川牛膝、麻牛膝及杂交牛膝多糖的分离、纯化和对猪小肠上皮细胞活性的比较研究
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摘要
研究旨在比较川牛膝及其常见伪品的多糖含量、组分和对猪小肠上皮细胞活性的差异。用乙醇除杂,水加热回流法得粗多糖;用阴离子交换层析法和凝胶柱层析法分离、纯化多糖,并测定多糖的相对分子量;用CCK-8试剂盒系统测定多糖对猪小肠上皮细胞活性的影响;用荧光定量PCR检测多糖作用后炎症细胞因子IL-6表达情况。试验结果:①川牛膝粗多糖得率为7.40%;麻牛膝粗多糖得率为1.67%;杂交牛膝粗多糖得率为0.38%;②川牛膝酸性多糖得率为94.40%,中性多糖得率为1.10%;麻牛膝酸性多糖得率为30.74%,中性多糖得率为23.00%;杂交牛膝酸性多糖得率为11.20%,中性多糖得率为10.00%;③川牛膝多糖相对分子量主要分布在7 500 Da左右、200 000～400 000 Da和1 200 000～1 700 000 Da;麻牛膝多糖的相对分子量主要集中在7 500 Da左右;杂交牛膝多糖相对分子量主要集中在6 000 Da左右;④川牛膝多糖和麻牛膝多糖的低剂量和高剂量组对猪小肠上皮细胞活性均无明显影响;川牛膝多糖和麻牛膝多糖均能显著抑制炎症细胞因子IL-6的表达。川牛膝多糖含量显著高于麻牛膝和杂交牛膝多糖,川牛膝和麻牛膝多糖对猪小肠上皮细胞活性无明显影响,但均能降低炎症细胞因子IL-6的表达量。
This study was conducted to compare content,component and porcine intestinal epithelial cells viability of polysaccharide of Cyathula officinalis and its' common spurious breed. Digestion with ethanol, use heating reflux method extract crude polysaccharide; use anion exchange chromatography and gel column chromatography to separate and purify the crude polysaccharide, determinate the molecular weight of the purified polysaccharide; use Cell Counting Kit-8 to measure the effect of polysaccharides on cell viability of IPEC-J2; use quantitative real-time PCR to measure the relative expression of inflammatory cytokine IL-6 after the treatment of polysaccharides. ① The crude polysaccharide yield of Cyathula officinalis is 7.40%; The crude polysaccharide yield of Cyathulae capitatae is 1.67%;The crude polysaccharide yield of C.officinalis yield of Cyathula officinalis is 94.40%, the neutral polysaccharide yield is 1.10%; The acidic polysaccharide yield of Cyathulae capitatae is 30.74%,the neutral polysaccharide yield is 23.00%; The acidic polysaccharide yield of C.officinalis×C.capitata is 11.20%, the neutral polysaccharide yield is 10.00%polysaccharide of Cyathula officinalis mainly distribute in 7 500, 200 000～400 000 Da and1 200 000～1 700 000 Da; The relative molecular weight of polysaccharide of Cyathulae capitatae mainly distribute in 7 500 Da; The relative molecular weight of polysaccharide of C.officinalis×C.capitata mainly distribute in 6 000 Da. ④ Both low dose and high dose of polysaccharides of Cyathula officinalis and Cyathulae capitatae have no obvious effect on cell viability of IPEC-J2; Polysaccharides of Cyathula officinalis and Cyathulae capitatae can significantly inhibit the expression of inflammatory cytokine IL-6. Polysaccharides content of Cyathula officinalis is much higher than Cyathulae capitatae and C.officinalis×C.capitata, polysaccharides of Cyathula officinalis and Cyathulae capitatae have no obvious effect on cell viability of IPEC-J2 and they both can significantly inhibit the expression of inflammatory cytokine IL-6.
This study was conducted to compare content,component and porcine intestinal epithelial cells viability of polysaccharide of Cyathula officinalis and its' common spurious breed. Digestion with ethanol, use heating reflux method extract crude polysaccharide; use anion exchange chromatography and gel column chromatography to separate and purify the crude polysaccharide, determinate the molecular weight of the purified polysaccharide; use Cell Counting Kit-8 to measure the effect of polysaccharides on cell viability of IPEC-J2; use quantitative real-time PCR to measure the relative expression of inflammatory cytokine IL-6 after the treatment of polysaccharides. ① The crude polysaccharide yield of Cyathula officinalis is 7.40%; The crude polysaccharide yield of Cyathulae capitatae is 1.67%;The crude polysaccharide yield of C.officinalis yield of Cyathula officinalis is 94.40%, the neutral polysaccharide yield is 1.10%; The acidic polysaccharide yield of Cyathulae capitatae is 30.74%,the neutral polysaccharide yield is 23.00%; The acidic polysaccharide yield of C.officinalis×C.capitata is 11.20%, the neutral polysaccharide yield is 10.00%polysaccharide of Cyathula officinalis mainly distribute in 7 500, 200 000～400 000 Da and1 200 000～1 700 000 Da; The relative molecular weight of polysaccharide of Cyathulae capitatae mainly distribute in 7 500 Da; The relative molecular weight of polysaccharide of C.officinalis×C.capitata mainly distribute in 6 000 Da. ④ Both low dose and high dose of polysaccharides of Cyathula officinalis and Cyathulae capitatae have no obvious effect on cell viability of IPEC-J2; Polysaccharides of Cyathula officinalis and Cyathulae capitatae can significantly inhibit the expression of inflammatory cytokine IL-6. Polysaccharides content of Cyathula officinalis is much higher than Cyathulae capitatae and C.officinalis×C.capitata, polysaccharides of Cyathula officinalis and Cyathulae capitatae have no obvious effect on cell viability of IPEC-J2 and they both can significantly inhibit the expression of inflammatory cytokine IL-6.
引文
[1]中国药典委员会.中华人民共和国药典[M].北京:中国医药科技出版社,2015:38-39.
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[10]刘浩民,孙恩江,南慧静.中药饲料添加剂应用现状和发展前景[J].广东畜牧兽医科技,2018,43(3):16-18.
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[12]曹日亮,车向荣,王红妍,等.中药添加剂对断奶仔猪小肠黏膜形态和养分消化率的影响[J].山西农业科学,2013,41(2):183-187.
[13]朱晓萍,尚秀国,杨永杰,等.中草药制剂大山楂丸对仔猪生长性能及消化率的影响[J].中国畜牧杂志,2017,53(7):95-97.
[14]戴亮.中草药饲料添加剂对断奶仔猪生产性能和腹泻率的影响[J].中兽医学杂志,2017(3):13-15.
[15]朱建国.中草药添加剂对哺乳母猪和断奶仔猪生产性能的影响[J].中国畜禽种业,2017,13(11):70-72.
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[17]赵新明.中草药添加剂对种用柴母鸡繁殖性能影响[J].中国畜禽种业,2017,13(5):141-142.
[18]严作廷,王东升,陈炅然,等.中草药饲料添加剂禽健宝对蛋鸡生产性能和防病效果观察[J].湖北农业科学,2011(20):4238-4239.
[19]孙燕杰,吴永继,刘增援,等.中药野菊花提取物与抗菌药联合对产ESBLs大肠杆菌抑制效果研究[J].中国畜牧兽医,2016,43(8):2170-2175.
[20]Huang C,Zhu Z,Cao X,et al.A Pectic Polysaccharide from Sijunzi Decoction Promotes the Antioxidant Defenses of SW480Cells[J].Molecules,2017,22(8):1341.
[21]吴向莉.川牛膝与麻牛膝的鉴别[J].贵阳中医学院学报,2000,22(1):62.
[22]赵磊.川牛膝及其常见混用品鉴别研究[J].亚太传统医药,2017,13(16):32-34.
[23]陈艾萌,潘洁,胡博,等.川牛膝多糖抗氧化活性测定和微波提取工艺优化[J].中成药,2017(1):80-84.
[24]张培全,刘盈萍,张超.川牛膝提取物清除自由基作用的研究[J].中药材,2013,36(3):458-461.
[25]封海波.川牛膝多糖对小鼠免疫反应的影响及机理研究[D].雅安:四川农业大学,2013.
[26]丁杰龙.川牛膝多糖的抗肿瘤作用初探[J].中国民族民间医药,2009,18(13):46-47.
[27]张亮,包海鹰.中药饲料添加剂的发展概况[J].经济动物学报,2017,21(3):177-180.
[28]张冰,关增春,于晓娜,等.中兽药饲料添加剂的功效研究[J].湖北畜牧兽医,2014,35(12):67-70.
[29]周明,汪炳红,周书苑,等.麻黄复方制剂在育肥猪中应用效果的试验研究[J].经济动物学报,2016,20(1):40-43.
[30]孙学亮,郭永军,季延滨,等.3种复方中草药对中华绒鳌蟹生长和部分抗氧化指标的影响[J].经济动物学报,2015,19(2):86-94.
[31]陈钢,王超儀,马斐斐,等.一种中药饲料添加剂的制备工艺和抑菌试验[J].经济动物学报,2012,16(2):71-75.
[32]Subramaniam M D,Kim I H.Clays as dietary supplements for swine:A review[J].Journal of Animal Science&Biotechnology,2015,6(1):38.
[2]张祎楠.川牛膝种质资源评价研究[D].成都:成都中医药大学,2013:1-129.
[3]丁杰龙.川牛膝多糖的抗肿瘤作用初探[J].中国民族民间医药,2009,18(13):46-47.
[4]宋军,杨金蓉,李祖伦,等.川牛膝多糖对小鼠肝癌细胞抑制作用研究[J].四川生理科学杂志,2002,24(3):118-119.
[5]曹颖莉,崔荣军,赵漩,等.螺旋藻多糖对老龄小鼠脑和肝中SOD,MDA的影响[J].中国初级卫生保健,2003,17(4):68-69.
[6]苗明.三五味子多糖对衰老模型小鼠的影响[J].中国医药学报,2002,17(3):187-188.
[7]王剑,蒲蔷,何开泽,等.川牛膝多糖的体外免疫活性研究[J].应用与环境生物学报,2008,14(4):481-483.
[8]王剑,付田,蒲蔷,等.川牛膝多糖的体内免疫活性研究[J].中药药理与临床,2007,23(6):31-33.
[9]李祖伦,石圣洪,陈红,等.川牛膝多糖促红细胞免疫功能研究[J].中药药理与临床,1999,15(4):26-27.
[10]刘浩民,孙恩江,南慧静.中药饲料添加剂应用现状和发展前景[J].广东畜牧兽医科技,2018,43(3):16-18.
[11]李玉娟.中草药饲料添加剂的特性及在养殖业中的应用[J].家畜生态学报,2015,36(1):76-79.
[12]曹日亮,车向荣,王红妍,等.中药添加剂对断奶仔猪小肠黏膜形态和养分消化率的影响[J].山西农业科学,2013,41(2):183-187.
[13]朱晓萍,尚秀国,杨永杰,等.中草药制剂大山楂丸对仔猪生长性能及消化率的影响[J].中国畜牧杂志,2017,53(7):95-97.
[14]戴亮.中草药饲料添加剂对断奶仔猪生产性能和腹泻率的影响[J].中兽医学杂志,2017(3):13-15.
[15]朱建国.中草药添加剂对哺乳母猪和断奶仔猪生产性能的影响[J].中国畜禽种业,2017,13(11):70-72.
[16]曾礼华.天然植物作为饲料添加剂的应用研究[J].西南农业学报,2004(2):257-261.
[17]赵新明.中草药添加剂对种用柴母鸡繁殖性能影响[J].中国畜禽种业,2017,13(5):141-142.
[18]严作廷,王东升,陈炅然,等.中草药饲料添加剂禽健宝对蛋鸡生产性能和防病效果观察[J].湖北农业科学,2011(20):4238-4239.
[19]孙燕杰,吴永继,刘增援,等.中药野菊花提取物与抗菌药联合对产ESBLs大肠杆菌抑制效果研究[J].中国畜牧兽医,2016,43(8):2170-2175.
[20]Huang C,Zhu Z,Cao X,et al.A Pectic Polysaccharide from Sijunzi Decoction Promotes the Antioxidant Defenses of SW480Cells[J].Molecules,2017,22(8):1341.
[21]吴向莉.川牛膝与麻牛膝的鉴别[J].贵阳中医学院学报,2000,22(1):62.
[22]赵磊.川牛膝及其常见混用品鉴别研究[J].亚太传统医药,2017,13(16):32-34.
[23]陈艾萌,潘洁,胡博,等.川牛膝多糖抗氧化活性测定和微波提取工艺优化[J].中成药,2017(1):80-84.
[24]张培全,刘盈萍,张超.川牛膝提取物清除自由基作用的研究[J].中药材,2013,36(3):458-461.
[25]封海波.川牛膝多糖对小鼠免疫反应的影响及机理研究[D].雅安:四川农业大学,2013.
[26]丁杰龙.川牛膝多糖的抗肿瘤作用初探[J].中国民族民间医药,2009,18(13):46-47.
[27]张亮,包海鹰.中药饲料添加剂的发展概况[J].经济动物学报,2017,21(3):177-180.
[28]张冰,关增春,于晓娜,等.中兽药饲料添加剂的功效研究[J].湖北畜牧兽医,2014,35(12):67-70.
[29]周明,汪炳红,周书苑,等.麻黄复方制剂在育肥猪中应用效果的试验研究[J].经济动物学报,2016,20(1):40-43.
[30]孙学亮,郭永军,季延滨,等.3种复方中草药对中华绒鳌蟹生长和部分抗氧化指标的影响[J].经济动物学报,2015,19(2):86-94.
[31]陈钢,王超儀,马斐斐,等.一种中药饲料添加剂的制备工艺和抑菌试验[J].经济动物学报,2012,16(2):71-75.
[32]Subramaniam M D,Kim I H.Clays as dietary supplements for swine:A review[J].Journal of Animal Science&Biotechnology,2015,6(1):38.