巴西榥榥木的药理作用探究

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中文摘要

  目的:针对巴西榥榥木这种研究信息较少的天然药材,本研究拟通过网络药理学、代谢组学和药理学实验相结合的方式,建立一种筛选其药效的实验模式,且为榥榥木后期的研究开发提供实验依据。

  方法:

  1.通过建立巴西榥榥木的网络药理学来对其药理作用进行预测:基于雨林数据库(http://www.rain-tree.com/muirapuama.htm#.W67_VfZKI2w)及文献检索等方式,获得巴西榥榥木成分 59 种,获取其 CAS 号与名称,使用 sciencefinder 数据库检索 59 个化合物的结构 ,采用 ACD/Lab Percepta 软件将以上述各成分结构进行类药性分析,根据运算结果选择化合物。利用Swisstarget(http://www.swisstargetpredictio.ch/)数据库,依据分子的 SMILES 号进行查找相应靶点,将其下载为 csv 格式并整理相关数据。以 Probability>0.5 为筛选条件,将满足条件的靶点与其对应成分进行保留。 通过基因间相互作用的预测网站 GeneMANIA(http://genemania.org/),将靶点信息导入其中,以此构建出PPI 网络。对所建立的 PPI 网络,利用 cytoscape 软件中的 mcode 聚类分析模块,进行核心子网的推断和构建,利用子网中的靶点进行疾病的富集分析,根据富集分析结果预测相关的疾病。

  2.运用超高效液相色谱串联四极杆飞行时间质谱(UHPLC-Q-TOF-MS)技术,结合多变量数据分析,探究巴西榥榥木对正常大鼠血浆内源性代谢物的影响及其代谢机制。实验采用巴西榥榥木的高剂量 5g/kg 生药量、低剂量 2.5g/kg 生药量干预正常SD大鼠 。在相同的饲养条件和给药条件下收集血清样本,利用UHPLC-Q-TOF-MS 技术,将双蒸水对照组与巴西榥榥木给药组大鼠血清样品进行代谢轮廓分析。结合正交偏最小二乘判别分析法(OPLS-DA)与偏最小二乘法分析(PLS-DA)等多元统计分析方法,分析巴西榥榥木对正常大鼠代谢标志物的影响,并借助 MetPA,分析药物对大鼠内源性代谢标志物代谢途径的影响,初步阐述巴西榥榥木的靶标范围。

  3.对巴西榥榥木醇提物展开急性毒理实验:观察小鼠给巴西榥榥木 281.84g/kg后,1h、24h 与 7d 的体态变化反应、体重变化及死亡状况。在此剂量下,小鼠未表现出毒性作用,可以初步认定巴西榥榥木基本没有毒性。按此剂量范围进行给药。在小鼠适应性负重游泳中,实验分为高剂量组(14g/kg 生药量)、中剂量组(7g/kg 生药量)、低剂量组(3.5g/kg 生药量)三组,持续灌胃 5 天后,将小鼠放入恒温水中进行 3 天的给药负重游泳实验,其负重条件为小鼠体重的 8%,从小鼠落入水中立即开始计时,至小鼠沉于水中,且 10 秒内不能自主上浮,这段时间为小鼠力竭时间。在第 9 天时,对给药后无负重游泳的小鼠摘眼球取血,同时取肝脏和肌肉待测,按试剂盒说明书测定小鼠肝脏匀浆中肝糖原含量、骨骼肌匀浆中肌糖原含量、血清乳酸、血清尿素氮含量与乳酸脱氢酶活性等指标。

  结果:

  1.将巴西榥榥木成分对接相关靶点,根据靶点预测相关疾病,并分析疾病网络富集结果:基于检索等方式获得巴西榥榥木成分 59 种,根据化合物的结构 ,采用ACD/Lab Percepta 软件分析化合物的类药性分析,将结构导入进行运算,最后导出类药性分析结果,根据结果选择具有类药性的化合物。利用 Swisstarget数据库,依据分子的 SMILES 号进行查找相应靶点,其主要靶点为 Tyrosyl-DNAphosphodiesterase 1 、 Muscleblind-like protein 3 、 Muscleblind-like protein 1 、5-anhydro-D-fructose reductase 等 67 种相应靶点。通过建立的 PPI 网络,利用cytoscape 软件中的 mcode 聚类分析模块,进行核心子网的推断和构建,利用子网中的靶点进行疾病的富集分析,可以得到 Digestive System Diseases、Neoplasms、Neoplasms by Site、Skin and Connective Tissue Diseases、Pathological Conditions, Signs and Symptoms 等 41 种相关疾病。

  2.巴西榥榥木乙醇提取物对正常大鼠的给药后血清代谢组学代谢分析:血清代谢组学稳定性分析结果表明,高剂量给药组大鼠的内源性代谢物更加稳定。低剂量组差异更大。在代谢组学研究中,经过血清代谢物检索后,富集并分析差异代谢物的代谢通路,得到 30 个有意义的代谢通路:β-丙氨酸代谢、丙酸盐代谢、酮体的合成与降解、类固醇合成、花生四烯酸代谢和柠檬酸循环等。

  3.通过对巴西榥榥木醇提物展开急性毒理实验,结果证实,巴西榥榥木醇提物的灌胃给药最大耐受量为 281.84g 生药/kg;并进行小鼠的负重游泳实验,结果显示与给双蒸水的空白对照组相比,巴西榥榥木乙醇提物给药组的小鼠,负重游泳时间相对更长且具有显著差异性,其中中剂量组和低剂量组能显著减少力竭游泳后小鼠血清中乳酸含量、增加乳酸脱氢酶的活性,且中剂量组血清尿素氮含量显著减少。

  结论:

  1.通过网络药理学对其药理作用进行分析预测:巴西榥榥木主要成分所对应的靶点主要为雄/雌激素类受体、肌类蛋白、脂蛋白受体、活化肽片段等。根据靶点初步推测,巴西榥榥木可能在能量代谢、性功能、脂存贮等方面有一定的药理作用;根据靶点相关疾病的分析结果,巴西榥榥木可能对于消化系统疾病、肿瘤、肝硬化、呼吸道疾病等有一定的治疗作用。

  2.依据代谢标志物变量的显著性和变化强度,初步筛选代谢标志物变量 49 个进行定性分析,通过差异代谢物的代谢通路富集,分析得到 30 个有意义的代谢通路,其中主要可以分为能量代谢、激素类作用、脂类的合成与代谢、氨基酸类代谢四种类型的代谢途径。其中大多直接或者间接参与能量代谢。因此可以说明,巴西榥榥木能够影响大鼠的能量的代谢、氨基酸代谢、脂类物质的代谢、激素合成等。基于血清代谢组学分析,榥榥木参与大鼠体内多条代谢通路,为后期的药效学实验提供可参考的研究方向,为巴西榥榥木的未来开发利用提供科学依据。

  3.巴西榥榥木的毒性实验结果证明了巴西榥榥木的安全性,为后期给药方案提供了一个剂量范围;小鼠力竭性游泳后,其体内能源物质的耗竭和某些代谢产物的增加。例如:ATP 的储量下降,血糖的显著性消耗。这些代谢物的变化,可引起中枢性疲劳。抗疲劳作用实验结果证明,巴西榥榥木醇提物在一定剂量范围内,具有的抗疲劳作用,经典药理学的实验结果与网络药理学与代谢组学预测的结果一致。

  关键词:巴西榥榥木;网络药理学;代谢组学;毒性研究;抗疲劳作用

A preliminary study on pharmacological action of Muira Pauma- From Brazil

Abstract

  Objective:This study aims to establish the composition, network pharmacology of thetarget and disease, and to preliminarily predict the pharmacological effect of paumapauma root, a natural herbal medicine of which there is little information.At the sametime, analysis of the metabonomic and pharmacological experiments of rat blood wasconducted, and the basis of the metabonomic analysis experiment of rats wasestablished to explore the core effect and mechanism of action of this root muirapauma root from Brazil., according to the result of prediction of networkpharmacology and metabonomics, common analysis further determine Brazil muirapauma was possible pharmacological effects, and finally, through a pharmacologicalexperiment Brazilian muira pauma was fatigue experiment, and establish a screeningits effectiveness experiment mode, and validation to confirm its pharmacologicalaction of the feasibility of network pharmacology, and metabolomics ideas, late muirapauma was late for the further study of the research and development of medicinalvalue to provide experimental basis for theoretical basis.

  Methods:

  1.Establish a network pharmacology of pauma pauma root of Brazil to evaluate thepharmacological effect: 50 components of pauma root, based on the rainforestdatabase (http://www.rain-tree.com/muirapuama.htm#.W67_VfZKI2w) and literatureretrieval, obtain the CAS number and name, and use sciencefinder database to retrievethe structure of 50 compounds, and use ACD/Lab PerceptaSoftware operations will beto import the above composition structure, the export as a result, choose according tothe results of compound using Swisstarget database(http://www.swisstargetpredictio.ch/), on the basis of molecular SMILES to find theappropriate targets, to download it to CSV format and organize related data toProbability>0.5 as the filter condition, will meet the conditions of the target and itscorresponding ingredients to retain GeneMANIA through interaction between geneprediction website (http://genemania.org/) import some targets information, to build aPPI network established on PPI network, using cytoscape McOde cluster analysismodule of software core subnet inference and building, using the target to analyze theenrichment of disease in the subnet, according to the result of enrichment of targetsand predict related disease.

  2. The application of super-high performance liquid chromatography coupled withtandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) combinedwith multivariate data analysis to explore the effect of endogenous metabolites inplasma of normal rats from Brazil, and the metabolic mechanism of muira Pauma rootwas studiedLow-dose intervention in normal SD rats, serum samples, using UHPLC -Q - TOF - MS technique to the control group and Brazil muira pauma was drugmetabolism of rat serum samples contour analysis, combined with partial least squaresanalysis (PLS - DA) and orthogonal partial least-squares discriminant analysis (OPLS- DA), multivariate statistical analysis methods such as analysis on the impact ofBrazil muira pauma was on normal rats metabolic marker and identify with theanalysis of MetPA herbs to endogenous metabolic markers of the metabolic pathwayof rats, this Brazilian muira pauma was the biological mechanism of effects on thebody.

  3. The Brazilian muira pauma was alcohol extract on acute toxicity experiment, theBrazilian muira pauma was toxic pharmacological research: a study of the toxicity of281.84g/kg given to a rat, the loss of body mass, weight, and death of a rat within 1hour, 24 hours and 7 days.Muira Pauma - This dose - is of no toxicity to mice andmice given by gavage, and a preliminary conclusion is that the Brazilian dose ofmuira pauma is of minimal toxicity.;The drug is administered within this doserange.In mice as a condition for the construction of fatigue in mice model (adaptiveweight loading swimming), the experiment is divided into high dose group (14 g/kg)content, middle dose group (7 g/kg) content, low dose group (3.5 g/kg) content inthree groups, continuous lavage for five days later, the mice in the constanttemperature water for three days of weight loading swimming experiment, its loadconditions for the 8% of body weight in mice, the mice fell into the waterimmediately began to timing, mice to sink in water, and cannot be independentbuoyancy for 10 seconds, the time can be identified as exhaustion time in mice., andin nine days, the dosage of weight loading swimming 1 h after an pick eyeball bloodin mice, at the same time take the liver and muscles under test, by pressing the kitmanual operation steps to determine the content of hepatic glycogen in liverhomogenate in mice, and muscle glycogen content in skeletal muscle homogenate,and serum lactic acid, serum urea nitrogen content and activity of lacticdehydrogenase.

  Results:

  1.The analysis of its pharmacological action network pharmacology prediction resultsshowed that through the establishment of the Brazilian muira pauma was networkpharmacology to forecast its pharmacological effects: based on the retrieval methodssuch as Brazil muira pauma was composition 59 species, according to the structure ofthe compounds, use ACD/Lab Percepta software operations will be to import theabove composition structure, the export as a result, choose according to the results ofcompound using Swisstarget database, on the basis of molecular SMILES to find theappropriate targets, the main targets of Tyrosyl - DNACutive-phodiesterase 1Muscleblind-like protein 3 Muscleblind-like protein 1 5-anhydro-d-Fructose reductaseand other 67 corresponding targets were used to infer and construct the core subnetthrough the ESTABLISHED PPI network and the McOde cluster analysis module inthe Cytoscape software, and the targets in the subnet were used for disease enrichmentanalysis to obtain Digestive systemSystem Diseases Neoplasms Neoplasms by SiteSkin and Connective Tissue Diseases Diseases, Signs and Symptoms, etc.

  2. Serum metabolomics muira pauma was ethanol extracts of rat serum metabolicanalysis: Brazil muira pauma was serum metabolomics stability analysis results showthat in the high dosage of medicine is endogenous metabolism of normal rats whichhave influence on the stability of low dose is relatively high dose difference biggersample peak out of liquid phase and mass spectrometry conditions suitable foranalysis of the experimental sample to QC sample repeatability characterize thestability of the samples and equipment, through the QC samples of different qualitycontrol during the period of coincidence detection has no obvious change, ion flowdiagram of sample during the test, the sample and instrument has a good stability,after the completion of metabolites retrieval in metabolic pathway analysis, find thebetaAlanine metabolism Propionate Metabolism Valine leucine and isoleucinedegradation of arachidonic acid metabolism Citric acid cycle and other 30 significantmetabolic pathways.

  3. An acute toxicity study of an extract of pauma pauma root - this is a study fromBrazil. The results confirm that the maximum tolerable dose of an oral dose of281.84g /kg of raw medicine - pauma pauma root - this root pauma root isnon-toxic.To construct an anti-fatigue model of weight-bearing swimming of a mouse,the results showed that the extract of pauma pauma root had a longer and significantdifference in the duration of a weight-bearing swim compared to a control groupgiven normal saline (P<0.05), in which the middle dose group and the low dosegroup can significantly reduce the lactate content and increase the activity of lactatedehydrogenase after the exercise of isoleucine mice, and the serum urea nitrogencontent of the middle dose group is also significantly reduced (P<0.05) Compared tothe control group, a medium dose of the extract, pauma pauma root, from Brazil,significantly increased the duration of swimming (P<0.01), the lactic acid contentwas significantly reduced (P<0.05) Lactate dehydrogenase activity increased (P<0.01), there was no significant difference between liver glycogen and muscleglycogen, and serum urea nitrogen content in the medium dose group wassignificantly reduced (P<0.05).

  Conclusion:

  1.Through the analysis of its pharmacological action network pharmacology predictedresults analysis: the Brazilian muira pauma was the main component of the targets aremainly male/estrogen receptor muscle protein lipoprotein receptor activation peptidefragment 1 aldehyde ketone reductase family members B10 according to targetpresumed likely role in energy metabolism, sexual function, its effect on the fatstorage, according to the analysis result forecast targets related diseases Brazil muirapauma was probably cirrhosis of the liver tumor in the digestive system diseasesThetherapeutic effect of respiratory diseases, etc., network pharmacology analysis of thepharmacological effect of the pauma root of Brazil provides a theoretical basis for thedevelopment and discussion of the later study.Therefore, the following differentialmetabolite analysis can be carried out.

  2.Based on metabolic marker variables significantly and preliminary screening ofmetabolic changes in intensity mark variable 49 on qualitative analysis of differentmetabolites of metabolic pathway enrichment analysis article 30 meaningfulmetabolic pathways, which mainly can be divided into four kinds of metabolicpathways, which are directly or indirectly involved in energy metabolism, Brazilmuira pauma was in rats serum level can affect the body's energy metabolismhormone synthesis of amino acid metabolism, etc. Has a certain degree ofcorrelationThe analysis of serum metabolomics that Pauma pauma root participates ina variety of metabolic pathways in a rat, which can reflect the internal process of itsinvolvement, thus providing theoretical basis for further research and scientific basisfor the development and utilization of new resources of traditional Chinese medicine.

  3.The toxicity study of This root muira pauma root, Brazil, proves that the safety ofthe safety of this root pauma root provides a dose scope for later administration andprovides a theoretical safety benefit to the development of its medicinal value.Afterexhaustion exercise mice, the increase of energy depletion and metabolites ofmaterials: ATP reserves decline, liver glycogen and muscle sugar original significantconsumption, even will cause the loss of blood sugar level, further central fatiguecaused by metabolic product during lactic acid accumulation and lactatedehydrogenase levels increase, the urea nitrogen content increased, therefore, as thebasis of distinguishing fatigue effect anti-fatigue function experimental results provethat its muira pauma was alcohol alcohol extract in certain dose range has theanti-fatigue function, which is consistent with the prediction of networkpharmacology, and metabolomics.

  Keywords: muira puama;Network pharmacology; Metabolomics; Toxicity study;Antifatigue action.

目 录

  引 言

  第一章 基于网络药理学的巴西榥榥木核心药效分析

  1.材料

  2.方法

  2.1 主要化学成分的收集与筛选

  2.2 类药性分析

  2.3 构建成分-靶点-疾病相互作用网络 

  2.4 蛋白质-蛋白质相互作用网络(PPI)的构建 

  2.5 靶点-疾病共同靶点的生物过程和通路分析

  3 结果

  3.1 化学成分信息分析

  3.2 类药性分析

  3.3 巴西榥榥木成分、靶点筛选结果分析

  3.4PPI 网络图构建

  3.4 核心子网分析

  4 讨论

  第二章 基于代谢组学研究巴西榥榥木对大鼠的代谢机制

  1 材料与仪器

  1.1 实验动物

  1.2 试剂

  1.3 受试药物

  1.4 主要设备与耗材

  2 实验方法

  2.1 药物配制

  2.2 实验动物处理

  2.3 血浆样品处理

  2.4 液相条件

  2.5 质谱条件

  2.6 数据处理

  2.7 方法稳定性考察

  3 实验结果

  3.1 血清总离子流图

  3.2 系统及样品稳定分析

  3.3 代谢轮廓的偏最小二乘法判别分析

  3.4 代谢标志物分析

  3.5 差异代谢物的代谢通路富集分析结果

  3.6 代谢通路分析

  4.讨论

  第三章 巴西榥榥木小鼠抗疲劳药理作用研究

  1.材料与仪器

  1.1 实验仪器

  1.2 实验药材

  1.3 实验试剂

  1.4 实验动物

  2 实验方法

  2.1 巴西榥榥木急性毒性实验

  2.2 巴西榥榥木对小鼠抗疲劳作用研究

  2.2.4 统计学处理

  3 实验结果

  3.1 巴西榥榥木醇提物急性毒性实验结果

  3.2 巴西榥榥木醇提物对小鼠适应性负重游泳时间影响

  3.3 巴西榥榥木醇提物对运动后小鼠生理指标影响

  4 讨论

  结论

  文献综述

  1.历史研究状况

  2.化学成分研究

  3.药理作用研究

  参考文献

引 言

  中医药在发展过程中不断地吸收国外传统医药体系里临床上有效的动植物药材,如西洋参、番泻叶、乳香、没药等,经过长期的临床实践和研究,如今这些药材及饮片均纳入了中医药体系,且进入了中国药典,极大地丰富了中医药的实践和理论。当今世界,国外传统医药体系里仍然有相当数量的传统药物,由于种种原因没有被充分认识和利用。在地理纬度上与中国没有重叠的巴西,就有这样一批这些的药用植物,在当地被认为是临床有效的,但研究不够充分[1]。本文仅对巴西榥榥木这种植物进行初步的活性探讨研究,以期对其核心药理学功效的阐明提供研究线索。传统的药效筛选方法多依据临床线索,对相应的疾病模型动物直接用药,观察其药效,然后进行统计分析,得出结论[2]。这种方法成功的把握不大,针对性不够强,人力、物力和时间成本均较高,效率比较低。尤其是当临床线索很多、哪个是其核心功效尚不清楚时,对每个功效逐一研究然后进行对比,这种研究令人生畏,似乎无从下手[3]。随着对植物成分、疾病成因、生理机制等系列研究的深入和研究成果的积累,人类攻克疾病可利用的资料越来越多,面临的研究数据越来越宠大[4]。如何合理选择已有研究成果,成为当今科研工作者必须首先解决的问题。随着计算机网络数据库的诞生和发展有了系列的数据库和数据分析工具可以利用,网络药理学应运而生。传统中药学的研究具有系统性和整体性的特点和注重药物之间的相关性,网络药理学的研究思路具有整体性和系统性的特点解决中药的多成分、多靶点和多作用途径的复杂系统[5]。而巴西榥榥木作为巴西以及亚马逊流域著名的道地药材,其在其本土的应用方法与我国传统的中药应用思想具有一定的相似性,因此,将网络药理学引入对巴西榥榥木的研究,对了解巴西榥榥木的药理作用的整体性和系统性有一定的帮助作用。

  巴西榥榥木是巴西榥榥木为铁青树 Olacaceae 科,Ptychopetalum 属,olacoides种植物,主要药用部位为树皮和根,常用名有: Muira puama, potency wood,marapuama,marapama,muirat?, muiratam,pau-homen, potenzholz。在当地也被称为“效能之木(potency wood)”[6],是一种原产于巴西亚马逊和亚马逊雨林区域的一种树木。研究显示巴西榥榥木的主要功效包括:缓解疲劳、增强性欲、 防止溃疡、提高性功能、抗抑郁、增强记忆力、有助于肠胃和生殖疾病、抗风湿等,也可用于缓解压力和治疗创伤,对神经刺激能够安神、 保护脑细胞 、保护脑细胞等,其他的功效包括:缓解疼痛、降低血压、减少疲劳等[7-8]。研究表明其中化学成分多为有机化合物,因此,其作为药用使用时多以酊剂和汤剂的形式。但目前为止,巴西榥榥木的药理作用研究尚不明确,且缺少足够的实验基础研究支持其认为的功效作用。

  本课题以巴西榥榥木为研究对象,研究内容分为三部分:1.基于网络药理学的巴西榥榥木的核心药效分析。2.基于血清代谢组学的巴西榥榥木代谢通路分析。3.巴西榥榥木的药效学研究。

  实验技术路线见下图:

技术路线图

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