以生物碱为主要部位的7种腰痛宁衍生方的细胞抗炎、免疫活性比较及优选组方的体内药效评价
|
摘要
目的探究以马钱子、麻黄生物碱为主要部位的7种腰痛宁衍生方的细胞药理活性,并对细胞模型下有优效的组方进行体内药效学评价。方法以50%马钱子生物碱+50%麻黄生物碱为组方基础,分别与腰痛宁胶囊组方药材及8种常用风湿骨病组方中药的总黄酮、总皂苷、总挥发油/水提物及总多糖有效部位组合成7个样品,同时以腰痛宁加黄酒药引为对照。测定各样品抑制小鼠巨噬细胞中前列腺素E2(PGE2)增殖的IC50及促进小鼠巨噬细胞中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)及肿瘤坏死因子(TNF-α)增殖的EC50,并进行比较。同时采用最小二乘优化方法,计算各样品的EC50或IC50叠加值,根据EC50或IC50实验值和叠加值之间的差异分析各有效部位间的相互作用关系。采用小鼠耳肿胀模型和小鼠皮肤迟发型超敏反应(DTH)模型进行最优组合的体内药效学评价。结果 25%马钱子生物碱+25%麻黄生物碱与总黄酮组合(4号样品)具有最佳的综合细胞抗炎、免疫调节活性,且各模型下该组方中有效部位间有极强的协同作用;25%马钱子生物碱+25%麻黄生物碱分别与总皂苷、总挥发油/水提物及总多糖组合样品(5、6、7号样品)的抗炎活性与4号样品相当;5和7号样品有良好的综合免疫调节活性;25%马钱子生物碱+25%麻黄生物碱以及马钱子生物碱或麻黄生物碱与总黄酮组合样品(1、2、3号样品)的药理活性均显著弱于4号样品;小鼠体内药效学实验表明将4号样品组合中25%马钱子生物碱+25%麻黄生物碱比例降为25%及5%时,具有较好的抗炎、免疫作用。结论马钱子生物碱不宜单独与麻黄生物碱配伍,2种生物碱不宜单独与总黄酮配伍,25%马钱子生物碱+25%麻黄生物碱与总黄酮、总皂苷、总挥发油/水提物及总多糖分别组合通常会产生协同或叠加作用,有助于增强25%马钱子生物碱+25%麻黄生物碱的细胞药理活性,25%马钱子生物碱+25%麻黄生物碱与总黄酮组合样品的细胞抗炎和免疫调节活性在小鼠体内亦得到体现,提示根据细胞实验结果筛选优化中药组方具有可行性。
Objective To investigate which kind of Chinese medicinal effective fractions will produce good comprehensive cellular pharmacological activities when they were combined with alkaloids from Strychnos nux-vomica and Ephedra sinica, and to evaluate in vivo pharmacological activities of the effective fraction combination screened by cellular experiments. Methods The equal proportion mixture of alkaloids from S. nux-vomica and E. sinica was set as a sample(sample 1, A); Two samples(samples 2 and 3) were designed by respectively adding the equal ratio mixture of five flavonoids(F) at 50% into alkaloids from S. nux-vomica or E. sinica; Four samples(samples 4—7) were designed by respectively adding F, equal proportion mixture of four saponins(S), equal ratio mixture of six volatile oils/aqueous(V), and equal proportion mixture of six polysaccharides(P) at 50% into A. Murine macrophage cells and chondrocytes were exposed to the new recipes, and then the half inhibitory concentration(IC50) of recipes for inhibiting(PGE2) in macrophages and the half effective concentration(EC50) for promotion of the secretion of IL-6, IL-1β, and TNF-α in macrophages were detected and compared. The interactions among the active fractions were evaluated by comparing the experimental EC50/IC50 values to their corresponding additive EC50/IC50 values calculated by the least square optimum method. The in vivo pharmacodynamics of the best combination was evaluated by the ear swelling model and delayed type hypersensitivity(DTH) model in mice. Results Sample 4 has good comprehensive activities of cellular anti-inflammation and immunoregulation. Moreover, strong synergistic effect among the effective fractions in this sample was observed; Cellular anti-inflammatory activities of samples 5, 6, and 7 were equivalent with sample 4; Samples 5 and 7 had good comprehensive cellular immunoregulation activity; But the alkaloids mixture(A) and the combinations of S. nux-vomica or E. sinica alkaloids with F(samples 2 and 3) were significantly weaker than sample 4 in cellular anti-inflammation, immunoregulation, and chondrocyte-proliferation. Sample 4 also exhibits a certain effect on in vivo anti-inflammation and immunity in mice when A was decreased at 25% or 5%. Conclusion It is not suitable to design a combination just by alkaloids from S. nux-vomica and alkaloids of E. sinica; Alkaloids from S. nux-vomica or alkaloids of E. sinica are also not appropriate to solely combine with the mixture of flavonoids(F). When A is combined with F, S, V, and P, respectively, synergistic or additive effects among the active fractions are usually observed. These active fractions help to strengthen comprehensive cellular pharmacological activities of A. Sample 4 not only has good cellular activities of anti-inflammation and immunoregulation but also has better in vivo effect on anti-inflammation and immunity, suggesting that it is feasible to screen the optimized Chinese medicine formula based on cellular pharmacological experiments.
Objective To investigate which kind of Chinese medicinal effective fractions will produce good comprehensive cellular pharmacological activities when they were combined with alkaloids from Strychnos nux-vomica and Ephedra sinica, and to evaluate in vivo pharmacological activities of the effective fraction combination screened by cellular experiments. Methods The equal proportion mixture of alkaloids from S. nux-vomica and E. sinica was set as a sample(sample 1, A); Two samples(samples 2 and 3) were designed by respectively adding the equal ratio mixture of five flavonoids(F) at 50% into alkaloids from S. nux-vomica or E. sinica; Four samples(samples 4—7) were designed by respectively adding F, equal proportion mixture of four saponins(S), equal ratio mixture of six volatile oils/aqueous(V), and equal proportion mixture of six polysaccharides(P) at 50% into A. Murine macrophage cells and chondrocytes were exposed to the new recipes, and then the half inhibitory concentration(IC50) of recipes for inhibiting(PGE2) in macrophages and the half effective concentration(EC50) for promotion of the secretion of IL-6, IL-1β, and TNF-α in macrophages were detected and compared. The interactions among the active fractions were evaluated by comparing the experimental EC50/IC50 values to their corresponding additive EC50/IC50 values calculated by the least square optimum method. The in vivo pharmacodynamics of the best combination was evaluated by the ear swelling model and delayed type hypersensitivity(DTH) model in mice. Results Sample 4 has good comprehensive activities of cellular anti-inflammation and immunoregulation. Moreover, strong synergistic effect among the effective fractions in this sample was observed; Cellular anti-inflammatory activities of samples 5, 6, and 7 were equivalent with sample 4; Samples 5 and 7 had good comprehensive cellular immunoregulation activity; But the alkaloids mixture(A) and the combinations of S. nux-vomica or E. sinica alkaloids with F(samples 2 and 3) were significantly weaker than sample 4 in cellular anti-inflammation, immunoregulation, and chondrocyte-proliferation. Sample 4 also exhibits a certain effect on in vivo anti-inflammation and immunity in mice when A was decreased at 25% or 5%. Conclusion It is not suitable to design a combination just by alkaloids from S. nux-vomica and alkaloids of E. sinica; Alkaloids from S. nux-vomica or alkaloids of E. sinica are also not appropriate to solely combine with the mixture of flavonoids(F). When A is combined with F, S, V, and P, respectively, synergistic or additive effects among the active fractions are usually observed. These active fractions help to strengthen comprehensive cellular pharmacological activities of A. Sample 4 not only has good cellular activities of anti-inflammation and immunoregulation but also has better in vivo effect on anti-inflammation and immunity, suggesting that it is feasible to screen the optimized Chinese medicine formula based on cellular pharmacological experiments.
引文
[1]倪力军,张强祖,朱立中,等.腰痛宁胶囊治疗腰腿痛、腰肌劳损和风湿性关节炎临床究的meta分析[J].中成药,2012,34(9):1653-1660.
[2]刘艳平,李引刚.腰痛宁胶囊治疗寒湿瘀阻型坐骨神经痛临床疗效观察[J].中草药,2015,46(19):2916-2918.
[3]徐阳平,杨功旭,李胜利,等.腰痛宁胶囊治疗腰肌纤维炎多中心临床试验研究[J].中草药,2015,46(18):2764-2767.
[4]林昌松,陈纪藩,刘晓玲,等.马钱子药理研究及临床应用概况[J].中药新药与临床药理,2006,17(2):158-160.
[5]魏世超,徐丽君,张秀桥.马钱子总生物碱对大鼠佐剂性关节炎的作用[J].中国药理学通报,2001,17(4):479-480.
[6]郑萍,戴贵东,李汉青.麻黄碱及伪麻黄碱药理作用研究进展[J].宁夏医学杂志,2002,24(2):126-127.
[7]蒋袁絮,闫琳,余建强,等.麻黄碱、伪麻黄碱及其水杨酸衍生物对小鼠中枢神经系统作用的比较[J].中草药,2004,35(11):1274-1277.
[8]药品生产质量管理规范[S].1998.
[9]张立国,赵丽丽,倪力军,等.以多糖为主要部位的6种腰痛宁衍生方对巨噬细胞中炎症因子表达及软骨细胞增殖的影响[J].中草药,2015,46(24):3710-3716.
[10]倪力军,徐晓玲,史万忠,等.腰痛宁胶囊活性部位组合对PGE2、IL-2、NO细胞因子的影响及其相互作用[J].中草药,2014,45(23):3424-3431.
[11]Ni L J,Xu X L,Zhang L G,et al.Quantitative evaluation of the in vitro effect and inter actions of active fractions in Yaotongning-based formulae on prostaglandin E2production[J].J Enthnopharmacol,2014,154(3):807-817.
[12]Feng H B,Du X G,Liu J,et al.Novel polysaccharide from Radix Cyathulae officinalis Kuan can improve immune response to ovalbumin in mice[J].Int J Biol Macromol,2014,65:121-128.
[13]刘俊峰,郭雪峰.甘草皂苷对卡拉库尔羊血液中免疫球蛋Ig A和Ig G的影响[J].江苏农业科学,2014,42(1):172-174.
[14]Xie F,Sakwiwatkul K,Zhang C R,et al.Atractylodis macrocephalae Koidz.polysaccharides enhance both serum Ig G response and gut mucosal immunity[J].Carbohydr Polym,2013,91(1):68-73.
[15]徐颖,牟孝硕,孙悦朋,等.淫羊藿多糖对免疫功能低下小鼠免疫功能的影响[J].沈阳药科大学学报,2000,17(6):434-437.
[16]Sun H X,Pan H J.Immunological adjuvant effect of Glycyrrhiza uralensis saponins on the immune responses to ovalbumin in mice[J].Vaccine,2006,24(11):1914-1920.
[17]Ni L J,Wang N N,Guo Y Z,et al.Evaluation of the effects of active fractions of Chinese medicine formulas on IL-1β,IL-6,and TNF-αrelease from ANA-1 murine macrophages[J].J Enthnopharmacol,2016,179:420-431.
[18]吴婷婷.风湿痹病候选复方祛风四味方的质量分析及其体内药效学评价[D].上海:华东理工大学,2015.
[2]刘艳平,李引刚.腰痛宁胶囊治疗寒湿瘀阻型坐骨神经痛临床疗效观察[J].中草药,2015,46(19):2916-2918.
[3]徐阳平,杨功旭,李胜利,等.腰痛宁胶囊治疗腰肌纤维炎多中心临床试验研究[J].中草药,2015,46(18):2764-2767.
[4]林昌松,陈纪藩,刘晓玲,等.马钱子药理研究及临床应用概况[J].中药新药与临床药理,2006,17(2):158-160.
[5]魏世超,徐丽君,张秀桥.马钱子总生物碱对大鼠佐剂性关节炎的作用[J].中国药理学通报,2001,17(4):479-480.
[6]郑萍,戴贵东,李汉青.麻黄碱及伪麻黄碱药理作用研究进展[J].宁夏医学杂志,2002,24(2):126-127.
[7]蒋袁絮,闫琳,余建强,等.麻黄碱、伪麻黄碱及其水杨酸衍生物对小鼠中枢神经系统作用的比较[J].中草药,2004,35(11):1274-1277.
[8]药品生产质量管理规范[S].1998.
[9]张立国,赵丽丽,倪力军,等.以多糖为主要部位的6种腰痛宁衍生方对巨噬细胞中炎症因子表达及软骨细胞增殖的影响[J].中草药,2015,46(24):3710-3716.
[10]倪力军,徐晓玲,史万忠,等.腰痛宁胶囊活性部位组合对PGE2、IL-2、NO细胞因子的影响及其相互作用[J].中草药,2014,45(23):3424-3431.
[11]Ni L J,Xu X L,Zhang L G,et al.Quantitative evaluation of the in vitro effect and inter actions of active fractions in Yaotongning-based formulae on prostaglandin E2production[J].J Enthnopharmacol,2014,154(3):807-817.
[12]Feng H B,Du X G,Liu J,et al.Novel polysaccharide from Radix Cyathulae officinalis Kuan can improve immune response to ovalbumin in mice[J].Int J Biol Macromol,2014,65:121-128.
[13]刘俊峰,郭雪峰.甘草皂苷对卡拉库尔羊血液中免疫球蛋Ig A和Ig G的影响[J].江苏农业科学,2014,42(1):172-174.
[14]Xie F,Sakwiwatkul K,Zhang C R,et al.Atractylodis macrocephalae Koidz.polysaccharides enhance both serum Ig G response and gut mucosal immunity[J].Carbohydr Polym,2013,91(1):68-73.
[15]徐颖,牟孝硕,孙悦朋,等.淫羊藿多糖对免疫功能低下小鼠免疫功能的影响[J].沈阳药科大学学报,2000,17(6):434-437.
[16]Sun H X,Pan H J.Immunological adjuvant effect of Glycyrrhiza uralensis saponins on the immune responses to ovalbumin in mice[J].Vaccine,2006,24(11):1914-1920.
[17]Ni L J,Wang N N,Guo Y Z,et al.Evaluation of the effects of active fractions of Chinese medicine formulas on IL-1β,IL-6,and TNF-αrelease from ANA-1 murine macrophages[J].J Enthnopharmacol,2016,179:420-431.
[18]吴婷婷.风湿痹病候选复方祛风四味方的质量分析及其体内药效学评价[D].上海:华东理工大学,2015.