Pharmacokinetics of Gelsemium elegans in female rats

Background:Gelsemium elegans Benth(G.elegans)is a poisonous perennial evergreen vine plant that has been applied in livestock production and veterinary clinical practice.Early studies found that the toxicity of G.elegans showed significant gender differences in rats,but the underlying reasons for this difference are still not well understood.Methods:In order to explore whether the gender differences in the toxicity of G.elegans are related to pharmacokinetic differences,based on the previous pharmacokinetic study of multiple components of G.elegans in male rats,this study used HPLC-MS/MS method established in the laboratory to conduct a pharmacokinetic study of multiple alkaloids in the plasma of female rats after a single gavage administration of G.elegans(dose of 0.1 g/kg).Results:Through detection,17 alkaloid components in the plasma of female rats were identified,and the pharmacokinetic parameters of 11 of these alkaloids were calculated.We find that in female rats.The T_(max)values were generally less than 0.5 h,and the T_(1/2)values exceeded 3 h,with the longest reaching up to 32.80 h half elimination time.Additionally,the C_(max)and AUC results indicated that female rats had generally higher absorption and exposure levels for most alkaloids.Conclusion:These results suggest that the reason for the differences in the toxicology of G.elegans may be related to the absorption and exposure of gelsemidine-type alkaloids in animals.

钩吻(Gelsemium elegans)生物碱结构类型增补探讨

根据对亚洲钩吻(Gelsemium elegans)的化学研究结果,探讨将已知5种生物碱骨架类型扩展为9种,其中新增补并命名为Geleboline-type,Gelselegine-type,Gelsemamide-type,以及首次报道在亚洲钩吻中得到的Yohimbane-type类型。

Two new benzofuran lignan glycosides from Gelsemium elegans

Two new benzofuran lignan glycosides, gelsemiunoside A and B, were isolated from the whole plant of Gelsemium elegans Benth. Their structures were elucidated on the basis of spectroscopic evidence. Furthermore, gelsemiunoside A and B were shown a potent cytotoxic activity by suppressing the proliferation of A375-S2 cells.

马钱科植物钩吻(Gelsemium elegans)的化学研究

研究钩吻(Gelsemium elegans)根茎的化学成分。采用色谱方法进行分离纯化,通过理化及波谱鉴定化学结构。经钩吻甲醇提取物的酸碱处理后的总碱分离得到13个生物碱,分别为五种结构类型,3个Sarpagine-type生物碱:Gardnerine(1),Koumidine(2),N-methoxyanhydrovo-basinediol(3);3个Humantenine-type生物碱:Humantenine(4),Humantenirine(5),Rankinidine(6),3个Gelsedine-type生物碱:Gelsedine(7),Gelsenicine(8),19-Oxo-gelsenicine(9),2个Koumine-type生物碱:Koumine(10),19S-Kouminol(11);2个Gelsemine-type生物碱:Gelsemine(12),Gelesevirine(13).这五种类型构成钩吻生物碱的基础生源途径。

STUDIES ON THE INDOLE ALKALOIDS OF GELSEMIUM ELEGANS

The modified Hofmann degradation of koumine(1) has proved abortive. The struture and stereochemistry of the main product(3) were deduced by spectral methods and confirmed by X-ray diffration analysis. From the roots of Gelsemium elegans, dihydrokounine was first isolated as a natural product.

Mechanism of Action of Low Dose Preparations from <i>Coffea arabica</i>, <i>Gelsemium</i>and <i>Veratrum</i>Based on <i>in Vivo</i>and <i>in Vitro</i>Neurophysiological Findings

Low dose remedies are widely administered in medicine. We used Tele-Stereo-EEG and the hippocampal slice preparation to measure physiological effects of orally given Coffea D6 (40 mg/kg), Gelsemium D4 (10 mg/kg) and Veratrum D6 (30 mg/kg) in rats. Adult rats were implanted with electrodes positioned stereotactically into four brain regions. Changes in field potentials were transmitted wirelessly. After frequency analysis data from 6 - 8 animals were averaged. For in vitro testing, preparations were superfused directly on hippocampal slices. Stimulation of Schaffer Collaterals by single stimuli (SS) or theta burst stimulation (TBS) resulted in stable population spike amplitudes. All three low dose preparations produced decreases of spectral power. Statistically significant changes were observed in delta, theta and alpha2 spectral power. In the hippocampal slice preparation Coffea facilitated signal transfer presumably by enhancing glutamate AMPA receptor transmission. Gelsemium showed a similar effect, but only after single shock stimulation. Opposite to this, attenuation of the electric pathway was recognized after theta burst stimulation due to AMPA receptor and glutamate metabotropic II receptor mediated transmission. Veratrum was able to attenuate glutamatergic due to receptor-mediated signalling sensitive to AMPA and NMDA. The results strongly speak in favour of the existence of biologically active molecules in these low dose preparations.

Preparative Separation of Four Alkaloids from Gelsemium elegans by High-speed Counter-current Chromatography

Objective To develop an efficient preparative method for the separation of Gelsemium alkaloids from Gelsemium elegans. Methods High-speed counter-current chromatography （HSCCC） with several two-phase solvent systems was investigated for the separation of Gelsemium alkaloids. The purity and structure identification of the purified compounds were performed with HPLC and NMR spectra, respectively. Results In a single operation, 206.6 mg of crude alkaloid sample was separated to yield 28.7 mg of koumine, 24.9 mg of gelsemine, 26.9 mg of humantenine, and 7.2 mg of gelsevirine, with the purities of 97.8%, 95.4%, 97.4%, and 93.5%, respectively. Conclusion A preparative HSCCC method is successfully established for the separation of four Gelsemium alkaloids from G. elegans with a modified two-phase solvent system com posed of n-hexane-ethyl acetate-ethanol-O. 5% triethylamine-H2O （3：5：3：4）.

Whole-genome sequencing and analysis of the Chinese herbal plant Gelsemium elegans

Gelsemium elegans(G.elegans)(2 n=2 x=16)is genus of flowering plants belonging to the Gelsemicaeae family.Here,a high-quality genome assembly using the Oxford Nanopore Technologies(ONT)platform and high-throughput chromosome conformation capture techniques(Hi-C)were used.A total of 56.11 Gb of raw GridION X5 platform ONT reads(6.23 Gb per cell)were generated.After filtering,53.45 Gb of clean reads were obtained,giving 160 x coverage depth.The de novo genome assemblies 335.13 Mb,close to the 338 Mb estimated by k-mer analysis,was generated with contig N50 of 10.23 Mb.The vast majority(99.2%)of the G.elegans assembled sequence was anchored onto 8 pseudo-chromosomes.The genome completeness was then evaluated and 1338 of the 1440 conserved genes(92.9%)could be found in the assembly.Genome annotation revealed that 43.16%of the G.elegans genome is composed of repetitive elements and 23.9%is composed of long terminal repeat elements.We predicted 26,768 protein-coding genes,of which 84.56%were functionally annotated.The genomic sequences of G.elegans could be a valuable source for comparative genomic analysis in the Gelsemicaeae family and will be useful for understanding the phylogenetic relationships of the indole alkaloid metabolism.

An integrated strategy toward comprehensive characterization and quantification of multiple components from herbal medicine: An application study in Gelsemium elegans

Objective: To develop a powerful integrated strategy based on liquid chromatography coupled with mass spectrometry(LC-MS) systems for the comprehensive characterization and quantification of multiple components of herbal medicines.Methods: Firstly, different mobile phase additives, analysis time, and MS acquisition modes were orthogonally tested with liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(LC-QTOF/MS) in order to detect as many components of Gelsemium elegans as possible with high peak intensity. Secondly, several data mining strategies, including database searching, diagnostic ion filtering and neutral loss filtering, were utilized to perform chemical profiling. Subsequently, this study focused on the quantification and validation of the performance of a liquid chromatography-triple mass spectrometry(LC-Qq Q/MS) assay based on derivative multiple reaction monitoring(De MRM).Results: A total of 147 components from G. elegans were characterized, among them 116 nontarget components were reported for the first time. A sensitive and reproducible LC-Qq Q/MS method was successfully developed and validated for the simultaneous relative quantification of 41 components of G. elegans.This LC-Qq Q/MS method was then applied to compare the contents of components in the roots, stems and leaves.Conclusion: The present integrated strategy would significantly contribute to chemical studies on herbal medicine, and its utility could be extended to other research fields, such as metabolomics, quality control,and pharmacokinetics.

胆汁制钩吻总碱抑制结肠癌细胞增殖的作用研究

目的探讨胆汁制钩吻总碱(胆钩吻总碱)体外抗肿瘤效果,旨在为钩吻减毒后存效的研究提供科学依据。方法通过CCK-8检测观察胆钩吻总碱对人结肠HCT-116细胞、人神经胶质瘤U87细胞、人肝癌HepG2细胞、人肺癌A549细胞的增殖作用,进一步以结肠癌HCT-116为研究对象,以不同浓度胆钩吻总碱(50、100、200μg·mL-1)干预HCT-116细胞,通过流式细胞技术检测其对细胞周期阻滞的影响;Annexin V FITC/PI流式细胞术检测HCT-116细胞凋亡情况;进一步从蛋白水平检测凋亡相关蛋白表达。结果在钩吻总生物碱IC 50浓度下,胆钩吻总碱对U87、A549、HepG2、HCT-116肿瘤细胞的增殖抑制率均高于钩吻总生物碱组,并且各组之间的差异具有统计学意义(P<0.01)。与空白组相比,不同浓度的胆钩吻总碱处理(50、100、200μg·mL-1)可有效降低HCT-116细胞的集落形成,并将细胞周期阻滞在G 2/M期。胆钩吻总碱还能引发结肠癌HCT-116细胞的晚期凋亡,并对凋亡相关蛋白Bax、Bcl-2、caspase-3起到调控作用。结论胆钩吻总碱可以通过调控结肠癌HCT-116细胞周期和凋亡相关蛋白的表达来抑制其增殖。

钩吻枝叶乙醇提取物的化学成分

从钩吻〔Gelsemium elegans(Gardn.et Champ.)Benth.〕枝叶乙醇提取物中共分离鉴定出6个化合物,分别为熊果酸、27-O-对-(E)-香豆酰基-乌索酸、27-O-对-(Z)-香豆酰基-乌索酸、16-表伏康树卡平碱、(Z)-阿枯米定碱、N(a)-去甲基嘌呤。其中,16-表伏康树卡平碱首次从钩吻中分离获得,其余5个化合物首次从钩吻属(Gelsemium Juss.)中分离获得。

钩吻总碱诱导慢性粒细胞白血病K562细胞凋亡的机制分析

目的 探讨钩吻总碱对慢性粒细胞白血病K562细胞的抑杀作用及其机制,为其抗白血病研究提供科学依据。方法 以不同浓度钩吻总碱(25、50、100、200、400μg·mL-1)干预K562细胞,MTT法测定其对K562细胞活力的影响;倒置相差显微镜观察其对K562细胞形态的影响;DAPI染色法观察K562细胞核形态变化;Annexin V FITC/PI流式细胞术检测K562细胞凋亡情况;比色法测定caspase-3活性变化;RT-PCR检测Bax和Bcl-2基因的表达水平。结果 钩吻总碱对K562细胞抑制效果呈时间、剂量依赖性,24 h时IC50为122μg·mL^(-1);TAG干预K562细胞后细胞形态逐渐不规则、胞质不清、胞核皱缩,最终胞膜的完整性破坏;DAPI染色发现细胞体积变小,整体皱缩,胞核固缩、生成典型的凋亡小体;Annexin V FITC/PI流式细胞测定显示K562细胞凋亡以早凋为主,存在量效关系;不同浓度钩吻总碱作用于K562细胞24 h后caspase-3的活性提高;RT-PCR检测发现TAG干预后会下调Bcl-2基因表达、上调Bax基因表达,二者均表现出量效关系。结论 钩吻总碱可能通过上调Bax基因表达、下调Bcl-2基因表达,活化caspase-3,从而诱导慢性粒细胞白血病K562细胞发生早期凋亡,抑制其细胞活力。

不同发酵天数赤芝钩吻的毒性及镇痛作用研究

目的比较2批不同发酵天数钩吻的急性毒性及镇痛药效。方法采用改良寇氏法测定发酵14天、21天钩吻半数致死量(LD_(50)),并获取2批不同发酵钩吻的最大安全给药剂量为药效学浓度。采用醋酸扭体法测定不同发酵钩吻对醋酸所致小鼠扭体反应疼痛阈值的影响,ELISA法检测各组小鼠血清中TNF-α、IL-1β和IL-6水平。结果发酵14、21天的赤芝钩吻的LD_(50)分别为10.91 g·kg^(-1)、15.4 g·kg^(-1);2批发酵钩吻对醋酸扭体镇痛模型的镇痛率分别为74%、79%,发酵钩吻显著降低血清炎症因子含量,且批次之间无显著差异。结论2批不同发酵天数赤芝钩吻毒性降低,并具有显著的镇痛作用,2批次之间镇痛药效无显著差异。

钩吻生物碱对肺癌的抑制作用及其机制

目的 探讨钩吻素子(koumine,KOU)及钩吻总生物碱(total alkaloids of Gelsemium elegans Benth,TAG)抗肺癌的作用及其机制。方法 采用活细胞动态分析系统、集落形成实验研究低、中、高浓度(100、150、200μg/mL)KOU对人肺腺癌细胞A549、SPCA1的增殖作用。构建肺癌细胞小鼠移植瘤模型,分为模型组(Model组)、环磷酰胺组(CTX组,剂量为20 mg/kg)、KOU组(剂量为2 mg/kg)和TAG组(剂量为0.5 mg/kg),CTX组和KOU、TAG组按0.1 mL/10 g隔天腹腔注射,给药10 d检测KOU和TAG对肺癌实体瘤生长的作用,采用HE、免疫组化(immunohistochemistry,IHC)及TUNEL染色观察肿瘤组织,并计算肿瘤体积和肿瘤抑制率。对TAG作用48 h的A549细胞进行RNA测序分析,筛选差异表达基因(differentially expressed genes,DEGs),并进行KEGG(Kyoto encyclopedia of genes and genomes,KEGG)和GO(Gene Ontology,GO)功能富集分析。采用实时荧光定量逆转录聚合酶链反应(real-time quantitative reverse transcription polymerase chain reaction,real-time-qPCR)进一步分析验证相关基因的表达。结果 活细胞动态分析系统拟合出A549、SPCA1细胞融合度下降,细胞数目减少,生长状态变差。集落形成实验结果证实KOU使细胞克隆数目形成减少,在高浓度200μg/mL时最为显著(P<0.01)。动物实验表明KOU组、TAG组的肿瘤抑制率依次可达24.55%、36.08%,与Model组相比,TAG组肿瘤体积显著下降(P<0.05)。RNA测序分析筛选出DEGs总数为2 793个,其中上调基因1 433个,下调基因1 360个。富集分析发现DEGs主要富集于IL-17信号通路、肿瘤坏死因子信号通路及p53信号通路等。RT-qPCR验证结果与RNA测序分析结果一致,GADD34、ZFP36、GADD45A、GADD45B、TP53INP2基因在TAG组显著表达增加(P<0.01)。结论 在体内、外水平钩吻生物碱均抑制肺癌增殖,转录组学发现其抑制肺癌作用的多个DEGs和通路。

钩吻总碱对肺腺癌细胞增殖和凋亡作用的研究

目的 以人肺腺癌细胞(A549、SPCA1)为研究对象,通过在培养液中加入不同浓度的钩吻总碱(TAG)研究其抗肿瘤作用。方法 采用加热回流、萃取等方法提取TAG,利用薄层色谱法鉴定TAG的提取,通过Incucyte S3活细胞动态分析系统拟合不同浓度的TAG作用A549、SPCA1细胞的细胞融合度并观察A549、SPCA1细胞的形态,采用CCK-8法、集落形成实验检测细胞增殖,Hoechst33258染色、Rhodamine123染色检测细胞凋亡,碘化丙啶单染法检测细胞周期,Western blotting检测凋亡指标蛋白Bax、Bcl-2、Caspase-3蛋白表达。结果 经薄层色谱法鉴定成功提取TAG。Incucyte S3活细胞动态分析系统拟合出TAG作用A549、SPCA1细胞的EC50值分别是99.2、82.0μg/mL,由此确定TAG作用A549、SPCA1细胞的低、中、高浓度依次为50、100、150μg/mL,且细胞拍照观察到随着药物浓度增加细胞融合度下降、细胞数目减少。CCK-8法和集落形成实验结果表明TAG抑制A549、SPCA1细胞增殖(P <0.05)。Hoechst 33258细胞核染色实验发现给药组细胞出现核碎裂;流式细胞术检测Rhodamine123探针发现给药组出现荧光信号强度增加,检测细胞周期发现TAG使A549、SPCA1细胞周期阻滞在G_2/M期;Western blot结果显示TAG诱导Bcl-2蛋白表达下调,Bax表达上调、Caspase-3蛋白切割活化增加,通过细胞染色实验、流式细胞术及Western blotting表明TAG促进A549、SPCA1细胞凋亡(P <0.05)。结论 TAG抑制肺腺癌细胞增殖,诱导细胞凋亡。

断肠草资源的开发与应用

断肠草(Gelsemium elegansBenth)是一种有毒植物,主要含有多种吲哚类生物碱,近年来在临床上广泛用于抗肿瘤、镇痛及免疫调节等方面.文章对断肠草在医药、农业、畜牧业等方面的应用进展进行了综述,并对其在应用中存在的问题及发展前景进行了讨论和展望.

钩吻素子体外诱导人结肠腺癌LoVo细胞凋亡的实验研究

目的探讨钩吻素子(Kou)体外能否诱导LoVo细胞凋亡。方法以Kou(50 μmol/L)作用于LoVo细胞,经光镜、电镜和荧光显微镜观察细胞凋亡情况,并用流式细胞仪检测细胞增殖周期状态。结果通过光镜、电镜和荧光显微镜观察均证实Kou可诱导LoVo细胞凋亡,且凋亡百分率随Kou作用时间的延长而升高。经Kou作用后的肿瘤细胞,细胞周期状态发生明显变化,G0/G1期细胞从31.3%上升到42.3%,S期细胞从62.0%下降到38.7%。结论Kou可诱导LoVo细胞凋亡,且存在时间依赖关系,Kou可抑制细胞DNA的合成,阻止细胞由G1期向S期转化。

钩吻总生物碱中钩吻素子的提取与分离

目的研究钩吻中总生物碱的提取、总量测定方法及其中主要成分钩吻素子的分离、鉴定方法。方法采用加热回流提取与氯仿萃取相结合的方法提取总生物碱;采用碱性硅胶柱层析、梯度洗脱法分离钩吻素子并用薄层层析法和紫外分光光度法定性鉴别。结果从钩吻中分离得到了无色棱柱状钩吻素子晶体。结论本实验所采用的提取钩吻总生物碱及分离钩吻素子的方法行之有效。

大孔吸附树脂分离纯化钩吻总生物碱的研究

目的:研究用大孔吸附树脂分离纯化钩吻总生物碱中主要成分的方法和工艺条件。方法:采用不同型号的HPD系列大孔吸附树脂对钩吻总生物碱进行提取纯化,以高效液相色谱法检测钩吻总生物碱中主要成分的峰面积为指标,考察最佳工艺和参数条件。结果:HPD-500、HPD-600、HPD-800型大孔吸附树脂对钩吻总生物碱提取效果好,并得到HPD-800的最佳工艺和参数条件。结论:采用HPD-800型大孔吸附树脂及不同浓度乙醇洗脱的方法,可用于钩吻总生物碱的提取纯化。

胡蔓藤中非生物碱类成分的分离与鉴定(Ⅲ)

目的研究胡蔓藤(Gelsemium elegans Benth.)中的化学成分。方法采用硅胶柱色谱和ODS柱色谱进行分离,Sephadex LH-20及制备液相进行纯化,根据理化性质和光谱分析进行结构鉴定。结果分离鉴定了8个化合物,分别为(+)-8-hydroxypinoresinol(1)、cleomiscosin C(2)、cleomiscosinA(3)、3,4-二羟基苯甲醛(3,4-dihydroxyphenyl aldehyde,4)、咖啡酸(caffeic acid,5)、1-O-咖啡酰基奎宁酸(1-O-caffeoylquinic acid,6)、4-O-咖啡酰基奎宁酸(4-O-caffeoylquinic acid,7)、1-O-咖啡酰基奎宁酸甲酯(1-O-caffeoylquinic acid methyl ester,8)。结论化合物1-8为首次从该属植物中分离得到。