František Bilka

Several novel copper (II) complexes of reduced Schiff bases containing fluoride substituents were prepared and structurally characterized by single-crystal X-ray diffraction. The complexes exhibited diverse structures, with the central atom in distorted tetrahedral geometry. The biological effects of the products were evaluated, specifically their cytotoxicity, antimicrobial, and antiurease activities, as well as affinity for albumin (BSA) and DNA (ct-DNA). The complexes showed marked cytotoxic activities in the HepG2 hepatocellular carcinoma cell line, considerably higher than the standard cisplatin. The cytotoxicity depended significantly on the substitution pattern. The best activity was observed in the complex with a trifluoromethyl group in position 4 of the benzene ring—the dichloro[(±)-trans-N,N′-bis-(4-trifluoromethylbenzyl)-cyclohexane-1,2-diamine]copper (II) complex, whose activity (IC₅₀ 28.7 μM) was higher than that of the free ligand and markedly better than the activity of the standard cisplatin (IC₅₀ 336.8 μM). The same complex also showed the highest antimicrobial effect in vitro. The affinity of the complexes towards bovine serum albumin (BSA) and calf thymus DNA (ct-DNA) was established as well, indicating only marginal differences between the complexes. In addition, all complexes were shown to be excellent inhibitors of the enzyme urease, with the IC₅₀ values in the lower micromolar region.

Rheumatoid arthritis (RA), an autoimmune disease, is characterized by inflammation that affects not only the liver but also other organs and the musculoskeletal system. The standard therapy for RA is methotrexate (MTX), which has safety limitations. The extract from Crocus sativus L. (saffron—SF) is also known for its anti-inflammatory effects. Therefore, we decided to investigate the potential benefit of SF in monotherapy via two doses (SF1—25 mg/kg of b.w.; SF2—50 mg/kg of b.w.) and in combination with MTX (0.3 mg/kg of b.w., twice a week) using adjuvant arthritis in rats. To evaluate these therapeutic settings, we used biometric, immunological, and biochemical parameters, as well as the relative gene expression of the mRNA in the liver. Our results showed a statistically significant increase in the experimental animals’ body weight and the arthritic score (AS) on day 14 for monotherapy with SF1 and SF2. The change of hind paw volume (CHPV) was significant only for SF2 monotherapy on the 14th day of the experiment. A combination of SF1 and SF2 with MTX significantly modulated all the biometric parameters during the experimental period. Additionally, AS and CHPV improved considerably compared to MTX monotherapy on day 21. Furthermore, all monotherapies and combination therapies were significant for the biochemical parameter γ-glutamyl transferase (GGT) in the joint. GGT activity in the spleen was less pronounced; only MTX in combination with SF1 significantly modified this parameter. The higher dose of SF monotherapy (SF2) was similarly significant with respect to immunological parameters, such as plasmatic IL-17A, IL-1β, and MMP-9 on day 21. The combination of both doses of SF with MTX significantly improved these immunological parameters, except for C-reactive protein (CRP), which was influenced only by the higher dose of SF2 in combination with MTX in plasma at the end of the experiment. A different effect was found for the relative expression of CD36 mRNA, where only SF1 significantly decreased gene expression in the liver. However, the relative gene mRNA expression of IL-1β in the liver was significantly reduced by the SF monotherapies and the combination of both SF doses with MTX. Our findings showed SF’s partial antiarthritic and anti-inflammatory potential in monotherapy, but the effect was stronger in combination with MTX.

A series of Schiff base ligands obtained by the condensation of trans-cyclohexane-1,2-diamine and fluorinated benzaldehydes were prepared, followed by their reduction with NaBH₄. The reduced ligands were employed in the synthesis of zinc complexes of the general formula [ZnCl₂(L)]. The structures of both the original and the reduced Schiff bases, as well as of the zinc complexes, were characterized by single-crystal X-ray analysis, along with NMR and IR spectroscopy. The antimicrobial activities of the reduced Schiff bases and their zinc complexes were evaluated in vitro against E. coli, S. aureus, and C. albicans. The compounds containing the 4-(trifluoromethylphenyl) moiety showed marked antibacterial activity. Interestingly, the antimicrobial effect of the zinc complex with this moiety was significantly higher than that of the corresponding free reduced ligand, comparable with ciprofloxacin used as standard. Thus, a synergic effect upon the complexation with zinc can be inferred.

This research focused on a three-step synthesis, analytical, physicochemical, and biological evaluation of hybrid molecules 6a–g, containing a lipophilic 3-trifluoromethylphenyl moiety, polar carbamoyloxy bridge, 2-hydroxypropan-1,3-diyl chain and 4-(substituted phenyl)-/4-diphenylmethylpiperazin-1-ium-1-yl fragment. The estimation of analytical and physicochemical descriptors (m/z_measured via HPLC-UV/HR-MS, log ε_{2 (Ch–T)} from UV/Vis spectrophotometry and log k_w via RP-HPLC) as well as in vitro antimycobacterial and cytotoxic screening of given compounds were carried out (i.e., determination of MIC and IC₅₀ values). These highly lipophilic molecules (log k_w = 4.1170–5.2184) were tested against Mycobacterium tuberculosis H₃₇R_a ATCC 25177 (Mtb H₃₇R_a), M. kansasii DSM 44162 (MK), M. smegmatis ATCC 700084 (MS), and M. marinum CAMP 5644 (MM). The impact of the 6a–g set on the viability of human liver hepatocellular carcinoma (HepG2) cells was also investigated. 1-[2-Hydroxypropyl-{(3-trifluoromethyl)- phenyl}carbamoyloxy]-4-(3,4-dichlorophenyl)piperazin-1-ium chloride (6e) and 1-[2-hydroxy- propyl-{(3-trifluoromethyl)phenyl}carbamoyloxy]-4-(4-diphenylmethyl)piperazin-1-ium chloride (6g) most effectively inhibited the growth of Mtb H₃₇R_a (MIC < 3.80 μM). The substance 6g also showed interesting activity against MM (MIC = 8.09 μM). All obtained data served as input values for structure-activity relationship evaluations using statistical principal component analysis. In fact, the toxicity of both 6e (IC₅₀ = 29.39 μM) and 6g (IC₅₀ = 22.18 μM) in HepG2 cells as well as selectivity index (SI) values (SI < 10.00) prevented to consider these promising antimycobacterials safe.

Phosphorus-containing heterocyclic cationic surfactants alkyldimethylphenylphospholium bromides with the alkyl chain length 14 to 18 carbon atoms were used for the stabilization of silver nanodispersions. Zeta potential of silver nanodispersions ranges from +35 to +70 mV, which indicates the formation of stable silver nanoparticles (AgNPs). Long-chain heptadecyl and octadecyl homologs of the surfactants series provided the most intensive stabilizing effect to AgNPs, resulting in high positive zeta potential values and smaller diameter of AgNPs in the range 50–60 nm. A comparison with non-heterocyclic alkyltrimethylphosphonium surfactants of the same alkyl chain length showed better stability and more positive zeta potential values for silver nanodispersions stabilized with heterocyclic phospholium surfactants. Investigations of biological activity of phospholium-capped AgNPs are represented by the studies of antimicrobial activity and cytotoxicity. While cytotoxicity results revealed an increased level of HepG2 cell growth inhibition as compared with the cytotoxicity level of silver-free surfactant solutions, no enhanced antimicrobial action of phospholium-capped AgNPs against microbial pathogens was observed. The comparison of cytotoxicity of AgNPs stabilized with various non-heterocyclic ammonium and phosphonium surfactants shows that AgNPs capped with heterocyclic alkyldimethylphenylphospholium and non-heterocyclic triphenyl-substituted phosphonium surfactants have the highest cytotoxicity among silver nanodispersions stabilized by the series of ammonium and phosphonium surfactants.

The basal production of secondary metabolites in medicinal plants is limited. One of the effective approaches that encourages plants to produce a remarkable amount of precious compounds is an application of elicitors. Our work was focused on the elicitation of Eschscholzia californica Cham. suspension cultures using various concentrations of MnCl₂ (5; 10; 15 mg/L) with the aim of evaluating its effect on sanguinarine, chelerythrine, and macarpine production and gene expression of enzymes involved in the biosynthesis of mentioned secondary metabolites (BBE, 4′-OMT, CYP80B1) or in defense processes (LOX). Suspension cultures were exposed to elicitor for 24, 48, and 72 h. The content of alkaloids in phytomass was determined on the basis of their fluorescence properties. The relative mRNA expression of selected genes was analyzed using the ΔΔCt value method. PCR products were evaluated by melting curve analysis to confirm the specific amplification. Our results demonstrated that Eschscholzia californica Cham. cell suspension cultures evince sensitivity to the presence of MnCl₂ in growth media resulting in the increased production of benzophenanthridine alkaloids and gene expression of selected enzymes. Manganese chloride seems to be a potential elicitor supporting natural biosynthetic properties in plant cell cultures and can be applied for the sustained production of valuable secondary metabolites.

Methotrexate (MTX) is still the gold standard for treatment of rheumatoid arthritis (RA). The therapeutic efficacy of low-dose of MTX can be increased by its combination with a natural substance, ferulaldehyde (FRA). The aim of this study was to evaluate the effect FRA and MTX administered alone or in combination in adjuvant arthritis. The disease was induced to Lewis male rats by intradermal injection, which contains a suspension of heat-inactivated Mycobacterium butyricum in incomplete Freund’s adjuvant. The experiment of 28 days included: healthy animals, arthritic animals, arthritic animals with administration of FRA at the oral daily dose of 15 mg/kg, arthritic animals with administration of MTX at the oral dose of 0.3 mg/kg twice a week, and arthritic animals administered with FRA and MTX. FRA in monotherapy decreased significantly only the level of interleukin-1β (IL-1β) and matrix metalloproteinase-9 in plasma. Combination of FRA and low-dose MTX was more effective than MTX alone when comparing body weight, hind paw volume, arthritic score, plasmatic levels of IL-1β, activity of γ-glutamyl transferase, and relative mRNA expression of IL-1β in the spleen. Therefore, the combination treatment was the most effective. The obtained results are interesting for future possible innovative therapy of patients with RA.

Intact plants of the Papaveraceae family are producers of a whole range of benzylisoquinoline alkaloids, which are used in pharmaceutical industry. In vitro cultures derived from plants of the Papaveraceae do not have the ability to produce such a broad spectrum of alkaloids, only the biosynthetic pathway leading to sanguinarine is active. This study deals with the preparation of in vitro cultures of Papaver somniferum, Eschscholtzia californica, Chelidonium majus and Macleaya cordata. Their sanguinarine production abilities were tested and compared. The lowest amounts of sanguinarine from all cultures tested were accumulated in suspension cultures of the opium poppy (0.45-0.55 μg in 1 g of fresh weight). Eschscholtzia californica, Chelidonium majus and Macleaya cordata cultures produced similar amounts of sanguinarine (18.0-22.7 μg; 20.5-26.3 μg; 15.4-20.3 μg in 1 g of fresh weight, resp.). The elicitation study used a biotic stressor, Botrytis cinerea hydrolysate. In all cultures treated, an increase in sanguinarine accumulation was observed. Of all cultures tested, the most intensive response was observed in the opium poppy cultures, although the amount of sanguinarine in the elicited poppy cultures was lower than in the non-elicited samples of the other cultures.

Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic acid (K_M = 1.78 mM and V_max = 11.4 μmol mg⁻¹ min⁻¹) and linolenic acid (K_M = 1.27 mM and V_max = 10.2 μmol mg⁻¹ min⁻¹). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products being about 1:1. The activity of purified LOX was stimulated by Mg²⁺ and Ca²⁺.

Greater celandine, similarly as other plants of the family Papaveraceae, produces benzylisoquinoline alkaloids, primarily benzophenanthridines. Polyphenoloxidase (PPO) is most probably involved in the formation of dopamine, which is one of the precursors of norcoclaurine, the first intermediate with the benzylisoquinoline structure. This study has revealed that PPO present in the latex of greater celandine is localized in the organelles, which serve to store alkaloids (the so-called 1000 g organelles). The enzyme was purified by means of affinity chromatography into electrophoretic homogeneity. It possesses a relative molecular mass of approximately 65 kDa and exerts two activities, the monophenolase and diphenolase ones. With the use of a polymerase chain reaction, it was possible to amplify a part of the PPO gene from the region of the active site.