Study on Processing Technology for Aconiti Kusnezoffii Radix by Bean Curd

[Objectives] The aim was to explore the best parameters for processing Aconiti Kusnezoffii Radix by bean curd. [Methods] Orthogonal test( L_(16)(4~3)) was designed to investigate the optimal soaking time,decocting time and bean curd proportion for processing Aconiti Kusnezoffii Radix by bean curd. The contents of monoester alkaloids and diester-alkaloids were measured as the evaluation indexes. [Results]The contents of monoester alkaloids were 0. 11%,0. 07%,0. 062% and 0. 048% respectively under the four levels of soaking time,were0. 081%,0. 066%,0. 074% and 0. 067% respectively under the four levels of decocting time,and were 0. 070%,0. 072%,0. 080% and0. 073% respectively under the four levels of bean curd proportion. The content of diester-alkaloids was all below 0. 034%. The influence intensity of the three factors on the content of monoester alkaloids ranked as soaking time > decocting time > bean curd proportion. Under any of the above conditions,the content of diester-alkaloids was below the limit of Chinese Pharmacopoeia. The content of diester-alkaloids was reduced obviously with the increased use of bean curd. [Conclusions] The optimum processing conditions for Aconiti Kusnezoffii Radix were A_3B_3C_2,i. e.,soaking time of 6 d,decocting time of 5 h and bean curd proportion of 25%.

Matrix Solid-phase Dispersion Extraction of Alkaloids from the Roots of Aconitum kusnezoffii Reichb

Matrix solid-phase dispersion（MSPD） was developed for the extraction of four alkaloids, including aconitine, mesaconitine, hypaconitine and deoxyaconitine, from the roots ofAconitum kusnezoffii Reichb. The determination of the analyte was carried out by high performance liquid chromatography with UV detection. The alkaline alumina was used as sorbent. The mixture of acetonitrile and water was used as elution solvent. Several extraction parameters, such as type of sorbent, the ratio of sample to solid support material, type of the elution solvent and the volume of the elution solvent were tested. Mean recoveries ranged from 93.16% to 102.73%, with relative standard deviations from 0.27% to 4.17%. With the extraction efficiency and time expenditure taken into account, MSPD extraction should be a comparatively good method.

Cytotoxicity of Mongolian Medicine <i>Aconitum kusnezoffii</i>

Aconitum kusnezoffii used as an analgesic and anti-inflammatory. It is often used not only in Mongolian medicine but also in traditional Chinese medicine. However, there are few studies on its impact of inflammatory cytokines. This study explored the effects of Aconitum kusnezoffii in vitro used human colon cancer (Caco-2) cells. Cytotoxicity was assessed by measuring effects on expression of IL-1β, IL-6, IL-8, and TNF-α used enzyme-linked immunosorbent assay (ELISA). In vitro study demonstrated that incubation of Caco-2 cells with some kinds of Aconitum kusnezoffii at some concentrations inhibit secretion of TNF-α and FAXIU ACONITUM KUSNEZOFFII could inhibit the secretion of IL-1β. While IL-6 and IL-8 levels were unaffected after 24 h of treatment with Aconitum kusnezoffii, qujian Aconitum kusnezoffii, faxiu Aconitum kusnezoffii.

Identification of Xunxi Shujin Decoction by TLC

[Objectives]To establish a TLC qualitative method for the determination of Radix Aconiti,Radix Aconiti Kusnezoffii,Caesalpinia sappan L.and Radix zanthoxyli.[Methods]TLC was used to study the quality of Radix Aconiti,Radix Aconiti Kusnezoffii and C.sappan L.in Xunxi Shujin Decoction,and the influencing factors were investigated to optimize the TLC conditions.[Results]In the TLC identification test of Radix Aconiti,Radix Aconiti Kusnezoffii,C.sappan L.and R.zanthoxyli in Xunxi Shujin Decoction,the spots were clear,the resolution was good,the specific value was moderate,the negative control was not disturbed,and the specificity was strong.[Conclusions]The established TLC method was simple,specific and reproducible,and could be used for the quality control of Xunxi Shujin Decoction.

草乌的研究进展及质量标志物预测

草乌药用价值极高,在中医、蒙医等领域具有悠久的用药历史,但因其中毒剂量和治疗剂量较为接近,治疗窗狭窄,其临床应用受到一定限制,因此全方位建立草乌的质量标准评价体系是十分必要的。从基源考证、化学成分和药理作用等几个方面对草乌的研究现状进行综述。基于中药质量标志物(Q-marker)理论,针对草乌活性毒性的药物特性,结合其化学成分、现代药理毒理活性研究等方面,推测乌头碱型二萜生物碱类和挥发油可作为其Q-marker,为草乌的质量评价研究提供科学依据。

草乌炮制研究概况

乌头类中药草乌是我国广泛使用的毒性药材,性热有大毒,归心、肝、脾、肾经,具有祛风散寒、温经止痛等功效。草乌毒性主要表现为可引发心律失常的心肌毒性,伴有心脏血管系统异常等问题,中毒严重时可导致死亡。因其毒性剧烈,乌头类药材生品不入药使用,需经炮制后入药。草乌炮制方法并无统一标准,差异表现在药材前期处理、蒸煮工艺、辅料选择等方面。本文对草乌毒性研究进展及炮制方法沿革进行归纳,为今后草乌的炮制方法等研究提供参考。

草乌致大鼠肝肾亚急性毒性的代谢组学研究

目的基于代谢组学技术分析和探讨草乌致大鼠肝肾亚急性毒性的生物标志物和可能的作用机制。方法以草乌为示例药物,以大鼠为研究对象,除正常组外,给药组灌胃相应溶液,连续给予28 d。通过代谢组学技术对采集的大鼠血清、肝、肾样本进行检测,用主成分分析结合偏最小二乘判别分析筛选肝肾亚急性毒性敏感标志物及代谢通路。结果代谢组学分析显示,与正常组比较,草乌组血清、肝、肾样本中分别鉴定得到34、23和30个差异代谢物,主要富集于ABC转运蛋白、精氨酸和脯氨酸代谢、癌症的中心碳代谢、烟酸和烟酰胺代谢等通路。结论初步认为草乌通过影响ABC转运蛋白、精氨酸和脯氨酸代谢、癌症的中心碳代谢等对肝肾造成损伤,亚精胺、羟脯氨酸、葡萄糖、L-亮氨酸和尿素可作为潜在的肝肾毒性敏感生物标志物。

草乌幼苗形成期叶绿体发育的转录组分析

为确定草乌(Aconitum kusnezoffii Reichb.)苗期生长发育过程中的转录组特征,本研究利用Illumina测序对草乌从种子萌发到幼苗建成涉及到的四个时期(T1,T2,T3,T4)进行转录分析,挖掘草乌幼苗形成期叶绿体发育关键基因。结果显示,4个时期的6个差异分类检测到的差异基因数量依次为:16 379,28 155,16 100,34 368,38 250和44 568个。GO富集分析显示T3vsT4差异表达基因主要富集到对几丁质的反应,光合系统Ⅰ等。KEGG富集分析显示差异表达基因显著富集的通路主要是光合生物中的碳固定,植物激素信号转导等。草乌幼苗形成期叶绿体发育过程中,19个差异表达基因注释光信号调控叶绿体发育通路上,如GLK1/2,PIFs,HY5基因等。44个差异表达基因注释植物激素信号转导通路上,如AUX/IAA,ARF,DELLA基因等。本研究挖掘到一部分草乌幼苗形成期叶绿体发育相关基因,为进一步研究草乌生长发育相关分子机制提供理论基础。

不同程度制草乌生物碱含量对大鼠迷走神经动作电位和心电图的影响

目的比较不同炮制程度的制草乌生物碱含量及对SD大鼠心电图、颈部迷走神经动作电位的影响,建立生草乌及其炮制品的毒性评价方法。方法采用HPLC测定生草乌及泡煮30 min、不泡煮4 h、泡煮4 h、泡煮8 h的制草乌中苯甲酰新乌头原碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、新乌头碱、次乌头碱、乌头碱6个生物碱成分的含量;SD大鼠舌下引流分别给予草乌生品及4种炮制品,记录给药前后大鼠迷走神经动作电位及心电图,对生物碱含量与迷走神经放电面积和心率进行相关性分析。结果4种制草乌双酯型生物碱含量为:泡煮30 min>不泡煮4 h>泡煮4 h>泡煮8 h;单酯型生物碱含量为:不泡煮4 h>泡煮30 min>泡煮4 h>泡煮8 h。与给药前比较,大鼠舌下给予不同炮制品后迷走神经放电幅度增加,心率升高,心律失常程度增加,且随炮制程度而变化;相关性分析显示,迷走神经放电面积与双酯型生物碱和总生物碱含量有明显相关性。结论大鼠舌下引流给药后迷走神经动作电位和心电图可反映草乌麻舌程度,可为草乌“麻舌感”的研究提供参考。

Chemical constituents and analgesic activity of Aconitum kusnezoffii Reichb.

In the present study,we aimed to investigate the chemical constituents and analgesic activity of Aconitum kusnezoffii Reichb. The isolation and purification of components were achieved by a series of chromatography, including silica gel, Sephadex LH-20 and HPLC. By using spectroscopic analysis, their structures were identified. Using PDE-4A as analgesic target, moleculardocking was conducted between isolated compounds by using Schrodinger software. Neoline is a typical non-ester diterpene alkaloid. It was studied by using the mouse torsion body method and hot plate method. A total of 12 diterpene alkaloids were obtainedand identified as Mesaconitine(1), Bewutine (2), Bewudine (3), Songoramine (4), Songorine (5), Neoline (6), Talasamine (7), isotalatizidine (8), Hokbusine A (9), Mesaconine (10), 8-OEt-14-benzoylmesaconine (11), 8-Methoxy-14-benzoyl-beiwutinine (12).Compounds 9 and 12 were isolated from Aconitum kusnezoffii Reichb. for the first time. Twelve diterpenealkaloids could act on the analgesic target. Neoline is a typical non-ester diterpene alkaloid. It had significant analgesic effect. Diterpene alkaloids were the main components of Aconitum kusnezoffiiReichb., and they had good analgesic activity.

诃子在藏药炮制中的应用

通过查阅近年来国内外文献,归纳、分析了诃子在藏药炮制中的应用以及诃子炮制一些矿物药和有毒植物药过程的研究进展,根据诃子化学成分推测诃子炮制铁粉、草乌的原理及减毒机制,为进一步深入探讨藏药诃子炮制矿物药、有毒植物药的机制提供参考。

UPLC-QTOF/MS结合网络药理学探讨诃子与草乌配伍“异病同治”类风湿性关节炎和痛风性关节炎作用机制

目的:探讨诃子与草乌配伍“异病同治”类风湿性关节炎(RA)和痛风性关节炎(GA)的作用机制。方法:采用数据挖掘技术找出治疗“图赖”病的核心药物组合(诃子-草乌),并使用超高效液相色谱-四极杆飞行时间质谱法(UPLC-QTOF/MS)检测大鼠灌胃诃子与草乌配伍药物后的入血成分;运用PubChem、Swiss Target Prediction、GeneCards、OMIM、DisGeNET、Metascape等多个数据库检索诃子与草乌化学成分对应靶点、RA和GA的疾病靶点,以及代谢通路等信息,利用Cytoscape3.8.2软件构建成分-靶点-通路-疾病网络,并利用分子对接方法证实诃子与草乌的主要成分与关键靶点之间的高亲合力。结果:诃子与草乌入血成分相应靶点与RA靶点交集后得到323个交集靶点、与GA靶点交集后得到24个交集靶点,两者共有靶点24个,主要有肿瘤坏死因子(TNF)、肿瘤蛋白p53(TP53)、基质金属蛋白酶-9(MMP-9)、前列腺素内过氧化物合酶2(PTGS2)和信号转导和转录激活因子3(STAT3),与十八碳四烯酸、绢酸、5-(3-羟基-4-乙酰氧基-1-丁基)-2,2'-联噻吩、灵芝酸H等有紧密联系,涉及通路有TNF信号通路、破骨细胞分化通路、癌症通路等。分子对接结果显示灵芝酸H和TNF、5-(3-羟基-4-乙酰氧基-1-丁基)-2,2'-联噻吩和MMP-9有强烈对接活性。结论:蒙古族药诃子与草乌配伍治疗RA和GA具有多靶点效应,可能通过炎症反应的调节、细胞对生物刺激的反应、对细菌源分子的反应、磷酸盐代谢的正调控和对激素的反应等通路发挥“异病同治”作用。

草乌化学成分、药理作用、毒性及蒙医学毒性控制方法概述

草乌所含的特征性成分二萜类生物碱是其主要毒性成分也是有效成分,具有抗炎、镇痛、抑菌、抗病毒、强心、抗肿瘤、局部麻醉等药理活性的同时也存在各种毒性作用。故传统医药通常通过炮制或配伍,降低其毒性,保障临床用药安全。作为中医药的重要组成部分蒙医药在这方面优势更为突出。

Rapid Differentiation of Aconiti Kusnezoffii Radix from Different Geographic Origins Using Ultra-Performance Liquid Chromatography Coupled with Time-of-Flight Mass Spectrometry

Objective:There are different geographic origins of Aconiti Kusnezoffii Radixs(AKRs)sold in the market with different quality.This study aims to establish a rapid analysis method to distinguish the different geographic origins of AKRs and to realize the rapid evaluation of their quality.Methods:An ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry(UPLC-Q-TOF MS)method was utilized to acquire the constituents'information of AKRs from different geographic origins.MSE data and Progenesis QI software were employed to identify the chemical constitutes.Principal component analysis(PCA)was applied to comparing MS data to find the chemical markers of AKRs from different geographic origins.Results:Twenty-three components were detected and 17 out of them were identified,including diester-diterpenoid alkaloids,monoester-diterpenoid alkaloids,and amine-diterpenoid alkaloids.Three pairs of isomers were detected and two of them were distinguished by the retention time of standard samples.Thirteen chemical markers were screened out through PCA and orthogonal partial least square discriminant analysis.Through detecting Napelline or isomer of Napelline(m/z 360.2530)and Aconifine(m/z 662.3170),AKRs from inner Mongolia autonomous could be screened.According to the existence of benzoylaconine(m/z 604.3108)and Indaconitine(m/z 630.3159),it could be confirmed that the AKRs are from Xinjiang Uygur autonomous.AKRs that cannot detect compounds above-mentioned could be from Liaoning or Shanxi Province.Conclusions:The chemical profile could be used not only to distinguish the AKRs from different geographic origins but also to identify the true and false of AKRs.This study lays a foundation for the study of efficacy and toxic of AKRs.

草乌叶生物碱成分及其抗炎活性研究

目的探讨蒙古族习用药材草乌叶抗炎作用的物质基础及作用机制。方法制备草乌叶生物碱组分,并利用LC-MS技术进行质谱鉴定;进一步检测鉴定生物碱对LPS诱导的RAW264.7细胞炎症模型的影响。首先,采用CCK-8法检测生物碱对RAW264.7细胞的增殖-毒性作用,确定最大给药浓度;其次,通过PCR实验检测TNF-α、IL-10、IL-1β和iNOS的基因表达情况;采用Griess法测定NO浓度,ELISA法测定炎症因子TNF-α、IL-10、IL-1β含量;最后通过Western blotting实验检测COX-2蛋白表达情况。结果通过LC-MS技术同时鉴定了乌头碱、次乌头碱、新乌头碱、苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱等6种生物碱;上述6种生物碱对LPS诱导的炎症因子TNF-α、IL-10、IL-1β及NO具有不同程度的抑制作用,且同时可抑制iNOS基因表达及COX-2蛋白表达。结论草乌叶生物碱组分可含双酯型生物碱乌头碱、次乌头碱、新乌头碱及单酯型生物碱苯甲酰乌头原碱、苯甲酰次乌头原碱、苯甲酰新乌头原碱等6种生物碱;而这6种生物碱具有不同程度的体外抗炎效应,抗炎作用机制可能与抑制iNOS/COX-2炎症信号传导有关。

草乌在蒙药中的应用区别于中药的特色研究

目的:从草乌性能、炮制、应用、毒性及中毒解救等方面探讨草乌在蒙药中的应用区别于中药的特色研究。方法:检索国内外数据库中相关报道及古籍文献中有关草乌应用、炮制方法、毒性、中毒解救等相关的记载,总结草乌在蒙药中应用与中药中应用区别,探讨在蒙药中的应用特色。结果:草乌在蒙药及中药中应用性能、炮制、配伍规律、用法用量等方面均存在着一定的差异。结论:由于蒙医药理论与中医药理论有所不同,在用药方面也存在一定差异,尤其具有较强毒性的草乌类药物在临床上的应用具有明显的差异,本研究对比草乌在蒙药与中药中的应用,探讨了蒙药用草乌特色,为蒙医临床安全用草乌及为进一步研究草乌在蒙药中特色应用的独特安全性评价及风险控制措施提供参考。

基于药效团和分子对接探究诃子制草乌通过瞬时受体电位香草酸1介导的减毒机制

目的:本研究利用药效团、分子对接及实时反转录PCR(RT-PCR)方法探究诃子制草乌通过瞬时受体电位香草酸1(TRPV1)介导的减毒机制。方法:采用Discovery Studio 4.0软件构建基于TRPV1激动剂分子共同特征的TRPV1激活药效团,虚拟筛选出草乌中的潜在活性成分;对草乌成分及诃子制草乌中引入的诃子成分进行Autodock vina分子对接研究;根据结果选出代表性成分,采用实时反转录PCR验证是否可促进心肌细胞中TRPV1 mRNA表达。结果:药效团共筛选出草乌中包括毒性较大的双酯型生物碱乌头碱、新乌头碱及次乌头碱在内的13个成分,分子对接结果表明这13个成分及诃子制草乌引入的诃子成分均有潜力与TRPV1结合。RT-PCR实验中没食子酸和新乌头碱在浓度≥25μmol/L时,均可明显促进大鼠H9c2心肌细胞中TRPV1 mRNA表达的增加,随着给药浓度的增大表达量增加,新乌头碱促进TRPV1 mRNA表达的作用更强。药效团、分子对接结果表明诃子和草乌中均含有多种具备TRPV1激活潜力的成分。结论:实验证明新乌头碱、没食子酸均可在一定程度上激活TRPV1通道,可作为TRPV1的激动剂。根据结果推测,诃子可能会通过拮抗草乌中的活性成分与TRPV1的结合,从而降低草乌通过TRPV1介导的毒性;也可能是诃子制草乌在引入没食子酸等诃子成分后,可在安全剂量范围内使TRPV1通道快速脱敏从而实现减毒。

SCX-SPE结合UHPLC-Q-Exactive Orbitrap MS分析草乌中的四类二萜生物碱

目的对草乌中的二萜生物碱进行表征分析。方法以强酸性阳离子交换树脂固相萃取(SCX-SPE)分离纯化草乌中的二萜生物碱,并结合超高效液相色谱串联四极杆静电场轨道阱质谱(UHPLC-Q-Exactive Orbitrap MS)进行结构鉴定。结果结合精确分子质量、多级质谱裂解规律、对照品对比、Clog P值及文献报道,共从草乌中鉴定了99个二萜生物碱类成分,包括双酯型二萜生物碱(DDA)27个,单酯型二萜生物碱(MDA)29个,酰胺型二萜生物碱(ADA)40个,多酯型二萜生物碱(PDA)2个,以及长链型二萜生物碱(LDA)1个。结论本文所建立的SCX-SPE结合UHPLC-Q-Exactive Orbitrap MS方法能快速地鉴定草乌中的二萜生物碱类成分,可为草乌的药效物质基础及质量控制研究提供科学依据。

草乌传粉过程中的广告效应与回报物质研究

虫媒传粉植物与其传粉者之间的相互作用被认为是被子植物花多样性的一个重要选择压力。这种相互作用体现在植物对传粉者的吸引以及传粉者行为对花粉的转运两个方面。本项研究通过去除不同的花部器官研究了草乌(Aconitum kusnezoffii)对其传粉者的吸引,并结合传粉者的访问行为和草乌花的生物学特性探讨了传粉过程与交配系统的适应。红光熊蜂(Bombus ignites)是草乌的有效传粉者。去除花萼片显著降低了红光熊蜂的访问频率,但去除特化成蜜腺叶的花瓣并没有显著改变红光熊蜂的访问频率,这表明草乌吸引红光熊蜂的主要结构是由5枚萼片组成的花部外观形态,而非花瓣。花蜜是草乌提供给红光熊蜂的回报物质,糖浓度为39.23%,组氨酸浓度为0.25μg/μL。草乌花较大、单花花期长、雌雄异熟、花粉寿命长,是一个自交亲和但需传粉者传粉完成繁殖过程的异交物种。草乌花序是无限花序,当上部的花处于雄性阶段时,下部的花正好处于雌性阶段。而红光熊蜂在草乌花序上的访问顺序通常自下而上,带来异交花粉为下部的花进行异花授精,同时又带走上部花的花粉,这就很好地促进了草乌的异交。草乌雄蕊自外向内逐渐成熟,是一种有效的限制传粉者单次访问浪费大量花粉的花粉装配策略,能提高植物通过花粉散布获得的雄性适合度。

考马斯亮蓝G-250染色法测定草乌中可溶性蛋白质含量

采用考马斯亮蓝G-250染色法对草乌药材中可溶性蛋白质含量进行了测定,建立了快速、灵敏的测定蛋白质的方法.结果表明,考马斯亮蓝G-250染色法简单、迅速,且相对较为准确,是测定低浓度蛋白质含量的有效方法.