats were investigated. Results showed that5mg/kg.bw ip scopolamine decreased SOD and GSH-Px activities, as well as ACh,5-HT, DA, and NE contents in hippocampus and cerebral cortex. Decrease of ACh content owed to decrease of ChAT activity and increase of AChE activity. Expressions of Connexin36, p-ERK and p-CaMK Ⅱ in hippocampus and cerebral cortex were also decreased by scopolamine. Compared with scopolamine injured group rats, FVP reversed the decrease of SOD and GSH-Px activities induced by scopolamine, and increased ACh,5-HT, DA, and NE contents, especially effect of highest concentration FVP on ACh, DA, and NE contents in hippocampus. FVP increased ChAT activity and decreased AChE activity to raise ACh content. Moreover, FVP increased the expressions of Connexin36, p-ERK and p-CaMK Ⅱ in hippocampus and cerebral cortex. FVP achieves its learning and memory improving function through modulation of all the above mechanism.
本文通过Box-Behnken响应面优化金针菇多糖(FVP)的超声波辅助提取工艺,并采用高效凝胶过滤色谱法(HPGFC)、气相色谱法(GC)、傅里叶红外光谱(FT-IR)、紫外扫描法光谱(UV)、核磁共振技术(NMR)等光谱技术和一些化学分析方法对金针菇多糖结构进行分析。进一步利用Morris迷宫试验隐藏平台试验和定位航行试验,研究金针菇多糖对东茛菪碱诱导大鼠记忆损伤的改善作用。并分析脑组织海马和大脑皮质中生理生化指标变化,通过检测超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)等抗氧化酶的活性,乙酰胆碱(ACh)、5-羟色胺(5-HT)、多巴胺(DA)、去甲肾上腺素(NE)等神经递质含量,胆碱乙酰转移酶(ChAT)和乙酰胆碱酯酶(AChE)活性,Ca2+/钙调蛋白依赖的蛋白激酶Ⅱ(CaMKⅡ)、细胞外信号调节激酶(ERK)、以及细胞间隙连接蛋白Connexin36等一些蛋白激酶分子的变化,研究金针菇多糖改善记忆功能作用机制。研究结果表明：
(1)通过研究金针菇四种不同溶剂提取物(水、乙醇、石油醚、乙酸乙酯)抑制AChE活性,水萃取物组分具有最强的AChE抑制活性,抑制率为19.75%。进一步研究水溶液中的多糖成分AChE抑制活性为20.0%(2.0mg/mL).通过Box-Behnken响应面优化金针菇多糖超声波辅助提取工艺条件(料液比(X1)、超声波功率(X2)、超声时间(X3)、超声温度(X4)),建立金针菇多糖提取模型方程为Y=-11.94+0.18·X1+0.03-X2+0.30·X3+0.25·X4+6.88×10-5·X1X2+1.0×10-3·X1X3+1.2×10-3·X1X4-2.59×10-4·X2X3-1.11×10-4·X2X4-1.33×10-3·X3X4-5.99×10-3·X12-1.86×10-5·X22-2.87×10-3·X32-2.06×10-3·X42.确定金针菇多糖超声波辅助提取最佳工艺条件为料液比为25mL/g、超声波功率为620W、超声时间为20min、超声温度为45℃,在此条件下金针菇多糖得率为8.33%,比普通热水浸提法的多糖得率提高了62.7%。
(2)金针菇粗多糖经DEAE纤维素-52离子交换色谱、Sephadex G-100葡聚糖凝胶色谱分离纯化得到FVP-1和FVP-2两种金针菇多糖组分,其总糖含量分别是98.65%和96.24%。经过高效凝胶过滤色谱测得分子量分别为28kD和268kD。FVP-1和FVP-2糖醛酸含量分别为1.56%和3.42%,硫酸基含量分别为0.09%和0.14%。FVP-1由葡萄糖、半乳糖、甘露糖和岩藻糖4种单糖组成,其摩尔比为葡萄糖：半乳糖：甘露糖：岩藻糖=81.3：12.1：3.6：3.0。FVP-2由葡萄糖、甘露糖、半乳糖、木糖和岩藻糖5种单糖组成,其摩尔比为葡萄糖：甘露糖：半乳糖：木糖：岩藻糖=57.9：12.0：15.1：9.5：5.5。FVP-1和FVP-2均是具有三股螺旋结构的多糖分子。紫外光谱图显示FVP-1和FVP-2不含有核酸和蛋白质类物质。红外光谱扫描结果,FVP-1和FVP-2含有多糖类物质的特征吸收峰,FVP-1属于吡喃糖,FVP-2属于呋喃糖。FVP-1和FVP-2均是以β-糖苷键链接的多糖分子。
(3)通过Morris水迷宫试验研究金针菇多糖对东莨菪碱诱导大鼠记忆损伤模型的改善作用。研究结果发现：腹腔注射5mg/kg.bw剂量东莨菪碱能够造成大鼠学习记忆功能损伤,显著延长大鼠在隐藏平台实验中的平均逃避潜伏期,增加大鼠的总游泳路程；并能够降低大鼠在空间探索实验中穿过原平台位置的次数和在原平台象限区域的游泳时间。200mg/kg.bw和400mg/kg.bw金针菇多糖能够显著降低大鼠在隐藏平台实验中的平均逃避潜伏期,减少逃避潜伏期的总路程长度,增加空间探索实验中大鼠穿过原平台位置的次数和在原平台象限区域的游泳时间。表明金针菇多糖对记忆功能损伤大鼠的空间学习和记忆能力有显著的修复作用,为金针菇辅助改善记忆功能的作用提供了科学的证明。
(4)通过分析多糖对大鼠海马和大脑皮质的生理生化指标的变化,研究金针菇多糖改善记忆机制。结果表明,5mg/kg.bw ip的东茛菪碱能够显著抑制海马和大脑皮质中SOD和GSH-Px活性,降低多巴胺、去甲肾上腺素、5-羟色胺、乙酰胆碱等神经递质含量,抑制ChAT活性,促进AChE的活性升高。同时,还能够显著降低海马和大脑皮质中Connexin36、p-ERK和p-CaMK Ⅱ蛋白的表达。最终导致学习记忆能力下降。与损伤模型组相比,金针菇多糖能够显著提高海马和大脑皮质中SOD和GSH-Px活性,修复东茛菪碱引起的海马和大脑皮质中DA、NE、5-HT和ACh等神经递质含量的降低,特别是高浓度的FVP对海马中DA、NE和ACh的含量效果最显著。FVP通过增强ChAT活性和降低AChE的活性以加速ACh的合成和降低ACh的分解速度,有效促进ACh含量的增加。此外,金针菇多糖能够显著增强海马和大脑皮质中Connexin36、p-ERK和p-CaMKⅡ蛋白的表达水平,从而达到改善大鼠学习记忆功能。
Flammulina velutipes is one of the most popular mushrooms in China, which contains plentiful nutrients and active substance and can be used for pharmaceutical and edible functions. A few reports have shown that F. velutipes was benefit for children growth and intellectual development. Although many researches have proved its improvement of learning and memory functions, the functional ingredients and mechanism haven't been elucidated.
In this study, response surface methodology, based on Box-Behnken design was used to optimize the ultrasonic extraction conditions of F. velutipes polysaccharides (FVP), and chromatographic and spectroscopy technologies such as high performance gel filtration chromatography (HPGFC), gas chromatography (GC), Fourier transform infrared spectrum (FT-IR), UV scanning spectrum (UV), and nuclear magnetic resonance (NMR), as well as chemical analysis methods were employed to characterized FVP structure. Hidden platform test and probe test of Morris water maze were applied to evaluate the improvement of scopolamine-induced memory impairments of FVP. Effects of FVP on the biochemical and neurological changes in hippocampus and cerebral cortex of rats were investigated, SOD and GSH-Px activities, content of some neurotransmitters such as acetylcholine (ACh),5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE), choline acetyltransferase (ChAT) and acetylchohnesterase (AChE) activities, phosphorylation of Calcium/calmodulin dependent protein kinase (CaMKⅡ) and extracellular regulated protein kinases (ERK), and expression of gap junction protein connexin36are were analyzed to clarify the mechanism. The following work was performed and the results are summarized:
(1) Four solvent extracts of F. velutipes were prepared for AChE inhibitory activities assay. Results showed that the AChE inhibitory activity of water extract was significantly better than the other solvents extracts (ethanol, petroleum ether, and ethyl acetate), with19.75%inhibition rate. FVP as the main component in water extract was further shown to have20.0%AChE inhibition rate (2.0mg/mL). Box Behnken design was used to optimize the ultrasonic extraction conditions of FVP and four independent variables (ratio of water to raw material, ultrasonic power, ultrasonic time, and ultrasonic temperature) were taken into consideration. A quadratic model was constructed between ultrasonic conditions and yield of FVP, Y=-11.94+0.18·X1+0.03·X2+0.30·X3+0.25X4+6.88×10-5·X1X2+1.0×10-3·X1X3+1.2×10-3·X1X4-2.59×10-4·X2X3-1.11×10-4·X2X4-1.33×10-3·X3X4-5.99×10-3·X12-1.86×10-5·X22-2.87×10-3X32-2.06×10-3·X42。A modified optimization condition was ratio of water to material of25ml/g, ultrasonic power of620W, ultrasonic time of20min, and ultrasonic temperature of45℃, and a yield of FVP of8.33%was obtained under this condition, which is62.7%higher than that obtained with hot water extraction.
(2) FVP was further purified by DEAE cellulose-52chromatography and Sephadex G-100size-exclusion chromatography to afford FVP-1and FVP-2, with total sugar content of98.65%and96.24%, respectively. Molecular weight of FVP-1and FVP-2was28kDa and268kDa, respectively. Uronic acid content was1.56%and3.42%, respectively. Sulfate content was0.09%and0.14%, respectively. FVP-1was composed of glucose, galactose, mannose and fucose in an approximate molar ratio81.3:12.1:3.6:3.0and FVP-2was composed of glucose, mannose, galactose, xylose and fucose in an approximate molar ratio57.9:12.0:15.1:9.5:5.5. Both FVP-1and FVP-2were triple helical structures. Ultraviolet spectrum indicated that FVP-1and FVP-2were without protein and nucleic acid. Characteristic absorptions of polysaccharides were observed in infrared spectra of FVP-1and FVP-2, which indicated the possible presence of pyranose ring in FVP-1and furanose ring in FVP-2, respectively, and glycosyl residues of FVP-1and FVP-2were mainly β-type glycosidic linkages.
(3) Morris water maze was employed to evaluate effect of FVP on scopolamine-induced learning and memory impairments. Results showed that, compared with control,5mg/kg.bw ip scopolamine can injure the learning and memory of rats, presenting an increase in the escape latency and total swimming distance in hidden platform test, and decreased the numbers of crossing the platform and distance of moving around platform of rats in probe test.200mg/kg.bw and400mg/kg.bw FVP significantly decreased the escape latency and total swimming distance of rats in hidden platform test, and increased the numbes of crossing the platform and distance of moving around platform of rats in probe test. All these results indicated that FVP could improve scopolamine-induced learning and memory impairments, which afford scientific proof for learning and memory function of F.velutipe.
(4) To clarify the learning and memory improvement mechanism of FVP, effects of FVP on the biochemical and neurological changes in hippocampus and cerebral cortex of rats were investigated. Results showed that5mg/kg.bw ip scopolamine decreased SOD and GSH-Px activities, as well as ACh,5-HT, DA, and NE contents in hippocampus and cerebral cortex. Decrease of ACh content owed to decrease of ChAT activity and increase of AChE activity. Expressions of Connexin36, p-ERK and p-CaMK Ⅱ in hippocampus and cerebral cortex were also decreased by scopolamine. Compared with scopolamine injured group rats, FVP reversed the decrease of SOD and GSH-Px activities induced by scopolamine, and increased ACh,5-HT, DA, and NE contents, especially effect of highest concentration FVP on ACh, DA, and NE contents in hippocampus. FVP increased ChAT activity and decreased AChE activity to raise ACh content. Moreover, FVP increased the expressions of Connexin36, p-ERK and p-CaMK Ⅱ in hippocampus and cerebral cortex. FVP achieves its learning and memory improving function through modulation of all the above mechanism.
引文
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