中药血清与血浆药理学方法商讨Rochester

,药理学实验分类整体（在体，in vivo）：复方或单味的疗效，副作用离体（体外，in vitro）：单体或单一组分的药理，毒理半离体（半体内，ex vivo）： 单体或单一组分; 有效成分未确定的中药药理，毒理 血清和血浆药理学方法属半离体实验 中药血清药理学方法系田代真一提出（现代东洋医学 1992） 中药血浆药理学方法系贺石林，葛金文等提出（中国药理学通报，2005；国外科学技术-中药现代化 2008） *罗焕敏的“血清药理学”与“血浆药理学”一文（中国药理学通报 2003）曽将未经抗凝全血直接离心20分钟后，所得的液体部分视为血浆， 这是一种误解。,田代真一提出中药血清药理学方法的背景 日本的汉方研究工作者都是中西医结合型专家，不可能缺乏生物医学基本知识。 可以设想：他们应该知道正常人与非DIC病人体内血液中真实存在的液体成分是血浆，而不是血清；从胃肠道吸收的中药成分是进入血浆，并非血清。 联系当时历史条件的局限性，许多人认为血浆等于血清加纤维蛋白原。他们提出中药血清药理学方法很可能是不了解血浆与血清的实际差异。 随着科学与技术发展，时至今日，血浆与血清多方面的差别已逐步揭开。,Crosstalk of coagulation 185:5628-5636),8，凝血和相关过程对核酸类的影响（1）RNA与刺激与损伤血细胞，引起血清中RNA Cheng HH, et al.,2013）,9， 凝血和相关过程对重要活性物质的影响（1）生长因子：来自血小板释放PDGF,VEGF, EGF, CTAP,TGF-等。（2）氧化与抗氧化体系：N释放O-2 二者生成ONOO- ； 而RBC & Plts释放GSH-Px, SOD, catalase, GSH-redutase(GR)。,（3） 细胞肽类（cytokines，细胞因子)： 在常测的15种细胞肽中，血清的IL-6含量明显低于血浆，CXCL-8含量明显高于血浆。 肝素抗凝血浆对细胞肽类检测的稳定性最好。（de Jager W, et al., 2009; Considerations for measuring cytokine levels in serum or plasma. Posted on May 28, 2013 by Andrea in PBMC Basics ）,10, 凝血和相关过程导致血浆与血清蛋白、 及 肽类差异二维凝胶电泳显示血清与血浆差异蛋白约53个，其中仅出现在血清中的有9个（Kim HJ, et al.，2007）；蛋白与肽组学研究发现血清中质谱信号约3000-7000，其中40%以上仅出现于血清中，并不见于血浆（Schulte I, et al.， 2005；Tammen H, et al.，2007）。（如将血清信号数量多视为优点是错误的） 39 例 Alzheimers 病(AD) 患者血清与血浆自身配对， 检测100 个蛋白，结果仅40个高度相关。（Huebinger RM., et al.,2012)HUPO和有关专家根据血浆与血清差异， 从反映体内实际情况考 虑 ，一致认为蛋白组学和肽组学研究应采用血浆。（Omenn GS, et al.，2005 & 2011；Thadikkamn I, et al.，2005） 在癌症蛋白 组学中，血清 标本易发生误导，以血浆 为宜。(Baggerly KA, et al., 2004),11, 凝血和相关过程导致血浆与血清中代谢产物的差异 有关专家根据血浆与血清差异， 从反映体内实际情况考 虑 ，认为代谢组学研究应采用血浆为宜。（Teahan O, et al.，2006） 有报道血浆代谢产物指纹谱的可重复性优于血清。（Yu Z, et al.，2011） 小细胞性肺癌患者血浆中的甘油磷酸胆硷(glycerophosphocholines)、赤藓醇（erythritol）、 棕榈酸（hexadecanic acid)和 谷氨酰胺（glutamine）含量与预后显著相关；但血清并无这种关系。（WedgeDC，et al., 2011） 对于胆固醇类指纹谱虽血浆与血清存在差别，二者相关性尚好；但在不同年龄段儿童研究中，采用 血浆更优。（Berentzen N.E., 2013） 国人报道血浆与血清代谢产物存在差异，但血清孵育对分析峰面积影响小于血浆， 故认为代谢组学研究以血清为好。（Liu L, et al., 2010）,小结 血浆是体内循环体内循环血流中的液体成分。 血清是 体外血液凝固后去除凝块（含纤维蛋白和血细胞）的液体成分。正常人与患者体内循环血流中的液体成分并非血清。 制备血清的凝血和相关过程,致使血清成分在多方面偏离血浆。有些成分减少甚至消失，同时又产生许多新的成分。 血清= 血浆- 纤维蛋白原与/或消耗因子 概念是错误的。 血浆较血清接近体内真实情况。,制备血清与预处理可能带耒的影响制备血清的影响 凝血酶和弹性蛋白可能对某些中药成分（尤蛋白-肽类）具有降解作用。 在除去纤维蛋白过程中可能会发生中药成分共沉淀现象。 在除去血细胞过程中可能会发生被吸附的中药成分丧失。 血清预处理的影响通常采用物理（加热）或化学（乙醇/丙酮）变性手段试图消除血清非药的活性。这些预处理可能导致以下影响中药成分损失-直接或间接（吸附于沉淀蛋白的成分）药物介导的活性成分损失血清成分(特别是蛋白类)*已有报道：以蛋白组学方法，观察560C1小时对血浆和血清中100个可溶性蛋白的影响。 结果：血浆热处理后，36个蛋白出现差异； 血清热处理后, 19个蛋白出现差异。 这些差异蛋白涉及趋化肽、生长因子、金属蛋白酶和粘附分子等类(Ayache S, et al., 2006),中药药学专家述评 没有一种实验方法是完美的,血清药理学从提出伊始就存在一些难以解决的问题。血清药理学要想真实反映药物在机体内的生物效应,就需要克服可能干扰实验结果和作出结论的多种不利因素。（韩笑, 等。2009） 新近报道从应用层面也指出血清药理学方法存在诸多具体问题，它并不是中药药理体外实验的适宜方法。（张红敏，等，2011）,(3) 含药血清与对照血清的 可比性可能存疑 对照是实验设计的首要原则 有效对照必须是实验组(处理组)与对照组之间的非处理因素 齐同； 只有这样， 才能排除非处理因素对实验结果分析/判断 的干扰。 设处理因素为T ，非处理因素为C, 则 施加因素 测试效应 (I) 实验组 : T + C ET + Ec (II)对照组 : C Ec - (I) (II) T = ET关键在于组间C相等,原因 凝血及相关过程产生的活性物质 非药效应 实验组 对照组Ec （违背齐同对比原则 丧失对照意义） 干扰实验结果 假阳性或假阴性结论,举例：血小板源生长因子（PDGF） 血小板能够表达血小板源生长因子（PDGF），其中包括VEGF等多种生长因子。 平时这些生长因子储存在夥粒中。血液凝固时，凝血酶刺激血小板释放生长因子。 据报道血清中VEGF含量约为血浆5-10 倍。（Verheul HM, et al., 1997;Webb NJ, etal.,1998; Maloney JP, et al.,1998; Wartiovaara U, et al.,1998; George ML, et al.,2000)（血小板裂解物促进伤口癒合；细胞培养时常加胎牛血清）具有促凝或抗凝的药物通过改变凝血酶生成量，均可影响血小板PDGF的释放量，使含药血清中VEGF含量不同于无药对照血清；从而改变细胞繁殖与生长的速度 ，因此干扰以细胞繁殖为指标的实验。,涉及中医“气”、“血”的中药可能影响凝血及其相关过程 Ex: 筛选抑制癌细胞生长复方 若复方中存在黄芪、赤芍、灵芝等具有抗凝作用。给药动物凝血酶生成减少, 使plts 释放PDGF 随之降低, 故含药血清中PDGF 低于对照血清, 可能导致假阳性结果。 如复方中存在促凝或促plts活化药物， 则含药血清中PDGF 增加，可能导致含药血清中PDGF 高于对照血清, 可能导致假阴性结果。,含药血浆药学实验的可行性1，药学实验采用血标本首选血浆的理由 血浆成分较血清接近体内真实情况 血浆制备较血清简单，干扰因素相对少 血浆获量较血清多15-20%2，西药（1）药代动力学研究 西药药代动力学(pharmacokinetics) 与 药效动力学(pharmacodynamics)研究通常首选血浆已成常规。若抗凝剂干扰实验，则采用血清。,（2）西药药理ex vivo研究方法 除in vivo，in vitro外，ex vivo实验如采用血标本时，通常采用血浆。 西药ex vivo药理 实验采用血浆举例 药理作用选择性 药效的影响因素 药物对靶细胞的影响 药物代谢产物及其相关酶,a，含药（西药）血浆进行药理作用选择性实验举例Ex vivo assay to determine the cyclooxygenase selectivity of non-steroidal anti-inflammatory drugs (NSAIDs)(Giuliano F, et al., Brit J Pharmacol 1999)Triad Treatment(factor): NSAIDs Rats Blood(pre- TXB2 as index of COX-1 activityProtocol(outline): preparations of plasmas & cells mixture incubation (30min) induction (15min) supernatant index determinations analysis*A549, a human epithelial carcinoma cell line expresses COX-2 when exposed to IL-1.Production of PGE2 by this cell line can be used as an index of COX-2 activity.*The production of thromboxane (TX) B2 by platelets was used as index of COX-1 activity.,Conclusions I. The selectivity of NSAIDs is divided into: (a) Main action on COX-1: sulindac sulphide, aspirin (b) Main action on COX-2: nimesulide, diclofenac (c) Equal action on COX-1 &-2: sod. Salicylate II. The research strategy: It could be extended by the analysis of plasma samples taken from humans similarly treated with other test drugs.,b，含药（西药）血浆研究药效的影响因素举例 Absolute Bioavailability of cis-Mirincamycin and trans-Mirincamycin in Healthy Rhesus Monkeys and Ex Vivo Antimalarial Activity against Plasmodium falciparum (Khemawoot P, et al., 2011)Triad Target factors: cis-mirincamycin vs trans-mirincamycin oral vs intravenous in vitro vs in vivo Object: Rhesus Monkeys Effect: antimalarial activity on W2 clone of Plasmodium falciparumDesign animal groups： cis oral, trans oral Cis i.V, trans i.v blood sampling: at 0, 15, 30, and 60 min, 2, 4, 6, 8, 12, and 24 h, and 2 and 7 days; heparin anticoagulation; preparation of plasma containing drugs preparation of W2 clone of Plasmodium falciparum（恶性疟原虫）,c， 含药（西药）血浆研究药物对靶细胞凋亡和染色体损伤影响的举例 Authors investigated whether high dose vitamin C influenced the viability of human lymphocytes in plasma, in the presence or absence of hydrogen peroxide (512 M) by scoring necrotic, apoptotic and micronucleated cells using the cytokinesis-block micronucleus assay. (Crott JW, et al., 1999),d, 含药（西药）血浆研究药物代谢产物及其相关酶的举例 EX VIVO METHODS TO IDENTIFY CIRCULATING DRUG METABOLITES WITH DRUG INTERACTION POTENTIAL (Patent Application number 20130330737) Plasma preparation Post-administration(clinical)plasma(P1): It is from subjects who have been orally administered an investigational drug in vivo. The clinical plasma comprises the administered drug at a first concentration and a plurality of metabolites of the drug. Control plasma (P2) It comprises the drug (added to blank plasma in vitro) at a second concentration. The control plasma is essentially free of metabolites of the drug, but contains the drug at a concentration that is substantially the same as the first concentration. Preparation of in vitro test system Drug-metabolizing enzymes or drug transporters / the cells expressing the drug-metabolizing enzymes/drug transporters from liver, lung, kidney.- to evaluate the changes in activity or expression of drug-metabolizing enzymes and/or drug transporters Add P1 & P2 to in vitro test system separately The changes in the activity or expression of drug-metabolizing enzymes and/or drug transporters in the first and second in vitro test systems is detected By comparing the clinical plasma sample to the drug-spiked control, the inhibitory and/or inducing effects on drug-metabolizing enzymes and/or drug transporters can be correctly attributed to the parent drug or its associated metabolites.,3, 中药含药血浆在 药学研究中应用 （1）,（2）中药药代动力学研究举例,总共664个蛋白斑点被分析。76个是含药血清特有，69个是含药血浆特有；其它许多斑点蛋白含量存在差异。,植物药利用血浆进行ex vivo 实验举例1, Effect of plasma metabolites of flavonoids on monocyte adhesion to human aortic endothelial cells (Koga T, et al., USA,2001) Research triad: Treatment pure flavonoid(1);blank plasma (2,rats); plasma containing drugs (3,rats) Object: HAEC, U937C Effects: cell adhesion, ROS Analysis: possible suggestions 2 vs 1-direct effects of parent components 3 vs 1-effects of metabolites from flavonoids 3 vs 2-total effects of original components & metabolites from flavonoids,Results: Pretreatment of HAEC with (+)-catechin metabolites inhibited U937 cell adhesion to IL-1stimulated cells, whereas pretreatment with intact (+)-catechin had no effect. Generation of ROS in hydrogen peroxidestimulated HAEC was inhibited by (+)-catechin, its metabolites, and control plasma extract,whereas ROS generation in IL-1stimulated HAEC was inhibitedby (+)-catechin metabolites only. Quercetin inhibited U937 cell adhesion to IL-1stimulated HAEC, whereas its metabolites were not effective.Conclusions: Metabolic conversion of flavonoids such as (+)-catechin and quercetin modifies the flavonoids biological activity. Metabolites of flavonoids, rather than their intact forms, may contribute to the reported effects of flavonoids on reducing the risk ofcardiovascular disease.,2, Inhibition of NF-B activation and MMP-9 secretion by plasma of human volunteers after ingestion of maritime pine bark extract (Pycnogenol) (Grimm T, et al., Germany, 2006)Research triad: Treatment-Pycnogenol (volunteers,oral) Object-Monocytes from blood donors Effects-NF-B activation, MMP-9 releaseResult analysis: before vs afterConclusion: Plasma of volunteers after Pycnogenol intake statistically significantly inhibited MMP-9 release from human monocytes and NF-B activation. The observed ex vivo effects with plasma containing drug are consistent with reported clinical anti-inflammatory effects in vivo.,3, The anti-inflammatory pharmacology of Pycnogenol in humans involves COX-2 and 5-LOX mRNA expression in leukocytes (Canalia R, et al., Switzerland, 2009) Research triad Treatment- Pycnogenol ( volunteers, oral) Object-Human PMNC from volunteers Effects-5-LOX, FLAP, Cox-1 & Cox-2 gene expression Analysis pre- vs post,Fig. 1.Effect of fMLP agonist stimulation on 5-LOX and FLAP gene expression in PMNL isolated before (A) and after (B) Pycnogenol supplementation. Before fMLP activation cells were primed with LPS for 30min at 37C. Data are expressed as fold of changes with respect to control. Control is gene expression before any treatments. P0.05 compared to PMNL isolated before Pycnogenol supplementation.,Fig. 2.Effect of fMLP agonist stimulation on LTB4 release in PMNL isolated before and after Pycnogenol supplementation. Before fMLP activation cells were primed with LPS for 30min at 37C. Data are expressed as % compared with control. Control is LTB4 released in the supernatant of PMNL before any treatments. Pre-PYC are PMNL isolated before Pycnogenol supplementation, post-PYC are PMNL isolated after Pycnogenol supplementation. P0.05 compared to pre-PYC PMNL.,Fig. 3.Effect of fMLP agonist stimulation on COX-1 gene expression in PMNL isolated before and after Pycnogenol supplementation. Before fMLP activation cells were primed with LPS for 30min at 37C. Data are expressed as fold of changes compared with control. Control is gene expression before any treatments. Pre-PYC are PMNL isolated before Pycnogenol supplementation, post-PYC are PMNL isolated after Pycnogenol supplementation.P0.05 compared to pre-PYC PMNL.,Fig. 4.Effect of fMLP agonist stimulation on COX-2 gene expression in PMNL isolated before and after Pycnogenol supplementation. Before fMLP activation cells were primed with LPS for 30min at 37C. Data are expressed as fold of changes compared with control. Control is gene expression before any treatments. Pre-PYC are PMNL isolated before Pycnogenol supplementation, post-PYC are PMNL isolated after Pycnogenol supplementation.P0.05 compared to pre-PYC PMNL.,Fig. 5.Effect of fMLP agonist stimulation on TXB2 release in PMNL isolated before and after Pycnogenol supplementation. Before fMLP activation cells were primed with LPS for 30min at 37C. Data are expressed as % compared with control. Control is TXB2 released in the supernatant of PMNL before any treatments. Pre-PYC are PMNL isolated before Pycnogenol supplementation, post-PYC are PMNL isolated after Pycnogenol supplementation. P0.05 compared to pre-PYC PMNL.,Fig. 6.Effect of fMLP agonist stimulation on PGE2 release in PMNL isolated before and after Pycnogenol supplementation. Before fMLP activation cells were primed with LPS for 30min at 37C. Data are expressed as % compared with control. Control is PGE2 released in the supernatant of PMNL before any treatments. Pre-PYC are PMNL isolated before Pycnogenol supplementation, post-PYC are PMNL isolated after Pycnogenol supplementation.P0.05 compared to pre-PYC PMNL.,Fig. 7.Effect of fMLP agonist stimulation on PLA2 enzyme activity in PMNL isolated before and after Pycnogenol supplementation. Before fMLP activation cells were primed with LPS for 30min at 37C. Data are expressed as % compared with control. Control is PLA2 activity before any treatments. Pre-PYC are PMNL isolated before Pycnogenol supplementation, post-PYC are PMNL isolated after Pycnogenol supplementation.P0.05 compared to pre-PYC PMNL.,ResultsPycnogenol supplementation inhibited 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) gene expression and phospholipase A2 (PLA2) activity. This effect was associated with a compensatory up-regulation of COX-1 gene expression. Pycnogenol suspended the interdependency between 5-LOX and 5-lipoxygenase activating protein (FLAP) expression. Pycnogenol supplementation reduced leukotriene production but did not leave prostaglandins unaltered, which we attribute to a decline of COX-2 activity in favour of COX-1.Conclusions Pycnogenol supplementation simultaneously inhibits COX-2 and 5-LOX gene expression and reduces leukotriene biosynthesis in human PMNL upon pro-inflammatory stimulation ex vivo.,4, Bioavailable constituents/metabolites of pomegranate (Punica granatum L) preferentially inhibit COX2 activity ex vivo and IL-1beta-induced PGE2 production in human chondrocytes in vitro (Shukla M, et al., USA, 2008)Research triad Treatment- pomegranate fruit (polyphenol rich) extract, rabbits (gavage), Object- chondrocytes (rabbits) Effects-cox-1, cox-2, NO & PGE2Analysis Comparisons between groups,ConclusionThese studies provide evidence to show that bioavailable constituents and/or metabolites of PFE exert an anti-inflammatory effect by inhibiting the activity of eicosanoid generating enzymes and the production of NO. This further suggests that consumption of PFE may be of value in inhibiting inflammatory stimuli-induced cartilage breakdown and production of inflammatory mediators in arthritis.,Biol. Pharm. Bull. 2005：28(6) 10311037 1031Expression Patterns of Plasma Proteins in Spontaneously Diabetic Rats after Oral Administration of a Kampo Medicine, Hachimi-jio-gan, Using SELDI ProteinChip PlatformChizuru KIGA, Takako NAKAGAWA, Keiichi KOIZUMI, Hiroaki SAKURAI, Yukari SHIBAGAKI,Kazuo OGAWA, Hirozo GOTO, Ikuo SAIKI*AbstractWe investigated the changes (increase or decrease in peak intensity) in the expression of plasma proteins in spontaneously diabetic WBN/Kob rats that were with complicated diabetic nephropathy, to determine multiple biomarkers in the plasma of diabetic rats. The present study using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) demonstrated that six peaks at mass/charge ratios (m/z) of 4678, 4732, 4808, 9058, 9323, and 9465, among approximately 80 peaks per spectrum in the 200010000 Damass range, had increased peak intensities with the development or progression of diabetic nephropathy in plasma of spontaneously diabetic WBN/Kob rats as compared with those of normal Wistar rats. Administration of the Kampo medicine Hachimi-jio-gan was effective at reducing the expression of diabetic nephropathy but not at reducing blood glucose levels. It also improved the increased levels of these plasma proteins. Other biomarker peaks at m/z 5067, 5279, 7598, and 7917 were not affected by Hachimi-jio-gan administration. Further study will be needed to identify these positive biomarkers and to evaluate the relationship between the efficacy and expression patterns of the plasma proteins in greater detail. The expression patterns of proteins and molecular-related ions revealed that several proteins in plasma may be involved in the development and/or progression of diabetic nephropathy in WBN/Kob rats and the efficacy of Hachimi-jio-gan. This study using ProteinChip technology may provide a useful basis in the search for multiple biomarkers in plasma for the diagnosis of disease and therapeutic evaluation of Kampo medicines.,Biol. Pharm. Bull. 28(10) 18691872 (2005)A Newly Devised Formulation for Self Medication Enhances Interferon- Production and Proliferation of Splenic LymphocytesEiji TEGA, Chizuru KIGA, Atsushi CHINO, Hiroaki SAKURAI, Keiichi KOIZUMI, Tadato TANI, Ikuo SAIKIAbstractA newly devised formulation for self-medication in Toyama, PanaWang, is a new herbal medicine (so called Toyama original brand formulation) developed based on traditional philosophy and scientific evidence. We here tried to examine the effect of oral administration of PanaWang on the balance of type I helper T cells (Th1) and Th2 cells. Splenic lymphocytes from normal mice were stimulated wi