Anticaries and antimicrobial activities of methanolic extract from leaves of <i>Cleistocalyx operculatus</i> L.

Objective: To investigate antimicrobial activities of methanolic extract of leaves of Cleistocalyx operculatus L. (C. operculatus) grown in Vietnam.Methods: The methanolic extract of C. operculatus leaves was phytochemically screened and tested for its antimicrobial activity against six Gram-positive bacteria (three of which were antibiotic multiresistant Staphylococcus spp.), two Gram-negative bacteria, and one fungal species using an agar diffusion method. Anticaries activity was tested using pH drop and biofilm assays formed in 96-well plastic plates.Results: Phytochemical screening revealed the presence of flavonoids and terpenes, in which flavonoid content was 6.8 mg/g dry material. Antibacterial activity of the C. operculatus extract was shown only against Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis and Streptococcus mutans GS-5 (S. mutans), and three multiresistant bacteria being Staphylococcus epidermidis 847, Staphylococcus haemolyticus 535 and Staphylococcus aureus North German epidemic strain. Interestingly, methanolic extract of C. operculatus leaves exhibited the anticaries activity against S. mutans in terms of inhibition of acid production and biofilm formation. Activity of two key enzymes responsible for acidogenicity of S. mutans, F-ATPase and phosphotransferase system were inhibited by the extract with IC50 of 51.0 and 98.0 mg/mL, respectively. Cytotoxicity of the extract against keratinocytes was found only for higher concentrations [IC50 = (119.98 ± 4.63) mg/mL]. Conclusions: The methanolic extract of C. operculatus leaves has the potential for development of antimicrobial preparations, especially anticaries products

Anticaries and antimicrobial activities of methanolic extract from leaves of <i>Cleistocalyx operculatus</i> L.

Objective: To investigate antimicrobial activities of methanolic extract of leaves of Cleistocalyx operculatus L. (C. operculatus) grown in Vietnam. Methods: The methanolic extract of C. operculatus leaves was phytochemically screened and tested for its antimicrobial activity against six Gram-positive bacteria (three of which were antibiotic multiresistant Staphylococcus spp.), two Gram-negative bacteria, and one fungal species using an agar diffusion method. Anticaries activity was tested using pH drop and biofilm assays formed in 96-well plastic plates. Results: Phytochemical screening revealed the presence of flavonoids and terpenes, in which flavonoid content was 6.8 mg/g dry material. Antibacterial activity of the C. operculatus extract was shown only against Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis and Streptococcus mutans GS-5 (S. mutans), and three multiresistant bacteria being Staphylococcus epidermidis 847, Staphylococcus haemolyticus 535 and Staphylococcus aureus North German epidemic strain. Interestingly, methanolic extract of C. operculatus leaves exhibited the anticaries activity against S. mutans in terms of inhibition of acid production and biofilm formation. Activity of two key enzymes responsible for acidogenicity of S. mutans, F-ATPase and phosphotransferase system were inhibited by the extract with IC50 of 51.0 and 98.0 μg/mL, respectively. Cytotoxicity of the extract against keratinocytes was found only for higher concentrations [IC50 = (119.98 ± 4.63) μg/mL]. Conclusions: The methanolic extract of C. operculatus leaves has the potential for development of antimicrobial preparations, especially anticaries products

Natural phenolic compounds as biofilm inhibitors of multidrug-resistant Escherichia coli – the role of similar biological processes despite structural diversity

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

Bestimmung der Immuntoxizität ausgewählter Naturstoffe mittels zellbasierter Untersuchungen

Neu isolierte und synthetisierte Wirkstoffe müssen neben ihrer biologischen Wirksamkeit auch auf ihre Unbedenklichkeit für den Menschen hin untersucht werden. Ein Bestandteil der Untersuchungen zur Unbedenklichkeit ist die Prüfung auf mögliche Immuntoxizität. Die Risikobewertung und -klassifizierung von (immun-)toxischen Substanzen erfolgt derzeit in Tierversuchen, die, abgesehen von ethischen Bedenken, zeit- und kostenintensiv sind und deren Übertragbarkeit auf den Menschen nicht vollständig gewährleistet ist. Im Fokus dieser Arbeit stand die Etablierung und Anwendung eines Methodensets basierend auf funktionalen in vitro Methoden zur Charakterisierung immunologischer Wirkungen ausgewählter Naturstoffe. Dieses sollte der Beurteilung der immuntoxischen Wirkungen der getesteten Naturstoffe und der Entwicklung eines Entscheidungsbaums, der die Vorhersage des immuntoxischen Potentials mithilfe von in vitro Versuchen gestattet, dienen. Dazu wurden zwei humane Immunzelllinien (Jurkat-Zellen als Beispiel für T-Zellen, THP-1-Zellen als Beispiel für Monozyten) und für einige Versuche vergleichsweise primäre Blutzellen eingesetzt. Es wurden Methoden zur Untersuchung folgender Parameter etabliert und angewendet: Vitalität, Zellzyklusverteilung, Induktion von Apoptose, iROS, DNA-Schäden (Genotoxizität), Zytokinfreisetzung und mitochondriale Funktion. Folgende Naturstoffe wurden für die Untersuchungen ausgewählt: Mannitol und Urethan als Negativkontrollen, Cyclosporin A, Deoxynivalenol und Mycophenolsäure als Positivkontrollen, ausgehend von Hinweisen auf Wirkungen im Immunsystem Tulipalin A, Helenalin, Vincristin, Cannabidiol, Agaritin und p-Tolylhydrazin als Testsubstanzen. Es zeigten sich nur geringfügige Unterschiede der Substanzwirkungen zwischen den Immunzelllinien, welche v.a. auf Zytokinebene nachweisbar waren. Die Substanzen besaßen zeit- und konzentrationsabhängige Effekte. Die Negativkontrolle Mannitol hatte eine geringe Wirkung auf die Immunzelllinien, während Urethan die Zytokinfreisetzung supprimierte/¬stimulierte. Die untersuchten Positivkontrollen zeigten einen Einfluss auf die Zytokinfreisetzung und führten weiterhin zu immuntoxischen Effekten durch eine konzentrationsabhängige Apoptoseinduktion. Die Testsubstanzen Vincristin, Agaritin und p-Tolylhydrazin besaßen nur eine geringe toxische Wirkung auf die Immunzellen. Weitere Substanzen wie Cannabidiol, Helenalin und Tulipalin A wiesen immunspezifisch und - unspezifisch vermittelte Immuntoxizität durch einen Einfluss auf die Zytokinfreisetzung, Apoptose und iROS auf. Funktionale in vitro Untersuchungen zur Vitalität, Zellzyklusverteilung, Apoptose und Zytokinfreisetzung waren zum Nachweis bzw. Ausschluss von Immuntoxizität geeignet und neben Proteom- und Metabolomanalysen wesentlicher Bestandteil eines Entscheidungsbaums zur Klassifizierung von direkt immuntoxischen Substanzen. Es zeigte sich, dass die Zytokinmessung der wichtigste Parameter zur Klassifizierung von immuntoxischen Substanzen im subtoxischen Bereich ist. Es konnte sowohl Cyclosporin A als Positivkontrolle als auch Mannitol als Negativkontrolle in beiden Zelllinien bestätigt werden. Von den hinreichend untersuchten Testsubstanzen wurde Cannabidiol, Helenalin und Tulipalin A in Jurkat-Zellen sowie Cannabidiol und Tulipalin A in THP-1-Zellen unter Verwendung des Entscheidungsbaums als immuntoxisch klassifiziert. Darüber hinaus konnte die hautsensibilisierende Wirkung von Farnesol und Tulipalin A durch Anwendung von weiteren in vitro Methoden bestätigt werden. Eine Validierung der Ergebnisse mit weiteren bekannten immuntoxischen und nicht-immuntoxischen Substanzen würde eine Anwendung als Vorscreening Testung neuer Substanzen ermöglichen und nicht nur zu einer Reduktion von Tierversuchen führen, sondern auch eine Zeit- und Kostenersparnis bedeuten.Newly isolated and synthesized active compounds have to be investigated besides their biological effectiveness on their safety for humans. One aspect of the safety evaluations is testing for possible immunotoxicity. The risk assessment and classification of (immuno-) toxic substances is currently being carried out in animal experiments which, apart from ethical concerns, are time-consuming and cost-intensive and whose transferability to humans is not fully guaranteed. The focus of this work was the establishment and application of a set of functional in vitro methods for the characterization of immunological effects of selected natural compounds. This should enable the assessment of the immunotoxic effects of the tested natural products and the development of a decision tree that allows the prediction of the immunotoxic potential. For this purpose, two human immune cell lines (Jurkat cells as an example of T cells, THP-1 cells as an example of monocytes) were used. Methods have been established to study the following parameters: viability, cell cycle distribution, induction of apoptosis, iROS, DNA damage (genotoxicity), cytokine release and mitochondrial function. The following natural products were selected for the tests: mannitol and urethane as negative controls, cyclosporin A, deoxynivalenol and mycophenolic acid as positive controls, based on indications of effects in the immune system tulipalin A, helenaline, vincristine, cannabidiol, agaritin and p-tolylhydrazine as test substances. There were only slight differences of the compounds’ effects between the immune cell lines that were mainly detectable at the cytokine level. The substances showed time- and concentration-dependent effects. The negative control mannitol had a small effect on the immune cell lines, while urethane suppressed / stimulated cytokine release. The tested positive controls showed an influence on the cytokine release and further led to immunotoxic effects through a concentration-dependent apoptosis induction. The test substances vincristine, agaritine and p-tolylhydrazine had only a slight toxic impact on the immune cells. Other substances such as cannabidiol, helenaline and tulipalin A showed immune-specific and –unspecific mediated immunotoxicity by influencing cytokine release, iROS and apoptosis. Functional in vitro studies on viability, cell cycle distribution, apoptosis and cytokine release were suitable for the detection or exclusion of immunotoxicity and in addition to proteome and metabolome analyses, an essential part of a decision tree for the classification of directly immunotoxic substances. It has been shown that cytokine measurement is the most important parameter for the classification of immunotoxic substances, investigating subtoxic concentrations. Both cyclosporin A as a positive control and mannitol as a negative control have been confirmed in both cell lines. Of the adequately investigated test substances, cannabidiol, helenaline and tulipalin A in Jurkat cells, and cannabidiol and tulipali A in THP-1 cells were classified as immunotoxic using the decision tree. In addition, the skin sensitizing effects of farnesol and tulipalin A could be confirmed by further in vitro methods. A validation of the results with other known immunotoxic and non-immunotoxic substances would allow an application as pre-screening testing of new compounds and not only lead to a reduction of animal testing, but also mean time and cost savings

Synergistic antimicrobial activities of epigallocatechin gallate, myricetin, daidzein, gallic acid, epicatechin, 3‐hydroxy‐6‐methoxyflavone and genistein combined with antibiotics against ESKAPE pathogens

Abstract 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

Ethiopian Medicinal Plants Traditionally Used for the Treatment of Cancer; Part 3: Selective Cytotoxic Activity of 22 Plants against Human Cancer Cell Lines

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