Impact of aliphatic acyl and aromatic thioamide substituents on the anticancer activity of Ru(II)-p-cymene complexes with acylthiourea ligands-in vitro and in vivo studies.

Six different acylthiourea ligands (L1-L6) and their corresponding Ru(II)-p-cymene complexes (P1-P6) were designed to explore the structure-activity relationship of the complexes upon aliphatic chain and aromatic conjugation on the C- and N-terminals, respectively. The compounds were synthesized and adequately characterized using various analytical and spectroscopic techniques. The structures of P2-P6, solved using single crystal X-ray diffraction (XRD), confirmed the neutral monodentate coordination of the S atoms of the acylthiourea ligands to Ru(II) ions. In silico studies showed an increase of lipophilicity for the ligands with an increase in alkyl chain length or aromatic conjugation at the C- or N-terminal, respectively. Subsequently, mitogen-activated protein kinases (MAPK) were predicted as one of the primary targets for the complexes, which showed good binding affinity towards extracellular signal-regulated kinases (ERK1, ERK2 and ERK5), c-Jun N-terminal kinase (JNK) and p38 of the MAPK pathway. Henceforth, the complexes were tested for their anticancer activity in lung carcinoma (A549) and cisplatin-resistant lung carcinoma (cisA549R) cells and human umbilical vein epithelial normal cells (HUVEC). Interestingly, an increase in chain length or aromatic conjugation led to an increase in the activity of the complexes, with P5 (7.73 and 13.04 µM) and P6 (6.52 and 14.45 µM) showing the highest activity in A549 and cisA549R cells, which is better than the positive control, cisplatin (8.72 and 44.28 µM). Remarkably, we report the highest activity yet observed for complexes of the type [(η6-p-cymene)RuIICl2(S-acylthiourea)] in the tested cell lines. Aqueous solution studies showed that complexes P5 and P6 are rapidly hydrolyzed to produce solely aquated species that remained stable for 24 h. Staining assays and flow cytometric analyses of P5 and P6 in A549 cells revealed that the complexes induced apoptosis and arrested the cell cycle predominantly in the S phase. In vivo studies demonstrated the higher toxicity of cisplatin and a comparatively higher survival rate of mice injected with the most active complex P6. Histological analyses revealed that treatment with P6 at high doses of up to 8 mg kg-1 did not cause any palpable damage to the tested organs.

PEI-coated PLA nanoparticles to enhance the antimicrobial activity of carvacrol.

Carvacrol (CAR) is a natural bioactive compound with antioxidant and antimicrobial activity that is present in essential oils. The application of CAR in food preservation is hampered by its high volatility, low solubility in water, and susceptibility to light, heat and oxygen degradation. Polylactide (PLA) is an FDA-approved polymer derived from renewable resources. Controlled release of CAR from PLA nanoparticles (NPs) could improve its antimicrobial efficacy and storage. In this study, negatively charged CAR-NPs and positively charged polyethylenimine (PEI)-coated CAR-(PEI)NPs were formulated by nanoprecipitation methods and characterised by dynamic light scattering, electron microscopy, encapsulation efficiency, and drug loading capacity. The positively charged (PEI)NPs enhanced the in vitro antimicrobial activity of CAR against Escherichia coli, Listeria monocytogenes, Salmonella enterica and Staphylococcus aureus. Bacterial uptake, evaporation tests, release studies and NP stability after storage were assessed to provide evidence supporting CAR-(PEI)NPs as a potential nanocarrier for further development in food preservation.

Cuminaldehyde potentiates the antimicrobial actions of ciprofloxacin against Staphylococcus aureus and Escherichia coli.

Escherichia coli and Staphylococcus aureus are important agents of urinary tract infections that can often evolve to severe infections. The rise of antibiotic-resistant strains has driven the search for novel therapies to replace the use or act as adjuvants of antibiotics. In this context, plant-derived compounds have been widely investigated. Cuminaldehyde is suggested as the major antimicrobial compound of the cumin seed essential oil. However, this effect is not fully understood. Herein, we investigated the in silico and in vitro activities of cuminaldehyde, as well as its ability to potentiate ciprofloxacin effects against S. aureus and E. coli. In silico analyses were performed by using different computational tools. The PASS online and SwissADME programmes were used for the prediction of biological activities and oral bioavailability of cuminaldehyde. For analysis of the possible toxic effects and the theoretical pharmacokinetic parameters of the compound, the Osiris, SwissADME and PROTOX programmes were used. Estimations of cuminaldehyde gastrointestinal absorption, blood brain barrier permeability and skin permeation by using SwissADME; and drug likeness and score by using Osiris, were also evaluated The in vitro antimicrobial effects of cuminaldehyde were determined by using microdilution, biofilm formation and time-kill assays. In silico analysis indicated that cuminaldehyde may act as an antimicrobial and as a membrane permeability enhancer. It was suggested to be highly absorbable by the gastrointestinal tract and likely to cross the blood brain barrier. Also, irritative and harmful effects were predicted for cuminaldehyde if swallowed at its LD50. Good oral bioavailability and drug score were also found for this compound. Cuminaldehyde presented antimicrobial and anti-biofilm effects against S. aureus and E. coli.. When co-incubated with ciprofloxacin, it enhanced the antibiotic antimicrobial and anti-biofilm actions. We suggest that cuminaldehyde may be useful as an adjuvant therapy to ciprofloxacin in S. aureus and E. coli-induced infections.

Carvacrol loaded nanostructured lipid carriers as a promising parenteral formulation for leishmaniasis treatment.

Leishmaniasis are a group of neglected infectious diseases caused by protozoa of the genus Leishmania with distinct presentations. The available leishmaniasis treatment options are either expensive and/or; cause adverse effects and some are ineffective for resistant Leishmania strains. Therefore, molecules derived from natural products as the monoterpene carvacrol, have attracted interest as promising anti-leishmania agents. However, the therapeutic use of carvacrol is limited due to its low aqueous solubility, rapid oxidation and volatilization. Thus, the development of nanostructured lipid carriers (NLCs) was proposed in the present study as a promising nanotechnology strategy to overcome these limitations and enable the use of carvacrol in leishmaniasis therapy. Carvacrol NLCs were obtained using a warm microemulsion method, and evaluated regarding the influence of lipid matrix and components concentration on the NLCs formation. NLCs were characterized by DSC and XRD as well. In addition, to the in vitro carvacrol release from NLCs, the in vitro cytotoxicity and leishmanicidal activity assays, and the in vivo pharmacokinetics evaluation of free and encapsulated carvacrol were performed. NLCs containing carvacrol were obtained successfully using a warm microemulsion dilution method. The NLCs formulation with the lowest particle size (98.42 ± 0.80 nm), narrowest size distribution (suitable for intravenous administration), and the highest encapsulation efficiency was produced by using beeswax as solid lipid (HLB=9) and 5% of lipids and surfactant. The in vitro release of carvacrol from NLCs was fitted to the Korsmeyer and Peppas, and Weibull models, demonstrating that the release mechanism is probably the Fickian diffusion type. Moreover, carvacrol encapsulation in NLCs provided a lower cytotoxicity in comparison to free carvacrol (p<0.05), increasing its in vitro leishmanicidal efficacy in the amastigote form. Finally, the in vivo pharmacokinetics of carvacrol after IV bolus administration suggests that this phenolic monoterpene undergoes enterohepatic circulation and therefore presented a long half-life (t1/2) and low clearance (Cl). In addition, C0, mean residence time (MRT) and Vdss of encapsulated carvacrol were higher than free carvacrol (p < 0.05), favoring a higher distribution of carvacrol in the target tissues. Thus, it is possible to conclude that the developed NLCs are a promising delivery system for leishmaniasis treatment.

Transdermal delivery enhancement of carvacrol from Origanum vulgare L. essential oil by microemulsion.

Carvacrol has been reported for analgesic and anti-inflammatory activity by cyclooxygenase inhibition but it could induce gastrointestinal toxicity because of its non-selective inhibition. Therefore, the present study aimed to develop transdermal microemulsion from Origanum vulgare essential oil to deliver carvacrol into and through the skin which would overwhelm the gastrointestinal problems. O. vulgare essential oil was extracted by hydrodistillation and its carvacrol content was determined using high performance liquid chromatography. Pseudoternary phase diagrams were constructed using water dilution method to investigate the suitable microemulsion components. Microemulsions were then characterized for external appearance, particle size, size distribution, zeta potential, electrical conductivity, refractive index, viscosity, transmittance, pH, and stability. Additionally, the irritation property of microemulsions were investigated by hen's egg on the chorioallantoic membrane assay. The release profile, percutaneous absorption, and skin retention were investigated using dialysis bag and Franz diffusion cell, respectively. The interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were investigated using the enzyme-linked immunosorbent assay. The results remarked that carvacrol was a major component of O. vulgare essential oil with high concentration of 83.7%. The most suitable microemulsion (ME 1), composing of 5% w/w O. vulgare essential oil, 25%w/w Tween 60, 25%w/w butylene glycol, and 45%w/w deionized water, had the smallest internal droplet size (179.5 ± 27.9 nm), the narrowest polydispersity index (0.30 ± 0.07), the highest transmittance (93.13 ± 0.04%), and Newtonian flow behavior with low viscosity of 0.30 ± 0.07 Pas. ME 1 could reduce the irritation effect of O. vulgare essential oil since ME 1 (IS = 3.1 ± 0.10) exhibited significantly lower irritation effect than its blank formulation (IS = 4.8 ± 0.02) and O. vulgare oil solution (IS = 5.0 ± 0.01) (p < 0.05). Furthermore, ME 1 sustain released carvacrol from the formulation, remarkedly deliver more carvacrol through the skin layer (2.6 ± 2.2%) and significantly retained carvacrol in the skin layer (2.60 ± 1.25%). Additionally, ME 1 significantly enhanced IL-6 inhibition of O. vulgaris oil and carvacrol (p < 0.05). Therefore, O. vulgaris oil microemulsion was suggested to be used for the transdermal delivery and anti-inflammatory activities enhancement of carvacrol.

Design, synthesis and pharmacological evaluation of CVIB, a codrug of carvacrol and ibuprofen as a novel anti-inflammatory agent.

The search for new drugs with anti-inflammatory properties remains a challenge for modern medicine. Among the various strategies for drug discovery, deriving new chemical entities from known bioactive natural and/or synthetic compounds remains a promising approach. Here, we designed and synthesized CVIB, a codrug developed by association of carvacrol (a phenolic monoterpene) with ibuprofen (a non-steroidal anti-inflammatory drug). In silico pharmacokinetic and physicochemical properties evaluation indicated low aqueous solubility (LogP ≥5.0). Nevertheless, the hybrid presented excellent oral bioavailability, gastrointestinal tract absorption, and low toxicity. CVIB did not present cytotoxicity in peripheral blood mononuclear cells (PBMCs), and promoted a significant reduction in IL-2, IL-10, IL-17, and IFN-γ cytokine levels in vitro. The LD50 was estimated to be approximately 5000 mg/kg. CVIB was stable and detectable in human plasma after 24 h. In vivo anti-inflammatory evaluations revealed that CVIB at 10 and 50 mg/kg i.p. caused a significant decrease in total leukocyte count (p < 0.01) and provoked a significant reduction in IL-1ß (p < 0.01). CVIB at 10 mg/kg i.p. efficiently decreased inflammatory parameters better than the physical mixture (carvacrol + ibuprofen 10 mg/kg i.p.). The results suggest that the codrug approach is a good option for drug design and development, creating the possibility of combining NSAIDs with natural products in order to obtain new hybrid drugs may be useful for treatment of inflammatory diseases.

Electrospun asymmetric membranes for wound dressing applications.

To accomplish a rapid wound healing it is necessary to develop an asymmetric membrane with interconnected pores consisting of a top layer that prevents rapid dehydration of the wound and bacteria penetration and a sub-layer with high absorption capacity and bactericidal properties. Polycaprolactone (PCL)/polyvinyl acetate (PVAc) asymmetric membranes loaded with the bactericidal monoterpene carvacrol (CRV) were synthesized and characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Mechanical properties in dry and wet conditions and fluid handling behavior were also assessed. In addition, biological studies regarding their bactericidal effects, cytocompatibility and wound closure properties were also developed. Loading efficiencies of 40-50% were achieved in the prepared samples and 85-100% of the loaded CRV was released in simulated wound pH evolution medium. The significant inhibition of Gram negative (Escherichia coli S17) and Gram positive (Staphylococcus aureus ATCC 25923) bacteria growth clearly showed the suitability of the fabricated membranes for wound healing applications. Furthermore, cytocompatibility of the loaded membranes was demonstrated both in 2D and 3D human dermal fibroblast cultures, as well as cell migration was not impaired by released carvacrol from the membranes. These results highlight the potential of these polymeric electrospun membranes for wound healing.

Novel cuminaldehyde self-emulsified nanoemulsion for enhanced antihepatotoxicity in carbon tetrachloride-treated mice.

OBJECTIVES: Cuminaldehyde self-emulsified nanoemulsion (CuA-SEN) was prepared and optimised to improve its oral bioavailability and antihepatotoxicity. METHODS: Cuminaldehyde self-emulsified nanoemulsion was developed through the self-nanoemulsification method using Box-Behnken Design (BBD) tool while appropriate physicochemical indices were evaluated. The optimised CuA-SEN was characterised via droplet size (DS), morphology, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, in-vitro release, and pharmacokinetic studies while its antihepatotoxicity was evaluated. KEY FINDINGS: Cuminaldehyde self-emulsified nanoemulsion with acceptable characteristics (mean DS-48.83 ± 1.06 nm; PDI-0.232 ± 0.140; ZP-29.92 ± 1.66 mV; EE-91.51 ± 0.44%; and drug-loading capacity (DL)-9.77 ± 0.75%) was formulated. In-vitro drug release of CuA-SEN significantly increased with an oral relative bioavailability of 171.02%. Oral administration of CuA-SEN to CCl4 -induced hepatotoxicity mice markedly increased the levels of superoxide dismutase, glutathione and catalase in serum. Also, CuA-SEN reduced the levels of tumour necrosis factor-alpha and interleukin-6 in both serum and liver tissues while aspartate aminotransferase, alanine aminotransferase and malonaldehyde levels were significantly decreased. CONCLUSIONS: These findings showed that the improved bioavailability of cuminaldehyde via SEN provided an effective approach for enhancing antioxidation, anti-inflammation and antihepatotoxicity of the drug.