Quality evaluation of Erigeron breviscapus from different origins and its related species by HPLC coupled with chemometrics / 中草药

Objective: To establish the HPLC fingerprint for effective quality control and scientific evaluation of Erigeron breviscapus. Methods: Separation was performed on a Zorbax SB-C18 column (150 mm × 4.6 mm, 5 μm) and the mobile phase was methanol-0.1% phosphoric acid with gradient elution. The flow rate at 1 mL/min, the column temperature at 30 oC, and the detection wavelength at 335 nm. A total of 19 batches of E. breviscapus and its related species were analyzed. Similarity evaluation combined with hierarchical clustering analysis (HCA) and principal components analysis (PCA) were used to evaluate the quality of herbs from different batches. Results: The HPLC fingerprint of E. breviscapus was established with 11 common peaks, and five peaks were identified. Similarities of the 19 batches of samples were 0.873-0.978. Two batches of samples from its related species were high similarity. These 19 batches of samples could be classified into three clusters. The PCA result was consistent with HCA. The comprehensive score of S5 was the highest and the quality was the best. There was possibility for using E. multiradiatus as herbs instead of E. breviscapus. Conclusion: The establishment of HPLC fingerprint and the recognition of chemical pattern can provide a more comprehensive reference for the quality control of herbs.

Identification of Salviae Yunnanensis Radix et Rhizoma and Its Allied Species Based on Chemical Composition by FTIR with Chemometric Analysis and HPLC Fingerprint / 中国实验方剂学杂志

Objective:To investigate the differences of chemical constituents of Salviae Yunnanensis Radix et Rhizoma and its closely related species,and to provide reference for the clinical application of Salviae Yunnanensis Radix et Rhizoma. Method:Fourier transform infrared (FTIR) spectroscopy combined with principal component analysis (PCA),cluster analysis (HCA) and partial least squares discriminant analysis (PLS-DA) were used to study the differences of components between Salviae Yunnanensis Radix et Rhizoma and its closely related species,and high performance liquid chromatography (HPLC) was used to compare the differences of water-soluble components between them. Result:There were some differences between Salviae Yunnanensis Radix et Rhizoma and its closely related species acrcording to FTIR spectroscopy and HPLC fingerprint,especially the water-soluble polar components were more abundant in Salviae Yunnanensis Radix et Rhizoma than other species. The chemical components of Salvia trijuga,S. przewalskii and S. bulleyana were more similar in terms of their genetic relationship. The result showed that the Salviae Yunnanensis Radix et Rhizoma and its closely related species can be clearly distinguished by FTIR combined with chemometrics method. Conclusion:Compared with its closely related species,Salviae Yunnanensis Radix et Rhizoma has a unique chemical composition,which has great therapeutic potential for blood stasis. The FTIR combined with chemometrics model can be used for rapid identification of Salviae Yunnanensis Radix et Rhizoma and its closely related species.

High-Performance Thin-layer Chromatographic Fingerprint Analysis of Alpinia katsumadai Hayata and Its Related Species / 中国药学杂志

OBJECTIVE: To establish the high-performance thin layer chromatographic(HPTLC) fingerprints of volatile oil and flavonoids in Alpinia katsumadai Hayata so as to provide scientific information for its quality control, and determine the fingerprints similarity of with its related species. METHODS: The separation was performed on the pre-coated HPTLC GF254 silica gel plates. The volatile oil was developed with solvent system of toluene-ethyl acetate(9: 1). The relative humidity was 18%. The spots were visualized with 5% vanillin sulfuric acid solution. The flavonoides compounds were developed with solvent system of toluene-ethyl acetate-acetic acid (8: 1.5: 0.5). The spots were visualized with 5% aluminium chloride ethanol solution which needed to be observed at 365 nm. The common patterns of HPTLC fingerprints were obtained by CHROMAP 1.5 solution software, and authentication and quality assessment were performed by similarity and principle component analysis. RESULTS: The common patterns of the volatile oil and flavonoids consisted of 10 characteristic peaks and 7 characteristic peaks, respectively. Eucalyptol and alpinetin were identified by chemical reference substances. CONCLUSION: The qualities of Alpinia katsumadai Hayata collected from different areas are not distinctly different. Obvious difference exists in the chemical compositions of the volatile oil and flavonoids between Alpinia katsumadai Hayata and its counterfeit and other related species. This method is simple and rapid, which can serve as an effective identification and quality assessment method for Alpinia katsumadai Hayata.

Identification of Panax japonicus and its related species or adulterants using ITS2 sequence / 中草药

Objective: To provide a scientific basis for the modern identification of Panax japonicus and ensure the clinical efficacy and medication accuracy, the molecular identification of P. japonicus and its related species or adulterants was carried out. Methods: ITS2 sequences of P. japonicus were amplified and sequenced directionally. ITS2 sequences of related species and adulterants were downloaded from GenBank. Cutting with ITS2 database, the final dataset consisted of 102 sequences from 24 species. DAMBE program was also implemented to detect substitution saturation of ITS2 sequences. MEGA 6.06 software was utilized for sequence alignment, calculating GC content, analyzing variation sites, estimating intra-specific and inter-specific genetic distances, and finally building NJ tree. Moreover, the secondary structure of ITS2 was predicted by using the ITS2 database. Results: The length of ITS2 sequences from P. japonicas was 230 bp and GC content was 63.7%. The average genetic distance analysis, NJ tree, and secondary structure characteristics of the ITS2 sequences showed that there were great differences between P japonicus and its non-identical adulterants or partial related species (P. stipuleanatus, P. pseudoginseng, P. trifolius, P. vietnamensis var. fuscidiscus, and P. vietnamensis). Howerer, the application of such method for the identification of P. japonicus and partial closely related species (P. quinquefolius, P. ginseng, P. notoginseng, P. japonicus var. major, P. japonicus var. bipinnatifidus, P. assamicus, P. variabilis, and P. japonicus var. angustifolius) had certain limitation. Conclusion: The ITS2 sequence can be used as one of the DNA barcodings for the identification of P. japonicus and its non-identical adulterants at high identification rate, however, its versatility of identification between P japonicus and related species needs to be further verified. Our study provides the basis for the identification of inter-specific genetic and affinity relationship of P. japonicas and its related species, the distinguishment between P. japonicas and non-identical adulterants, and the clinical safety of P japonicas.

Molecular identification of Cynanchum auriculatum and its closely related species based on ITS2 sequence / 中草药

Objective To identify Cynanchum auriculatum and its closely related species using the ITS2 barcode. Methods A total of 54 samples of C. auriculatum and its related species were collected. Genomic DNAs were extracted from these samples. The ITS2 sequences of these samples were amplified and bidirectional sequenced by PCR. The obtained sequences were submitted to the Gen Bank and the ITS2 sequences of 47 samples belonging to 15 species were downloaded from the GenBank, and ITS2 sequences were annotated by ITS2 database. A total of 101 ITS2 sequences were aligned and the intraspecific and interspecific distances were calculated using the MEGA 5.0. Identification analyses were performed using the similarity search method and nearest distance method, and were presented intuitively by constructing neighbor-joining (NJ) tree. Results The length of all ITS2 sequences of C. auriculatum was 249 bp, which was a haplotype and was close to Cynanchum. There was a significant difference between the interspecific and intraspecific genetic distances of the ITS2 sequences. The NJ tree showed that C. auriculatum obviously differed from its closely related species, which showed high monophyly. According to the secondary structure of ITS2, it was also possible to distinguish between C. auriculatumi and Asclepiadaceae related species. Conclusion As a DNA barcode, ITS2 sequences can stably and accurately distinguish C. auriculatum from its closely related species and also provide a new technique to ensure the clinical safety in utilization of Chinese materia medica.

High-performance thin-layer chromatographic fingerprint analysis of Amomum villousm and its related species / 中国药学杂志

OBJECTIVE: To study the high-performance thin layer chromatographic (HPTLC) fingerprint of volatile oil constituents from Amomum villousm and its related species so as to set up the identification protocol of the medicinal plant and provide scientific information for its quality control. METHODS: TLC was used to analyze comparatively 10 batches of Amomum villosum Lour.samples, 10 batches of Amomum villousm crude drugs collected from different producing areas and stored for different time, 10 batches of the fruits of counterfeit species and 10 kinds of related species in the Zingiberaceae family. The samples were separated on silica gel G precoated plates with a mixture of cyclohexane-chloroform-ethyl acetate (13:2:2) as developing solvent system. The relative humidity was 67%. The spots were visualized with 5% vanillin sulfuric acid solution, then were analyzed by utilizing CHROMAP 1.5 solution software. RESULTS: The fingerprint of volatile oil of Amomum villosum, with 9 specific bands examined under natural light, was set up. The quality of Amomum villosum stored for different time or collected from different areas was distinctly variable. Obvious difference existed in the chemical composition of the volatile oils between Amomum villosum and its counterfeit and other related species. CONCLUSION: The HPTLC fingerprint analysis method can be used for rapid identification and quality control ofAmomum villosum.

Molecular identification of Atractylodes lancea and its closely related species based on ITS2 sequence / 中草药

Objective: The aim of present study was to identify Atractylodes lancea and its closely related species (Atractylodes chinesis and Atractylodes macrocephala) using the ITS2 barcode. Methods: The total genomic DNAs were extracted from twenty-nine samples of A. lancea and its closely related species from different habitats. The ITS2 sequences of these samples were amplified and bidirectional sequenced by PCR. The obtained sequences were submitted to the GenBank and the ITS2 sequences of 45 samples belonging to ten species were downloaded from the GenBank. Total 73 ITS2 sequences were aligned and the genetic distances were analyzed using the MEGA 5.1. Identification analyses were performed using the BLAST1 and the nearest distance methods, and were presented intuitively by constructing Neighbor-joining (NJ) tree. Results: The lengths of all ITS2 sequences of A. lancea were 229 bp presented as one haplotype pattern. There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 sequences. The NJ tree showed that A. lancea could differed obviously from its closely related species, which showed high monophyly. The secondary structures of ITS2 in the helical regions displayed clear differences in stem loop number, size, position, and screw angle among the medicinal plants of Compositae. Conclusion: As a DNA barcode, ITS2 sequences can stably and accurately distinguish A. lancea from its closely related species and also provide a new technique to ensure the clinical safety in utilization of Chinese materia medica.

Caracterização do complexo Candida glabrata em isolados de hemocultura e avaliação da sensibilidade a antifúngicos / Characterization of the complex Candida glabrata in blood culture isolates and evaluation of sensitivity to antifungals

Candida glabrata assumiu grande importância na clínica médica, desde que, sua resistência adquirida ao fluconazol foi descrita. Além disso, estudos mostraram a fraca atividade in vitro de outros fármacos azólicos contra isolados dessa espécie. C. glabrata é agente de infecções invasivas e o monitoramento da eficácia de antifúngicos usados na prática médica frente a isolados dessa espécie tem relevância clinica. C. bracarensis e C. nivariensis são espécies relacionadas, fenotipicamente, à C. glabrata para as quais há necessidade de métodos moleculares para sua identificação. Na América Latina, a ocorrência de infecções em corrente sanguínea por C. glabrata e espécies correlatas não é tão alta quanto na América do Norte e, por isso, pouco é conhecido sobre sua distribuição e perfil de suscetibilidade a antifúngicos nessa região. Neste estudo, foram analisados 75 isolados com características morfológicas e bioquímicas de C. glabrata, obtidos da corrente sanguínea de pacientes atendidos em hospitais do estado de São Paulo, entre 2007 e 2013. As concentrações inibitórias mínimas (CIM) de cinco fármacos antifúngicos: anfotericina B, caspofungina, voriconazol, fluconazol e itraconazol, foram determinadas pela metodologia de microdiluição de referencia M27-A3 do Clinical and Laboratory Standards Institute (CLSI). A ação fungicida de anfotericina B foi avaliada por método de curva de morte. A pesquisa das duas espécies correlatas foi realizada com metodologia de reação em cadeia da polimerase (PCR) em todos os isolados. Nenhum isolado de C. bracarensis e C. nivariensis foi encontrado neste estudo. Resistência a itraconazol foi encontrada em 18,6 % (14) das cepas de C. glabrata. Altos valores de CIM de voriconazol (> 0,5 mg/L), de acordo com cutoff (ponto de corte) epidemiológico, foram observados para duas cepas...

Candida glabrata has assumed great importance in clinical medicine, since acquired resistance to fluconazole had been described. Furthermore, studies have shown the weak in vitro activity of other azole drugs against isolates of this species. Since C. glabrata is an agent of invasive infections, monitoring the effectiveness of antifungal agents used in medical practice against isolates of this species has become of great importance. C. bracarensis and C. nivariensis are phenotypically related species to C. glabrata and so it is necessary molecular methods to identify properly these members. In Latin America, the occurrence of bloodstream infections in C. glabrata and related species is not as high as in North America, and little is known about their distribution and antifungal susceptibility profile in this region. In this study, we analyzed 75 isolates with morphological and biochemical features of C. glabrata obtained from the bloodstream of patients treated in hospitals in the state of São Paulo, between 2007-2013. The minimum inhibitory concentrations (MIC) of five antifungal drugs, namely: amphotericin B, caspofungin, voriconazole, fluconazole and itraconazole were determined by microdilution reference method M27-A3 from the Clinical and Laboratory Standards Institute (CLSI). The fungicidal action of amphotericin B was evaluated by the method of time-kill curves. The investigation of the two related species was performed with polymerase chain reaction (PCR) in all isolates. No isolate of C. bracarensis and C. nivariensis was found in this study. Resistance to itraconazole was found in 18.6% (14) strains of C. glabrata. High MIC values of voriconazole (> 0.5 mg/L), according to epidemiological cut-off were observed for two strains. The fluconzole-MICs ranged from 4mg/L to 16mg/L, caspofungin-MIC were between 0.03 mg/L and 0.5 mg/L, and amphotericin B-MIC were between 0.12 mg/L and 1 mg/L...

Identification of Bamboo Shavings, Concretio Silicea Bambusae and Their Closely Related Species based on the psbA-trnH Intergenic Region / 世界科学技术-中医药现代化

In this study, the psbA-trnH sequence was used as DNA barcoding to evaluate the accuracy and stability for identification of Bamboo shavings, Concretio Silicea Bambusae and their closely related species. 56 samples were collected and conducted the DNA extraction. Obtained sequences were assembled using the CodonCode Aligner V4.2, the genetic distances and NJ tree were computed and constructed using MEGA 5.0. The results shows that the maximum intra-specific K2P distances were less than the minimum inter-specific K2P distances. The NJ tree indicated that Bamboo shavings, Concretio Silicea Bambusae and their closely related species can be distinguished from each other clearly, except for Bambusa tuldoides Munro and Bambusa textilis, which perhaps attributed to their closely genetic relationship.

Identification of Inulae Herba, Inulae Flos and Their Closely Related Species Based on ITS2 Sequence / 世界科学技术-中医药现代化

This study aimed to identify Inulae Herba, Inulae Flos and their closely related species using the ITS2 bar-code, and secure the quality and clinical curative effect of these medicinal materials. DNA was extracted from Inula linariifolia, I. japonica, I. britanica, which are the original species of Inulae Herba and Inulae Flos, together with their closely related species. The ITS2 sequence was amplified by PCR and sequenced bidirectionally. Sequence assembly and generation of consensus sequence were conducted by the CodonCode Aligner. The genetic distances were comput-ed using MEGA 5.0 in accordance with the Kimura-2-parameter (K2P) model, and the phylogenetic tree constructed by the neighbor-joining (NJ) method. The results showed that the ITS2 sequences of the various species have stable variable sites. The intraspecific genetic distances among Inulae Herba and Inulae Flos were obviously lower than the interspecific genetic distance among the above two medicinal materials and its adulterants. The NJ tree based on ITS2 sequences can clearly differ from Inulae Herba, Inulae Flos and their closely related species. It is concluded that ITS2 sequence can be used as DNA barcode to identify Inulae Herba, Inulae Flos and their closely related species.

Niche enlargement as a consequence of co-existence: a case study / Ampliação do nicho como consequência da co-existência: um estudo de caso

Spatio-temporal changes in the diet, niche breadth and niche overlap of two species of Characidium from three different sites along a Neotropical coastal stream were studied during a dry and rainy season. Seasonal changes were restricted to the occurrence of plant items in the stomach contents. The relative importance of food items in the diet of both species varied across sites, but Diptera, Ephemeroptera, Simuliidae, Trichoptera and Coleoptera larvae were always the main prey items. Contrary to the expected pattern, values of the niche breadth were higher at the site where Characidium species co-existed and niche overlapped at this site indicated 52% (p = 0.52) of feeding overlap.

Variações espaço-temporal na dieta, na amplitude e na sobreposição de nicho foram estudadas para duas espécies de Characidium de três localidades distintas de um riacho costeiro da região Neotropical, considerando-se as estações seca e chuvosa. As alterações sazonais na dieta foram restritas à presença/ausência de itens vegetais nos conteúdos estomacais. A importância relativa dos itens alimentares, de ambas as espécies, variou entre as localidades de estudo; porém, as larvas de Diptera, Ephemeroptera, Simuliidae, Trichoptera e Coleoptera foram sempre as presas mais consumidas. Em oposição ao padrão esperado, os valores de amplitude de nicho foram maiores na localidade em que as duas espécies de Characidium ocorreram em sintopia e a sobreposição de nicho, nessa localidade, indicou 52% (p = 0,52) de sobreposição alimentar.

Environmental influences on the spatial and temporal distribution of the puffer fish Sphoeroides greeleyi and Sphoeroides testudineus in a Brazilian subtropical estuary

The spatial and temporal distribution of Sphoeroides greeleyi and Sphoeroides testudineus were established from collections (biological material and environmental data) conducted on a monthly basis from May 2000 to April 2001 in intertidal areas along the north-south axis of the estuarine complex of Paranaguá, Paraná State. In addition to characterizing a north-south spatial gradient, which fluctuates seasonally, the variation in the abiotic factors made possible the division of the estuary into three regions: north, central and south. Spatially, it was found that the number of individuals declines significantly for both species in the north-south direction of the estuary. Moreover, significant differences were found in the size of individuals across the estuarine regions. The largest S. greeleyi individuals were caught in the north, as well as the smallest S. testudineus individuals. The catches with the highest numbers of puffer fish occurred from late spring to early autumn, coinciding with the occurrence of specimens of smaller size and lower mean body mass. The results indicate that spatial and temporal variations in the environment impact the distribution patterns of both puffer fish species, suggesting that the co-occurrence of closely related species functions as a modulating factor in that distribution.

A distribuição espacial e temporal de Sphoeroides greeleyi e Sphoeroides testudineus foram estabelecidas a partir de coletas (material biológico e dados ambientais) realizadas mensalmente de maio/2000 a abril/2001 em áreas intertidais, no eixo norte-sul, do complexo estuarino de Paranaguá, Estado do Paraná. A variação dos fatores abióticos coletados, além de caracterizar um gradiente espacial, no sentido norte-sul, que varia sazonalmente, possibilitou a divisão do estuário em três regiões: norte, central e sul. Espacialmente, verificou-se para ambas as espécies que o número de indivíduos decresce, significativamente, no sentido norte-sul estuarino. Ainda, foram encontradas diferenças significativas no porte dos indivíduos entre as regiões do estuário, no norte ocorreram os maiores indivíduos de S. greeleyi e os menores de S. testudineus. As maiores capturas dos baiacus ocorreram do final da primavera ao início do outono, coincidindo com a ocorrência de exemplares de menor tamanho e de menor massa corporal média. Os resultados indicam que as variações espaciais e temporais do ambiente afetam os padrões de distribuição de ambas as espécies de baiacus, sugerindo que a co-ocorrência de espécies aparentadas age como um fator modulador nesta distribuição.

The neotropical biting midges related to Culicoides paraensis (Diptera: Ceratopogonidae)

Culicoides paraensis (Goeldi), a common and widespread American bloodsucking midge that has been incriminated in the transmission of Mansonellosis and Oropouche Fever of humans in South America, is redescribed and figured. All published records are listed and new distribution is based on examination of extensive collections from throughout its range. Three closely related species of the subgenus Haematomyidium that have been confused with C. paraensis are briefly redescribed and figured, and a key is presented for their identification.