RESUMO
Monographs of the European Pharmacopoeia (Ph. Eur.) are the basis for quality control of medicinal plants and therefore important to ensure the consistency, quality, safety, and efficacy of phytopharmaceuticals. The traditional medicinal plant sundew (Drosera sp.) has disappeared from therapy due to nature conservation, but can now be cultivated sustainably on rewetted peatland. However, currently there is no valid Ph. Eur. monograph for the quality control of Droserae herba. In this study, sundew material from different species and sources was investigated with the aim of developing quality control methods based on the Ph. Eur. and defining a uniform quality standard for Droserae herba. It was possible to distinguish between sundew species of different quality, using macroscopic, microscopic, and chromatographic methods. Special emphasis was laid on the content of flavonoids and naphthoquinones as important quality parameters as their content differed between the sundew species. The differences in content and toxicity result in the recommendation that only round-leaved sundew (Drosera rotundifolia L.) should be used as a medicinal plant for the production of phytopharmaceuticals in the future.
Assuntos
Drosera , Plantas Medicinais , Drosera/química , Relação Estrutura-Atividade , FlavonoidesRESUMO
AIM: To verify synergistic effects, we investigated the antimicrobial activity of seven phenolic phytochemicals (gallic acid; epicatechin; epigallocatechin gallate; daidzein; genistein; myricetin; 3-hydroxy-6-methoxyflavone) in combination with six antibiotics against multidrug-resistant isolates from the ESKAPE group. METHODS AND RESULTS: To investigate single phytochemicals and combinations, initial microdilution and checkerboard assays were used, followed by time-kill assays to evaluate the obtained results. The research revealed that phenolic compounds on their own resulted in little or no inhibitory effects. During preliminary tests, most of the combinations resulted in indifference (134 [71.3%]). In all, 30 combinations led to antagonism (15.9%); however, 24 showed synergistic effects (12.8%). The main tests resulted in nine synergistic combinations for the treatment of four different bacteria strains, including two substances (3-hydroxy-6-methoxyflavone, genistein) never tested before in such setup. Time-kill curves for combinations with possible synergistic effects confirmed the results against Acinetobacter baumannii as the one with the greatest need for research. CONCLUSIONS: The results highlight the potential use of antibiotic-phytocompound combinations for combating infections with multi-resistant pathogens. Synergistic combinations could downregulate the resistance mechanisms of bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The aim of this study is to demonstrate the potential use of phenolic natural compounds in combination with conventional antibiotics against multidrug-resistant bacteria of the ESKAPE group. Due to synergistic effects of natural phenolic compounds combined with antibiotics, pathogens that are already resistant to antibiotics could be resensitized as we were able to reduce their MICs back to sensitive. In addition, combination therapies could prevent the development of resistance by reducing the dose of antibiotics. This approach opens up the basis for future development of antimicrobial therapy strategies, which are so urgently needed in the age of multidrug-resistant pathogens.
Assuntos
Acinetobacter baumannii , Catequina , Antibacterianos/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Farmacorresistência Bacteriana Múltipla , Sinergismo Farmacológico , Flavonas , Flavonoides , Ácido Gálico/farmacologia , Genisteína/farmacologia , Isoflavonas , Testes de Sensibilidade MicrobianaRESUMO
Screening for biofilm inhibition by purified natural compounds is difficult due to compounds' chemical diversity and limited commercial availability, combined with time- and cost-intensiveness of the laboratory process. In silico prediction of chemical and biological properties of molecules is a widely used technique when experimental data availability is of concern. At the same time, the performance of predictive models directly depends on the amount and quality of experimental data. Driven by the interest in developing a model for prediction of the antibiofilm effect of phenolic natural compounds such as flavonoids, we performed experimental assessment of antibiofilm activity of 320 compounds from this subset of chemicals. The assay was performed once on two Escherichia coli strains on agar in 24-well microtiter plates. The inhibition was assessed visually by detecting morphological changes in macrocolonies. Using the data obtained, we subsequently trained a Bayesian logistic regression model for prediction of biofilm inhibition, which was combined with a similarity-based method in order to increase the overall sensitivity (at the cost of accuracy). The quality of the predictions was subsequently validated by experimental assessment in three independent experiments with two resistant E. coli strains of 23 compounds absent in the initial data set. The validation demonstrated that the model may successfully predict the targeted effect as compared to the baseline accuracy. Using a randomly selected database of commercially available natural phenolics, we obtained approximately 6.0% of active compounds, whereas using our prediction-based substance selection, the percentage of phenolics found to be active increased to 34.8%.
Assuntos
Biofilmes , Escherichia coli , Teorema de Bayes , Fenóis/farmacologiaRESUMO
Medicinal plants have been traditionally used to treat cancer in Ethiopia. However, very few studies have reported the in vitro anticancer activities of medicinal plants that are collected from different agro-ecological zones of Ethiopia. Hence, the main aim of this study was to screen the cytotoxic activities of 80% methanol extracts of 22 plants against human peripheral blood mononuclear cells (PBMCs), as well as human breast (MCF-7), lung (A427), bladder (RT-4), and cervical (SiSo) cancer cell lines. Active extracts were further screened against human large cell lung carcinoma (LCLC-103H), pancreatic cancer (DAN-G), ovarian cancer (A2780), and squamous cell carcinoma of the esophagus (KYSE-70) by using the crystal violet cell proliferation assay, while the vitality of the acute myeloid leukemia (HL-60) and histiocytic lymphoma (U-937) cell lines was monitored in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) microtiter assay. Euphorbia schimperiana, Acokanthera schimperi, Kniphofia foliosa, and Kalanchoe petitiana exhibited potent antiproliferative activity against A427, RT-4, MCF-7, and SiSo cell lines, with IC50 values ranging from 1.85 ± 0.44 to 17.8 ± 2.31 µg/mL. Furthermore, these four extracts also showed potent antiproliferative activities against LCLC-103H, DAN-G, A2780, KYSE-70, HL-60, and U-937 cell lines, with IC50 values ranging from 0.086 to 27.06 ± 10.8 µg/mL. Hence, further studies focusing on bio-assay-guided isolation and structural elucidation of active cytotoxic compounds from these plants are warranted.
Assuntos
Medicinas Tradicionais Africanas/métodos , Extratos Vegetais/análise , Plantas Medicinais/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Etiópia , Humanos , Concentração Inibidora 50 , Extratos Vegetais/químicaRESUMO
Tricholoma populinum Lange is an edible basidiomycete from the family Tricholomataceae. Extracts, fractions, and different metabolites isolated from the fruiting bodies of this mushroom were tested for degranulation-inhibiting activities on RBL-2H3 cells (rat basophils). Dichloromethane extracts decreased degranulation significantly, as did a fraction after column chromatography. In addition, the extract decreased the IL-2 release from Jurkat T cells and the release of IL-8 from HMC-1 human mast cells. The results show the significant effects of extracts of T. populinum on cells of the innate (basophils and mast cells) and adaptive (T cells) immune system and indicate the influence of the mushroom on different immunological processes. As one fraction showed activity, it seems to be possible that it includes an active principle. The compounds responsible for this effect, however, could not be identified as the contents oleic acid (1), ergosterol peroxide (2), and 9,11-dehydroergosterol peroxide (3) showed no effects. Nevertheless, the mushroom could be used for supporting allergy treatment in future studies.
Assuntos
Basófilos/efeitos dos fármacos , Produtos Biológicos/farmacologia , Degranulação Celular/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Tricholoma/química , Animais , Basófilos/fisiologia , Produtos Biológicos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Gel/métodos , Carpóforos/química , Humanos , Interleucina-2/metabolismo , Interleucina-8/metabolismo , Células Jurkat , Espectroscopia de Ressonância Magnética , Mastócitos/metabolismo , Cloreto de Metileno/química , Ratos , Sílica Gel/químicaRESUMO
The immune system is permanently exposed to several environmental influences that can have adverse effects on immune cells or organs leading to immunosuppression or inappropriate immunostimulation, called direct immunotoxicity. The natural compound Tulipalin A (TUPA), a lactone with α-methylene-γ-butyrolactone moiety, can influence the immune system and lead to allergic contact dermatitis. This in vitro study focused on effects of TUPA using two immune cell lines (Jurkat T cells and THP-1 monocytes). To evaluate the immunotoxic potential of the compound, a proteomic approach applying 2D gel electrophoresis and MALDI-TOF/TOF-MS in combination with metabolomic analysis was used after exposure of the cells to IC10 of TUPA. THP-1 cells showed a strong robustness to TUPA treatment since only five proteins were altered. In contrast, in Jurkat T cells an increase in the abundance of 66 proteins and a decrease of six proteins was determined. These intracellular proteins were mapped to biological processes. Especially an accumulation of chaperones and an influence on the purine synthesis were observed. The changes in purine synthesis were confirmed by metabolomic analysis. In conclusion, the data indicate possible target processes of low doses of TUPA in Jurkat T cells and provides knowledge of how TUPA affects the functionality of immune cells.
Assuntos
4-Butirolactona/análogos & derivados , Proteômica/métodos , 4-Butirolactona/imunologia , 4-Butirolactona/toxicidade , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Dermatite Alérgica de Contato/etiologia , Eletroforese em Gel Bidimensional , Humanos , Células Jurkat/efeitos dos fármacos , Células Jurkat/imunologia , Células Jurkat/metabolismo , Metaboloma , Dobramento de Proteína/efeitos dos fármacos , Purinas/biossíntese , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Testes de Toxicidade/métodosRESUMO
Multidrug-resistant gram-negative pathogens such as Escherichia coli have become increasingly difficult to treat and therefore alternative treatment options are needed. Targeting virulence factors like biofilm formation could be one such option. Inhibition of biofilm-related structures like curli and cellulose formation in E. coli has been shown for different phenolic natural compounds like epigallocatechin gallate. This study demonstrates this effect for other structurally unrelated phenolics, namely octyl gallate, scutellarein and wedelolactone. To verify whether these structurally different compounds influence identical pathways of biofilm formation in E. coli a broad comparative RNA-sequencing approach was chosen with additional RT-qPCR to gain initial insights into the pathways affected at the transcriptomic level. Bioinformatical analysis of the RNA-Seq data was performed using DESeq2, BioCyc and KEGG Mapper. The comparative bioinformatics analysis on the pathways revealed that, irrespective of their structure, all compounds mainly influenced similar biological processes. These pathways included bacterial motility, chemotaxis, biofilm formation as well as metabolic processes like arginine biosynthesis and tricarboxylic acid cycle. Overall, this work provides the first insights into the potential mechanisms of action of novel phenolic biofilm inhibitors and highlights the complex regulatory processes of biofilm formation in E. coli.
RESUMO
Beta glucans are complex glucose polymers well known for their immune modulatory properties. Therefore they are used and advertised in dietary supplements. Unfortunately there is no standardized test system for quality control of such health-related foods. This approach combined wet chemical and enzyme-based quantification methods (e.g. aniline blue, Glucatell®) with a cytokine secretion assay as parameter for immune activation to resolve this problem and to establish a quality control system for ß-glucan containing products and extracts. Commercially available pure ß-glucans with different origin and structure were used in this study. None of the methods allowed an accurate ß-glucan quantification. Most promising was the test kit K-EBHLG (Megazyme). However, cytokine secretion from whole blood was detectable under the influence of ß-glucans, but there was no correlation with the quantification results using the commercially available kits. Therefore, the quality control of ß-glucan containing products needs further efforts.
Assuntos
beta-Glucanas , Bioensaio , Controle de QualidadeRESUMO
The immune system is an important target of various xenobiotics, which may lead to severe adverse effects including immunosuppression or inappropriate immunostimulation. Mitochondrial toxicity is one possibility by which xenobiotics exert their toxic effects in cells or organs. In this study, we investigated the impact of three natural compounds, cyclosporine A (CsA), deoxynivalenol (DON) and cannabidiol (CBD) on mitochondrial functions in the THP-1 monocytic cell line. The cells were exposed for 24h to two different concentrations (IC10 and IC50 determined by MTT) of each compound. The cells showed concentration-dependent elevated intracellular reactive oxygen species (iROS) and induction of apoptosis (except DON) in response to the three test compounds. Mitochondrial functions were characterized by using bioenergetics profiling experiments. In THP-1 monocytes, the IC50 of CsA decreased basal and maximal respiration as well as ATP production with an impact on spare capacity indicating a mitochondrial dysfunction. Similar reaction patterns were observed following CBD exposure. The basal respiration level and ATP-production decreased in the THP-1 cells exposed to the IC50 of DON with no major impact on mitochondrial function. In conclusion, impaired mitochondrial function was accompanied by elevated iROS and apoptosis level in a monocytic cell line exposed to CsA and CBD. Mitochondrial dysfunction may be one explanation for the cytotoxicity of CBD and CsA also in other in immune cells.
Assuntos
Produtos Biológicos/toxicidade , Canabidiol/toxicidade , Ciclosporina/toxicidade , Mitocôndrias/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Tricotecenos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Mitocôndrias/metabolismo , Monócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Immunotoxicity, defined as adverse effects of xenobiotics on the immune system, is gaining increasing attention in the approval process of industrial chemicals and drugs. In-vivo and ex-vivo experiments have been the gold standard in immunotoxicity assessment so far, so the development of in-vitro and in-silico alternatives is an important issue. In this paper we describe a widely applicable, easy-to use computational approach which can serve as an initial immunotoxicity screen of new chemical entities. Molecular fingerprints describing chemical structure were used as parameters in a machine-learning approach based on the Naïve-Bayes learning algorithm. The model was trained using blood-cell growth inhibition data from the NCI database and validated externally with several in-house and literature-derived data sets tested in cytotoxicity assays on different types on immune cells. Both cross-validations and external validations resulted in areas under the receiver operator curves (ROC/AUC) of 75% or higher. The classification of the validation data sets occurred with excellent specificities and fair to excellent selectivities, depending on the data set. This means that the probability of actual immunotoxicity is very high for compounds classified as immunotoxic, while the fraction of false negative predictions might vary. Thus, in a multistep immunotoxicity screening scheme, the classification as immunotoxic can be accepted without additional confirmation, while compounds classified as not immunotoxic will have to be subjected to further investigation.
Assuntos
Linfócitos B/efeitos dos fármacos , Biologia Computacional/métodos , Linfócitos T/efeitos dos fármacos , Xenobióticos/toxicidade , Linfócitos B/imunologia , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/imunologia , Linfócitos T/imunologiaRESUMO
Extracts of Arnica spp. are traditionally used due to their anti-inflammatory effects for the topical treatment of e.g. haematoma or muscle distortions. One of the main active compounds is Helenalin, a sesquiterpene lactone that can be found in various Asteraceae. However, immunotoxic effects of the compound are only poorly analysed. In this study, a 2D gel electrophoresis based proteomic approach together with a membrane based proteomic assay, metabolomics and the detection of intracellular reactive oxygen species (iROS) were used to investigate potential immunotoxic properties of Helenalin on the human immune cell lines Jurkat and THP-1 and on human peripheral blood mononuclear cells (PBMC). The study revealed a dose-dependent cytotoxicity towards both tested cell lines and the PBMC. However, the cell lines were less sensitive to the Helenalin treatment than the PBMC. The proteomic assays showed strong effects on the carbohydrate metabolism and the protein folding in THP-1 cells but only weak impact on Jurkat cells. Metabolomic studies as well as iROS detection in THP-1 cells verified the results of the proteomic analysis. In summary, the approaches used in this study were able to identify target pathways of Helenalin especially in THP-1 monocytes and thus enable a risk assessment of the substance.