基于多指标抗炎活性检测的注射用血塞通生物评价方法研究
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摘要
[目的]建立基于抗炎活性检测的血塞通批次间一致性生物评价方法。[方法]采用脂多糖(lipopolysaccharide,LPS)刺激骨髓来源巨噬细胞向M1型极化,以M1型巨噬细胞为细胞模型,以一氧化氮(nitric oxide,NO)、白细胞介素-6(interleukin 6,IL-6)以及高迁移率族蛋白B1(high-mobility group box 1,HMGB1)分泌量作为抗炎活性指标,考察血塞通抗炎活性的量效关系,找出其中主要的抗炎活性成分,并检测24个正常批次和4个异常批次血塞通样品的抗炎活性。[结果]血塞通能够剂量依赖性降低M1型巨噬细胞NO、IL-6以及HMGB1分泌量,人参皂苷Rb1、人参皂苷Rd能显著抑制NO、IL-6以及HMGB1分泌,是其主要抗炎活性物质。24个正常批次血塞通样品对NO、IL-6、HMGB1分泌的平均抑制率分别为(75.47±7.68)%、(78.34±8.07)%、(45.84±8.45)%,而异常批次抑制率明显低于正常批次。综合多个指标初步建立了血塞通批次间一致性的生物活性评价标准,为批次间一致性评价提供了新方法。[结论]基于抗炎活性检测的血塞通生物评价方法,能够快速检测其抗炎有效性,可用于批次间生物活性的一致性评价。
[Objective] To establish a biological evaluation method for batch-to-batch consistency assessment of Xuesaitong injection(XST) based on antiinflammatory activity.[Method] The M1 macrophages, which was polarized from bone marrow-derived macrophages with the stimulation of lipopolysaccharide(LPS),was adopted as a cell model.The secretions of nitric oxide(NO), interleukin 6(IL-6) and high-mobility group box 1(HMGB1) in the cell model were considered as indicators of anti-inflammatory activity. The anti-inflammatory effectiveness of different doses of XST, the main active components of XST,24 normal batches and 4 abnormal batches XST were evaluated by those indicators.[Results] XST could reduce the secretions of NO, IL-6, HMGB1 on M1 macrophages in a dose-dependent manner. Ginsenoside Rb1 and ginsenoside Rd were the main active components of XST against inflammation.The average inhibition rates of normal batches XST against NO, IL-6 and HMGB1 were 75.47 ±7.68%, 78.34±8.07% and 45.84 ±8.45% respectively, which were significantly higher than abnormal batches XST.Therefore, a preliminary bio-activity evaluation criteria was established based on multi-indicators anti-inflammatory activity, which provided a new method for batch-to-batch consistency evaluation of XST. [Conclusion] The biological evaluation method based on anti-inflammatory activity could reflect the anti-inflammatory effectiveness of XST and be suitable for the batch-to-batch consistency assessment of XST.
[Objective] To establish a biological evaluation method for batch-to-batch consistency assessment of Xuesaitong injection(XST) based on antiinflammatory activity.[Method] The M1 macrophages, which was polarized from bone marrow-derived macrophages with the stimulation of lipopolysaccharide(LPS),was adopted as a cell model.The secretions of nitric oxide(NO), interleukin 6(IL-6) and high-mobility group box 1(HMGB1) in the cell model were considered as indicators of anti-inflammatory activity. The anti-inflammatory effectiveness of different doses of XST, the main active components of XST,24 normal batches and 4 abnormal batches XST were evaluated by those indicators.[Results] XST could reduce the secretions of NO, IL-6, HMGB1 on M1 macrophages in a dose-dependent manner. Ginsenoside Rb1 and ginsenoside Rd were the main active components of XST against inflammation.The average inhibition rates of normal batches XST against NO, IL-6 and HMGB1 were 75.47 ±7.68%, 78.34±8.07% and 45.84 ±8.45% respectively, which were significantly higher than abnormal batches XST.Therefore, a preliminary bio-activity evaluation criteria was established based on multi-indicators anti-inflammatory activity, which provided a new method for batch-to-batch consistency evaluation of XST. [Conclusion] The biological evaluation method based on anti-inflammatory activity could reflect the anti-inflammatory effectiveness of XST and be suitable for the batch-to-batch consistency assessment of XST.
引文
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[9]鄢丹,任永申,骆骄阳,等.中药质量生物测定的思考与实践---以板蓝根为例[J].中国中药杂志,2010,35(19):2637-2640.YAN Dan,REN Yongshen,LUO Jiaoyang,et al.Application of bioassay in quality control of Chinese materia medicataking Radix Isatidis as an example[J].China Journal of Chinese Materia Medica,2010,35(19):2637-2640.
[10]韩冰,毛鑫,韩淑娴,等.基于抑制血小板聚集活性检测的血栓通胶囊质量控制研究[J].中国中药杂志,2015,40(23):4597-4602.HAN Bing,MAO Xin,HAN Shuxian,et al.Anti-platelet aggregation bioassay based quality control for XST capsules[J].China Journal of Chinese Materia Medica,2015,40(23):4597-4602.
[11]谭鹏,张海珠,张定堃,等.基于化学表征和生物效价检测的大黄配方颗粒质量评价研究[J].中国中药杂志,2017,42(14):2683-2690.TAN Peng,ZHANG Haizhu,ZHANG Dingfang,et al.Quality evaluation of rhubarb dispensing granules based on multi-component simultaneous quantitative analysis and bioassay[J].China Journal of Chinese Materia Medica,2017,42(14):2683-2690.
[12]Li J P,Liu Y,Guo J M,et al.A Comprehensive strategy to evaluate compatible stability of Chinese medicine injection and infusion solutions based on chemical analysis and bioactivity assay[J].Front Pharmacol,2017,8:833.
[13]Lee H H,Shin J S,Lee W S,et al.Biflorin isolated from the flower buds of Syzygium aromaticum L.,suppresses LPS-induced inflammatory mediators via STAT1inactivation in macrophages and protects mice from endotoxin shock[J].J Nat Prod,2016,79(4):711-720.
[14]Forstermann U,Kleinert H.Nitric oxide synthase:expression and expressional control of the three isoforms[J].Naunyn Schmiedebergs Arch Pharmacol,1995,352(4):351-364.
[15]Gabay C.Interleukin-6 and chronic inflammation[J].Arthritis Res Ther,2006,8(Suppl 2):S3.
[16]Ramadori P,Ahmad G,Ramadori G.Cellular and molecular mechanisms regulating the hepatic erythropoietin expression during acute-phase response:A role for IL-6[J].Lab Invest,2010,90(9):1306-1324.
[17]Wang H,Bloom O,Zhang M,et al.HMG-1 as a late mediator of endotoxin lethality in mice[J].Science,1999,285(5425):248-251.
[18]Park S Y,Lee S W,Kim H Y,et al.HMGB1 induces angiogenesis in rheumatoid arthritis via HIF-1alpha activation[J].Eur J Immunol,2015,45(4):1216-1227.
[19]Hirata Y,Kurobe H,Higashida M,et al.HMGB1 plays a critical role in vascular inflammation and lesion formation via toll-like receptor 9[J].Atherosclerosis,2013,231(2):227-233.
[20]王立平,刘翠梅,张红.复元醒脑汤联合西药治疗腔隙性脑梗死疗效及对IL-6、TNF-α和NO的影响[J].现代中西医结合杂志,2015,24(29):3207-3209.WANG Liping,LIU Cuimei,ZHANG Hong.Effect of Fuyuanxingnao Decoction combined with western medicine on lacunar infarction and their impact on the levels of IL-6,TNF-αand NO[J].Modern Journal of Integrated Traditional Chinese and Western Medicine,2015,24(29):3207-3209.
[21]曹广平,周永勤,马磊,等.血清HMGB1、NSE在脑梗死及多梗死性痴呆患者中的临床意义[J].中华全科医学,2010,8(10):1226-1227.CAO Guangping,ZHOU Yongqin,MA Lei.Assay of high mobility group box-1 in serum of patients with cerebral infarction and multi-infarct dementia[J].Chinese Journal of General Practice,2010,8(10):1226-1227.
[22]张朝华,贺文奇.活血养心汤对心梗PCI后患者心功能、心肌酶以及血清SOD、ET、NO等指标的影响[J].陕西中医,2018,39(8):1025-1028.ZHANG Chaohua,HE Wenqi.The effect of Huoxue Yangxin decoction on cardiac function,myocardial enzymes and serum SOD,ET,NO and so on after myocardial infarction PCL[J].SHAANXI ZHONGYI,2018,39(8):1025-1028.
[23]孟繁宇,赵学忠.不同剂量阿托伐他汀对急性心梗患者血脂、C反应蛋白及IL-6的影响[J].中国实验诊断学,2010,14(1):54-56.MENG Fanyu,ZHAO Xuezhong.Effects of different doses of atorvastatin on serum lipid,inflammatory factor HsCRP and IL-6 AMI patients[J].Chin J Lab Diagn,2010,14(1):54-56.
[24]杨萍,陆林,朱天奇,等.急性ST段抬高型心梗患者血清HMGB1检测及其临床意义[J].国际心血管病杂志,2012,39(4):239-241.YANG Ping,LU Lin,ZHU Tianqi.Serum HMGB1 levels are associated with the severity of ST-segment elevation myocardial infarction[J].International Journal of Cardiovascular Disease,2012,39(4):239-241.
[25]Huang H,Qu H.In-line monitoring of alcohol precipitation by near-infrared spectroscopy in conjunction with multivariate batch modeling[J].Anal Chim Acta,2011,707(1-2):47-56.
[2]汶医宁,刘利.血塞通注射液治疗急性心肌梗塞80例[J].陕西中医,2007,28(10):1289-1290.WEN Yining,LIU Li.Xuesaitong injection in the treatment of 80 cases on acute myocardial infarction[J].Shaanxi TCM,2007,28(10):1289-1290.
[3]王洪涛,韩建娜.血塞通治疗颈动脉粥样硬化症30例[J].中西医结合心脑血管病杂志,2005,3(10):932-933.WANG Hongtao,HAN Jianna.Xuesaitong injection in the treament of 30 cases on carotid atherosclerosis[J].Chinese Journal of Integrative Medicine on Cardio/Cerebrovascular Disease,2005,3(10):932-933.
[4]Yao H,Shi P,Shao Q,et al.Chemical fingerprinting and quantitative analysis of a Panax notoginseng preparation using HPLC-UV and HPLC-MS[J].Chin Med,2011(6):9.
[5]靳慧娟.高效液相法测定血塞通注射液中皂苷含量[J].黑龙江科技信息,2017(6):170.JIN Huijuan.Determining the saponins content of Xuesaitong injection by HPLC[J].Heilongjiang Science and Technology Information,2017(6):170.
[6]国家药典委员会.中华人民共和国药典-四部[M].北京:中国医药科技出版社,2015:381.Chinese Pharmacopoeia Committee.Pharmacopoeia of People's Republic of China:Vol.4[M].Beijing:Chemical Industry Press,2015:381.
[7]Wang L,Yu Y,Yang J,et al.Dissecting Xuesaitong's mechanisms on preventing stroke based on the microarray and connectivity map[J].Mol Biosyst,2015,11(11):3033-3039.
[8]Wang L,Li Z,Zhao X,et al.A network study of chinese medicine xuesaitong injection to elucidate a complex mode of action with multicompound,multitarget,and multipathway[J].Evid Based Complement Alternat Med,2013,2013:652373.
[9]鄢丹,任永申,骆骄阳,等.中药质量生物测定的思考与实践---以板蓝根为例[J].中国中药杂志,2010,35(19):2637-2640.YAN Dan,REN Yongshen,LUO Jiaoyang,et al.Application of bioassay in quality control of Chinese materia medicataking Radix Isatidis as an example[J].China Journal of Chinese Materia Medica,2010,35(19):2637-2640.
[10]韩冰,毛鑫,韩淑娴,等.基于抑制血小板聚集活性检测的血栓通胶囊质量控制研究[J].中国中药杂志,2015,40(23):4597-4602.HAN Bing,MAO Xin,HAN Shuxian,et al.Anti-platelet aggregation bioassay based quality control for XST capsules[J].China Journal of Chinese Materia Medica,2015,40(23):4597-4602.
[11]谭鹏,张海珠,张定堃,等.基于化学表征和生物效价检测的大黄配方颗粒质量评价研究[J].中国中药杂志,2017,42(14):2683-2690.TAN Peng,ZHANG Haizhu,ZHANG Dingfang,et al.Quality evaluation of rhubarb dispensing granules based on multi-component simultaneous quantitative analysis and bioassay[J].China Journal of Chinese Materia Medica,2017,42(14):2683-2690.
[12]Li J P,Liu Y,Guo J M,et al.A Comprehensive strategy to evaluate compatible stability of Chinese medicine injection and infusion solutions based on chemical analysis and bioactivity assay[J].Front Pharmacol,2017,8:833.
[13]Lee H H,Shin J S,Lee W S,et al.Biflorin isolated from the flower buds of Syzygium aromaticum L.,suppresses LPS-induced inflammatory mediators via STAT1inactivation in macrophages and protects mice from endotoxin shock[J].J Nat Prod,2016,79(4):711-720.
[14]Forstermann U,Kleinert H.Nitric oxide synthase:expression and expressional control of the three isoforms[J].Naunyn Schmiedebergs Arch Pharmacol,1995,352(4):351-364.
[15]Gabay C.Interleukin-6 and chronic inflammation[J].Arthritis Res Ther,2006,8(Suppl 2):S3.
[16]Ramadori P,Ahmad G,Ramadori G.Cellular and molecular mechanisms regulating the hepatic erythropoietin expression during acute-phase response:A role for IL-6[J].Lab Invest,2010,90(9):1306-1324.
[17]Wang H,Bloom O,Zhang M,et al.HMG-1 as a late mediator of endotoxin lethality in mice[J].Science,1999,285(5425):248-251.
[18]Park S Y,Lee S W,Kim H Y,et al.HMGB1 induces angiogenesis in rheumatoid arthritis via HIF-1alpha activation[J].Eur J Immunol,2015,45(4):1216-1227.
[19]Hirata Y,Kurobe H,Higashida M,et al.HMGB1 plays a critical role in vascular inflammation and lesion formation via toll-like receptor 9[J].Atherosclerosis,2013,231(2):227-233.
[20]王立平,刘翠梅,张红.复元醒脑汤联合西药治疗腔隙性脑梗死疗效及对IL-6、TNF-α和NO的影响[J].现代中西医结合杂志,2015,24(29):3207-3209.WANG Liping,LIU Cuimei,ZHANG Hong.Effect of Fuyuanxingnao Decoction combined with western medicine on lacunar infarction and their impact on the levels of IL-6,TNF-αand NO[J].Modern Journal of Integrated Traditional Chinese and Western Medicine,2015,24(29):3207-3209.
[21]曹广平,周永勤,马磊,等.血清HMGB1、NSE在脑梗死及多梗死性痴呆患者中的临床意义[J].中华全科医学,2010,8(10):1226-1227.CAO Guangping,ZHOU Yongqin,MA Lei.Assay of high mobility group box-1 in serum of patients with cerebral infarction and multi-infarct dementia[J].Chinese Journal of General Practice,2010,8(10):1226-1227.
[22]张朝华,贺文奇.活血养心汤对心梗PCI后患者心功能、心肌酶以及血清SOD、ET、NO等指标的影响[J].陕西中医,2018,39(8):1025-1028.ZHANG Chaohua,HE Wenqi.The effect of Huoxue Yangxin decoction on cardiac function,myocardial enzymes and serum SOD,ET,NO and so on after myocardial infarction PCL[J].SHAANXI ZHONGYI,2018,39(8):1025-1028.
[23]孟繁宇,赵学忠.不同剂量阿托伐他汀对急性心梗患者血脂、C反应蛋白及IL-6的影响[J].中国实验诊断学,2010,14(1):54-56.MENG Fanyu,ZHAO Xuezhong.Effects of different doses of atorvastatin on serum lipid,inflammatory factor HsCRP and IL-6 AMI patients[J].Chin J Lab Diagn,2010,14(1):54-56.
[24]杨萍,陆林,朱天奇,等.急性ST段抬高型心梗患者血清HMGB1检测及其临床意义[J].国际心血管病杂志,2012,39(4):239-241.YANG Ping,LU Lin,ZHU Tianqi.Serum HMGB1 levels are associated with the severity of ST-segment elevation myocardial infarction[J].International Journal of Cardiovascular Disease,2012,39(4):239-241.
[25]Huang H,Qu H.In-line monitoring of alcohol precipitation by near-infrared spectroscopy in conjunction with multivariate batch modeling[J].Anal Chim Acta,2011,707(1-2):47-56.