HPLC法测定蟾皮中蟾毒配基类成分的含量

目的用HPLC法测定蟾皮中蟾蜍灵、脂蟾毒配基、华蟾蜍精等成分的含量。方法样品经乙醇室温浸渍提取,采用HPLC法测定。色谱柱为DiamonsilTMC18(250 mm×4.6 mm,5μm),流动相为甲醇-乙腈-水(体积比为4∶1∶5),流速为1.0 mL.min-1,检测波长为296 nm,柱温为40℃。结果蟾蜍灵、脂蟾毒配基和华蟾蜍精分别在0.0848~0.5088、0.0884~0.5304和0.2000~1.2000μg内与色谱峰面积线性关系良好,相关系数r分别为0.9999、0.9992和1.0000。蟾蜍灵、脂蟾毒配基和华蟾蜍精的平均回收率分别为99.3%、99.6%和99.0%,RSD分别为0.5%、0.2%和0.6%(n=9)。结论HPLC法可用于蟾皮中蟾蜍灵、脂蟾毒配基和华蟾蜍精的含量测定。     

花背蟾蜍耳后腺分泌物的药效学研究

<正> 据调查国产蟾酥及干蟾大宗产品的原动物基本是大蟾蜍中华亚种(Bufo bufo gargarizans Cantor).近二十年来出版的有关中药材方面的部分书籍多将花背蟾蜍(Bufo raddei Staueh)列为蟾酥及干蟾的原动物。为了澄清蟾酥及干蟾的药材品种及其原动物间的关系,作者对花背蟾蜍耳后腺分泌物的药效作了研究,并与中华大蟾蜍耳后腺分泌物作了比较。     

干蟾皮及其炮制品的指纹图谱建立与化学模式识别

目的:建立干蟾皮生品及炮制品UPLC指纹图谱,并同时测定干蟾皮中指标性成分日蟾毒它灵、沙蟾毒精、蟾蜍它里定、蟾毒它灵、华蟾毒它灵、蟾毒灵、华蟾酥毒基、脂蟾毒配基、沙蟾毒精-3-辛二醇辛酯的含量,通过化学模式识别控制干蟾皮质量。方法:干蟾皮的甲醇提取液的分析采用Waters Acquity UPLC BEH色谱柱(100 mm×2.1 mm, 1.7μm),以乙腈-0.5%乙酸为流动相,梯度洗脱,流速为0.2 mL·min^-1,柱温为30℃,检测波长为296 nm。结果:12批生品和12批炮制品的总含量分别为3.18～4.69 mg·g^-1和0.91～2.77 mg·g^-1,指纹图谱中均有9个共有峰,相似度分别为0.963～0.991和0.692～0.988,主成分分析和聚类分析能很好地识别干蟾皮的生品和炮制品。结论:该方法稳定、可靠,可为干蟾皮的质量控制、炮制工艺,进一步开发研究提供科学依据。     

中华大蟾蜍皮化学成分的研究

从中华大蟾蜍(Bufo bufo gargarizans Cantor)皮水溶性部分应用制备型反相HPLC分离得到一个新蟾蜍毒素,根据光谱(UV,IR,¹HNMR,¹³CNMR,EIMS,FDMS)、氨基酸分析及化学性质,确定其结构为Ⅰ所示,命名为蟾毒它灵3-丁二酰精氨酸酯,同时还分到三个已和蟾蜍毒素;蟾毒灵3-丁二酰精氨酸酯(Ⅱ),华蟾毒精3-丁二酰精氨酸酯(Ⅲ),脂蟾毒配基3-丁二酰精氨酸酯(Ⅳ)。     

毒性中药蟾酥质量检测方法研究

目的:建立蟾蜍中多种药效及毒性成分蟾蜍毒素的定性定量检测方法。方法:采用液相色谱-质谱/质谱联用方法检测蟾酥中多种蟾蜍毒素成分。结果:建立了华蟾毒它灵、蟾蜍灵、华蟾毒配基及脂蟾毒配基的LC-MS/MS检测方法。比较了蟾酥样本的液相色谱-质谱/质谱联用分析数据。对4个不同来源中华大蟾蜍及黑眶蟾蜍的蟾酥样本进行检测,检出华蟾毒它灵、蟾蜍灵、华蟾毒配基及脂蟾毒配基的含量范围为0.05%~5.12%(w/w)。结论:液相色谱-质谱/质谱联用方法可用于蟾酥定性定量检测,并为质量评价提供参考。     

蟾皮中蟾毒配基类成分的分离与鉴定

目的研究中华大蟾蜍(Bufo bufo gargarizansCantor)皮中的蟾毒配基类成分。方法利用反复硅胶柱色谱、Sephadex LH-20凝胶柱色谱、半制备高效液相色谱、重结晶等手段分离纯化蟾皮中的蟾毒配基类成分,根据化合物的理化性质和各种波谱学数据鉴定其化学结构。结果分离得到11个蟾毒配基类化合物,分别鉴定为脂蟾毒配基(resibufogenin,1)、华蟾毒精(cinobufagin,2)、蟾毒灵(bufalin,3)、远华蟾毒精(telocinobufagin,4)、蟾毒它灵(bufotalin,5)、去乙酰华蟾毒它灵(desace-tylcinobufotalin,6)、嚏根草苷元(hellebrigenin,7)、沙蟾毒精(arenobufagin,8)、日蟾毒它灵(gam-abufotalin,9)、11β-羟基脂蟾毒配基(11β-hydroxylresibufogenin,10)、华蟾毒它灵(cinobufotalin,11)。结论化合物10和11为首次从中华大蟾蜍皮中分离得到。     

HPLC法同时检查华蟾素片中蟾毒灵、华蟾酥毒基及酯蟾毒配基限量和测定蟾蜍噻咛的含量

目的 采用HPLC法对华蟾素片中有毒成分蟾毒灵、华蟾酥毒基及酯蟾毒配基进行限量检查;并利用HPLC法测定其有效成分蟾蜍噻咛的含量.方法 限量检查:色谱柱为Agela C18柱,流动相为乙腈-0.5％磷酸二氢钾溶液(50∶50)(用磷酸调节pH为3.2),检测波长为296nm.含量测定:色谱柱为Kromasil C18柱,流动相为乙腈-水(10∶90),检测波长为226nm.结果 在与蟾毒灵、华蟾酥毒基及酯蟾毒配基对照品色谱峰相同的保留时间处,5批样品出现的色谱峰面积之和小于对照品峰面积之和.蟾蜍噻咛在0.1045～1.0448μg范围内呈良好的线性关系,r=0.9998;平均回收率为98.9％,RSD=1.6％;每片含量不得少于0.15mg.结论 方法快速、准确、重复性好,可用于华蟾素片质量标准的控制.     

蟾酥的镇痛活性成分

药效学实验结果表明：蟾酥镇痛的活性成分主要存在于氯仿提取物中．分离蟾酥的氯仿提取物得到了脂蟾毒配基等６种单体化合物．６种单体化合物均具有一定的镇痛作用．华蟾毒精和脂蟾毒配基有非常显著的镇痛作用；蟾毒灵镇痛作用较平稳；南美蟾毒精镇痛作用较弱     

HPLC法同时测定蟾酥中11个蟾毒配基成分的含量

目的:建立HPLC法同时测定蟾酥中11个蟾毒配基成分(伪异沙蟾毒精、日蟾毒它灵、沙蟾毒精、蟾蜍它里定、远华蟾蜍精、蟾毒它灵、脂蟾毒精、南美蟾毒精、蟾毒灵、脂蟾毒配基和华蟾酥毒基)的含量。方法:采用Agilent InfinityLab Poroshell 120 EC-C₁₈色谱柱(150 mm×3.0 mm 2.7μm),以0.3%乙酸水溶液-乙腈为流动相,梯度洗脱,流速为0.5 mL·min^-1,柱温为30℃,检测波长为296 nm。结果:11个蟾毒配基成分的线性范围分别为2.024～20.24μg·mL^-1(r=0.999 8)、18.44～184.4μg·mL^-1(r=0.999 9)、12.40～124.0μg·mL^-1(r=0.999 5)、3.720～37.20μg·mL^-1(r=0.999 5)、10.28～102.8μg·mL^-1(r=0.999 8)、22.16～221.6μg·mL^-1(r=...     

蟾皮化学成分的分离与结构鉴定

蟾皮为蟾蜍科动物中华大蟾蜍Bufo bufo gargarizans Cantor的干燥皮，在我国民间多用来治疗原发性肝癌、肺癌、肠癌等，其水溶性成分制剂华蟾素注射液临床上用于消化道中晚期癌症，疗效确切，体外抗肿瘤活性筛选也表明蟾皮水提取物对结肠癌细胞HCT-8、肺癌细胞A-549生长均有明显的抑制作用。为了阐明其活性成分，对其水溶性成分进行了系统研究，采用Sephadex LH-20、硅胶柱色谱结合结晶法，从其水提物中分离得到6个化合物，分别鉴定为4-氨基-3-羟甲基-环辛酰胺骈四氢-α-呋喃酮(蟾蜍环酰胺B，I)、蟾蜍环酰胺C(II)、蟾蜍噻咛(III)、去氢蟾蜍色氨氢溴酸盐(IV)、辛二酸(V)和丁二酸(VI)，其中化合物I和II为新化合物。     

Effects of tissue cultured ginseng on gastric secretion and pepsin activity]

Effects of the tissue cultured and cultivated ginseng on gastric secretion and pepsin activity were investigated. Fifty percent ethanol extracts of both cultured and cultivated ginsengs reduced gastric secretion and acid output in pylorus-ligated rats. They did not affect pepsin activity. The tissue cultured ginseng inhibited histamine and pentagastrin-induced acid secretion in rats, whereas the cultivated ginseng showed no such effect. They also suppressed acid secretion induced by 2-deoxy-D-glucose and baclofen [beta-(p-chlorophenyl)-gamma-aminobutyric acid], which are known to stimulate gastric acid secretion via the central nervous system. However, they had no effect on acid secretion induced by vagal stimulation. These results suggest that both tissue cultured and cultivated ginsengs may have an inhibitory effect on gastric secretion. The effect seems to be due to the inhibition of acid secretion via the central nervous system.     

The role of reactive oxygen species in the herbicide acetochlor-induced DNA damage on Bufo raddei tadpole liver

After exposure of Bufo raddei tadpoles to acetochlor (ACETO) for 14 days, malondialdehyde (MDA) and DNA-single strand break (DNA-SSB) in livers were analyzed. An enhanced accumulation of MDA suggests that ACETO causes oxidative stress, and the significant increase in the level of DNA-SSB indicates that ACETO induces DNA damage in a dose-dependent manner as well. On the basis of the fact that oxidative stress is caused by excessive production of reactive oxygen species (ROS), and the present results, we speculate that ACETO-induced DNA damage may be a consequence of the generation of ROS. To evaluate this hypothesis, tadpoles were treated with ROS scavenger, N-acetyl-L-cysteine (NAC) or melatonin (MEL), prior to ACETO exposure. The decrease of DNA-SSB level and the increase of total antioxidant capability (TAC) show that ACETO-caused DNA damage can be attenuated by NAC and MEL. In addition, a negative correlation was observed between the extent of DNA damage and the level of TAC in tadpole liver. In conclusion, the results suggest that ACETO-induced DNA damage is mediated by ROS.     

Effect of Ilex extracts and isolated compounds on peroxidase secretion of rat submandibulary glands.

Free radicals are involved in diverse disorders such as tumoral, central nervous system alterations, immunological and inflammatory pathologies. Peroxidase is an oral enzyme involved in the defense of the oral cavity. Ilex species such as Ilex paraguariensis St. Hil. and the commercial product made with it "Yerba Mate" are used traditionally as antirheumatics and for the treatment of gastrointestinal diseases among others and also as a beverage with nutritional and stimulant properties. The presence of polyphenolic derivatives and flavonoids in the aqueous extract has been determined by HPLC analysis. In this study, the activity of aqueous extracts of I. paraguariensis and "Yerba Mate" on peroxidase secretion in female rat submandibular glands was investigated. The contribution to this pharmacological activity by some major hydrocynnamic acid derivatives present in the crude extracts, such as chlorogenic acid and caffeic acid and the most abundant methylxanthine, caffeine, was also evaluated. Spectrophotometrical determination of peroxidase activity showed that both extracts produced a significant increase in both secreted peroxidase and total peroxidase activity, though "Yerba Mate" showed a higher activity (EC(50) "Yerba Mate": 148+/-10 microg/ml; EC(50)I. paraguariensis: 841+/-20 microg/ml). The HPLC/DAD analysis of the crude extracts was performed and chlorogenic acid, caffeic acid and caffeine were identified and quantified. The results (expressed as W/W percentage of dried material) were as follows: I. paraguariensis: chlorogenic acid: 2.80+/-0.30, caffeic acid: 0.023+/-0.004, caffeine: 1.06+/-0.06; "Yerba Mate": chlorogenic acid: 1.98+/-0.37; caffeic acid: 0.020+/-0.003, caffeine: 0.70+/-0.06. Caffeine and chlorogenic acid were proved to play an important role in the induction of peroxidase secretion induced by the extracts.     

干蟾皮药材质量标准研究

本文旨在完善干蟾皮药材的质量标准.采用显微鉴别法、薄层色谱法鉴别干蟾皮;按《中国药典》规定方法检测干蟾皮的水分和醇溶性浸出物;用高效液相色谱(HPLC)法测定华蟾酥毒基和脂蟾毒配基的含量.初步建立了干蟾皮显微鉴别法、薄层色谱方法及HPLC含量测定方法;制定了水分、醇溶性浸出物的限度.本研究为干蟾皮药材质量标准的提高提供参考.     

Antitumor activity of extracts and compounds from the skin of the toad Bufo bufo gargarizans Cantor

The skin of the toad Bufo bufo gargarizans Cantor is known to be rich in bufadienolides, peptides and alkaloids. It has been found to be a source of some extracts and biologically active compounds with antitumor activity. Cinobufacini (Huachansu), a Chinese medicine prepared from the dried toad skin, has been widely used in clinical therapy for various cancers in China. Bufadienolides, such as bufalin, cinobufagin, resibufogenin, and telocinobufagin, are the major active compounds derived from the toad skin. They are the maker biologically active compounds of cinobufagin while the antitumor activity of cinobufagin may be due to this kind of components. Experimental research has suggested that cinobufacini and its active compounds (e.g. bufalin and cinobufagin) exhibit significant antitumor activity, including inhibition of cell proliferation, induction of cell differentiation, induction of apoptosis, disruption of the cell cycle, inhibition of cancer angiogenesis, reversal of multi-drug resistance, and regulation of the immune response. Clinical data have indicated that cinobufacini may have effective anticancer activity with low toxicity and few side effects. Data to date suggest it may also enhance quality of life for patients with cancer. Thus, this review briefly summarizes recent studies on the anticancer activity of cinobufacini and some of its active compounds from the skin of the toad Bufo bufo gargarizans Cantor. This might provide additional evidence for further study of the extracts and active compounds from the toad skin in cancer treatment.     

Effect of gallic acid derivatives on secretion of Th1 cytokines and Th2 cytokines from anti CD3-stimulated spleen cells]

As reported previously (Kosuge et al., Yakugaku Zasshi, 120, 408 (2000)), methyl gallate, a gallic acid derivative, which has been one of compounds isolated from extracts of Psidium geneus Myrtaceae, selectively suppresses Th2 cytokine secretion. In the present study, to examine more effective compounds than methyl gallate, the effects of various gallic acid derivatives on the secretion of helper T cell subtype specific cytokines from anti CD3-stimulated spleen cells were investigated. Ten micrograms/ml of methyl gallate and ethyl gallate remarkably suppressed the secretion of IL-4 and IL-5, Th2 cytokines, but did not suppress meaningfully the secretion of IFN-gamma, a Th1 cytokine. On the other hand, the other gallic acid derivatives suppressed the secretion of both IL-4 and IFN-gamma. Ten micrograms/ml of methyl gallate suppressed the secretion of IL-2, a Th1 cytokine, but the same concentration of ethyl gallate did not suppress it. In conclusion, it seemed that ethyl gallate was the most selective inhibitor for the secretion of Th2 cytokines among gallic acid derivatives used in this study.     

Effect of hypobaric hypoxia on spermatogenesis, Leydig cells and delta 5-3 beta-hydroxysteroid dehydrogenase activity in toad

The effect of different grades of hypobaric hypoxia for 48 hours was studied on spermatogenesis, Leydig cells and delta 5-3 beta-hydroxysteroid dehydrogenase activity in toad (Bufo melanostictus). Maximum inhibition of testicular activity was noted in 7,315 m exposed animals. The impairment of testicular function at high altitude is possibly due to inhibition of gonadotrophin secretion.