液质联用技术评估汞接触污染蟾蜍泌酥中蟾蜍甾烯水平

目的:运用液质联用技术研究重金属汞皮肤接触中华大蟾蜍后生理状态及泌酥中蟾蜍甾烯水平变化,为中药蟾酥品质评价提供参考。方法:配制不同质量浓度的氯化汞溶液(0.5,0.75,1.0 g·L~(-1))对蟾蜍进行半静水式处理,观察其生理状态,且在30 d后收集蟾蜍耳后腺分泌液,进一步采用液相-质谱联用技术测定蟾蜍泌酥中游离型蟾蜍甾烯的水平,结合Hem I热图软件进行数据分析。结果:氯化汞接触明显影响蟾蜍泌酥中蟾蜍甾烯水平,和正常组比较,低质量浓度(0.5 g·L~(-1))汞污染组有20种蟾蜍甾烯水平明显上调,其中有12个物质发生1.5倍变化。而中质量浓度(0.75 g·L~(-1)),高质量浓度(1.0 g·L~(-1))污染组分别有15,13种物质发生下调,且大部分为中低脂溶性蟾蜍甾烯(异沙蟾毒精、去乙酰华蟾毒精等)。结论:重金属汞污染对蟾蜍甾烯水平有潜在影响,且随着浓度的升高呈现明显降低趋势。     

蟾酥化学成分及其人工合成的研究进展

蟾酥为我国传统名贵中药,其化学成分复杂,具有广泛的药理活性。蟾酥的化学成分主要包括蟾蜍内酯类、吲哚类生物碱、甾醇类及其他类化合物。现代药理学研究表明,蟾酥具有抗肿瘤、强心、抗炎、局麻、镇痛等多种药理活性。对近年来蟾酥化学成分、药理活性及其人工合成等方面的研究进展进行综述,以期为蟾酥的物质基础研究、药效机制阐释及其开发利用提供参考。     

蟾酥脂质微球注射液对四种荷瘤裸鼠肿瘤生长的抑制作用

目的:观察蟾酥脂质微球注射液对荷人肝癌Hep G2、人食管癌EC9706、人结肠癌HCT-8和人胃癌BGC 803瘤株裸鼠肿瘤生长的抑制作用.方法:在BALB/c-nu裸鼠右前肢腋下接种4种人癌细胞株建立荷瘤动物模型,移植10 d后采用均衡法随机分为5组,即蟾酥脂质微球注射液高剂量组(2.50 mg/kg)、中剂量组(1.25 mg/kg)、低剂量组(0.63 mg/kg)以及阳性药组(氟尿嘧啶注射液,1.25 mg/kg)和阴性对照组(等体积脂质微球空白注射液),每组7只裸鼠,给药体积0.2 ml.采用尾静脉注射方法给药,各组动物于分组后每周给药2次,连续给药6次.于给药后动态检测并计算各组肿瘤体积(Vt)、肿瘤相对体积(VRT)、肿瘤增殖率(T/C),末次给药后3 d检测并计算各组肿瘤质量和抑瘤率.结果:各组裸鼠给药前肿瘤体积均无显著性差异.荷人肝癌Hep G2细胞瘤裸鼠给药7 d后,各剂量组肿瘤体积和相对肿瘤体积均较阴性对照组明显降低(P<0.05),末次给药后中、高剂量组T/C值<40%,各剂量组的平均肿瘤质量均较阴性对照组明显降低(P<0.05),高剂量组的抑瘤率为74.07%.荷人食管癌EC9706瘤裸鼠末次给药后,各剂量组肿瘤体积和相对肿瘤体积均较阴性对照组明显降低(P<0.05),中、高剂量组T/C值<40%,各剂量组的平均肿瘤质量均较阴性对照组明显降低(P<0.05),高剂量组的抑瘤率为70.38%.荷人结肠癌HCT-8细胞瘤裸鼠末次给药后,各剂量组肿瘤体积和相对肿瘤体积均较阴性对照组明显降低(P<0.05),高剂量组T/C值<40%,各剂量组的平均肿瘤质量均较阴性对照组明显降低(P<0.05),高剂量组的抑瘤率为52.42%.荷人胃癌BGC 803细胞瘤裸鼠末次给药后,中、高剂量组的肿瘤体积和相对肿瘤体积均较阴性对照组明显降低(P<0.05),各剂量组T/C值>40%,各剂量组的平均肿瘤质量均较阴性对照组明显降低(P<0.05),高剂量组的抑瘤率为47.42%.结论:蟾酥脂质微球注射液具有确切的抑制人肝癌Hep G2、食管癌EC9706、结肠癌HCT-8和胃癌BGC 803瘤株增殖的作用,作用效果以抑制人肝癌Hep G2和人食管癌EC9706瘤株生长作用为最佳.     

蟾毒灵、华蟾酥毒基及酯蟾毒配基大鼠体内药动学研究

目的 建立检测大鼠血浆中3种蟾蜍甾烯类化合物蟾毒灵、华蟾酥毒基及酯蟾毒配基的HPLC法,并用于蟾酥在大鼠体内的药动学研究。方法 分别于大鼠尾iv给予蟾酥提取物0.8 mg/kg后2、5、10、15、20、30、45、60、90 min,眼眶取血,采用乙腈沉淀蛋白与液液萃取相结合方法进行血浆样品预处理,以HPLC法测定大鼠血浆中蟾毒灵、华蟾酥毒基及酯蟾毒配基的质量浓度,以Kinetica软件拟合药动学参数。结果 蟾毒灵、华蟾酥毒基及酯蟾毒配基均得到很好的分离,重现性、精密度、线性关系良好,达到体内分析要求;经非房室模型拟合,得到蟾毒灵、华蟾酥毒基及酯蟾毒配基在大鼠体内主要药动学参数;iv给药30 min时,血药浓度均降至C_max的1/5以下。结论 建立的蟾毒灵、华蟾酥毒基及酯蟾毒配基血药浓度测定方法操作简单、结果准确可靠;蟾蜍甾烯类成分在大鼠体内代谢较为迅速,所得数据可为蟾酥提取物的药动学研究提供参考。     

Cardioactive steroid poisoning: A comparison of plant- and animal-derived compounds

Introduction

Cardioactive steroids (CASs) are found in plants, animals, and insects. Their affinity for Na+−K+ ATPase is attenuated by the type of lactone at carbon 17 (C17) of the steroid backbone: those with 5-membered lactone rings, or cardenolides, are derived mostly from plants with 6-membered rings or from animals with bufadienolides. A systematic review of CAS poisoning was performed to compare the mortality rate of cardenolides and bufadienolides.

Methods

MEDLINE was searched for articles using commonly reported names of CASs, and keywords were limited to human cases only. We searched cases from 1982 to 2003, so that supportive care was similar and digoxin-specific Fab was available. Identified reports of CAS poisoning were read to exclude cases involving licensed pharmaceuticals. Inclusion criteria included hyperkalemia, gastrointestinal symptoms, electrocardiographic evidence of CAS toxicity, digoxin serum concentration, or history of exposure to a substance containing a CAS. Clinical data was collected, including information about treatment with digoxin-specific Fab and treatment outcome.

Results

Fifty-nine articles, describing 924 patients, were identified. Eight hundred ninety-seven patients (97%) ingested a CAS with a 5-membered lactone ring, and mortality was 6% (n = 54). Twenty-seven patients (2.9%) ingested a CAS with a 6-membered lactone ring, and mortality was 29.6% (n = 8). The difference in mortality rates was statistically significant (p < 0.001, [X²]). CASs with 6-member rings accounted for the highest percentage of nonsuicidal exposures.

Conclusion

Although cardenolides accounted for the majority of exposures, bufadienolides were five times more lethal than cardenolides.     

细胞亲和-UPLC-QTOF测定蟾蜍甾烯与宫颈癌HeLa细胞的亲和及与化合物计算分子属性的相关性分析

目的测定蟾蜍甾烯类化合物(Bu)与人宫颈癌HeLa细胞的亲和率,分析其与化合物计算分子属性的相关性。方法蟾酥氯仿提取物与HeLa细胞亲和,采用UPLC-QTOF测定亲和后HeLa细胞裂解物和上清液中蟾蜍甾烯的水平,计算Bu与细胞的总亲和率。Pubchem数据库采集蟾蜍甾烯化合物的10种计算分子属性。统计分析两者间相关性。结果Bu与细胞的总亲和率与化合物脂水分配参数(XLogP3)、氢键供体(H-Bond Donor)、拓扑极性表面积(topological polarsurface area,TPSA)呈相关性,其相关系数分别是r=0.865(P<0.01),r=-0.901(P<0.01),r=-0.657(P<0.05)。与其它计算分子属性间不具有相关性(P>0.05)。结论蟾蜍甾烯与HeLa细胞发生不同程度亲和。化合物的脂水性、氢键供体个数和渗透性是影响蟾蜍甾烯与HeLa细胞亲和的非特异性因素。     

Biotransformation of resibufogenin by Actinomucor elegans

Resibufogenin (RB), a major bioactive bufadienolide, has the potential anticancer activity. In the present work, biotransformation of RB by Actinomucor elegans AS 3.2778 yielded five products, namely 3-oxo-resibufogenin (1), 3-epi-resibufogenin (2), 3-epi-12-oxo-hydroxylresibufogenin (3), 3α-acetoxy-15α-hydroxylbufalin (4), and 3-epi-12α-hydroxylresibufogenin (5), respectively. Among them, metabolites 3 and 4 are previously unreported. The chemical structures of metabolites 1–5 were fully elucidated on the basis of 2D NMR and HR-MS. The highly stereo- and regio-specific isomerization, hydroxylation, and esterification reactions were observed in the biotransformation process of RB by A. elegans. Their cytotoxicities against A549 and H1299 cells were evaluated.     

Microbial transformation of resibufogenin by Curvularia lunata AS 3.4381

In this paper, the microbial transformation of resibufogenin (1) by Curvularia lunata AS 3.4381 was investigated, and four transformed products were isolated and characterized as 3-epi-resibufogenin (2), 12α-hydroxy-3-epi-resibufogenin (3), 12-oxo-16β-hydroxy-3-epi-resibufogenin (4), and 12β,15-epoxy-3-epi-bufalin-14,15-ene (5). Among them, 4 and 5 are new compounds, and isomerization, hydroxylation, and oxidation reactions in microbial transformation process were observed. Additionally, the cytotoxicities of transformed products (2–5) were also investigated.     

Antiproliferative activity and new argininyl bufadienolide esters from the “cururú” toad Rhinella (Bufo) schneideri

Ethnopharmacological relevance

Toads known as “cururú” (Rhinella schneideri) have been used in the Brazilian Pantanal and Paraguayan Chaco wetlands to treat erysipelas and cancer. The aim of the study was to assess the antiproliferative effect of the venom obtained from Rhinella schneideri and to identify its constituents by spectroscopic and spectrometric methods.

Materials and methods

The venom was obtained by gentle pressing the parotid glands of the toads. The dry crude drug was analyzed by HPLC–MS–MS and chromatographed on Sephadex LH-20 to obtain purified compounds and fractions for spectroscopic analysis. The venom and fractions were evaluated for antiproliferative activity towards normal human lung fibroblasts (MRC-5) and four human cancer cell lines: gastric epithelial adenocarcinoma (AGS), lung cancer (SK–MES-1), bladder carcinoma (J82) and promyelocytic leukemia (HL-60).

Results

From the Rhinella schneideri venom, 29 compounds were isolated and/or identified by spectroscopic and spectrometric means. Three known alkaloids and five argininyl diacids were identified in the complex mixture by HPLC–MS–MS. Nine out of fifteen argininyl diacid derivatives of the bufadienolides bufalin, marinobufagin and telocinobufagin are reported for the first time and four argininyl diacids are described for the first time as natural products. The venom and the fractions 9–13 showed a remarkable antiproliferative effect, with IC₅₀ values in the range 0.019–0.022, 0.035–0.040, 0.028–0.064, 0.042–0.056 and 0.044–0.052 µg/mL for MRC-5, AGS, SK-MES-1, J82 and HL-60 cell lines, respectively. Under the same experimental conditions, IC₅₀ values of the reference compound etoposide were 2.296, 0.277, 1.295, 1.884 and 1.059 µg/mL towards MRC-5, AGS, SK-MES-1, J82 and HL-60 cells, respectively.

Conclusions

The venom showed a strong antiproliferative effect towards human cancer cells and presented a high chemical diversity in its constituents, supporting its use as anticancer agent. These findings encourage further work on the chemistry and bioactivity of South American toad venoms.     

中华大蟾蜍蟾酥中蟾毒内酯类化学成分研究

采用硅胶柱色谱、ODS柱色谱、凝胶柱色谱、制备型高效液相等方法从蟾酥的二氯甲烷萃取部位中分离得到12个蟾毒内酯类化合物,根据化合物的理化性质和波谱数据分别鉴定为脂蟾毒配基（1）、蟾蜍它灵（2）、去乙酰华蟾蜍精（3）、19-氧去乙酰华蟾蜍它灵（4）、华蟾蜍它灵（5）、1β-羟基蟾蜍灵（6）、12α-羟基蟾蜍灵（7）、蟾蜍它里灵（8）、嚏根草配基（9）、远华蟾蜍精（10）、嚏根草醇（11）及华蟾蜍精-3-辛二酰甲酯（12）。化合物 7和12 为首次从蟾酥中分离得到,且为新天然产物。