苗药头花蓼提取物血清药理学研究方法的建立及验证
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摘要
目的建立并验证头花蓼提取物血清药理学研究方法。方法以大鼠为含药血清的供体,MTT法确定空白血清及含药血清对小鼠单核巨噬细胞(RAW264.7细胞)活性的影响,采用10 ng/m L脂多糖(LPS)建立RAW264.7细胞炎症损伤模型,Griess试剂法检测细胞上清液中一氧化氮(NO)的释放量,以确定体系中的血清添加量和大鼠取血时间。以NO、TNF-α为指标测定含药血清的抗炎活性。结果大鼠按人临床用药等效剂量的27倍给予头花蓼水提醇沉提取物,与模型组相比,体系中加入1.5%(V/V)末次给药后90 min的含药血清能够显著抑制LPS诱导的RAW264.7细胞释放NO(P<0.001)。同时,该方法制备的含药血清能够显著抑制细胞释放NO和TNF-α(P<0.01或P<0.001)。结论本实验条件下制备的头花蓼含药血清具有明显的抗炎作用,该血清药理学方法可用于头花蓼提取物含药血清药效及作用机制研究。
Objective To establish and validate a serum pharmacological method for Polygonum capitatum extract. Methods Rats were used as the provider to prepare the drug-containing serum. Cell viability was detected to determine the effect of blank and drug-containing serum on murine mononuclear macrophage cells(RAW264.7 cells) by MTT assay. The inflammatory cell model was produced by 10 ng/m L lipopolysaccharide(LPS) treated RAW264.7 cells.Nitric oxide(NO) production was detected by the Griess assay to determine the suitable concentration of serum in culture system and the time of sample collection. NO and TNF-α were selected to validate the anti-inflammatory activity of the drug-containing serum. Results Rats were given oral administration of protein-free water extract of Polygonum capitatum(PCWEP) by 27 times dose of adults. 1.5%(V/V) serum after 90 min of the last administration could significantly inhibit the release of NO in LPS-induced RAW264.7 cells compared with the model group(P<0.001). Drug-containing serum prepared by this method could significantly inhibit the productions of NO and TNF-α in RAW264.7cells(P<0.01, P<0.001). Conclusion Drug-containing serum prepared by this method possesses obvious effect of anti-inflammatory in macrophage cells, and this serum pharmacological method can be applied to evaluate the pharmacological effect and mechanism of PCWEPS.
Objective To establish and validate a serum pharmacological method for Polygonum capitatum extract. Methods Rats were used as the provider to prepare the drug-containing serum. Cell viability was detected to determine the effect of blank and drug-containing serum on murine mononuclear macrophage cells(RAW264.7 cells) by MTT assay. The inflammatory cell model was produced by 10 ng/m L lipopolysaccharide(LPS) treated RAW264.7 cells.Nitric oxide(NO) production was detected by the Griess assay to determine the suitable concentration of serum in culture system and the time of sample collection. NO and TNF-α were selected to validate the anti-inflammatory activity of the drug-containing serum. Results Rats were given oral administration of protein-free water extract of Polygonum capitatum(PCWEP) by 27 times dose of adults. 1.5%(V/V) serum after 90 min of the last administration could significantly inhibit the release of NO in LPS-induced RAW264.7 cells compared with the model group(P<0.001). Drug-containing serum prepared by this method could significantly inhibit the productions of NO and TNF-α in RAW264.7cells(P<0.01, P<0.001). Conclusion Drug-containing serum prepared by this method possesses obvious effect of anti-inflammatory in macrophage cells, and this serum pharmacological method can be applied to evaluate the pharmacological effect and mechanism of PCWEPS.
引文
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[11]张晓红,董莉,杨雅欣,等.紫金龙乙醇组分对脂多糖诱导的RAW264.7细胞分泌炎症因子的影响[J].中国实验方剂学杂志,2014,20(21):149-152.
[2]马风伟.热淋清颗粒的体内代谢动力学研究[D].贵阳:贵州师范大学,2014.
[3]Goran B,Bjorn W,Catharina S,et al.Escherichia coli,fimbriae,bacterial persistence and host response induction in the human urinary tract[J].Int J Med Microbiol,2005,295(6-7):487-502.
[4]Yin JJ,Luo YQ,Deng HL,et al.Hugan Qingzhi medication ameliorates hepatic steatosis by activating AMPK and PPARαpathways in L02 cells and Hep G2 cells[J].J Ethnopharmacol,2014,154(1):229-239.
[5]Liao SG,Zhang LJ,Sun F,et al.Antibacterial and anti-inflammatory effects of extracts and fractions from Polygonum capitatum[J].J Ethnopharmacol,2011,134(3):1006-1009.
[6]张丽娟.苗药头花蓼抗菌物质基础研究[D].贵阳:贵阳医学院,2012.
[7]Liao SG,Zhang LJ,Sun F,et al.Identification and haracterization of phenolics in Polygonum capitatum by ultrahigh-performance liquid chromatography with photodiode array detection and tandem mass spectrometry[J].Phytochem,Anal,2013,24(6):556-568.
[8]田友清,尚靖,何婷,等.基于中药血清化学及血清药理学方法探讨香青兰保护心肌细胞缺氧/复氧损伤物质基础[J].中国中药杂志,2012,37(5):620-624.
[9]Anju B,Sanjay C,Kusum H.Pseudomonas quinolone signaling system:A component of quorum sensing cascade is a crucial player in the acute urinary tract infection caused by Pseudomonas aeruginosa[J].Int J Med Microbiol,2014,304(8):1199-1208.
[10]代艳文,杨莉,万静枝,等.竹节参醇提物对LPS诱导RAW264.7细胞炎症的保护作用[J].中国实验方剂学杂志,2014,20(2):163-166.
[11]张晓红,董莉,杨雅欣,等.紫金龙乙醇组分对脂多糖诱导的RAW264.7细胞分泌炎症因子的影响[J].中国实验方剂学杂志,2014,20(21):149-152.