基于凝集活性检测的板蓝根颗粒质量生物测定方法研究

建立可用于板蓝根颗粒质量生物测定的新方法。采用血红细胞凝集活性检测法, 对20批板蓝根颗粒抗病毒活性进行生物测定, 并以流感病毒神经氨酸酶 (NA) 抑制试验进行药效学验证。结果显示, 所建立方法简便易行, 重复性良好 (RSD = 2.0%); 所有测试样品均为阳性反应 (效价值U介于2～11之间), 具有较好的适用性; 血红细胞凝集活性测定结果与流感病毒NA抑制活性测定结果之间相关性良好 (r² = 0.878 3)。结果表明, 血红细胞凝集活性测定法具有与药效相关、重复性好、经济安全、专属灵敏等特点, 可用于表征板蓝根颗粒质量生物活性信息, 以补充和完善板蓝根颗粒现行质量控制标准。     

X-射线单晶衍射法研究双炔失碳醇α-差向异构体的构型

本文报道有抗生育作用和雌激素活性的双炔失碳醇α-差向异构体的构型研究。采用重结晶法、低压硅胶柱色谱与HPLC相结合的方法,制得单晶纯品。经HPLC,IR,MS,¹HNMR证实。经Ⅹ-射线单晶衍射法测定,晶体属正交晶系,空问群为P222₁,晶胞参数为a=6.777(2),b=12.125(4),c=25.292(8),V=2078.5(1.2)。晶体结构采用直接法和Fourier技术得到,经最小二乘法修正,最后的偏离因子R=0.039。结构表明:其相对构型中,C₂,C¹⁷之乙炔基为α-型,结合合成原料构型推定C₂为R型,C₁₇为R型。     

间尼索地平对5-羟色胺诱导的大鼠肺动脉平滑肌细胞增殖中Ca2+/CaM/CaN通路的影响

探讨Ca²⁺/CaM/CaN信号通路在5-羟色胺 (5-HT) 诱导的大鼠肺动脉平滑肌细胞 (PASMCs) 增殖中的作用以及间尼索地平 (m-Nis) 对此的影响。采用细胞培养、噻唑蓝 (MTT) 比色检测、激光共聚焦显微镜及反转录聚合酶链反应 (RT-PCR) 等方法, 研究5-HT (1 μmol·L⁻¹) 对大鼠PASMCs细胞增殖的影响以及m-Nis对此的抑制作用, 并通过检测m-Nis对5-HT诱导的大鼠PASMCs增殖中Ca²⁺/CaM/CaN通路的影响深入探讨其作用机制。结果显示, 5-HT (1 μmol·L⁻¹) 可明显诱导大鼠PASMCs增殖 (P < 0.01), m-Nis对此有明显的抑制作用, 并呈现一定的浓度依赖性 (P < 0.05或P < 0.01); 另外, m-Nis还明显抑制了5-HT引起的[Ca²⁺]_i的升高 (P < 0.01)、CaM和CaN mRNA的表达以及CaN活性的升高 (P < 0.05或P < 0.01)。结果表明, Ca²⁺/CaM/CaN信号通路在5-HT诱导的大鼠PASMCs增殖中起重要作用, m-Nis抗5-HT诱导的增殖作用可能与抑制[Ca²⁺]_i增高从而阻断了Ca²⁺/CaM/CaN信号通路有关。     

柴胡皂甙和甘草甜素抑制Na+,K+-ATP酶活性的构效关系

研究在离体条件下各种单体柴胡皂甙和甘草甜素抑制Na⁺,K⁺-ATP酶活性的构效关系。实验结果表明,各种柴胡皂甙抑制Na⁺,K⁺-ATP酶活性的作用强度依次为:b₁>d>b₂>b₄>a>b₃>e>c。柴胡皂甙化学结构中的C₂₃-OH,C₁₆-OH及C₁₁和C₁₃的共轭双烯可能对其抑制活性起重要作用。甘草甜素(GL),甘草次酸(GA)和生胃酮(18-β-甘草次酸半琥珀酸双钠盐,CX)抑制Na⁺,K⁺-ATP酶活性的作用强度依次为GA≥CX>GL。研究还证明,柴胡皂甙d对Na⁺,K⁺-ATP酶的抑制为非竟争性抑制。     

结肠镜联合腹腔镜根治术对老年结直肠癌炎性因子、 T淋巴细胞亚群的影响

目的 分析行结肠镜联合腹腔镜对根治术老年结直肠癌病人炎性因子、T淋巴细胞亚群的影响。方法 选取2014年9月至2017年3月在南阳医学高等专科学校第一附属医院诊断且符合纳入标准的80例结直肠癌老年病人为研究对象,按照随机数字表法将其分为研究组和对照组,每组各40例,对照组病人采用腹腔镜结直肠癌根治术治疗,研究组病人采用结肠镜联合腹腔镜结直肠癌根治术治疗,比较两组病人术前、术后1 d、术后5 d的炎性因子及T淋巴细胞亚群水平。结果 研究组病人术后1 d、5 d的肿瘤坏死因子-α(TNF-α)水平[(104.27±20.42)ng/L、(91.26±13.01)ng/L]显著低于对照组[(125.32±27.08)ng/L、(104.30±20.39) ng/L](均P=0.001);两组病人的TNF-α、白细胞介素(IL)-6、IL-8及T淋巴细胞亚群CD⁺₃、CD⁺₄、CD⁺₈水平存在组间效应(F=5.894,P=0.017;F=14.683,P=0.000;F=216.181,P=0.000;F=64.658,P=0.000;F=177.909,P=0.000;F=55.526,P=0.000),且术后1d、5d研究组各炎性因子水平显著低于对照组(P<0.05),T淋巴细胞亚群水平显著高于对照组(P<0.05);两组病人的TNF-α、IL-6、IL-8及T淋巴细胞亚群CD⁺₃、CD⁺₄、CD⁺₈水平存在时间效应(F=65.946,P=0.000;F=113.858,P=0.000;F=265.156,P=0.000;F=324.475,P=0.000;F=326.190,P=0.000;F=348.601,P=0.000),且术后1 d、5 d两组各炎性因子水平均显著高于术前(P<0.05),T淋巴细胞亚群水平显著低于术前(P<0.05);两组病人各指标的组间因素与时间因素存在交互效应(F=5.253,P=0.006;F=5.262,P=0.006;F=34.329,P=0.000;F=15.709,P=0.000;F=32.101,P=0.000;F=11.298,P=0.000)。结论 结肠镜联合腹腔镜根治术对老年结直肠癌病人的炎症反应及免疫功能影响较小。     

非核苷类逆转录酶抑制剂的研究XVⅢ: 4-烯丙基和4-叠氮基取代的三嗪类化合物的合成及抗HIV活性

在已有工作基础上, 基于分子对接设计合成了8个新的4-烯丙基取代和4-叠氮基取代的二芳基三嗪类衍生物。抗HIV-1活性的测试结果表明所有新化合物均具有抗HIV-1活性。其中化合物7c不仅抑制HIV-1野生株的复制 (IC₅₀ = 0.034 μmol×L^-1, SI = 6 475), 且对Y181C和K103C双突变酶显示出较强的抑制活性, 其IC₅₀值为9.39 μmol×L^-1, 高于奈韦拉平。     

蒺藜皂苷激活PKCε抗氧化应激诱导心肌细胞凋亡的机制

观察蒺藜皂苷 (gross saponins of Tribulus terrestris, GSTT) 在过氧化氢 (hydrogen peroxide, H₂O₂) 诱导新生大鼠心肌细胞凋亡过程中对蛋白激酶C Eplison亚型 (protein kinase C Eplison, PKCε) 及凋亡相关蛋白的影响, 探讨蒺藜皂苷抗心肌细胞凋亡的作用机制。新生大鼠心肌细胞原代培养, H₂O₂建立心肌细胞凋亡模型, 流式细胞术检测细胞凋亡率; Western blotting法检测phospho-PKCε及Bcl-2、Bax的蛋白含量; 免疫细胞化学技术检测cleaved caspase-3蛋白含量。与模型组比较, GSTT (100 mg·L^-1) 预处理可明显降低H₂O₂诱导的心肌细胞凋亡率 (P < 0.01), 并增加phospho-PKCε及Bcl-2的蛋白含量 (P < 0.01), 降低Bax及cleaved caspase-3的蛋白含量 (P < 0.01); PKC抑制剂白屈菜红碱 (chelerythrine, Che) 可部分阻断蒺藜皂苷抗心肌细胞凋亡作用, 与蒺藜皂苷组比较具有统计学意义 (P < 0.05, P < 0.01)。GSTT抗心肌细胞凋亡的作用机制可能与激活PKCε后抑制线粒体依赖的凋亡有关。     

氯雷他定血药浓度的HPLC荧光检测法及生物等效性研究

目的建立高效液相色谱法荧光检测血浆中氯雷他定(loratadine)含量的方法,以评价氯雷他定的相对生物利用度。方法色谱柱为Alltech C₁₈,4.6 mm×150 mm;流动相为乙腈-水-冰醋酸-三乙胺(90∶100∶6∶0.15);流速为1 mL·min^-1;荧光检测器测定波长,E_x＝274 nm,E_m＝450 nm。结果HPLC测定线性范围为0.2～30 μg·L^-1,最低定量限0.2 μg·L^-1,方法回收率为96％～98％。人体生物利用度结果表明,实验片、胶囊与对照片间的AUC,t_max,C_max和t_1/2β均无显著性差异(P>0.05),两者的相对生物利用度分别为107%±17%和100%±14%。AUC和C_max经可信区间法检验生物等效。结论3种制剂生物等效。     

HPLC-ELSD同时测定跌打丸中三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1的含量

目的 建立同时测定跌打丸中三七皂苷R₁、人参皂苷Rg₁和人参皂苷Rb1含量的HPLC-ELSD检测方法。方法 采用Phenomenex Kinetex C₁₈色谱柱(100 mm×4.6 mm,2.6 μm),柱温30 ℃,流速0.5 mL·min^-1,流动相为乙腈-水,梯度洗脱,ELSD检测器,漂移管温度110 ℃,载气(空气)体积流量3.0 L·min^-1。结果 三七皂苷R₁、人参皂苷Rg₁、人参皂苷Rb1分别在0.158~3.16 μg(r=0.999 8),0.407~8.14 μg(r=0.999 5),0.446~8.92 μg(r=0.999 9)内呈良好的线性关系,平均加样回收率分别为97.55%(RSD=1.04%),98.09%(RSD=1.03%),97.34%(RSD=0.81%)。结论 该方法简便、快速、准确,可用于跌打丸的质量控制。     

反相高效液相色谱法测定重组人干扰素α1b多剂量注射液中间甲酚含量

目的: 建立测定重组人干扰素α1b多剂量注射液中间甲酚含量的高效液相色谱(HPLC)检测方法,并对方法进行验证。方法: 采用反相高效液相色谱(RP-HPLC)法,选用Venusil, ASB-C18(5μm,4.6mm×150mm)色谱柱,流动相为0.2mol.L_^-1硫酸盐缓冲液(pH=2.3)-乙腈(74﹕26),流速1mL.min_^-1,进样量20μL,检测波长270nm,柱温40℃,等度洗脱。对方法进行专属性、线性、准确度、精密度、重复性、检测限、定量限等一系列验证。结果: 该方法在间甲酚含量为100.0～300.0μg.mL_^-1范围内线性良好,r_²值为1.000。相关辅料及干扰素α1b对间甲酚检测无干扰。该方法检测高、中、低3种浓度9组样品回收率(n=9)在96.2%～101.5%;精密度 RSD(n=9)为0.78%;定量下限为4.3mg.mL_^-1,检测下限为1.4mg.mL_^-1。对 6批次重组人干扰素α1b多剂量注射液中间甲酚含量均在其标示量的90～110%之间,质量合格。结论: 建立的RP-HPLC方法专属性良好,精密度、准确性高,可用于检测重组人干扰素α1b多剂量注射液中的间甲酚含量。     

蒙药好得乐胶囊的检测方法研究

采用薄层色谱法对好得乐胶囊中黄柏、龙胆、连翘及板蓝根进行了定性鉴别，建立了好得乐胶囊的定性检测方法。     

板蓝根药材HPLC指纹图谱研究

目的 研究板蓝根饮片HPLC指纹图谱。方法 应用RP-HPLC法，梯度洗脱分析市售的11批板蓝根饮片的醋酸乙酯提取物，建立板蓝根饮片的指纹图谱。结果 不同来源的板蓝根饮片的醋酸乙酯提取物的色谱图相近，特征共有峰12个，相对峰面积相差很大。结论 建立的方法用于板蓝根药材的鉴别和质量评价简便、准确，为深入研究板蓝根的质量标准提供了实验基础。     

Approaches to advancing quantitative human health risk assessment of environmental chemicals in the post-genomic era

The contribution of genomics and associated technologies to human health risk assessment for environmental chemicals has focused largely on elucidating mechanisms of toxicity, as discussed in other articles in this issue. However, there is interest in moving beyond hazard characterization to making more direct impacts on quantitative risk assessment (QRA) — i.e., the determination of toxicity values for setting exposure standards and cleanup values. We propose that the evolution of QRA of environmental chemicals in the post-genomic era will involve three, somewhat overlapping phases in which different types of approaches begin to mature. The initial focus (in Phase I) has been and continues to be on “augmentation” of weight of evidence — using genomic and related technologies qualitatively to increase the confidence in and scientific basis of the results of QRA. Efforts aimed towards “integration” of these data with traditional animal-based approaches, in particular quantitative predictors, or surrogates, for the in vivo toxicity data to which they have been anchored are just beginning to be explored now (in Phase II). In parallel, there is a recognized need for “expansion” of the use of established biomarkers of susceptibility or risk of human diseases and disorders for QRA, particularly for addressing the issues of cumulative assessment and population risk. Ultimately (in Phase III), substantial further advances could be realized by the development of novel molecular and pathway-based biomarkers and statistical and in silico models that build on anticipated progress in understanding the pathways of human diseases and disorders. Such efforts would facilitate a gradual “reorientation” of QRA towards approaches that more directly link environmental exposures to human outcomes.     

辛兰芷喷雾剂质量标准研究

目的:建立辛兰芷喷雾剂的质量标准。方法:采用TLC法对方中辛夷、板蓝根、白芷进行定性分析;用HPLC法对连翘苷进行含量测定。结果:TLC法可准确地对辛夷、板蓝根、白芷进行定性鉴别;连翘苷进样量在0.2~10.0μg范围内与峰面积积分值呈良好线性关系(r=0.9997);平均回收率为99.71%,RSD=1.85%(n=6)。结论:本方法简便、快速、重现性好,所建标准可用于本制剂的质量控制。     

组分板蓝根药材的研究

目的建立通过不同产地和批次板蓝根药材中尿苷、腺苷、鸟苷、表告依春的组分调配达到组分最优化效果的方法。方法用高效液相色谱法测定各批次板蓝根药材含量,再用统计学方法计算特定预测含量下的各批样品的调配比例,按此比例调配混合样品,最后测定混合样品的含量。结果组分调配的样品含量与经计算预测含量符合程度中,尿苷、腺苷、鸟苷、表告依春含量的RSD分别为1．63％,1．17％,1．64％,1．95％。结论建立通过不同产地和批次板蓝根药材中尿苷、腺苷、鸟苷、表告依春的组分调配达到组分最优化效果的方法可行,对组分板蓝根的发展意义重大。     

The cardiovascular and subjective effects of methamphetamine combined with γ-vinyl-γ-aminobutyric acid (GVG) in non-treatment seeking methamphetamine-dependent volunteers

γ-Vinyl-γ-aminobutyric acid (GVG) elevates central nervous system γ-aminobutyric acid (GABA) levels by irreversibly inhibiting GABA transaminase. An open-label clinical trial in humans suggested that GVG may reduce cocaine and methamphetamine use. To test safety and to obtain preliminary data on efficacy of GVG for treating methamphetamine dependence, we conducted a double-blind, placebo-controlled, parallel group study of GVG interaction with the cardiovascular and subjective effects produced by methamphetamine. Non-treatment seeking methamphetamine-dependent volunteers received either GVG (N = 8) or placebo (N = 9) by random assignment. GVG treatment was initiated at 1 g/day and increased to 5 g/day. After reaching the target dose of 5 g/day, participants received methamphetamine (15 + 30 mg, IV), and cardiovascular and subjective effects were assessed. No serious adverse events were noted, and the total number of adverse events was similar between the treatment groups. Considering the full time course and peak effects independently, no significant differences were detected between the groups for systolic or diastolic blood pressures, or heart rate, following methamphetamine exposure. Some methamphetamine-induced cardiovascular changes approached significance (p < 0.10) and may warrant attention in future trials. Methamphetamine-induced subjective effects (“any drug effect”, “high”, “crave methamphetamine”) were statistically similar between GVG and placebo treatment groups. Pharmacokinetic data indicate that GVG treatment did not alter methamphetamine or amphetamine plasma levels, and there was no association between methamphetamine or amphetamine plasma levels and peak cardiovascular effects. Taken together, the data indicate that GVG treatment is generally well tolerated but not efficacious in attenuating the positive subjective effects of methamphetamine in the laboratory.     

Ultrasonic-assisted precolumn derivatization-HPLC determination of acrylamide formed in Radix Asparagi during heating process

An ultrasonic-assisted precolumn derivatization-HPLC method was established and validated for the determination of acrylamide formed in traditional Chinese herb Radix Asparagi during heating process. This method entails extraction with water, ultrasonic-assisted derivatization with 2-mercaptobenzoic acid. The final extracted acrylamide derivative was separated on C₁₈ column by using a mixture of acetonitrile and acetic acid (1 g L⁻¹ water solution) (20:80, v/v) as mobile phase. The flow rate was 0.7 mL min⁻¹ and the detection wavelength was set at 238 nm. Factors influencing the derivative reaction were evaluated and the optimum derivatization conditions were as follows: molar ratio of derivative reagent to acrylamide was 35:1, pH 8–12, ultrasonic-assisted reaction time was 100 min. The calibration curve of acrylamide showed good linearity in the range of 0.015–4.5 μg mL⁻¹ with correlation coefficient of 0.9999. The limit of detection was estimated to be 25 μg kg⁻¹ based on the signal-to-noise ratio of 3 recorded at 238 nm. Recovery of acrylamide from the sample was 106.6 ± 6.6%. Relative standard deviation of five duplicate determinations for the same sample solution was 1.59%.     

板蓝根微量元素与(R,S)-告依春含量的相关性分析

目的:以甘肃不同产地板蓝根为研究对象,分析板蓝根中(R,S)-告依春与微量元素含量的相关关系。方法:采用火焰原子吸收光谱法测定不同产地板蓝根样品中铁(Fe)、铜(Cu)、钙(Ca)、锰(Mn)、锌(Zn)、镁(Mg)、镉(Cd)、铬(Cr)、钴(Co)、钠(Na)、锂(Li)、镍(Ni)、钾(K)元素含量,HPLC法测定板蓝根中(R,S)-告依春的含量,并应用SPSS 13.0进行相关性分析。结果:板蓝根指标性成分(R,S)-告依春与钾、钴、锂含量之间呈显著正相关,与铜含量呈极显著正相关性,与镉含量呈显著负相关;与经度呈显著的正相关,与纬度呈极显著的负相关。结论:板蓝根富集的微量元素之间存在协同或拮抗作用;地理指标是影响板蓝根富集微量元素的因素之一;板蓝根中(R,S)-告依春的产生不仅与经纬度等地理指标有关系,而且与板蓝根富集的微量元素含量有关;(R,S)-告依春含量的高低不是某一单一因子的影响,而是地理指标、微量元素等众多因子的综合作用造成的。     

板蓝根挥发油成分的GC/MS分析

目的:建立以气相色谱-质谱联用技术(GC/MS)分析鉴定板蓝根挥发油成分的方法。方法:采用水蒸气蒸馏法从板蓝根中提取挥发油,色谱柱为OV-1柱,流速为1.0mL.min-1,质谱采用EI离子源;通过NIS107数据系统分析鉴定板蓝根挥发油成分。结果:共分离得到22个色谱峰,鉴定出19个化合物,占总量的90.51%,其中含量最高的是十六酸,约占总量的38.52%。结论:本试验为板蓝根的综合利用提供了初步的试验数据。