Molluscicidal Activity of Nerium indicum Mill,Pterocarya stenoptera DC,and Rumex japonicum Houtt on Oncomelania hupensis

Objective To evaluate the molluscicidal activities of three Chinese plants N. indicum Mill, P. stenoptera DC, and R. japonicum Houtt, and to clarify the molluscicidal mechanism. Methods N-butanol extracts and water extracts of the three plants were obtained. The reactions of EST isozyme, glycogen and total protein of snails to the plant extracts were studied. Results EST electrophoresis showed that EST was an important antidotal enzyme system and reacted strongly to environment EST changed greatly during the whole exposure period so that it could be viewed as a pathological index of toxicity. Extracts decreased the glycogen content of the snails＇ soft tissues greatly, and also the protein content. ConcIusion All extracts show strong molluscicidal activity. The LD50 value of the water extract of N. indicum Mill is as low as 13.2 mg/L. EST can be viewed as a pathological index of toxicity. The energy metabolism abnormity is the key reason fnr the molluscicidal activities. The biochemical mechanism needs further research.

Reaction of Oncomelania Hupensis to the Allelopathic Triterpene Sapogenin from Nerium Indicum

This paper has analyzed allelopathic effects ofNerium indicum on Oncomelania hupensis through triterpene sapogenins, a potential molluscicide. The snails were treated under six various concentrations （0, 20, 40, 60, 80, 100 mg/L） of triterpene sapogenins and five periods （1, 2, 3, 4, 5 days）. The mortality of snails was positively correlated with the concentration of triterpene sapogenins and exposure time. The results ofprobit analysis showed that the LD50 （Lethal Dose, 50%） oftriterpenoid saponins from N. indicum by immersion for 2, 3, 4, 5 days were 78.31, 30.26, 20.50, 14.19 mg/L, respectively. And the corresponding 95% confidence intervals were 63.60-108.19, 9.49-44.42, 2.86-30.90, 0.23-22.79 mg/L, respectively. The observations of both scanning electron microscope and transmission electron microscope proved that 40 mg/L triterpene sapogenins could cause apparent damage to the structure of soft tissue, liver and intestine of O. hupensis. The esterase （EST） isozyme electrophoresis in liver of O. hupensis treated by 40 mg/L of the concentrations lixivium of the triterpene sapogenins from N. indicum was analyzed for 24, 48, 72, 96, 120 h, respectively. The activity of enzyme was higher than control water group after been treated up to 24-48 h, and then lowered and disappeared after 72 h. It was implicated that the extracted triterpene sapogenin from N. indicum were promising for controlling the snail, which were also providing the foundation for constructing plant community of oleander to control O. hupensis.

Insecticidal Effect of Water Extract of Oleander (Nerium indicum) against the Larvae of Enmonodia vespertilio Fabriciu

[Objective] The paper was to study the insecticidal effect of water extract of oleander （Nerium indicum） against the larvae of Enmonodia vespertilio Fabriciu. [Method] Through feeding experiment on the larvae of E. vespertilio, the insecticidal effect of water extract of oleander against the larvae was studied. [Result] The 1/20-1/200 times water extract of dry leaf weight of oleander had better insecticidal effect against the larvae of E. vespertilio, and the feeding mortality rate reached 100%. [Conclusion] The paper provided reference for development of new botanical pesticides with high efficiency, environmental protection and low carbon.

Reduction in Fertility by Nerium indicum Leaves Extract in Adults of Red Flour Beetle, Tribofium castaneum （Coleoptera： Tenebrionidae） Compared with Coopex （Bioallethrin： Permethrin）

Recent trend for the control of insect pests has been towards the use of substances of plant origin. Reductionin fertility was determined in red flour beetle, Tribolium castaneum （Herbst.） by Nerium indicum Millleaves extract compared with coopex applied by contact method under laboratory condition. When adults were placed on filter paper treated With 7.86, 9.43, 11.01, 12.58 and 14.15 μL/cm2 doses of N. indicum leaves extract, fertility was 154 ± 15.2, 139 ± 15.0, 126 ± 6.0, 89 ± 12.2 and 72 ± 13.6 larvae/pair, compared with untreated and controls fertility （233 ± 16.0 and 190 ± 15.1 larvae/pair, respectively）. However, the reduction in fertility was 33.2 ± 6.4%, 39.7 ± 3.7%, 45.7 ± 2.9%, 58.0 ± 4.4% and 66.3 ± 5.1% by the same doses ofN. indicum extract. Moreover, adults treated with the same doses of coopex with the same method, fertility was 160 ± 14.6, 154 ± 9.3, 140 ± 11.2, 113 ± 7.5 and 71 ±- 4.8 larvae/pair, compared with controls fertility （211 ± 8.5 larvae/pair）, respectively. Furthermore, reduction in fertility was 24.3 ± 5.2%, 30.2 ± 6.4%, 37.2 ± 8.0%, 51.3 ± 1.4% and 58.8 ± 4.7% by the same doses of coopex. Minimum fertility was found in August and maximum was in July in both, N. indicum extract and coopex treated batches. The trend of reduction in fertility was the same in both N. indicum leaves extract and coopex, i.e., it was increased gradually from lower to higher doses. Nerium indicum leaves extract reduced fertility significantly compared with coopex.

New indole derivatives from endophytic fungus Colletotruchum sp. HK-08 originated from leaves of Nerium indicum

Objective: To study secondary metabolites from endophytic fungus Colletotruchum sp. HK-08 originated from the leaves of Nerium indicum.Methods: The compounds were isolated by various column chromatographic techniques, and their structures were elucidated by spectroscopic techniques [high resolution electrospray ionization mass spectroscopy(HRESIMS), one-dimensional(1D) and two-dimensional(2D) nuclear magnetic resonance spectroscopy(NMR)], as well as comparison with literature data. The Ellman method was used to determine the acetylcholinesterase(ACh E) inhibitory activity.Results: Four indole derivatives were identified from Colletotruchum sp. HK-08, including 6’-hydroxymonaspiloindole(1), 2-(2-oxoindolin-3-yl)ethyl 2-(4-hydroxyphenyl) acetate(2), 2-(2-oxoindolin-3-yl)ethyl 2-(2-hydroxyphenyl)acetate(3), and monaspiloindole(4). Compound 4 presented weak ACh E inhibitory activity with IC50value of(69.30 ± 6.27) μmol/L [tacrine as the positive control, with IC50value of(0.61 ± 0.07) μmol/L].Conclusion: Compounds 1–3 were new compounds, and compound 4 had weak ACh E inhibitory activity.

盐胁迫下夹竹桃生理光合特性及生态修复应用

海洋盐分主要通过地下水入侵、浪花飞溅、盐雾等方式对滨海植物的根、茎和叶产生影响。采用长势良好2年生夹竹桃(Nerium indicum),分别设置对照0%、0.3%、0.6%、1%、2%、3%多个浓度梯度,开展根系和叶片耐盐实验。盐胁迫处理13周后正常浇水4周。在盐胁迫末期及正常浇水后测定叶片净光合速率(Pn)、蒸腾速率(Tr)、气孔导度(Gs)、水分利用率(WUE)等光合指标;同时测定细胞膜透性、叶片饱和水分亏缺(WSD)等理化指标。结果表明:根系盐胁迫和叶片盐胁迫均会影响夹竹桃植株形态和生长,根系盐胁迫≥1.0%时,夹竹桃植株生长状态向着小于2的趋势发生不可逆下降;叶片盐胁迫≤3.0%时,夹竹桃生长状态普遍大于2。根系盐胁迫极显著降低夹竹桃叶片Pn、Tr和Gs;叶片盐胁迫对夹竹桃植株的Pn和WUE影响极显著,对Tr和Gs无显著影响。夹竹桃Tr与Gs之间极显著相关;高浓度根系盐胁迫下,气孔因素对夹竹桃光合影响较为显著。叶片盐胁迫下,夹竹桃细胞膜透性和WSD变化不显著,高浓度根系盐胁迫下,夹竹桃细胞膜透性和WSD发生不可逆改变;这与生长状态和光合特性的结果可以很好对应,说明膜系统受损反过来影响夹竹桃生长和光合作用。总体上,夹竹桃可耐受3.0%以下的叶片盐胁迫和1.0%以下的根系盐胁迫。将根系和叶片耐盐特性分别研究,可以更好地为夹竹桃在滨海生态和植被修复应用中提供理论指导。

夹竹桃和龙须藤合剂对正常大鼠的痛觉调节作用

为研究夹竹桃和龙须藤合剂对正常大鼠的痛觉调节作用,选取6只雄性Wistar大鼠,提前进行适应性训练,以灌胃生理盐水作为夹竹桃和龙须藤合剂的对照组,合剂配置质量浓度为5、20、40 mg/mL,对大鼠进行灌胃,并分别在5、10、15、20、30、45、60 min分别测定大鼠后爪缩爪反应潜伏期(Hindpaw Withdrawal Latency, HWL)。结果表明,5 mg/mL夹竹桃与龙须藤合剂灌胃后大鼠右爪热刺激HWL与对照组相比有极显著差异(P<0.01),左爪机械刺激HWL与对照组相比有显著差异(P<0.05);20 mg/mL夹竹桃与龙须藤合剂灌胃后大鼠左爪热刺激与机械刺激HWL与对照组相比有显著差异(P<0.05);40 mg/mL夹竹桃与龙须藤合剂灌胃后大鼠左右爪热刺激HWL与对照组相比有显著差异(P<0.05),右爪的热刺激HWL与对照组相比有显著差异(P<0.05)。夹竹桃和龙须藤合剂能够延长正常大鼠的HWL,表明合剂具有镇痛作用,且在15 min效果达到最佳,即夹竹桃和龙须藤合剂参与了正常大鼠的痛觉调节作用。

夹竹桃强心总甙灭螺活性与机理

血吸虫病（Schistosomiasis）是严重危害人民身体健康的疾病.钉螺是血吸虫的唯一中间寄主,是血吸虫生活周期中最脆弱环节,灭螺能有效控制血吸虫病,保护湿地和水资源.按强心总甙提取方法,用有机溶剂提取,从118.1940g（相当于100g干材料）夹竹桃新鲜叶中得到0.9815g夹竹桃强心总甙.将强心总甙按5、10、15、20、25、30mg/L配制成6个不同浓度梯度的处理溶液处理钉螺.取活钉螺100只为1组,每20个装入一个尼龙网袋中,分别将各组（5袋/组;网孔2mm）钉螺浸入盛有2000g不同浓度的夹竹桃强心总甙灭螺剂水溶液的玻璃缸中,设1mg/L氯硝柳胺水溶液和无氯清水对照.分别于处理1、2、3、4、5d后,将各处理中的钉螺随机取出1袋,用无氯水冲洗数次,再放入清水中静置1d,然后作存活检查：在解剖镜下针刺无反应的证实死亡.结果显示夹竹桃强心总甙具有很好的毒杀钉螺活性,用20mg/L的夹竹桃强心总甙的水溶液处理3～4d的效果与1mg/L氯硝柳胺溶液处理2～3d的灭螺效果相当;经统计分析：用其水溶液灭螺的.LD50和LD90浓度分别是4.0500mg/L和22.2500mg/L.用LD50浓度的夹竹桃强心总甙溶液处理50个钉螺之后,取活螺用聚丙烯酰胺凝胶电泳（PAGE）进行测试,在处理6h时,显示各处理样品的酶活高于对照组,表现为酶带颜色加深,有新的酶带出现;而在处理12h和24h后,各处理的样品酶带浅于对照组,酶带颜色逐渐变浅,直到消失.采用蒽酮显色法测定经夹竹桃强心总甙处理后钉螺软体的糖原含量糖原含量显著下降,减少幅度从9.9%到32.6%;钉螺经夹竹桃强心总甙处理后蛋白质含量的变化不明显.分析了强化感作用植物夹竹桃植物新鲜叶中强心总甙的灭螺活性,并在微观领域探究其化感作用导致钉螺的形态病理以及糖代谢、蛋白质代谢等生理变化等方面所表现出的杀伤钉螺的机理,由此获得强化感作用植物夹竹桃灭螺的化学生态学证据,为研制新的具中国特色的植物成份灭螺剂打下了基础,并为合成仿生灭螺剂以及最终构建生态工程中强化感作用植物群落灭螺提供理论依据.

生态工程中群落模型植物灭螺机理研究

研究了夹竹桃、枫杨和羊蹄灭螺活性较强的正丁醇提取物和水提物对钉螺酯酶同工酶(EST)、糖原(Gn)和总蛋白(TPr)的生理生化影响。同工酶电泳分析表明,EST是动物体内一种重要的解毒酶系统,且是钉螺对外界刺激反应最强烈的同工酶之一,EST在整个提取物作用阶段变化明显,可作为钉螺中毒的病理指标。提取物能够显著地降低钉螺体内的糖原含量,在整体情况下,影响糖代谢的因素错综复杂,进行机制分析较为困难,可能是提取物对钉螺肝脏、消化道或酶综合作用的结果。提取物能够在一定程度上降低钉螺体内的蛋白质含量,但减少量不及糖原的减少那样显著,这表明灭螺药物对钉螺的致死作用可能更多地与使钉螺组织细胞中的能量代谢异常有关,但其生化机制还需进一步研究。实验从微观领域探究其对钉螺的杀伤机理,为最终构建人工灭螺植物群落提供理论依据。

夹竹桃对钉螺肝损伤的超微结构观察

钉螺经夹竹桃叶水浸液处理后 ,用扫描电镜和透射电镜分别观察了其肝表面及肝细胞结构的变化。处理后的肝表肿胀 ,明显的沟纹消失 ,其上的孔隙破损 ;2 4h后肝细胞核肿胀 ,核仁消失 ,核质稀疏 ,内质网断裂 ,线粒体增多 ,且随处理时间的延长 (48h) ,损伤加重 ,内质网几乎全部囊泡化 ,细胞核开始破裂 ,线粒体也破裂。文中也讨论了钉螺可能致死的原因 。

钉螺对夹竹桃化感物质三萜总皂甙毒理作用的反应

本文分别用0.02mg/L-0.1mg/L系列浓度的夹竹桃三萜总皂甙水溶液处理钉螺,设无氯清水饲养与浓度为1mg/L氯硝柳胺溶液浸杀对照,研究夹竹桃三萜总皂甙是否为夹竹桃灭螺的化感物质及其作用机理。结果表明:用不同浓度梯度的夹竹桃三萜总皂甙水溶液处理钉螺均有一定的杀灭作用,对试验数据进行概率单位回归分析,表明浸杀处理2、3、4和5d的LC50分别为78.31、30.26、20.50、14.19mg/L,其对应的95%置信区间分别为63.60-108.19、9.49-44.42、2.86-30.90、0.23-22.79mg/L;以不同处理浓度、时间下钉螺的死亡率为因变量,对夹竹桃三萜总皂甙水溶液浸杀灭螺效果进行方差分析,结果显示夹竹桃三萜总皂甙的浓度、处理时间的长短对钉螺的死亡率均有极显著的作用效果,并且药液浓度与处理时间之间的交互效应也极显著。扫描电镜观察结果显示40mg/L浓度处理后钉螺软体表层呈现明显的损伤;透射电镜观察40mg/L浓度处理后钉螺样品发现24h后肝细胞核肿胀,核仁消失,核质稀疏,内质网断裂,线粒体增多;当处理时间延长到48h时,损伤加重,内质网几乎全部囊泡化,细胞核开始破裂,线粒体也破裂;处理24h后钉螺小肠表面的微绒毛排列有些不整齐,有一小部分微绒毛出现脱落,肠周缘细胞出现肿胀,细胞核出现少量小空泡;处理48h时的钉螺小肠表面微绒毛排列明显不整齐,大部分微绒毛脱落,肠周缘细胞肿胀明显,有大量空泡,有时还出现游离核,胞质内的细胞器有大小不等的空泡样变性,细胞器结构已无法辨别,严重变性,胞膜不完整。采用聚丙稀酰胺电泳技术分离经40mg/L夹竹桃三萜总皂甙水溶液处理1、2、3、4、5d后的钉螺样品的酯酶同工酶,处理1-2d后酶的活性比清水对照增强,应激反应明显,而处理达3d时酶的活性开始下降,5d时,酶谱带数及其色泽与用1mg/L氯硝柳胺水溶液处理1d的对照相当。本研究获得化感作用植物夹竹桃灭螺的超微结构证据。

夹竹桃、枫杨、羊蹄皂甙类成分灭螺活性分析

正丁醇提取夹竹桃、枫杨和羊蹄的皂甙类成分 ,通过浸杀灭螺实验 ,观察提取物对钉螺上爬的抑制作用 ,直线回归法确定灭螺量效和时效关系 ,通过方程预测提取物达LD50 和LD90 的剂量和使用时间 ,为实际应用提供参考。

夹竹桃灭钉螺活性成分的分离纯化

夹竹桃叶提取液对钉螺具有明显的毒杀作用．采用溶剂提取和高效液相色谱的方法，分离了新鲜夹竹桃叶中的成分经过灭钉螺实验，得到一种有灭钉螺作用的成分，其浓度为5mg·L-1时24h灭钉螺效果为63％夹竹桃及其提取物在钉螺的防治中显示着潜在的应用前景．

夹竹桃内生真菌J14次生代谢产物的分离和抑菌活性

为进一步开发、利用和保护夹竹桃植物资源,对从秦岭山区夹竹桃茎中分离得到活性较高的内生真菌J14进行鉴定,并对其次生代谢产物的化学成分进行研究。采用形态学特征和ITS序列分析对菌株J14进行鉴定,利用硅胶柱色谱、Sephadex LH-20柱色谱、重结晶等方法对该菌株的发酵产物进行分离、纯化,根据化合物的理化性质和1 H-NMR、13C-NMR数据鉴定其结构,利用金黄色葡萄球菌、乳酸链球菌、大肠杆菌、绿脓杆菌以及小麦赤霉病菌、番茄灰霉病菌、烟草枯萎病菌、白菜黑斑病菌对所得化合物进行抑菌活性测试。结果表明:经形态学特征和ITS序列分析,鉴定菌株J14为链格孢霉(Alternariasp.SPS-04)。从其发酵产物中分离得到8个化合物,分别鉴定为交链孢甲醚(1)、交链孢酚(2)、malformin A1(3)、胸腺嘧啶(4)、尿嘧啶(5)、黄嘌呤(6)、赤藓醇(7)和甘露醇(8)。化合物3、4、5、6、7首次从链格孢属真菌发酵产物中得到。化合物1对金黄色葡萄球菌的最小抑菌浓度为1.95μg/mL,对番茄灰霉病菌的最小抑菌浓度为3.91μg/mL。

Nispex体内外抑制白血病细胞HL-60的生长

背景与目的:植物多酚类成分不仅有防癌抗癌作用,也可增强放化疗的效果,还可以保护正常细胞免受放化疗的毒性。本研究探讨一种主要成分为多酚的寄主为夹竹桃的红花桑寄生提取物(简称Nispex)的体内外抗急性髓性白血病HL-60的效果。方法:采用MTT法研究Nispex的体外抑瘤作用;以对小鼠的近似LD50值评估Nispex的急性毒性;以HL-60细胞裸鼠移植瘤模型为对象,研究Nispex及其与多柔比星(阿霉素)联用的体内抑瘤效果。以相对瘤体积和瘤重来评价药物的体内抑瘤效果,以药物相互作用指数(CDI)来评价药物联合作用疗效。结果:Nispex显著抑制HL-60细胞的增殖,存在明显的量效关系和时效关系,24、48、72和96h的IC50值分别是0.75、0.70、0.54和0.48μg/ml。Nispex对小鼠的近似LD50值为126.81mg/kg。Nispex(ip)-剂量为30mg/kg时,肿瘤生长抑制率达60.6%;10mg/kg与多柔比星联用时,肿瘤生长抑制率达94.4%,CDI=0.21<0.7,均有统计学意义。结论:Nispex体内外有一定的抑瘤效果,与多柔比星联用的协同作用非常显著,可能是一种有潜力的抗肿瘤植物提取物。

夹竹桃不同溶剂提取物对福寿螺的毒杀作用评价

采用浸杀试验法,评价了夹竹桃叶不同溶剂提取物对10日龄福寿螺的毒杀作用。生物活性测定结果表明,夹竹桃鲜叶水提液和干叶水提液对福寿螺均有较高的毒杀作用,其中干叶水提液的杀螺作用更大。用鲜叶水提液和干叶水提液处理12 h,对福寿螺的LC50分别为10.98和1.20 g/L。夹竹桃叶五种有机溶剂(正己烷、甲醇、氯仿、乙酸乙酯、乙醇)提取物对福寿螺均有很高的杀螺活性,其中正己烷提取物的毒杀活性最高,甲醇提取物的毒杀活性最低。以各溶剂提取物200 mg/L处理12 h,死螺率均高于80%。正己烷、氯仿、乙酸乙酯、乙醇和甲醇提取物处理24 h,对福寿螺的LC50分别为32.98,83.62,71.71,75.78和117.07 mg/L。综合考虑各溶剂提取物的杀螺活性、提取效率、有机溶剂价格、毒性等因素,我们认为,乙醇是一种比较理想的提取夹竹桃叶中杀螺活性成分的有机溶剂。

夹竹桃叶乙醇提取物对斑马鱼的毒性评价

研究表明,夹竹桃(Nerium indicum Mill.)及其提取物对多种害虫具有较强的毒杀作用,已在害虫生物防治中显示出较大作用。为了合理开发夹竹桃植物,需要进一步评价夹竹桃植物对水生生物的安全性和对动物的毒理学。文章利用索氏提取法提取夹竹桃叶乙醇粗提物,并进一步用氯仿萃取浓缩、硅胶柱层析方法提取分离乙醇精提物,经鉴定为强心甙组分。在此基础上,委托浙江省医学科学院评价了乙醇粗提物对实验动物的毒理学,并采用静态法评价了这二种提取物对斑马鱼(Brachydanio rerio)的急性毒性和慢性效应。结果表明:夹竹桃叶乙醇粗提物的提取率为45%,乙醇精提物(主要成分为强心甙组分)的提取率为0.25%。乙醇粗取物对大鼠经口和经皮毒性为低毒级,对家兔皮肤和眼无刺激,对豚鼠的致敏反应属弱致敏性。这说明夹竹桃叶乙醇粗提物对哺乳动物十分安全。在急性毒性试验中,发现斑马鱼的死亡率与提取物处理浓度和处理时间均呈明显的正相关。处理浓度越大,斑马鱼的死亡率越高;同一浓度处理时间越长,死亡率越高。用24 mg/L乙醇粗提物处理6、12、24和48h的死亡率分别为26.67%、60%、91.11%和95.56%;用0.5 mg/L乙醇精提物处理12、24、48、72和96h的死亡率分别为6.25%、33.33%、52.08%、54.17%和60.42%;用24 mg/L乙醇粗提物处理96h或32 mg/L处理48h,或者用1 mg/L乙醇精提物处理24h,斑马鱼全部死亡。在慢性毒性试验中,用3.33—10.0 mg/L乙醇粗提物处理斑马鱼28d,其死亡率为12%—20%。乙醇粗提物处理斑马鱼后,急性毒性LC50=12.52 mg/L(药后96h)>10 mg/L,慢性毒性LC50=199.51 mg/L(药后28d);乙醇精提物处理斑马鱼后,急性毒性LC50=0.46 mg/L(药后96h)<1 mg/L,根据《化学农药环境安全评价试验准则》(1990)中农药对鱼类的毒性分级标准,说明夹竹桃叶乙醇粗提物对斑马鱼的毒性属于低毒,比较安全;乙醇精提物对斑马鱼的毒性则为高毒,十分不安全。

夹竹桃叶提取物对福寿螺的杀螺活性

从夹竹桃叶中筛选杀灭福寿螺的活性成分。采用浸渍法研究了夹竹桃叶的乙醇粗提物和组分Ⅰ对不同日龄福寿螺的毒杀和抑制作用。结果表明:组分Ⅰ浸渍10、30和50日龄螺6h的LC50值分别为2.86、5.87和8.68mg/L;浸渍24h的LC50值分别为0.98、2.31和3.16mg/L,其中2mg/L的组分Ⅰ浸渍10日龄螺24h的死亡率为100%。乙醇粗提物对福寿螺的毒杀效果较差,用其100mg/L浸渍10日龄和30日龄螺72h的死亡率均为100%,浸渍50日龄螺120h时,死亡率100%时的质量浓度达到200mg/L。但乙醇粗提物和组分Ⅰ各浓度对福寿螺的活动均无抑制作用,反而有促进其上爬的作用。