生脉饮多糖提取工艺优化及对脾虚模型大鼠肠道功能调节作用的研究
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摘要
目的:优化生脉饮多糖的提取工艺,并探讨生脉饮及其多糖对脾虚模型大鼠肠道功能的调节作用。方法:采用硫酸苯酚法测定多糖含量,并计算多糖提取率;以此为考察指标,采用单因素试验和正交试验,对生脉饮多糖提取工艺的料液比、提取时间、提取温度、提取次数进行优化,并进行验证试验。将80只雄性SD大鼠随机分为空白组,模型组,生脉饮低、中、高剂量组(350、700、1 400 g/L,按生药量计)和生脉饮多糖低、中、高剂量组(24.5、49、98 g/L,按生药量计),每组10只。除空白组外,其余各组大鼠均灌胃大黄水煎液10 mL/kg复制脾虚模型,每天1次,连续15 d。自第16天起,空白组和模型组大鼠均灌胃等容水,其余各组大鼠均灌胃相应药液,每天1次,连续10 d。观察各组大鼠的一般情况,记录体质量,并采用间苯三酚法或酶联免疫吸附测定法检测其血清D-木糖、胃泌素(GAS)、血管活性肠肽(VIP)含量。结果:生脉饮多糖最优提取工艺为料液比1∶10(g/mL)、提取时间45 min、提取温度80℃、提取次数1次。验证试验结果显示,3次提取的多糖提取率分别为7.43%、7.64%、7.80%(RSD=1.01%,n=3)。造模后,除空白组外的其余各组大鼠均出现了大便溏稀、体形消瘦、食量减小等症状,且其体质量和血清D-木糖含量均较空白组显著降低(P<0.01)。末次给药后,各给药组大鼠上述症状均有不同程度的改善,且除模型组外的其余各组大鼠的体质量以及各组大鼠血清D-木糖含量均较同组造模前或给药前显著增加(P<0.05或P<0.01);与空白组比较,模型组大鼠体质量和血清GAS含量均显著降低,血清VIP含量显著增加(P<0.01);与模型组比较,生脉饮中剂量组和生脉饮多糖低、高剂量组大鼠体质量以及生脉饮中、高剂量组和生脉饮多糖低、中剂量组大鼠血清D-木糖、GAS含量均显著增加,生脉饮各剂量组和生脉饮多糖低、中剂量组大鼠血清VIP含量均显著降低(P<0.05或P<0.01)。结论:优化的生脉饮多糖提取工艺稳定、可行。生脉饮及其多糖可有助于恢复脾虚模型大鼠的肠道功能,这种作用可能与其调节肠道GAS、VIP等因子分泌有关。
OBJECTIVE: To optimize the extraction technology of Shengmaiyin polysaccharide, and to investigate the regulation effects of Shengmaiyin and its polysaccharide on intestinal function of spleen deficiency model rats. METHODS:The contents of polysaccharide were determined by phenol-sulfuric acid method, and the extraction rate of polysaccharide was calculated. Using extraction rate of Shengmaiyin polysaccharide as investigation index,singel factor and orthogonal tests were used to optimize material-liquid ratio,extraction time,extraction temperature and extraction times of Shengmaiyin polysaccharide.Validation test was also conducted. Totally 80 male SD rats were randomly divided into blank group,model group,Shengmaiyin low-dose,medium-dose and high-dose groups (350,700,1 400 g/L,by crude drug),Shengmaiyin polysaccharide low-dose,medium-dose and high-dose groups (24.5,49,98 g/L,by crude drug),with 10 rats in each group. Except for blank group,other groups were given Rheum palmatum water decoction 10 mL/kg to induce spleen deficiency model,once a day,for consecutive 15 d. Since the 16 th day,blank group and model group were given isovolumic water intragastrically,while other groups were given corresponding drugs,once a day,for consecutive 10 d. The general status of rats and body weights were recorded in each group. The serum contents of D-xylose,gastrin (GAS) and vasoactive intestinal peptide (VIP) were detected by phloroglucinol method or ELISA. RESULTS: The optimal extraction technology of Shengmaiyin polysaccharide was material-liquid ratio 1∶10 (g/mL),extraction time 45 min,extraction temperature 80 ℃,extracting for 1 time. Results of validation test showed that extraction rates of the polysaccharide in 3 times were 7.43%,7.64%,7.80% (RSD=1.01%,n=3). After modeling,except for blank group,other groups suffered from loose stools,thin body and reduced food intake,and the body weight and serum level of D-xylose were decreased significantly compared with blank group (P<0.01). After last medication,above symptoms of administration groups were improved to different extents. Except for model group,body weight and serum contents of D-xylose in other groups were increased significantly than those before modeling or before medication (P<0.05 or P<0.01). Compared with blank group,body weight and serum content of GAS were decreased significantly in model group,while serum content of VIP was increased significantly (P<0.01). Compared with model group,body weight of Shengmaiyin medium-dose group and Shengmaiyin polysaccharide low-dose and high-dose groups,serum contents of D-xylose and GAS in Shengmaiyin medium-dose and high-dose groups and Shengmaiyin polysaccharide low-dose and medium-dose groups were increased significantly, while serum contents of VIP in Shengmaiyin groups and Shengmaiyin polysaccharide low-dose and medium-dose groups were all decreased significantly (P<0.05 or P<0.01).CONCLUSIONS:The optimized extraction technology of Shengmaiyin polysaccharide is stable and feasible. Shengmaiyin and its polysaccharide contribute to the recovery of intestinal function of spleen deficiency model rat,the effects of which may be associated with the secretion regulation of GAS and VIP.
OBJECTIVE: To optimize the extraction technology of Shengmaiyin polysaccharide, and to investigate the regulation effects of Shengmaiyin and its polysaccharide on intestinal function of spleen deficiency model rats. METHODS:The contents of polysaccharide were determined by phenol-sulfuric acid method, and the extraction rate of polysaccharide was calculated. Using extraction rate of Shengmaiyin polysaccharide as investigation index,singel factor and orthogonal tests were used to optimize material-liquid ratio,extraction time,extraction temperature and extraction times of Shengmaiyin polysaccharide.Validation test was also conducted. Totally 80 male SD rats were randomly divided into blank group,model group,Shengmaiyin low-dose,medium-dose and high-dose groups (350,700,1 400 g/L,by crude drug),Shengmaiyin polysaccharide low-dose,medium-dose and high-dose groups (24.5,49,98 g/L,by crude drug),with 10 rats in each group. Except for blank group,other groups were given Rheum palmatum water decoction 10 mL/kg to induce spleen deficiency model,once a day,for consecutive 15 d. Since the 16 th day,blank group and model group were given isovolumic water intragastrically,while other groups were given corresponding drugs,once a day,for consecutive 10 d. The general status of rats and body weights were recorded in each group. The serum contents of D-xylose,gastrin (GAS) and vasoactive intestinal peptide (VIP) were detected by phloroglucinol method or ELISA. RESULTS: The optimal extraction technology of Shengmaiyin polysaccharide was material-liquid ratio 1∶10 (g/mL),extraction time 45 min,extraction temperature 80 ℃,extracting for 1 time. Results of validation test showed that extraction rates of the polysaccharide in 3 times were 7.43%,7.64%,7.80% (RSD=1.01%,n=3). After modeling,except for blank group,other groups suffered from loose stools,thin body and reduced food intake,and the body weight and serum level of D-xylose were decreased significantly compared with blank group (P<0.01). After last medication,above symptoms of administration groups were improved to different extents. Except for model group,body weight and serum contents of D-xylose in other groups were increased significantly than those before modeling or before medication (P<0.05 or P<0.01). Compared with blank group,body weight and serum content of GAS were decreased significantly in model group,while serum content of VIP was increased significantly (P<0.01). Compared with model group,body weight of Shengmaiyin medium-dose group and Shengmaiyin polysaccharide low-dose and high-dose groups,serum contents of D-xylose and GAS in Shengmaiyin medium-dose and high-dose groups and Shengmaiyin polysaccharide low-dose and medium-dose groups were increased significantly, while serum contents of VIP in Shengmaiyin groups and Shengmaiyin polysaccharide low-dose and medium-dose groups were all decreased significantly (P<0.05 or P<0.01).CONCLUSIONS:The optimized extraction technology of Shengmaiyin polysaccharide is stable and feasible. Shengmaiyin and its polysaccharide contribute to the recovery of intestinal function of spleen deficiency model rat,the effects of which may be associated with the secretion regulation of GAS and VIP.
引文
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[19]赵冉,王育苗,黄树杰,等.金仁陈子方对小鼠脾虚模型肠道菌群的影响研究[J].医学研究杂志,2014,43(6):59-62.
[20]张仲林,臧志和,钟玲,等.六君子汤对脾虚证大鼠胃肠激素影响的实验研究[J].中成药,2010,32(4):659-661.
[21]陆英杰,连志诚.胃肠激素对胃肠动力的影响[J].免疫学杂志,2006,22(3):94-96.
[22]金敬善,王广才,张绳祖,等.血清中胃泌素水平与脾虚证的关系[J].中西医结合杂志,1982,2(1):25-26、62.
[23]刘玉强,许晨曦,贾蕊,等.苍术中三种单体化合物对脾虚模型大鼠疗效研究[J].中华中医药学刊,2017,35(2):382-386.
[2]ZHOU SS,XU J,ZHU H,et al.Gut microbiota-involved mechanisms in enhancing systemic exposure of ginsenosides by coexisting polysaccharides in ginseng decoction[J].Sci Rep,2016.DOI:10.1038/srep22474.
[3]SHI LL,LI Y,WANG Y,et al.MDG-1,an Ophiopogon polysaccharide,regulate gut microbiota inhigh-fat diet-induced obese C57BL/6 mice[J].Int J Biol Macromol,2015DOI:10.1016/j.ijbiomac.2015.08.057.
[4]王令仪,王源,阮克锋,等.麦冬多糖MDG-1对非糖尿病小鼠糖耐量及肠道菌群的影响[J].中国新药与临床杂志,2011,30(6):453-457.
[5]王令仪,王硕,王源,等.麦冬多糖MDG-1对糖尿病小鼠糖耐量及肠道菌群的影响[J].世界华人消化杂志,2011,19(19):2058-2062.
[6]石林林,王源,冯怡.麦冬多糖MDG-1对膳食诱导肥胖模型小鼠肠道益生菌群多样性影响的研究[J].中国中药杂志,2015,40(4):716-721.
[7]周卫东,项磊,陈泽伟,等.五味子多糖对化疗性肠道黏膜炎小鼠的保护作用[J].中国实验方剂学杂志,2016,22(22):124-128.
[8]田碧文,胡宏.阿胶、五味子、刺五加、枸杞对双歧杆菌生长的影响[J].中国微生态学杂志,1996,8(2):11-13.
[9]王法琴,陆兔林,毛春芹,等.3种比色法测定五味子中多糖[J].中成药,2015,37(4):814-818.
[10]赵娟.生脉散对Ⅱ型糖尿病心肌纤维化的影响及机制研究[D].上海:上海中医药大学,2016.
[11]倪青,王阶,赵安斌,等.生脉散对2型糖尿病性心肌病大鼠心肌的保护作用[J].中国中医基础医学杂志,2010,16(7):572-576.
[12]王丹凤,刘玉强,才谦.苍术麸炒前后健脾作用研究[J].时珍国医国药,2013,24(1):155-156.
[13]高笑一,鲍建才,黄建军.人参多糖化学的国内研究新进展[J].人参研究,2018,30(4):44-46.
[14]王润平,冉夏雨.人参多糖提取的正交优化[J].广州化工,2018,46(10):63-65.
[15]卢少玲,宋照风,江龙章,等.微波提取麦冬多糖工艺的探究[J].广东化工,2018,45(9):102-104.
[16]王彤,李德坤,周大铮,等.麦冬总糖提取工艺考察[J].辽宁中医药大学学报,2018,20(2):37-40.
[17]沈丽波,钱会南.脾虚模型实验研究方法概述[J].中国中医药信息杂志,2005,12(1):93-95.
[18]吴三明,张万岱.脾虚泄泻患者肠道微生态学的初步研究[J].中国中西医结合脾胃杂志,1996,4(4):203-204.
[19]赵冉,王育苗,黄树杰,等.金仁陈子方对小鼠脾虚模型肠道菌群的影响研究[J].医学研究杂志,2014,43(6):59-62.
[20]张仲林,臧志和,钟玲,等.六君子汤对脾虚证大鼠胃肠激素影响的实验研究[J].中成药,2010,32(4):659-661.
[21]陆英杰,连志诚.胃肠激素对胃肠动力的影响[J].免疫学杂志,2006,22(3):94-96.
[22]金敬善,王广才,张绳祖,等.血清中胃泌素水平与脾虚证的关系[J].中西医结合杂志,1982,2(1):25-26、62.
[23]刘玉强,许晨曦,贾蕊,等.苍术中三种单体化合物对脾虚模型大鼠疗效研究[J].中华中医药学刊,2017,35(2):382-386.