Caffeic Acid Phosphanium Derivatives: Potential Selective Antitumor, Antimicrobial and Antiprotozoal Agents.

Caffeic acid (CA) is one of the most abundant natural compounds present in plants and has a broad spectrum of beneficial pharmacological activities. However, in some cases, synthetic derivation of original molecules can expand their scope. This study focuses on the synthesis of caffeic acid phosphanium derivatives with the ambition of increasing their biological activities. Four caffeic acid phosphanium salts (CAPs) were synthesized and tested for their cytotoxic, antibacterial, antifungal, and amoebicidal activity in vitro, with the aim of identifying the best area for their medicinal use. CAPs exhibited significantly stronger cytotoxic activity against tested cell lines (HeLa, HCT116, MDA-MB-231 MCF-7, A2058, PANC-1, Jurkat) in comparison to caffeic acid. Focusing on Jurkat cells (human leukemic T cell lymphoma), the IC50 value of CAPs ranged from 0.9 to 8.5 µM while IC50 of CA was >300 µM. Antimicrobial testing also confirmed significantly higher activity of CAPs against selected microbes in comparison to CA, especially for Gram-positive bacteria (MIC 13-57 µM) and the yeast Candida albicans (MIC 13-57 µM). The anti-Acanthamoeba activity was studied against two pathogenic Acanthamoeba strains. In the case of A. lugdunensis, all CAPs revealed a stronger inhibitory effect (EC50 74-3125 µM) than CA (>105 µM), while in A. quina strain, the higher inhibition was observed for three derivatives (EC50 44-291 µM). The newly synthesized quaternary phosphanium salts of caffeic acid exhibited selective antitumor action and appeared to be promising antimicrobial agents for topical application, as well as potential molecules for further research.

Green tea leaf constituents inhibit the formation of lysozyme amyloid aggregates: An effect of mutual interactions.

Amyloidoses represent a group of pathological conditions characterized by amyloid fibrils accumulating in the form of deposits in intra- or extracellular space, leading to tissue damage. The lysozyme from hen egg-white (HEWL) is often used as a universal model protein to study the anti-amyloid effects of small molecules. The in vitro anti-amyloid activity and mutual interactions of green tea leaf constituents: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), gallic acid (GA), caffeine (CF) and their equimolar mixtures were studied. The inhibition of HEWL amyloid aggregation was monitored by a Thioflavin T fluorescence assay and atomic force microscopy (AFM). The interactions of the analyzed molecules with HEWL were interpreted by ATR-FTIR and protein-small ligand docking studies. EGCG was the only substance efficiently inhibiting amyloid formation (IC50 âˆ¼193 µM), slowing the aggregation process, reducing the number of fibrils and partially stabilizing the secondary structure of HEWL. Compared to EGCG alone, EGCG-containing mixtures displayed lower overall anti-amyloid efficacy. The decrease in efficiency results from (a) the spatial interference of GA, CF and EC with EGCG while binding to HEWL, (b) the propensity of CF to form a less active adduct with EGCG, which participates in interactions with HEWL in parallel with pure EGCG. This study confirms the importance of interaction studies, revealing the possible antagonistic behavior of molecules when combined.

Synergy evaluation of non-normalizable dose-response data: Generalization of combination index for the linear effect of drugs.

The study of drug synergy plays a prominent role in the search for drug combinations with beneficial interactions. Firstly, in this process, the drug-effect response of individual parts and the mixture needs to be derived. This function is usually well described by Hill (or other logistic or sigmoid) curve. Due to its boundedness, it allows the measured data to be normalized. The normalized data can then be processed by interaction analysis using the Loewe, Bliss, or other models to evaluate possible synergy or antagonism of two or more drugs. However, sometimes, the drug-effect responses observed in pharmaceutical research do not appear to be bounded. Theoretically, the drug-effect curve cannot grow to infinity, but it may be impossible to determine its upper bound within the observed region. In this case, standard models cannot be used, since they assume that data are normalized. The approach of this article bypasses the need to normalize the data, allowing its broader application and usefulness in finding potential synergies in pharmaceutical research.

Amyloid Aggregation of Insulin: An Interaction Study of Green Tea Constituents.

Exogenous insulin, used as a therapeutic agent for diabetes, forms insoluble deposits containing amyloid fibrillar structures near the administration site. We have analyzed the in vitro anti-amyloid activity of four green tea constituents: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), gallic acid (GA), caffeine (CF), and their equimolar mixtures. Regarding individually tested compounds, only EGCG inhibited the fibrillization process. The individual EC, GA, and CF molecules were ineffective. The presence of EGCG in equimolar combinations with GA, EC, or CF was required for the inhibitory activity of most mixtures. Molecular docking revealed that EGCG interacts with an essential amyloidogenic region of insulin chain B. Individually inactive GA had a potentiating effect on the activity of EGCG. In contrast, EC and CF had a negative impact on the activity of the mixtures. We have observed diverse morphology and the amount of insulin amyloid aggregates formed in the presence of studied compounds. The distinct types of amyloid aggregates created in vitro in the presence of EGCG and other green tea constituents were characterized. Results indicate that the biological activity of individual molecules is not directly applicable to the pooled samples effects prediction.

Antimicrobial and Antioxidant Properties of Four Lycopus Taxa and an Interaction Study of Their Major Compounds.

The compositions of leaf infusions of three genotypes of Lycopus europaeus L. with origins in central Europe, namely L. europaeus A (LeuA), L. europaeus B (LeuB), and L. europaeus C (LeuC), and one genotype of L. exaltatus (Lex), were examined by LC-MS-DAD (Liquid Chromatography Mass Spectrometry and Diode Array Detection) analysis. This revealed the presence of thirteen compounds belonging to the groups of phenolic acids and flavonoids, with a predominance of rosmarinic acid (RA) and luteolin-7-O-glucuronide (LGlr). The antimicrobial activity of leaf infusions was tested on the collection strains of Gram-positive and Gram-negative bacteria, and on the clinical Staphylococcus aureus strains. We detected higher activity against Gram-positive bacteria, of which the most susceptible strains were those of Staphylococcus aureus, including methicillin-resistant and poly-resistant strains. Furthermore, we examined the antioxidant activity of leaf infusions using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) methods, and on NIH/3T3 cell lines using dichlorodihydrofluorescein diacetate (DCFH-DA). We also studied the mutual interactions between selected infusions, namely RA and/or LGlr. In the mixtures of leaf infusion and RA or LGlr, we observed slight synergism and a high dose reduction index in most cases. This leads to the beneficial dose reduction at a given antioxidant effect level in mixtures compared to the doses of the parts used alone. Therefore, our study draws attention to further applications of the Lycopus leaves as a valuable alternative source of natural antioxidants and as a promising topical antibacterial agent for medicinal use.

Mentha Rhizomes as an Alternative Source of Natural Antioxidants.

Unlike its aerial parts, the underground parts of Mentha have so far been studied only marginally. By examining the polyphenolic fingerprint, the antioxidant efficacy and the mutual antioxidant behaviour of mixtures of mint rhizomes, our study presents a modest contribution to addressing this gap. Firstly, we examined the composition of the mint rhizomes: Mentha × piperita cv. 'Perpeta' (MPP), M. longifolia (ML), and M. × villosa cv. 'Snezna' (MVS). Our LC-MS-DAD analysis revealed the presence of ten compounds belonging to groups of phenolic acids and flavonoids, of which the rosmarinic acid (RA) and lithospermic were most strongly represented. Secondly, we evaluated the antioxidant activity of rhizome infusions by DPPH and ABTS and on NIH/3T3 cell lines by DCFH-DA. Thirdly, we determined, examined, and explained the mutual interactions of rhizome infusions mixtures. While most of the combinations acted additive, synergy was observed in ternary infusion mixtures. The synergic action was also detected in the combination of MPP rhizome infusion and RA in the DCFH-DA test. The combinations of mint rhizomes and rosmarinic acid displayed a high dose-reduction index. This leads to beneficial dose reduction at a given antioxidant effect level in mixtures, compared to the dose of the parts used alone. So far, the pharmaceutical and food industry has not used mint rhizomes in commercial products. Hence, our study draws attention to further applications of the Mentha rhizomes as a valuable alternative source of natural antioxidants.

Amyloid aggregation of lysozyme: the synergy study of red wine polyphenols.

The amyloidoses are diseases associated with nonnative folding of proteins and characterized by the presence of protein amyloid aggregates. The ability of quercetin, resveratrol, caffeic acid, and their equimolar mixtures to affect amyloid aggregation of hen egg white lysozyme in vitro was detected by Thioflavin T fluorescence assay. The anti-amyloid activities of tested polyphenols were evaluated by the median depolymerization concentrations DC50 and median inhibition concentrations IC50 . Single substances are more efficient (by at least one order) in the depolymerization of amyloid aggregates assay than in the inhibition of the amyloid formation with IC50 in 10(-4) to 10(-5) M range. Analyzed mixture samples showed synergic or antagonistic effects in both assays. DC50 values ranged from 10(-5) to 10(-8) M and IC50 from 10(-5) to 10(-9) M, respectively. We observed that certain mixtures of studied polyphenols can synergistically inhibit production of amyloids aggregates and are also effective in depolymerization of the aggregates. Synergic or antagonistic effects of studied mixtures were correlated with protein-small ligand docking studies and AFM results. Differences in these activities could be explained by binding of each polyphenol to a different amino acid sequence within the protein. Our results indicate that synergic/antagonistic anti-amyloid effects of studied mixtures depend on the selective binding of polyphenols to the known amyloidogenic sequences in the lysozyme chain. Our findings of the effective reduction of amyloid aggregation of lysozyme by polyphenol mixtures in vitro are of the utter physiological relevance considering the bioavailability and low toxicity of tested phenols.

In vitro antioxidant activities of three red wine polyphenols and their mixtures: an interaction study.

The well-known antioxidant activity of red wine is explained mostly by its polyphenols content, where the final effect is based on the wine components' interaction. The aim of our work was the study of the interaction of three red wine polyphenols--quercetin, resveratrol and caffeic acid--alone and in their equimolar binary and ternary mixtures in different antioxidant/scavenging assays (inhibition of 2-deoxy-D-ribose degradation by hydroxyl radical, FRAP, Fe(III) reducing power, DPPH, ABTS and NO scavenging, respectively). Interaction analysis, based on median effect equation, was performed for the determination of synergy and/or antagonism. The obtained results indicate that the mutual interactions of tested polyphenols in their mixtures are markedly different from each other, depending on the reaction mechanism of the assay used. The measured antioxidant activity of individual polyphenols is not a constant value when other substances are present in the mixture with this polyphenol. Interactions can cause the finally observed synergy/antagonism/additive effects without any possibility of predicting them from the known activities of single compounds. This "unpredictability" claim based on in vitro assay results should be very important in multiple systems and processes in Nature, where the interactions among compounds in mixtures need to be take into account.

Synergy study of the inhibitory potential of red wine polyphenols on vascular smooth muscle cell proliferation.

Vascular smooth muscle cell (VSMC) proliferation contributes to the development of atherosclerosis. Red wine consumption due to the polyphenol content has been reported to counteract atherosclerosis progression possibly through inhibition of VSMC proliferation, among other mechanisms. In this study we investigate the antiproliferative activity of four wine polyphenols: resveratrol, quercetin, ethyl gallate, and (+)-catechin in rat aortic VSMC. All four polyphenols inhibited serum-induced VSMC proliferation when applied as a single treatment. To further address a potential synergistic action of the investigated polyphenols, the antiproliferative effect of different combinations in equimolar, as well as equipotent ratios were quantified. The IC50 values of single polyphenols regarding the inhibition of VSMC proliferation ranged from 49.58 µM to 86.06 µM. However, apparent inhibitory efficacy of each compound increased by a factor of 10.4 in the quadruple equipotent mixture, as calculated from the dose-reduction index. Thus, the effective IC50 values of each of the four mixture constituents ranged from 4.76 µM to 8.27 µM. The calculated combination index (CI, where CI <, =, or > 1 indicate synergy, additivity, or antagonism, respectively) values of equimolar combinations of the polyphenols indeed indicated mainly synergy (CI ranging from 0.24 ± 0.01 to 1.51 ± 0.13). Optimized equipotent mixture showed enhanced synergy (CI ranging from 0.18 ± 0.04 to 1.36 ± 0.26). In conclusion, we show for the first time that four major polyphenols from wine synergistically inhibit VSMC proliferation.