中国南海桶形芋螺毒液成分及其活性的研究

从海南岛采集了桶形芋螺标本１０ｋｇ。比较了“超低温脆化法”,“匀浆法”,“剥离法”的取毒效果。经凝胶过滤色谱分离后,共得粗毒液 ５０４ｍｇ,其中,Ｐ１,２１０ｍｇ,分子量 Ｍｗ＞５０００;Ｐ２,１８４ｍｇ,Ｍｗ＝１０００～５０００;Ｐ３,５０ｍｇ,Ｍｗ＜１０００。用高效液相色谱（ＨＰＬＣ）和高效毛细管电泳（ＨＰＣＥ）对 Ｐ２,Ｐ３进行了比较研究。毒液经 ＨＰＬＣ分离后得到数十个成分,各成分多数在２０ｍｇ以内。药理实验表明,它们具有一系列中枢神经活性（ＣＮＳ）。     

疣缟芋螺毒素的二维液相色谱分离方法

目的建立一种新的基于二维液相色谱技术的疣缟芋螺毒素分离方法,了解其毒素组构成特点。方法从疣缟芋螺毒管中提取芋螺毒素总肽,在传统的凝胶色谱和反相色谱方法的基础上,根据芋螺毒素的等电点和疏水性,利用目标蛋白快速分离系统(Proteome Lab TMPF2D),对其进行二维分离。结果经过传统分离方法,能够检测得到40个左右的芋螺毒素条带,且分离效果不明显。而通过二维液相色谱分离方法,pI/UV图谱显示共检测到约200个芋螺毒素条带。结论同传统分离方法相比,采用Proteome LabTMPF2D系统对毒素分离更快速、分辨率更高,因此更利于鉴定芋螺毒素肽组分,也为下游序列结构特征与生物活性研究奠定了良好的基础。     

作用于电压门控钠离子通道的芋螺毒素研究进展

电压门控钠离子(Na+)通道(VGSCs)对兴奋细胞动作电位具有调节作用,许多生物毒素能与VGSCs相互作用。近年来研究发现,热带海洋肉食性软体动物芋螺中含有多种多肽类毒素(芋螺毒素或芋螺肽),其中一大类芋螺毒素能与Na+通道各种亚型特异结合,改变Na+通道的功能,对于研究Na+通道的结构和功能以及研发作用于Na+通道的相关药物或其先导化合物具有重要作用。本文就近年来发现的作用于Na+通道的芋螺毒素的研究进展进行综述。     

中国南海织锦芋螺毒素的分离及鉴定

目的从中国南海织锦芋螺中分离新的芋螺毒素,为药物发现、药物设计提供新的活性多肽或前体多肽,比较不同地域织锦芋螺毒素的构成,研究环境对织锦芋螺毒素分泌的影响。方法织锦芋螺毒液经预处理后,凝胶层析分离粗毒,用HPLC进一步分离,氨基酸测序。结果分离得到了10余个织锦芋螺毒素组分,确定了10个织锦芋螺毒素的氨基酸序列,其中4个为新型织锦芋螺毒素,6个与已报道的相同。结论中国南海织锦芋螺毒液中含有丰富的M族芋螺毒素,毒素成分与菲律宾宿务岛(Cebu)、马尼拉岛的织锦芋螺相似。     

蚓激酶活性成分的分离纯化

目的本实验采用色谱分离的方法从赤子爱胜蚓（EiseniaFoelide）中分离纯化出一种纯度达到95％以上的蚯蚓纤溶酶。方法采用匀浆抽提、热变性、超滤、色谱分离、SDS—PAcE和高效液相色谱法等技术对蚓激酶进行分离纯化及鉴定,并用纤维平板法测定其活性。结果通过一系列纯化步骤所得的蚓激酶比活120000u／mg,经sDs—PAGE分析得一条带,相对分子量为28000Da;高效液相色谱分析其纯度达95％以上。结论本实验从蚯蚓中分离纯化出一种高活性、高纯度的蚓激酶。     

蚯蚓纤溶酶的分离纯化及抗栓活性研究

目的摸索蚯蚓纤溶酶的分离纯化工艺和研究蚯蚓纤溶酶的抗血栓活性。方法采用分步盐析、透析、凝胶色谱和亲和色谱法分离纯化蚯蚓纤溶酶 ;此酶的体内抗栓活性检测采用动静脉旁路血栓形成抑制实验法。结果分离纯化得到的蚯蚓纤溶酶比活高 ,为 16 0 5IU/mg ;蚯蚓纤溶酶的体内血栓形成抑制效果比尿激酶作用显著。 结论该法分离纯化工艺简单 ,特异性好 ,纯化的纤溶酶比活高 ;体内抗栓活性比尿激酶作用显著     

电压门控钠通道亚型及相关疾病的研究进展

目的综述近年国内外电压门控钠通道亚型及相关疾病的研究新进展,为今后相关疾病的诊断和治疗提供理论依据。方法通过检索国内外相关文献,从钠通道的类别、亚型及相关疾病等方面进行综述。结果钠通道的主要作用是参与动作电位的形成和信号传导,临床许多疾病的发生和发展与钠离子通道的改变有关。钠通道结构或功能异常通常会导致如心血管系统、神经系统和肌肉等相关疾病的发生。结论不同类型的钠通道在机体生理病理过程中的不同环节发挥着不同的作用,近期研究取得了很大进展,其在相关疾病诊断和治疗方面的应用前景值得期待。     

舟山眼镜蛇毒细胞毒素的分离纯化及其体外抗肿瘤活性

目的从眼镜蛇毒中分离纯化细胞毒素 F(CTX F)并鉴定其活性。方法应用凝胶过滤、离子交换柱色谱及疏水柱色谱等方法从舟山眼镜蛇毒中分离纯化CTX F ,以SRB法观察CTX F对体外培养癌细胞的杀伤作用。结果眼镜蛇毒粗毒经凝胶过滤获得 4个蛋白峰 ,将CTX所在第Ⅳ峰用阳离子交换柱色谱获A、B、C、D、E、F和G等7个组分 ,其中E、F和G具CTX活性 ,将F组分再经凝胶过滤和疏水色谱进一步纯化得不含磷酯酶A2 (PLA2 )的CTX纯品 ,暂定名为CTX F ,它对多种癌细胞株有杀伤作用。结论应用凝胶过滤、离子交换和疏水色谱等方法可从眼镜蛇毒中获得不含PLA2 的CTX ,其组分F有抗肿瘤活性     

4种ω-芋螺毒素的合成及活性研究

目的 合成新型ω-芋螺毒素,测定其作用靶标,获得新型镇痛分子并为N-型钙离子通道抑制剂设计提供依据。方法 采用固相法合成线性肽,然后进行折叠、富集和纯化得到纯肽;利用膜片钳技术,测定其对N、P/Q及L-型钙离子通道的抑制活性;利用金鱼测定毒副作用,采用热板法和醋酸扭体法测定活性高毒素的镇痛活性。结果 Ac6.4对N-型钙离子通道抑制活性高,IC50为0.42 μmol/L;C6.4、Castu-c2及Di7.5的抑制活性较低,10 μmol/L的C6.4、Castu-c2及Di7.5的抑制活性分别为（35.58 ± 12.32）%、（31.09 ± 12.24）%及（14.03 ± 4.84）%。该4种ω-芋螺毒素对P/Q、L-型钙离子通道的抑制活性低。1.0~3.0 μg/kg的Ac6.4显著增加小鼠对热板法的痛阈时间,1.0~10.0 μg/kg的Ac6.4显著减少小鼠对醋酸扭体次数,但其镇痛活性均低于齐考诺肽（MVIIA）。Ac6.4的金鱼的毒性显著低于ω-芋螺毒素MVIIA。结论 Ac6.4选择性抑制CaV2.2,且具有较高的镇痛活性及较低毒副作用。     

肝素钠精制工艺研究

目的：为改进肝素纳的精制工艺，运用离心技术和改进的丙酮脱水法，使肝素钠的回收率得到提高。方法：将肝素钠粗品制成10％左右的浓度，用盐酸调节pH，离心脱去大部分酸性蛋白，氧化后所得产物慢慢加入丙酮中脱水。精制后测定其回收率和效价并与传统方法进行对比。结果：回收率由传统工艺的（86.±0.9）％提高到（90.0±0.6）％，相关杂质的吸收度值下降；效价由传统工艺的（168.7±2.4）uspu·mg-1提高到（179.0±1.8）uspu·mg-1。与传统工艺比较，经t检验，回收率和效价P值均＜0.01。结论：这一方法确能提高肝素钠的质量。     

石杉碱甲的分离纯化工艺

目的:研究一种石杉碱甲的工业化分离纯化工艺,以达到批量生产之目的。方法:通过对石杉碱甲理化性质的分析,以中性氧化铝为填料对石杉碱甲粗品进行柱色谱分离纯化,采用薄层色谱-柱色谱考察了各因素的影响,以石杉碱甲回收率和含量为考察指标,寻找最佳工艺参数。结果:柱色谱最佳分离条件为:洗脱剂体系为氯仿-甲醇(v/v)=100∶5,100∶10,流速为3mL.min-1,柱床体积比为25∶1,进料量为10 mL,柱温为室温,在此条件下石杉碱甲回收率达97.6%,含量达99.2%。结论:该工艺条件能够在简单的条件下实现石杉碱甲批量分离纯化,具有工业应用价值。     

头孢哌酮钠他唑巴坦钠的体内抗菌作用研究

目的与方法　用Bliss法检测头孢哌酮钠他唑巴坦钠(4:0 5 )的抗菌活性 ,为临床研究和治疗提供依据。结果　头孢哌酮钠他唑巴坦钠 (4:0 5 )对产酶的金黄色葡萄球菌和埃希氏大肠杆菌感染的小鼠体内抗菌活性ED50 分别为 92 0mg·kg-1和 3 4mg·kg-1,头孢哌酮钠舒巴坦钠 (1∶1)的ED50 值为 16 4 0mg·kg-1和 6 9mg·kg-1,单组分头孢哌酮钠的ED50 值分别为 2 5 6 5mg·kg-1和 38 9mg·kg-1。头孢哌酮钠他唑巴坦钠 (4∶0 5 )对绿脓杆菌感染小鼠的体内抗菌活性ED50 为 10 3 3mg·kg-1,头孢哌酮钠舒巴坦钠 (1∶1)的ED50 为 16 5 2mg·kg-1。结论　国产头孢哌酮钠他唑巴坦钠对产酶的金黄色葡萄球菌、埃希氏大肠杆菌和绿脓杆菌感染小鼠有明显抗菌作用 ,且抗菌作用明显优于头孢哌酮钠单组分和海舒必     

Phencyclidine increases the affinity of dihydropyridine calcium channel antagonist binding in rat brain

Summary Phencyclidine (PCP) significantly reduces the apparent dissociation constant (K _D) of the dihydropyridine (DHP) calcium channel antagonist, [³H]nitrendipine, in synaptosomal membranes of rat and mouse brain without significantly effecting the maximum binding capacity (B _max). At an optimum concentration of PCP (10 M) the apparentK _D of [³H]nitrendipine was reduced from 178±9 pM to 112±9 pM in rat forebrain, a 58% increase in affinity. The structural derivatives of PCP, P-Br-PCP {1-[1-(4-bromophenyl-cyclohexyl)piperidine]}, m-NH₂-PCP {1-[1-(3-anilo)-cyclohexyl]piperidine}, (±)-PCMP [1-(1-phenyl)-cyclohexyl-3-methylpiperidine] also increased the apparent affinity of [³H]nitrendipine in the following order, p-Br-PCP PCMp>PCP>m-NH₂-PCP. Local anesthetics either reduced the apparent affinity of [³H]nitrendipine or had no effect. Kinetic analysis revealed that PCP both increased the microassociation rate constant and decreased the microdissociation rate contant of [³H]nitrendipine. The magnitude of this enhanced binding varied with the brain region studied; the greatest increase in apparent affinity of [³H]nitrendipine was observed in striatum, while no significant increase in affinity was observed in brainstem. In some brain areas, PCP was more effective in reducing theK _D in crude homogenates than in washed tissue. PCP (10 M) did not alter theK _D of [³H]nitrendipine to rat cardiac tissue. Both Ca²⁺ and Mg²⁺ inhibited the effect of PCP, while monovalent ions were ineffective in this regard. These data are consistent with an allosteric modulation of DHP calcium channel antagonist binding sites by PCP and structural derivatives that is not mediated through the brain PCP binding site. This modulation of DHP binding sites may account for some of the psychopharmacologic actions PCP and related compounds in vivo.     

The effect of ethanol extract of Glycyrrhiza uralensis on the voltage-gated sodium channel subtype 1.4

To investigate the inhibitory effect of Glycyrrhiza uralensis (G. uralensis) and its monomeric compounds on Nav1.4 voltage-gated sodium channels (VGSCs) and analyze the relationship between the content of its marker compounds and the inhibitory rate. Based on this study, we found that 4 mg/ml ethanol extract of G. uralensis at 30%, 50%, 70% and 90% (v/v) exhibited 77.00 ± 0.03%, 34.75 ± 0.09%, 100.00 ± 0.01% and 2.00 ± 0.01% inhibitory rates on I_Nav1.4 respectively, and 8 mg/ml ethanol extract of G. uralensis at 30%, 50%, 70% and 90% (v/v) exhibited 99.00 ± 0.01%, 97.10 ± 0.02%, 100.00 ± 0.01% and 17.00 ± 0.04% inhibitory rates on I_Nav1.4 respectively. Isoliquiritigenin, echinatin, liquiritin and glycyrrhizic acid exhibited higher inhibitory rates of 39.98 ± 4.55%, 33.20 ± 1.61%, 22.62 ± 0.30% and 20.54 ± 4.82% respectively. However, liquiritigenin, formononetin, neoisoliquiritin and glycyrrhetinic acid exhibited lower inhibitory rates of less than 20%. Further, liquiritin apioside, isoliquiritin and neoliquiritin exhibited almost no effect on I_Nav1.4. These findings showed that glycyrrhizic acid reached a maximum concentration of 49.15 μg/ml, while echinatin had the lowest concentration. The ethanol extract of G. uralensis has significant inhibitory effects on Nav1.4 VGSCs. This may be an important mechanism in the treatment of gastrocnemius spasm and could guide further research regarding material basis and mechanism of the treatment of gastrocnemius spasm with peony and licorice decoction.     

克隆的人神经母细胞瘤细胞株鲀毒素抵抗型钠通道的功能分析

目的 作者已经克隆了人神经母细胞瘤（NB-1）细胞株鲀毒素抵抗型（TTX-R）钠通道α亚单位及其选择性剪切体，分别命名为hNbR1和hNbR1-2，本实验观察这两个钠通道同型异构体的电生理特性。方法 使用全细胞膜片钳技术在转染了hNbR1和hNbR1-2的人胚肾细胞（HEK-293）上记录这两个钠通道的钠电流。结果 这两个变构体在稳态激活和失活方面略微不同，hNbR1和hNbR1-2的半数激活电压分别为－(37.8±1.3)和－(43.5±1.7)mV，半数失活电压分别为－(74.4±1.1)和－(81.5±1.1)mV，hNbR1-2的稳态激活和稳态失活曲线较hNbR1分别负向移位5.7 mV和 7.1 mV。TTX阻断hNbR1和hNbR1-2的IC₅₀分别为(7.9±2.3)和(8.3±2.5)μmol·L^-1，二者对TTX的敏感性无显著性差异。结论 两个钠通道变构体hNbR1和hNbR1-2均可在HEK-293细胞上表达并产生钠电流，二者在稳态激活和失活方面略微不同，对TTX的敏感性无显著性差异。     

克隆的人神经母细胞瘤细胞株鲀毒素抵抗型钠通道的功能分析

目的作者已经克隆了人神经母细胞瘤(NB-1)细胞株毒素抵抗型(TTX-R)钠通道α亚单位及其选择性剪切体,分别命名为hNbR1和hNbR1-2,本实验观察这两个钠通道同型异构体的电生理特性。方法使用全细胞膜片钳技术在转染了hNbR1和hNbR1-2的人胚肾细胞(HEK-293)上记录这两个钠通道的钠电流。结果这两个变构体在稳态激活和失活方面略微不同,hNbR1和hNbR1-2的半数激活电压分别为-(37.8±1.3)和-(43.5±1.7)mV,半数失活电压分别为-(74.4±1.1)和-(81.5±1.1)mV,hNbR1-2的稳态激活和稳态失活曲线较hNbR1分别负向移位5.7mV和7.1mV。TTX阻断hNbR1和hNbR1-2的IC50分别为(7.9±2.3)和(8.3±2.5)μmol·L-1,二者对TTX的敏感性无显著性差异。结论两个钠通道变构体hNbR1和hNbR1-2均可在HEK-293细胞上表达并产生钠电流,二者在稳态激活和失活方面略微不同,对TTX的敏感性无显著性差异。     

Epilepsy and sodium channel blockers

Epilepsy is one of the most frequent neurological diseases and although most patients nowadays respond well to anti-epileptic drugs (AED), 30 - 40% of them still present seizures despite treatment with 2 or more AED. This illustrates the need for discovery and testing of new molecules designed for specific brain targets. Several voltage-gated Na⁺ channel gene mutations were shown to be associated with genetic and hereditary epilepsy. Thus, while it is noteworthy that some currently available AED act through Na⁺ channels, the identified mutations in Na⁺ channel genes strongly support the development of new Na⁺ channel blockers used as AED. This review focuses on the pathophysiology of epilepsy and particularly on insight gained from the identification of mutations in idiopathic epilepsy (IE). All of the IE mutations identified concern genes coding for ion channels, suggesting a possible role of these integral membrane proteins in epileptogenesis. The recent identification of mutations in genes encoding the α1, α2 and β1 subunits of the voltage-gated Na⁺ channel (SCN1A, SCN2A, SCN1B) and their association with febrile seizures (FS) further illustrates the contribution of these channels in epilepsy. Modes of action of AED supposed to act through the voltage-gated Na⁺ channels are examined in an attempt to propose alternative routes to discover new active compounds.     

Inhibition of brevetoxin binding to the voltage-gated sodium channel by gambierol and gambieric acid-A.

Brevetoxins (BTXs) and ciguatoxins (CTXs) bind to site 5 of the voltage-gated sodium channel of excitable membranes. In the present study, we performed a competitive inhibition assay with other structurally distinct naturally occurring polyethers using isotope-labeled dihydro BTX-B ([3H]PbTx-3), which showed, for the first time, that gambierol and gambieric acid-A inhibit the binding of [3H]PbTx-3 while yessotoxins are inactive in this assay. The inhibition assay also suggested that there is a significant relationship between the size of the polycyclic region and inhibitory activity. Interestingly, the acute mouse toxicities of the compounds do not correspond directly to their inhibitory activities. These observations will serve as a guide for designing artificial polyethers with desired activity.