Quality evaluation of traditional Chinese drug toad venom from different origins through a simultaneous determination of bufogenins and indole alkaloids by HPLC.

A simple and efficient HPLC method has been developed to evaluate the quality of traditional Chinese medicine toad venoms. The major bioactive ingredients, including 10 bufogenins and 4 indole alkaloids in the drug, were separated and quantified on a phenyl-hexyl column with a UV detector. A total of 27 toad venom samples from two species, Bufo bufo gargarizans CANTOR and Bufo melanostictus SCHNEIDER, from the different drug production regions of China, were analyzed. The chromatograms showed significant differences with respect to the samples from different origins. These toad venom samples can be distinctly classified into 4 groups by cluster analysis using the contents of the 14 main constituents, including toad venom samples from B. bufo gargarizans from north China, median China and south China, and samples from B. melanostictus from south China. Toad venom samples from B. bufo gargarizans from median China were considered to be of the highest quality.

Evidence for biosynthesis of pseudophrynamine alkaloids by an Australian myobatrachid frog (pseudophryne) and for sequestration of dietary pumiliotoxins.

Australian myobatrachid frogs of the genus Pseudophryne have only two classes of alkaloids in skin extracts, pseudophrynamines (PSs) and pumiliotoxins (PTXs). The former are unique to such Australian frogs, while the PTXs occur worldwide in all other genera of frogs/toads that contain lipophilic alkaloids. The major alkaloid of wild-caught frogs from one population of Pseudophryne semimarmorata was PTX 267C, while PSs were only minor or trace alkaloids. Captive-raised frogs from the same parental stock had no PTXs, but had larger amounts of PSs. A PTX fed to captive-raised frogs accumulated into skin along with dihydro and hydroxy metabolites. Thus, Pseudophryne frogs appear to biosynthesize PSs, but to sequester into skin dietary PTXs. In addition, biosynthesis of PSs appears reduced when high levels of dietary PTXs have accumulated into skin. This is the first evidence indicating that certain frogs are capable of synthesizing rather than merely sequestering alkaloids. A wide range of PSs, including many with molecular weights >500, were detected using both GC-mass spectral and LC-mass spectral analysis.

The evolution of coloration and toxicity in the poison frog family (Dendrobatidae).

The poison frogs (family Dendrobatidae) are terrestrial anuran amphibians displaying a wide range of coloration and toxicity. These frogs generally have been considered to be aposematic, but relatively little research has been carried out to test the predictions of this hypothesis. Here we use a comparative approach to test one prediction of the hypothesis of aposematism: that coloration will evolve in tandem with toxicity. Recently, we developed a phylogenetic hypothesis of the evolutionary relationships among representative species of poison frogs, using sequences from three regions of mitochondrial DNA. In our analysis, we use that DNA-based phylogeny and comparative analysis of independent contrasts to investigate the correlation between coloration and toxicity in the poison frog family (Dendrobatidae). Information on the toxicity of different species was obtained from the literature. Two different measures of the brightness and extent of coloration were used. (i) Twenty-four human observers were asked to rank different photos of each different species in the analysis in terms of contrast to a leaf-littered background. (ii) Color photos of each species were scanned into a computer and a computer program was used to obtain a measure of the contrast of the colors of each species relative to a leaf-littered background. Comparative analyses of the results were carried out with two different models of character evolution: gradual change, with branch lengths proportional to the amount of genetic change, and punctuational change, with all change being associated with speciation events. Comparative analysis using either method or model indicated a significant correlation between the evolution of toxicity and coloration across this family. These results are consistent with the hypothesis that coloration in this group is aposematic.

Serous cutaneous glands of the western spade-foot toad Pelobates cultripes (Amphibia, Anura): an ultrastructural study on adults and juveniles.

The venom glands of the western spade-foot toad Pelobates cultripes were studied under light and electron microscopes. The glands exhibit the structural patterns usual in anurans, including the typical secretory syncytium. The peripheral cytoplasm contains a single row of nuclei and secretory organelles related to proteosynthesis. The inner cytoplasm is filled with large vesicles holding a thin product which originates from the merging of smaller ones containing a thicker material derived from the Golgi apparatus. The appearance and maturation of P. cultripes venom have been compared with patterns of biosynthesis and secretory evolution described in serous cutaneous glands of several anuran species. Following these criteria, the traditional trends in the terminology and classification of serous glands in anuran skin are discussed and reviewed.

Alkaloids from a panamanian poison frog, Dendrobates speciosus: identification of pumiliotoxin-A and allopumiliotoxin class alkaloids, 3,5-disubstituted indolizidines, 5-substituted 8-methylindolizidines, and a 2-methyl-6-nonyl-4-hydroxypiperidine.

Dendrobates speciosus is a small red or orange frog that occupies a small geographic range in the highlands of western Panama, where it occurs abundantly in some cloud forest habitats. Gc-ms analysis indicated the presence of at least 30 alkaloids in MeOH skin extracts from population samples at the extreme eastern end of the known geographic range. Eleven alkaloids were isolated by cc in quantities sufficient for 2D-nmr spectral analysis, which in some cases confirmed their identity with alkaloids known from other species and in other cases led to assignment of structures. Pumiliotoxin 251D, pumiliotoxin A [307A], pumiliotoxin B [323A], and allopumiliotoxin 267A were identified as major constituents. N-Oxides of 323A and 267A were also isolated. Indolizidines 195B and 223AB with 3-butyl-5-methyl and 3-butyl-5-propyl substituents, respectively, were identified. The 5-substituents of the 8-methyl-indolizidines 207A and 235B' were assigned as -(CH2)3CH = CH2 and -(CH2)5CH = CH2, respectively; indolizidine 235B' from D. speciosus is, thus, a positional double-bond isomer of indolizidine 235B previously isolated from a closely related poison frog, Dendrobates pumilio. A piperidine 241D was isolated and assigned the structure cis-cis-2-methyl-6-nonyl-4-hydroxypiperidine.