目的探讨绿原胶囊浸出液对成人外周血来源的树突状细胞(Dendritic cells,DCs)增殖及成熟的影响。方法采集健康男性(4名)外周血各100mL,采用密度梯度离心法获取外周血单个核细胞,贴壁法获取弱贴壁的DCs前体细胞,用添加重组人粒细胞-巨噬...目的探讨绿原胶囊浸出液对成人外周血来源的树突状细胞(Dendritic cells,DCs)增殖及成熟的影响。方法采集健康男性(4名)外周血各100mL,采用密度梯度离心法获取外周血单个核细胞,贴壁法获取弱贴壁的DCs前体细胞,用添加重组人粒细胞-巨噬细胞集落刺激因子(recombinant human GM-CSF,rhGM-CSF)和重组人白细胞介素4(recombinant human IL-4,rhIL-4)的RPML-1640的DCs培养基培养DCs前体细胞7天,可获得未成熟的DCs。以在DCs培养体系中加入1%浓度的绿原胶囊浸出液共培养未成熟DCs2天作为实验组,以20ng/mL重组人肿瘤坏死因子(Recombinant human TNF-α,rh TNF-α)共培养未成熟DCs2天作为对照组。倒置相差显微镜观察细胞形态,细胞计数法检测成熟DCs增殖能力,流式细胞仪检测成熟DCs表达的特异性标记物CD83。结果与对照组相比,实验组培养2天后,DCs的增殖能力提高,DCs表面呈现出比对照组更多和更长的树突状突起,实验组CD83表达[(84.1±0.3)%]明显高于对照组[(58.7±0.2)%],两组比较差异有统计学意义(P<0.05)。结论 1%浓度的绿原胶囊浸出液可促进外周血来源的DCs成熟和增殖,且作用较20ng/mL的rh TNF-α更强。展开更多
The determination method of chlorogenic acid in traditional Chinese prescription Shuanghuanglian capsule was established by using quantitative nuclear magnetic resonance spectroscopy(q NMR) in combination with solid p...The determination method of chlorogenic acid in traditional Chinese prescription Shuanghuanglian capsule was established by using quantitative nuclear magnetic resonance spectroscopy(q NMR) in combination with solid phase extraction(SPE). As the capsule’s main active component, chlorogenic acid comes from the extraction of Chinese herb medicine Flos Lonicerae. The chlorogenic acid in capsule was ultrasonically extracted at room temperature using pure water as solvent. The extracting solution was enriched and cleaned using HC-C18 SPE cartridge. The effect of ultrasonic extraction, sample pretreatment conditions via SPE and q NMR experimental conditions were investigated. The q NMR experiment conditions were selected using deuterated DMSO as solvent, calibrated 1,4-phthalaldehyde as internal standard, and P1(pulse width) = 14.4 μs, d1(pulse delay time) = 1 s, NS(number of scan) = 512. The 1 H NMR peaks of δ 6.138–6.182(H-8’, d, 1 H) of chlorogenic acid was chosen as the quantitative peaks. Method validation was performed, including precision(the intra-day RSD = 1.2% and the inter-day RSD = 1.5%), linearity(correlation coefficient r>0.9999), LOD(0.0017 mg/g) and LOQ(0.079 mg/g). The recovery of the SPE-q NMR was within the range of 100.2%–103.2%. The result showed that the method was stable, accurate and reliabile. Determined by the method, the chlorogenic acid in a real Shuanghuanglian capsule was within the range of 9.68–10.35 mg/g.展开更多
文摘目的探讨绿原胶囊浸出液对成人外周血来源的树突状细胞(Dendritic cells,DCs)增殖及成熟的影响。方法采集健康男性(4名)外周血各100mL,采用密度梯度离心法获取外周血单个核细胞,贴壁法获取弱贴壁的DCs前体细胞,用添加重组人粒细胞-巨噬细胞集落刺激因子(recombinant human GM-CSF,rhGM-CSF)和重组人白细胞介素4(recombinant human IL-4,rhIL-4)的RPML-1640的DCs培养基培养DCs前体细胞7天,可获得未成熟的DCs。以在DCs培养体系中加入1%浓度的绿原胶囊浸出液共培养未成熟DCs2天作为实验组,以20ng/mL重组人肿瘤坏死因子(Recombinant human TNF-α,rh TNF-α)共培养未成熟DCs2天作为对照组。倒置相差显微镜观察细胞形态,细胞计数法检测成熟DCs增殖能力,流式细胞仪检测成熟DCs表达的特异性标记物CD83。结果与对照组相比,实验组培养2天后,DCs的增殖能力提高,DCs表面呈现出比对照组更多和更长的树突状突起,实验组CD83表达[(84.1±0.3)%]明显高于对照组[(58.7±0.2)%],两组比较差异有统计学意义(P<0.05)。结论 1%浓度的绿原胶囊浸出液可促进外周血来源的DCs成熟和增殖,且作用较20ng/mL的rh TNF-α更强。
基金National Natural Science Foundation of China(Grant No.31671928)Natural Science Foundation of Shanghai(Grant No.15ZR1440800).
文摘The determination method of chlorogenic acid in traditional Chinese prescription Shuanghuanglian capsule was established by using quantitative nuclear magnetic resonance spectroscopy(q NMR) in combination with solid phase extraction(SPE). As the capsule’s main active component, chlorogenic acid comes from the extraction of Chinese herb medicine Flos Lonicerae. The chlorogenic acid in capsule was ultrasonically extracted at room temperature using pure water as solvent. The extracting solution was enriched and cleaned using HC-C18 SPE cartridge. The effect of ultrasonic extraction, sample pretreatment conditions via SPE and q NMR experimental conditions were investigated. The q NMR experiment conditions were selected using deuterated DMSO as solvent, calibrated 1,4-phthalaldehyde as internal standard, and P1(pulse width) = 14.4 μs, d1(pulse delay time) = 1 s, NS(number of scan) = 512. The 1 H NMR peaks of δ 6.138–6.182(H-8’, d, 1 H) of chlorogenic acid was chosen as the quantitative peaks. Method validation was performed, including precision(the intra-day RSD = 1.2% and the inter-day RSD = 1.5%), linearity(correlation coefficient r>0.9999), LOD(0.0017 mg/g) and LOQ(0.079 mg/g). The recovery of the SPE-q NMR was within the range of 100.2%–103.2%. The result showed that the method was stable, accurate and reliabile. Determined by the method, the chlorogenic acid in a real Shuanghuanglian capsule was within the range of 9.68–10.35 mg/g.
