发形霞水母触手提取物的蛋白稳定性及其溶血活性

目的探讨不同环境因素和处理方法对发形霞水母（Cyanea capillata）触手提取物（tentacle extract,TE）蛋白稳定性及其溶血活性的影响。方法结合蛋白浓度测定、溶血活性检测和SDS-PAGE分析等方法研究不同环境因素和处理方法对TE蛋白稳定性及其溶血活性的影响。结果 TE溶血活性呈明显的剂量依赖关系,其半数溶血分数HU50=226μg/ml;40℃水浴1 h,能去除TE中大量杂蛋白,但仍保持显著溶血活性;TE在4℃放置28 d,其溶血活性保持稳定,25℃下3 d内其溶血活性变化不明显;p H值对TE溶血活性的影响呈钟形曲线,在p H 6.0-11.0之间活性相对稳定,p H 8.0是TE保持溶血活性的最优p H条件;不同缓冲液对TE稳定性及其溶血活性影响显著,浓度大于26%的硫酸铵溶液对TE溶血蛋白具有良好的盐析作用。结论 40℃水浴1 h预处理可显著减少TE中非活性蛋白组分,并降低样品黏性;4℃和p H 8.0是TE保持蛋白稳定性和溶血活性的最适条件;浓度大于26%的硫酸铵盐析有利于溶血蛋白组分富集。     

蛋白酶抑制剂对霞水母毒素溶血活性的影响

目的研究蛋白酶抑制剂对霞水母毒素溶血活性的影响。方法首先制备霞水母刺丝囊细胞,再利用Mini-Beadbeater组织研磨器破碎细胞提取毒素,然后向获得的霞水母毒素中加入不同种类及剂量的蛋白酶抑制剂,如金属酶抑制剂EDTA、酸性蛋白酶抑制剂Pepstantin A、丝氨酸蛋白酶抑制剂PMSF和Aprotinin,巯基蛋白酶抑制剂Leupeptin,并且测定蛋白酶抑制剂对霞水母毒素溶血活性的影响。结果 EDTA和Pepstantin A能够明显抑制蛋白酶的活性从而增强霞水母毒素的溶血活性,1 mmol.L-1EDTA和4μg.ml-1 Pepstantin A使其溶血率分别从40%上升到93%和5%上升到78%;而PMSF、Aprotinin和Leupeptin却对霞水母毒素溶血活性的影响较小。结论金属蛋白酶抑制剂EDTA和酸性蛋白酶抑制剂Pepstantin A能有效地保护霞水母毒素的溶血活性,为深入研究霞水母毒素提供帮助。     

发形霞水母毒素溶血活性研究

目的 探讨发形霞水母毒素(Cyanea capillata full venom,CFV)的体外溶血活性及其影响因素.方法 超声破碎法分离得到CFV,测定其半数溶血分数(HU50),探讨温度、pH、放置时间对溶血活性的影响,并进一步研究不同动物红细胞对溶血活性的敏感性.结果 CFV溶血活性的HU50为5.82 μg·mL-1;溶血活性对温度和pH变化耐受性差,在40℃和pH 8.0时有最大溶血活性;25℃和37℃放置12 h CFV溶血活性迅速下降至18.6%和17.4%;对不同动物红细胞敏感性差异较大,其中以家犬红细胞最为敏感.结论 CFV有显著的溶血活性.     

发形霞水母共附生微生物的分离鉴定及其发酵液的抑菌活性研究

目的对发形霞水母(Cyanea capillata)共附生微生物进行分离鉴定并测定其抑菌活性,以期获得具有较高抑菌活性的菌株。方法利用4种分离培养基从C.capillata各部位分离共附生微生物,首先对挑选的单个菌落进行反复的划线分离,获得各菌株的纯培养;利用形态学观察、生理生化特征检测和16SrDNA序列测定和分析等手段对获得的菌株进行鉴定;以金黄色葡萄球菌、大肠杆菌、枯草芽孢杆菌作为指示菌,利用滤纸片扩散法测定菌株发酵液的抑菌活性;通过正交试验方法进一步优化发酵条件,提高菌株发酵产生抑菌活性物质的能力。结果从C.capillata触手、胃囊、伞部、肌肉4个部位共分离出83株菌株,其中放线菌...     

地龙中镇痛活性物质的分离纯化与表征

目的分离纯化并表征地龙中镇痛活性物质。方法采用硫酸铵高渗液提取活地龙粗提液,比较交联葡聚糖（Sephadex G15,G25,G50）凝胶过滤层析的分离效果,通过小鼠缩足潜伏期（PWTL）和甩尾潜伏期（TFL）实验对G25分离所得的4个主要色谱峰（P1、P2、P3、P4）进行镇痛活性表征,并对具有活性的P4进行反相高效液相色谱（HPLC）分析。结果Sephadex G25的分离效果较好,具有显著镇痛活性的组分为P4,其镇痛效果与阳性对照扶他林乳胶剂相当;HPLC主要色谱条件：乙腈-去离子水为流动相,梯度洗脱（20min内乙腈梯度由0升高至100%）,紫外检测波长215nm,此时可得5个主要色谱峰,色谱峰分离度及峰形良好。结论地龙提取物具有显著的镇痛活性。     

蝰蛇(缅甸亚种)毒促凝—纤溶双相活性组分FⅥbb的分离纯化及生物活性

目的从蝰蛇(缅甸亚种)毒分离纯化一种促凝—纤溶双相活性组分FⅥbb,并研究其理化性质和生物活性。方法应用CM-Sephadex C-50阳离子交换层析Sephadex G-150(超细)凝胶过滤层析Chelating Sepharose Fast Flow金属离子螯合亲和层析分离纯化蛇毒,反相HPLC检测组分FⅥbb纯度,采用MALDI质谱测定法测定分子量,以发色底物法、纤维平板法和SDS-PAGE测定FⅥbb的酶学特征和生物活性。结果从蝰蛇(缅甸亚种)毒分离得到的促凝—纤溶双相活性组分FⅥbb为单体蛋白,相对分子质量为59 138,最适温度为40℃,最适pH为10.0,为金属蛋白酶。结论应用CM-Sephadex C-50阳离子交换层析和Chelating Sepha-rose Fast Flow金属离子螯合亲和层析等方法可以从蝰蛇(缅甸亚种)毒纯化出促凝—纤溶双相活性组分FⅥbb,具有促凝—纤溶双相活性。     

叶下珠抗补体活性研究

目的研究叶下珠的抗补体活性及活性成分。方法以活性筛选跟踪为向导,利用溶剂萃取和色谱方法对叶下珠提取物进行分离纯化,采用~(13)C-NMR波谱法结合文献数据鉴定化合物结构,测定其抑制补体的活性及可能的机制。结果筛选出叶下珠醇提取物的乙酸乙酯部位为抗补体活性部位,从中分离得到2个抗补体活性成分,经鉴定为柯里拉京和鞣花酸。乙酸乙酯部位和2个化合物均明显抑制补体经典途径的溶血活性,其IC50分别为53.77、176.54、102.23 mg·L~(-1),但对补体旁路途径溶血的抑制作用不明显。进一步研究表明,乙酸乙酯部位和2个化合物干扰了补体经典途径成分C1、C2、C4形成C3转化酶。结论叶下珠的多酚组分是其主要的抗补体活性成分,其机制与这些组分影响经典途径C3转化酶的形成有关。     

小续命汤氯仿提取物的极性分离及其活性组分的筛选

目的:筛选、分离中药复方小续命汤的活性组分.方法:小续命汤氯仿提取物进行极性划断,用MTT的方法筛查氯仿提取物中5组分不同浓度分别对HEK293细胞、PC12细胞活力的影响.结果:氯仿提取物分离得到(A-B-C-D-E)5个组分.A组分不溶于DMSO去除;B 组分在浓度为2.5ug/ml 到100ug/ml时可能促进P...     

广东紫珠超临界提取物的GC-MS成分分析及体外抗菌活性

采用超临界CO2萃取法提取广东紫珠低极性成分,应用气相色谱-质谱法分析提取物的化学组成,结合计算机检索技术初步鉴定所分离的化合物,应用色谱峰面积归一化法计算各成分的相对百分含量,并考察各分离釜提取物对金黄色葡萄球菌、大肠杆菌和白色念珠菌的体外抗菌活性。结果从超临界CO2流体萃取分离釜Ⅰ提取物中初步鉴定了27个化合物,占总离子流图峰面积的81.12%;超临界CO2流体萃取分离釜Ⅱ提取物初步鉴定了20个化合物,占总离子流图峰面积的74.60%。广东紫珠超临界CO2萃取分离釜Ⅰ、Ⅱ提取物及二者1︰1混合物对金黄色葡萄球菌、大肠杆菌和白色念珠菌显示出抗菌活性。     

桑枝颗粒提取物对α-葡萄糖苷酶的抑制作用研究

目的初步分离桑枝颗粒提取物中对α-葡萄糖苷酶的抑制活性成分。方法利用超滤工艺对桑枝颗粒提取物进行纯化。结果在同样的样品浓度下,桑枝颗粒提取物中相对分子量在5×105D～10×105D的组分的抑制活性最高,抑制率达68.0%。结论相对分子量在5×105D～10×105D的组分对α-葡萄糖苷酶的抑制活性最高。     

Direct cardiac toxicity of the tentacle-only extract from the jellyfish Cyanea capillata demonstrated in isolated rat heart

Previous studies in our laboratory have shown that the cardiotoxicity is the main reason for rat death caused by tentacle-only extract from jellyfish Cyanea capillata. However, the direct cardiotoxicity in vitro and its mechanisms of toxic action remain unclear. The current studies were performed by using the Langendorff-perfused isolated heart model, which showed a dose-dependent hemodynamic and electrocardiogram changes. Heart injury-related enzymes increased. Histopathological analysis showed early ischemic damage in the myocardium. The Ca channel blockers nifedipine and verapamil led to a marked improvement in recovery of cardiac function, including heart rate, left ventricular developed pressure, positive and negative first derivatives of intraventricular pressure, coronary flow, left ventricular end-diastolic pressure, and electrocardiogram changes. Tentacle-only extract-induced cardiac dysfunction could be partly improved by the pretreatments of both propranolol and phentolamine, but not by either atropine or neostigmine at all. In conclusion, we have verified the direct cardiotoxicity of tentacle-only extract from jellyfish C. capillata by the Langendorff isolated heart model, which consisted of 3 separate parts: sinoatrial node malfunction, cardiomyocyte injury, and coronary spasm. The potential mechanism might be attributed to the overactivation of L-type Ca channel, β- and α-adrenergic receptors, but not cholinergic receptors.     

发形霞水母触手提取物对大鼠离体心脏传导系统的影响

目的观察水母触手提取物（TOE）对大鼠离体心脏传导功能的影响,探讨其心脏毒性可能的作用机制。方法72只SD雄性大鼠随机分为6组（n=12）,利用大鼠离体心脏Langendorff模型,分别给予不同剂量的TOE,观察其对大鼠心脏传导系统的影响。结果给予TOE后,与空白组比较,心率减慢和心律失常的几率明显增大,并呈现剂量依赖性。结论TOE对心脏的传导系统具有显著影响,是其心脏毒性的原因之一。     

In vitro and in vivo haemolytic studies of tentacle-only extract from jellyfish Cyanea capillata

To approach the real haemolytic process of jellyfish toxins, both in vitro and in vivo haemolysis of tentacle-only extract (TOE) from jellyfish Cyanea capillata has been studied. Dose–response curves of the haemolytic activity of TOE in vitro were sigmoid shaped in both erythrocyte suspension and diluted whole blood, with the former more sensitive to TOE. The in vivo haemolysis increased sharply in the first 10 min and was followed by a gradual increase in the following 3 h, with increasing blood potassium and lactic acid accordingly. SC5b-9 complexes were significantly up-regulated in vitro, but not in vivo. These results showed that the haemolysis of TOE in diluted whole blood and in vivo is not totally consistent with that in the erythrocyte suspension, and blood plasma might play a protective role against haemolysis. Thus we suggested that erythrocyte suspension can be used to test the damage of toxin on erythrocyte membrane, while the diluted whole blood may be more suitable to test the haemolysis of toxins.     

Cyanea capillata tentacle-only extract as a potential alternative of nematocyst venom: Its cardiovascular toxicity and tolerance to isolation and purification procedures

As the proteins with cardiovascular toxicity in jellyfish nematocyst venom and tentacle-only extract (TOE) are probably encoded by the same gene, TOE provides a potential alternative of nematocyst venom with much richer source for acquisition of such proteins. In this study, Cyanea capillata nematocyst venom and TOE (5 mg/kg) both exhibited cardiovascular toxicity in rats, and TOE caused blood pressure reduction in slightly greater amplitude than nematocyst venom 3 min after intravenous administration. SDS-PAGE suggested high likeliness that they both contained the same bioactive protein. The activity of TOE was dose-dependent within 1.25-5 mg/kg, but not at higher concentrations. The cardiovascular activity of TOE sustained a major loss after exposure to 60 °C, and was totally abolished after exposure to 80 °C. Within the pH range of 7-11, the activity of TOE was well preserved, and rapidly attenuated in pH below 5. At 4 °C, TOE lost cardiovascular toxicity after preservation for 7 days, which occurred only after an 8-h preservation at 20 °C. Repeated freeze-thawing and freeze-drying did not significantly affect the toxicity of TOE. Buffer solutions obviously affected the toxicity of TOE, and 0.02 mol/L HAc (pH 6.0) was optimal. These results provide experimental data for optimizing the conditions for isolating the proteins with cardiovascular toxicity from jellyfish TOE, which serves as a promising alternative source of nematocyst venom.     

The lethality of tentacle-only extract from jellyfish Cyanea capillata is primarily attributed to cardiotoxicity in anaesthetized SD rats

Previous studies in our laboratory have shown that tentacle-only extract (TOE) has similar hypotensive effects with nematocyst venom from jellyfish Cyanea capillata, and the experimental studies on the in vivo cardiovascular effects of TOE were further performed to explore the leading cause of death and analyze the basic physiopathologic change in anaesthztized SD rats. Plots of TOE dose versus time to death showed dose-dependent curvilinear relationship. ECG changed in a dose- and time-dependent manner. Haemodynamic parameters, including the heart rate, mean femoral arterial pressure, left ventricular developed pressure and the first derivative of left ventricular pressures, decreased, but left ventricular end-diastolic pressure did not increase. Arterial partial pressure of oxygen and oxygen saturation did not change. Lactate dehydrogenase, creatine kinase and MB isoenzyme of creatine kinase increased significantly. Histopathological examination showed congestion, haemorrhage, edema and denaturation in the heart; congestion, haemorrhage in the lung and acute congestion in the liver. Transmission electron microscopy examination found that parts of sarcomeric filaments disrupted, dissolved and disappeared, and parts of mitochondria swelled in cardiocytes. Laser scanning confocal microscope examination found that ventricular myocytes from adult rat were deformed and ultimately died within 30 min after TOE treatment. Our results reveal that cardiodepressive effect of C. capillata TOE is the leading cause of death and acute total heart failure is the basic physiopathologic change in anaesthetized SD rats.     

低剂量番泻叶预防肺癌患者化疗后便秘疗效观察

目的 探讨低剂量番泻叶预防肺癌患者化疗后便秘疗效.方法 80例肺癌患者,采用随机,交叉法将患者分为A、B 两组,A 组第一周期化疗同时服用低剂量3 g番泻叶组,第二周期单纯化疗,B组第一周期单纯化疗,第二周期在化疗同时服用低剂量3 g番泻叶组,自身交叉对照.比较番泻叶对A,B组化疗后缓解便秘的有效率;观察服用番泻叶后患者腹痛率情况.结果 A组患者预防使用低剂量番泻叶对便秘的有效率达90%,而不预防使用对便秘有效率仅15%,两者比较差异有统计学意义(P< 0.05).B组患者预防使用低剂量番泻叶对便秘的有效率达92.5%,而不预防使用对便秘有效率仅17.5%,两者比较差异有统计学意义(P<0.05).A组患者预防使用低剂量番泻叶,而B组患者不预防使用,A组对便秘的有效率达90%,B组队便秘的有效率仅17.5%,两者比较差异有统计学意义(P<0.05).结论 低剂量番泻叶预防肺癌化疗后便秘具有确切疗效.     

新疆胀果甘草粗提物和精制物对宫颈癌细胞的影响

目的研究新疆胀果甘草(Glycyrrhiza inflata Batalin)粗提物和精制物体外抗宫颈癌的活性。方法利用超声辅助乙醇对新疆胀果甘草进行粗提取,得到粗提物后,进一步利用大孔树脂柱层析法对粗提物进行精制,得到精制物,并利用紫外分光光度法对粗提物及精制物总黄酮的含量进行测定,测得粗提物总黄酮含量为5.6%,精制物总黄酮含量为56.4%。将粗提物和精制物分别作为试药,顺铂为阳性药。采用人宫颈癌HeLa和SiHa细胞作为体外实验对象,用MTT法测定对宫颈癌细胞抑制率,用流式细胞仪测定胀果甘草精制物对人宫颈癌细胞的凋亡作用。结果新疆胀果甘草粗提物和精制物均能明显抑制宫颈癌HeLa和SiHa细胞的生长,且2种药的抑制效应呈时间和质量浓度依赖性,但在同一质量浓度下,精制物的抑制率更高。流式细胞测定结果显示,甘草粗提物、精制物能够显著诱导宫颈癌HeLa和SiHa细胞凋亡。结论新疆胀果甘草粗提物和精制物能够有效抑制宫颈癌细胞增殖,促进宫颈癌细胞凋亡,且精制物的抗癌活性明显优于粗提物。     

Bronchodilating and Uterine Activities of Ageratum conyzoides Extract

Ageratum conyzoides root and aerial part extracts induced relaxation on isolated trachea. There was no significant difference between the activities of root and aerial part extracts. When Histamine (hist) and the plant extract were introduced into the tissue bath simultaneously, 14% of the activity of the plant extract was inhibited. When the same procedure was used, 5-hydroxytryptamine (5-HT) inhibited 21% of the plant extract activity. Conversely, 86% and 79% activities of hist and 5-HT, respectively, were inhibited by the plant extract. This could be the mechanism of its activity as a tracheal relaxant. Acetylcholine (ACh) and 5-HT induced contractions on isolated rat uterus. The treatment of isolated rat uterus with the plant extract inhibited uterine contractions induced by 5-HT, suggesting that the plant extract exhibited specific antiserotonergic activity on isolated uterus. However, the uterine contraction caused by ACh was unaffected by the plant extract.     

Pharmacological Activities of Vernonia glabra

A 70% methanol aerial part extract of Vernonia glabra caused a fall in blood pressure in anaesthetised rats. After the 50 µg dose, systolic, diastolic, and mean blood pressures decreased by 56%, 66%, and 61% respectively below the baseline levels. The adrenaline-like activity of the extract was not antagonised by L(-) propranolol on isolated rabbit heart. The extract caused contraction on the isolated rabbit ileum and was abolished with atropine, suggesting the presence of a compound with acetylcholine-like activity. The extract on isolated guinea pig ileum caused contraction which was dose related.     

加入胡桃醌与敲除胡桃醌的胡桃楸水提物的抗肿瘤活性

目的明确胡桃醌对胡桃楸抗肿瘤作用的贡献。方法将胡桃醌及胡桃楸所有成分作为整体考虑,利用胡桃醌易挥发的性质,通过水煎煮敲除胡桃醌,比较加入和敲除胡桃醌的胡桃楸水提物在小鼠体内的抗肿瘤活性。采用荷H22肝癌小鼠模型,以瘤质量、抑瘤率、免疫器官指数和外周血常规参数评价不同样品的抗肿瘤活性及对荷瘤机体的影响。结果加入胡桃醌的胡桃楸水提物低、高剂量组能显著抑制小鼠H22肝癌细胞的生长,抑瘤率分别为31.96%和50.61%,呈量效关系。敲除胡桃醌的胡桃楸水提物低、高剂量组的抑瘤率分别为37.01%和12.35%,不呈量效关系。胡桃楸水提物中加入胡桃醌高剂量后,抑瘤率显著提高;加入胡桃醌低剂量后能改善荷瘤小鼠红细胞数量的异常;敲除胡桃醌高剂量组能改善荷瘤小鼠淋巴细胞百分比和中性粒细胞百分比的异常;其余各胡桃楸提取物对荷瘤小鼠免疫器官和外周血常规的异常无明显改善作用。结论胡桃楸水提物具有较好的抗肿瘤活性;胡桃醌高剂量组对胡桃楸水提物的抗肿瘤作用贡献较大。