Development of a novel, entirely herbal-based mouthwash effective against common oral bacteria and SARS-CoV-2.

BACKGROUND: Parallel to the growth of the oral healthcare market, there is a constantly increasing demand for natural products as well. Many customers prefer products that contain fewer toxic agents, therefore providing an environmentally friendly solution with the benefit of smaller risk to the user. Medieval and early modern medicinal knowledge might be useful when looking for natural, herbal-based components to develop modern products. Along with these considerations we created, tested, and compared an entirely natural mouthwash, named Herba Dei. METHODS: The manufacturing procedure was standardized, and the created tincture was evaluated by GC/MS analysis for active compounds, experimentally tested in cell-based cytotoxicity, salivary protein integrity, cell-free antioxidant activity, anti-bacterial and anti-viral assays, and compared with three market-leading mouthwashes. RESULTS: Our tincture did not show significant damage in the cytotoxicity assays to keratinocyte and Vero E6 cells and did not disrupt the low molecular weight salivary proteins. Its radical scavenging capacity surpassed that of two tested, partly natural, and synthetic mouthwashes, while its antibacterial activity was comparable to the tested products, or higher in the bacterial aerobic respiratory assay. The active compounds responsible for the effects include naturally occurring phenylpropanoids, terpenes, and terpenoids. Our mouthwash proved to be effective in vitro in lowering the copy number of SARS-CoV-2 in circumstances mimicking the salivary environment. CONCLUSIONS: The developed product might be a useful tool to impede the transmission and spread of SARS-CoV-2 in interpersonal contact and aerosol-generating conditions. Our mouthwash can help reduce the oral bacterial flora and has an antioxidant activity that facilitates wound healing and prevents adverse effects of smoke in the oral cavity.

Quality Assessment of Goldenrod, Milkweed and Multifloral Honeys Based on Botanical Origin, Antioxidant Capacity and Mineral Content.

The goal of the study was to evaluate the pollen spectrum, antioxidant capacity and mineral content of four Hungarian honey types, using multivariate statistical analysis. The light colored honeys were represented by milkweed honey and a multifloral (MF) honey with dominant pollen frequency of linden (MF-Tilia); the darker ones were goldenrod honey and a multifloral honey with Lamiaceae pollen majority (MF-Lamiaceae). The pollen spectrum of the samples was established with melissopalynological analysis. The absorbance of the honeys positively correlated with the antioxidant capacity determined with three of the used methods (TRC, TEAC, DPPH), but not with ORAC. The latter method correlated negatively also with other antioxidant methods and with most of the mineral values. MF-Tilia had high ORAC value, K and Na content. The MF-Lamiaceae had the highest K, Mg, P, S, Cu and Zn content, the last five elements showing strict correlation with the TRC method. The darker goldenrod honey had higher SET values and total mineral content, than the milkweed honey. The above character-sets facilitate identification of each honey type and serve as indicators of variety. The antioxidant levels and mineral content of honeys allowed their clear separation by principal component analysis (PCA).

Quality Evaluation of Light- and Dark-Colored Hungarian Honeys, Focusing on Botanical Origin, Antioxidant Capacity and Mineral Content.

Melissopalynology, antioxidant capacity and mineral and toxic element contents were analyzed in eight types of Hungarian honeys. Based on color, two groups were distinguished: light honeys comprised acacia, amorpha, phacelia and linden honeys; while dark honeys included sunflower, chestnut, fennel and sage honeys, with 100 to 300 and 700 to 1500 mAU, respectively. The unifloral origin of each sample was supported using pollen analysis. The absorbance of honey correlated positively with antioxidant capacity determined by three different methods (TRC, DPPH, ORAC), and also with mineral content. The exception was the light amber linden honey with significantly higher K content and antiradical activity than other light honeys. The Mn, Zn and Fe contents were the highest in chestnut, sunflower and fennel honeys, respectively. The black meadow sage honey performed best regarding the content of other elements and antioxidant activity. The concentrations of several toxic elements were below the detection limit in the samples, indicating their good quality. The principal component analysis (PCA) revealed correlations between different antioxidant assays and minerals, and furthermore, confirmed the botanical authentication of the honeys based on the studied parameters. To our best knowledge, the present study is the first to provide a complex analysis of quality parameters of eight unifloral Hungarian honeys.

Formulation of Tioconazole and Melaleuca alternifolia Essential Oil Pickering Emulsions for Onychomycosis Topical Treatment.

Onychomycosis is a disease that affects many adults, whose treatment includes both oral and topical therapies with low cure rates. The topical therapy is less effective but causes fewer side effects. This is why the development of an effective, easy to apply formulation for topical treatment is of high importance. We have used a nanotechnological approach to formulate Pickering emulsions (PEs) with well-defined properties to achieve site-specific delivery for antifungal drug combination of tioconazole and Melaleuca alternifolia essential oil. Silica nanoparticles with tailored size and partially hydrophobic surface have been synthesized and used for the stabilization of PEs. In vitro diffusion studies have been performed to evaluate the drug delivery properties of PEs. Ethanolic solution (ES) and conventional emulsions (CE) have been used as reference drug formulations. The examination of the antifungal effect of PEs has been performed on Candida albicans and Trichophyton rubrum as main pathogens. In vitro microbiological experimental results suggest that PEs are better candidates for onychomycosis topical treatment than CE or ES of the examined drugs. The used drugs have shown a significant synergistic effect, and the combination with an effective drug delivery system can result in a promising drug form for the topical treatment of onychomycosis.

Antimicrobial Activity of Different Artemisia Essential Oil Formulations.

The extreme lipophilicity of essential oils (EOs) impedes the measurement of their biological actions in an aqueous environment. We formulated oil in water type Pickering Artemisia annua EO nanoemulsions (AEP) with surface-modified Stöber silica nanoparticles (20 nm) as the stabilizing agent. The antimicrobial activity of AEP and its effects on mature Candida biofilms were compared with those of Tween 80 stabilized emulsion (AET) and ethanolic solution (AEE) of the Artemisia EO. The antimicrobial activity was evaluated by using the minimum inhibitory concentrations (MIC90) and minimum effective concentrations (MEC10) of the compounds. On planktonic bacterial and fungal cells beside growth inhibition, colony formation (CFU/mL), metabolic activity, viability, intracellular ATP/total protein (ATP/TP), along with reactive oxygen species (ROS) were also studied. Artemisia annua EO nanoemulsion (AEP) showed significantly higher antimicrobial activity than AET and AEE. Artemisia annua EO nanoemulsions (AEP) generated superoxide anion and peroxides-related oxidative stress, which might be the underlying mode of action of the Artemisia EO. Unilamellar liposomes, as a cellular model, were used to examine the delivery efficacy of the EO of our tested formulations. We could demonstrate higher effectiveness of AEP in the EO components' donation compared to AET and AEE. Our data suggest the superiority of the AEP formulation against microbial infections.

Cytotoxic, Antimicrobial, Antioxidant Properties and Effects on Cell Migration of Phenolic Compounds of Selected Transylvanian Medicinal Plants.

Medicinal plants are widely used in folk medicine but quite often their composition and biological effects are hardly known. Our study aimed to analyze the composition, cytotoxicity, antimicrobial, antioxidant activity and cellular migration effects of Anthyllis vulneraria, Fuchsia magellanica, Fuchsia triphylla and Lysimachia nummularia used in the Romanian ethnomedicine for wounds. Liquid chromatography with mass spectrometry (LC-MS/MS) was used to analyze 50% (v/v) ethanolic and aqueous extracts of the plants' leaves. Antimicrobial activities were estimated with a standard microdilution method. The antioxidant properties were evaluated by validated chemical cell-free and biological cell-based assays. Cytotoxic effects were performed on mouse fibroblasts and human keratinocytes with a plate reader-based method assessing intracellular adenosine triphosphate (ATP), nucleic acid and protein contents and also by a flow cytometer-based assay detecting apoptotic-necrotic cell populations. Cell migration to cover cell-free areas was visualized by time-lapse phase-contrast microscopy using standard culture inserts. Fuchsia species showed the strongest cytotoxicity and the highest antioxidant and antimicrobial activity. However, their ethanolic extracts facilitated cell migration, most probably due to their various phenolic acid, flavonoid and anthocyanin derivatives. Our data might serve as a basis for further animal experiments to explore the complex action of Fuchsia species in wound healing assays.

Antimicrobial Activity of Chamomile Essential Oil: Effect of Different Formulations.

Essential oils (EOs) are highly lipophilic, which makes the measurement of their biological action difficult in an aqueous environment. We formulated a Pickering nanoemulsion of chamomile EO (CPe). Surface-modified Stöber silica nanoparticles (20 nm) were prepared and used as a stabilizing agent of CPe. The antimicrobial activity of CPe was compared with that of emulsion stabilized with Tween 80 (CT80) and ethanolic solution (CEt). The antimicrobial effects were assessed by their minimum inhibitory concentration (MIC90) and minimum effective (MEC10) concentrations. Besides growth inhibition (CFU/mL), the metabolic activity and viability of Gram-positive and Gram-negative bacteria as well as Candida species, in addition to the generation of oxygen free radical species (ROS), were studied. We followed the killing activity of CPe and analyzed the efficiency of the EO delivery for examined formulations by using unilamellar liposomes as a cellular model. CPe showed significantly higher antibacterial and antifungal activities than CT80 and CEt. Chamomile EOs generated superoxide anion and peroxide related oxidative stress which might be the major mode of action of Ch essential oil. We could also demonstrate that CPe was the most effective in donation of the active EO components when compared with CT80 and CEt. Our data suggest that CPe formulation is useful in the fight against microbial infections.

Antioxidant and antimicrobial properties of randomly methylated ß cyclodextrin - captured essential oils.

Free essential oils and their active components have a low physiochemical stability and low aqueous solubility which limit their applications as food preservatives and in packaging industry. The aim of this study was to characterize the physicochemical properties, antioxidant activities and antimicrobial activity of randomly methylated ß cyclodextrin (RAMEB) encapsulated thyme oil, lemon balm oil, lavender oil, peppermint oil and their active components that include thymol, citral, linalool, menthol and borneol. Inclusion complex formation of essential oils (EOs) and RAMEB were evaluated by several methods. Antioxidant capacities of RAMEB-EOs/components were reported to be more stable than free EOs/components (P < 0.05). Rapid SYBR green I/propidium iodide live/dead microbial cellular discrimination assay for Schizosaccharomyces pombe, Escherichia coli and Staphylococcus aureus showed similar results when compared with flow cytometry analysis (P < 0.01) suggesting that our novel microplate fluorescence method could be applied for the fast live/dead microbial discrimination in antimicrobial assays.

A novel luminol-based enhanced chemiluminescence antioxidant capacity microplate assay for use in different biological matrices.

INTRODUCTION: Reactive oxygen species (ROS) are normal metabolic products of living cells. However, a decrease of the defense mechanisms against the effects of ROS or increased ROS production maybe one important causative factor of cellular damage. A non-enzymatic scavenger system is considered to be responsible for the maintenance of total antioxidant capacity (TAC) as a protection against oxidative injuries that exist in all higher plants and in mammals as well. METHODS: In our work, we optimized and validated a luminol-peroxidase-4-iodophenol-H2O2 enhanced chemiluminescence-based (ECL) TAC measurement technique. BSA was applied in the reagent to prevent peroxidase from auto-oxidation. The ECL method was suitable for plant extracts and for human blood serum as well. Our TAC technique was adapted to microplates and compared to ORAC assay using plant extracts. RESULTS AND DISCUSSION: The ECL method is fast (10min) with an interassay precision of <10% as CV. TAC values of ethanolic extracts of 10 plant species did correlate (ECL vs ORAC assay data: r=0.84, 95% confidence interval, CI=0.78-0.89, P<0.001) but with systematic bias. Analysis of serum samples obtained from septic and control patients showed significantly higher TAC values in the patient group compared to those of controls (p<0.01). Moreover, we could discriminate between surviving and non-surviving patients, based on their TAC values (p<0.01). Pearson's statistics showed the strongest positive correlation with serum uric acid (r=0.73). Besides the routine laboratory parameters, our novel TAC method might give complementary information on the severity of systemic inflammation.

Antioxidant activities and polyphenolic contents of three selected Micromeria species from Croatia.

Antioxidant activities of three selected Micromeria species growing in Croatia (M. croatica, M. juliana and M. thymifolia) were evaluated using five different antioxidant assays, in comparison with plant polyphenolic constituents and reference antioxidants. All studied ethanolic extracts exhibited considerable activity to scavenge DPPH and hydroxyl free radicals, reducing power, iron chelating ability and total antioxidant capacity in the order: M. croatica > M. juliana > M. thymifolia. Total polyphenol (9.69-13.66%), phenolic acid (5.26-6.84%), flavonoid (0.01-0.09%) and tannin (3.07-6.48%) contents in dried plant samples were determined spectrophotometrically. A strong positive correlation between antioxidant activities and contents of phenolic acids and tannins was found, indicating their responsibility for effectiveness of tested plants. Our findings established Micromeria species as a rich source of antioxidant polyphenols, especially the endemic M. croatica.

Effects of pentoxifylline and pentosan polysulphate combination therapy on diabetic neuropathy in type 2 diabetes mellitus.

Vascular dysfunction, including impaired perfusion has a pivotal role in the pathogenesis of microvascular complications in diabetes mellitus. Both pentoxifylline (PF) and pentosan polysulphate (PPS) are known to improve microcirculation. Antioxidant and antiproteinuric effects of PF are also known. In a placebo-controlled study, we determined the possible efficacy of PF-PPS combination therapy on diabetic neuropathy and nephropathy in type 2 diabetic patients. Patients in Verum group (n = 77) received PF-PPS infusions (100-100 mg/day) for 5 days. Control diabetics (Placebo group; n = 12) were given only saline infusions. Specialized cardiovascular autonomic reflex tests, vibration threshold values and urinary albumin excretion were assessed before and after therapy. In Verum group, autonomic score, indicating the severity of cardiac autonomic dysfunction, decreased after therapy (p < or = 0.001). Of the reflexes, deep breath and handgrip tests also improved after therapy (p < or = 0.001). Vibration threshold values, an indicator of the loss of sensory nerve function, were increased after therapy (p < or = 0.001). Results of cardiac autonomic tests and vibration threshold values remained unaltered in Placebo group. Majority of patients had normalbuminuria, which was not affected by PF-PPS. In conclusion, short-term PF-PPS therapy was effective on cardiovascular autonomic function and vibration perception, whereas it failed to reduce albuminuria within normal range in type 2 diabetic patients.