Copaiba Oil-Loaded Polymeric Nanocapsules: Production and In Vitro Biosafety Evaluation on Lung Cells as a Pre-Formulation Step to Produce Phytotherapeutic Medicine.

Copaiba oil has been largely used due to its therapeutic properties. Nanocapsules were revealed to be a great nanosystem to carry natural oils due to their ability to improve the bioaccessibility and the bioavailability of lipophilic compounds. The aim of this study was to produce and characterize copaiba oil nanocapsules (CopNc) and to evaluate their hemocompatibility, cytotoxicity, and genotoxicity. Copaiba oil was chemically characterized by GC-MS and FTIR. CopNc was produced using the nanoprecipitation method. The physicochemical stability, toxicity, and biocompatibility of the systems, in vitro, were then evaluated. Β-bisabolene, cis-α-bergamotene, caryophyllene, and caryophyllene oxide were identified as the major copaiba oil components. CopNc showed a particle size of 215 ± 10 nm, a polydispersity index of 0.15 ± 0.01, and a zeta potential of -18 ± 1. These parameters remained unchanged over 30 days at 25 ± 2 °C. The encapsulation efficiency of CopNc was 54 ± 2%. CopNc neither induced hemolysis in erythrocytes, nor cytotoxic and genotoxic in lung cells at the range of concentrations from 50 to 200 µg·mL-1. In conclusion, CopNc showed suitable stability and physicochemical properties. Moreover, this formulation presented a remarkable safety profile on lung cells. These results may pave the way to further use CopNc for the development of phytotherapeutic medicine intended for pulmonary delivery of copaiba oil.

The role of aggregation and ionization in the chemical instability of Amphotericin B in aqueous methanol.

Amphotericin B (AmB) is a potent antimicrobial agent used in clinical practice. Nevertheless, the mechanism of its aqueous instability remains not yet fully understood, especially the role that its aggregation state plays in this process. Therefore, the current study used an aqueous methanol media to evaluate the AmB instability as a function of pH-, organic solvent- and concentration-dependent ionization and aggregation. To reach this goal, the aggregation status and instability were determined using UV-vis spectroscopy, LC-MS and HPLC. Moreover, not only the hydrolytic degradation products were identified by UV-vis spectroscopy and LC-MS, but also, the degradation rate constants were estimated by nonlinear regression. The results indicated that monomeric AmB was the predominant species under pH conditions, wherein the substrate was cationic (pH < 4) or anionic (pH > 9). On the other hand, aggregated AmB form was the predominant species for the zwitterionic substrate (at methanol concentration < 30 %(v/v)). Anionic substrate degraded by specific base-catalyzed lactone hydrolysis. Oxidation accounted for the loss of zwitterionic substrate. Aggregated zwitterionic AmB exhibited lower stability than monomeric zwitterionic AmB under neutral pH conditions. These studies are a step forward in comprehending the degradation kinetics of AmB in aqueous medium. In fact, along with our previous research on AmB instability in oils, it leads to a better understanding of the AmB stability in complex systems with an oil-water interface, such as disperse lipid systems.

Anti-Inflammatory Activity of Bullfrog Oil Polymeric Nanocapsules: From the Design to Preclinical Trials.

BACKGROUND: Although bullfrog oil (BFO) exerts anti-inflammatory effects, it has undesirable properties limiting its use. METHODOLOGY: BFO nanocapsules (BFONc) were produced through nanoprecipitation, and their physicochemical and morphological properties were characterized. To evaluate the biocompatibility of the formulation, a mitochondrial activity evaluation assay was conducted, and cell uptake was assessed. The in vitro anti-inflammatory activity was evaluated by measuring reactive oxygen species (ROS), nitric oxide (NO), type-6 interleukin (IL-6), and tumor necrosis factor (TNF) levels. The in vivo anti-inflammatory effect was assessed by quantifying myeloperoxidase (MPO) levels using the carrageenan-induced paw edema model. RESULTS: BFONc showed a particle size of 233 ± 22 nm, a polydispersity index of 0.17 ± 0.03, and a zeta potential of -34 ± 2.6mV. BFONc revealed remarkable biocompatibility and did not induce changes in cell morphology. Furthermore, BFONc decreased ROS levels by 81 ± 4%; however, NO level increased by 72 ± 18%. TNF and IL-6 levels were reduced by approximately 10% and 90%, respectively. Significant in vivo anti-inflammatory activity was observed compared to dexamethasone. MPO levels were reduced up to 2 MPOs/mg. CONCLUSION: Taken together, the results pointed out the remarkable biocompatibility and anti-inflammatory effects of BFONc.

Two phytocompounds from Schinopsis brasiliensis show promising antiviral activity with multiples targets in Influenza A virus.

Influenza A virus, the main flu agent, affects billions of people worldwide. Conventional treatments still present limitations related to drug-resistance and severe side effects. As a result, natural product-derived molecules have been increasingly investigated as prospect drug candidates. Therefore, the aim of this study was to investigate the possible anti-flu activity and to evaluate the toxicity and pharmacokinetic parameters, by in silico approaches, of the Schinopsis brasiliensis Engl. phytochemical compounds. Nine phytocompounds and six antiviral drugs (Amantadine, Umifenovir, Favipiravir, Nitazoxanide, Oseltamivir, Zanamivir) were selected for the analyses against four Influenza A proteins: neuraminidase, polymerase basic protein 2, hemagglutinin and M2 ion channel protein. The molecular docking, the predicted antiviral activity, the predicted toxicity and the pharmacokinetics investigations were conducted. The obtained results demonstrated that Syringaresinol and Cycloartenone display promising in silico antiviral activity (binding energy < 5.0 and ≥ 9.0 kcal/mol) and safety (low toxicity than commercial anti-flu drugs). Overall, this study corroborated the hypothesis that S. brasiliensis barks extract has a biological activity against Influenza A virus. Additionally, Syringaresinol and Cycloartenone have multiple targets in Influenza A virus and showed themselves as the most promising phytocompounds to be isolated and considered for the therapeutic arsenal against the flu.

Polishing the Therapy of Onychomycosis Induced by Candida spp.: Amphotericin B-Loaded Nail Lacquer.

Onychomycosis induced by Candida spp. has several limitations regarding its treatment. Nail lacquers display the potential to overcome these drawbacks by providing therapeutic compliance and increasing local drug bioavailability. Thus, this work aimed to produce a nail lacquer loaded with Amphotericin B (AmB) and evaluate its performance. The AmB-loaded nail lacquer was produced and preliminarily characterized. An AmB quantification method was developed. Stability, drug release, permeability and anti-Candida activity assays were conducted. The analytical method validation met the acceptance criteria. The drug loading efficiency was 100% (0.02 mg/g of total product), whereas the AmB stability was limited to ≅7 days (≅90% remaining). The nail lacquer displayed a drying time of 187 s, non-volatile content of around 20%w/w, water-resistance of approximately 2%w/w of weight loss and satisfactory in vitro adhesion. Moreover, the in vitro antifungal activity against different Candida spp. strains was confirmed. The AmB release and the ex vivo permeability studies revealed that AmB leaves the lacquer and permeates the nail matrix in 47.76 ± 0.07% over 24 h. In conclusion, AmB-loaded nail lacquer shows itself as a promising extemporaneous dosage form with remarkable anti-Candida activity related to onychomycosis.

Could natural products modulate early inflammatory responses, preventing acute respiratory distress syndrome in COVID-19-confirmed patients?

BACKGROUND: The ARDS (Acute Respiratory Distress Syndrome) is a severe respiratory syndrome that was recently associated as the main death cause in the COVID-19 pandemic outbreak. Hence, in order to prevent ARDS, the pulmonary function maintenance has been the target of several pharmacological approaches. However, there is a lack of reports regarding the use of effective pharmaceutical active natural products (PANPs) for early treatment and prevention of COVID-19-related ARDS. Therefore, the aim of this work was to conduct a systematic review regarding the PANPs that could be further studied as alternatives to prevent ARDS. Consequently, this work can pave the way to spread the use of PANPs on the prevention of ARDS in COVID-19-confirmed or -suspected patients. METHODS: The search strategy included scientific studies published in English from 2015 to 2020 that promoted the elucidation of anti-inflammatory pathways targeting ARDS by in vitro and/or in vivo experiments using PANPs. Then, 74 studies regarding PANPs, able to maintain or improve the pulmonary function, were reported. CONCLUSIONS: The PANPs may present different pulmonary anti-inflammatory pathways, wherein (i) reduction/attenuation of pro-inflammatory cytokines, (ii) increase of the anti-inflammatory mediators' levels, (iii) pulmonary edema inhibition and (iv) attenuation of lung injury were the most observed biological effects of such products in in vitro experiments or in clinical studies. Finally, this work highlighted the PANPs with promising potential to be used on respiratory syndromes, allowing their possible use as alternative treatment at the prevention of ARDS in COVID-19-infected or -suspected patients.

Unveiling the Amphotericin B Degradation Pathway and Its Kinetics in Lipid-Based Solutions.

The purpose of this work was to determine the degradation pathway of Amphotericin B (AmB) and its kinetics in lipid-based solutions. Mixtures of AmB in lipophilic solvent media were stored under different conditions, such as surface area, temperature, light exposure, presence of antioxidants and other co-solutes. AmB was quantified by HPLC and UV-Vis spectrometry. Empirical models were proposed, and degradation rate constants were estimated by nonlinear regression. The HPLC method was precise and accurate with linearity from 4.45 to 52.0 nM. Surface area studies revealed that adsorption to glass did not affect AmB loss. Unsaturated oils and methanol better preserved AmB compared to medium chain-triglyceride. Temperature increased AmB loss in a nonlinear behavior and the presence of antioxidants reduced its degradation. Under dark conditions, autoxidation was the predominant degradation pathway of AmB in oil, which undergoes a complex degradation. Under light exposure, photo-oxidation accounted for AmB loss, which appeared to be of pseudo-first order. AmB oily samples should be preferably stored in glass vials protected from light with the addition of antioxidants. Furthermore, this work encourages further investigation in other media for future complex modeling and estimation of AmB degradation and kinetics in lipid-based formulations.

Use of Natural Products in Asthma Treatment.

Asthma, a disease classified as a chronic inflammatory disorder induced by airway inflammation, is triggered by a genetic predisposition or antigen sensitization. Drugs currently used as therapies present disadvantages such as high cost and side effects, which compromise the treatment compliance. Alternatively, traditional medicine has reported the use of natural products as alternative or complementary treatment. The aim of this review was to summarize the knowledge reported in the literature about the use of natural products for asthma treatment. The search strategy included scientific studies published between January 2006 and December 2017, using the keywords "asthma," "treatment," and "natural products." The inclusion criteria were as follows: (i) studies that aimed at elucidating the antiasthmatic activity of natural-based compounds or extracts using laboratory experiments (in vitro and/or in vivo); and (ii) studies that suggested the use of natural products in asthma treatment by elucidation of its chemical composition. Studies that (i) did not report experimental data and (ii) manuscripts in languages other than English were excluded. Based on the findings from the literature search, aspects related to asthma physiopathology, epidemiology, and conventional treatment were discussed. Then, several studies reporting the effectiveness of natural products in the asthma treatment were presented, highlighting plants as the main source. Moreover, natural products from animals and microorganisms were also discussed and their high potential in the antiasthmatic therapy was emphasized. This review highlighted the importance of natural products as an alternative and/or complementary treatment source for asthma treatment, since they present reduced side effects and comparable effectiveness as the drugs currently used on treatment protocols.

Bullfrog Oil Reduces the Carrageenan-induced Edema in Wistar Rats by in vitro Reduction of Inflammatory Mediators.

Bullfrog oil (BFO) is a natural product from the adipose tissue of the amphibian Rana catesbeiana Shaw, a bio-product rich in polyunsaturated fatty acids, which claims anti-inflammatory activity. The objective of this work was to evaluate the cytotoxicity and the anti-inflammatory activity of BFO using in vivo and in vitro assays. Thus, the in vitro cytotoxicity was assessed by the MTT assay. Additionally, the in vivo anti-inflammatory activity was performed by the carrageenan-induced paw edema model in Wistar rats, followed by histological analysis. Moreover, the BFO effect on inflammatory pathways was investigated by in vitro evaluation of the nitric oxide (NO) synthesis, and type-6 interleukin (IL-6) and tumor-necrosis-factor (TNF) levels. In vivo experiments showed that BFO administered by intragastric route produced a significant anti-inflammatory effect, which was as substantial as indomethacin, the positive control. Histopathological analysis confirmed these results, showing the absence of the edema and minimal signs of inflammation in the paws of rats treated with BFO. The MTT results showed that BFO at all tested concentrations had no toxic effect against a macrophage cell line, not affecting the cell viability. In addition, after 48 hours of treatment, the BFO itself and its blend with Cetiol®-V (1:1v/v) at 200 µg.mL-1 were able to reduce the NO synthesis, and the IL-6 and TNF levels up to 35 ± 2%, 40 ± 6%, and 12 ± 3%, respectively. Therefore, these results provide unprecedented scientific evidence of the anti-inflammatory effect of BFO, suggesting its potential as a new candidate for the development of pharmaceutical products with anti-inflammatory activity.

Bullfrog oil (Rana catesbeiana Shaw) induces apoptosis, in A2058 human melanoma cells by mitochondrial dysfunction triggered by oxidative stress.

Bullfrog oil, an animal oil extracted from the adipose tissue of Rana catesbeiana Shaw, showed promising cytotoxic activity against melanoma cells and, therefore, has the potential to become a pharmaceutical active compound. However, there is a lack of information regarding the pathways involved in its pharmacological activity. Thus, the aim of this study was to investigate and elucidate the cytotoxic effect of this oil against A2058 human melanoma cells. The cytotoxic potential was evaluated by the MTT assay, the cell cycle analysis and the cell death assay. In addition, the apoptotic potential was investigated by (i) the DNA fragmentation using propidium iodide staining analysis, (ii) the evaluation of mitochondrial membrane potential and (iii) the determination of intracellular Reactive Oxygen Species (ROS) level. The results showed that the bullfrog oil was able to promote a time-dependent cytotoxic effect, decreasing cell viability to 38% after 72 h of treatment without affecting the cell cycle. Additionally, the bullfrog oil induced the apoptosis in A2058 cells, increasing up to 50 ±â€¯13% of the intracellular ROS level, maintaining the DNA integrity and promoting an approximate decrease of 35 ±â€¯5% in the mitochondrial membrane potential. It can be concluded that the in vitro cytotoxic effect of the bullfrog oil in A2058 human melanoma cells is mediated by oxidative stress that induces mitochondrial dysfunction, triggering the apoptosis. These unprecedented results highlight the pharmacological potential of bullfrog oil and provide important information to support studies on the development of new pharmaceutical products for complementary and alternative treatments for melanoma.

Buccal Bullfrog (Rana catesbeiana Shaw) Oil Emulsion: A Mucoadhesive System Intended for Treatment of Oral Candidiasis.

Oral candidiasis (OC) is an infectious disease caused by microorganisms of the genus Candida, leading to lesions in the buccal cavity. Its treatment consists of the administration of topical or systemic antifungal agents, which may compromise the patient compliance due to its side effects, highlighting the need for alternative treatments. In this scenario, bullfrog oil, an animal oil composed of a pool of saturated and unsaturated fatty acids, is introduced as a potential antifungal raw material. Thus, the aim of this work was to produce a mucoadhesive emulsified system able to deliver the bullfrog oil in the buccal cavity to treat the OC. The emulsion was produced and characterized by visual inspection, droplet size, polydispersity index (PdI), and zeta potential over the course of 60 days. In addition, its mucoadhesive ability was evaluated using an in vitro mucin model. The antifungal activity, evaluated by the broth microdilution assay and the biocompatibility, performed against human erythrocytes, were also carried out. The emulsion showed a droplet size of 320.79 ± 35.60 nm, a PdI of 0.49 ± 0.08, and a zeta potential of -38.53 ± 6.23 mV, with no significant changes over 60 days. The mucoadhesive properties of the system was improved by the use of pharmaceutical excipients. The antifungal activity showed that the bullfrog oil and the emulsion were able to inhibit the growth of different Candida species. Furthermore, the emulsion showed no significant hemolytic effect. Overall, the system showed suitable physicochemical characteristics and biocompatibility, with substantial in vitro antifungal activity, suggesting that this system can be further investigated for OC treatment.

Getting the Jump on the Development of Bullfrog Oil Microemulsions: a Nanocarrier for Amphotericin B Intended for Antifungal Treatment.

Amphotericin B (AmB), a potent antifungal drug, presents physicochemical characteristics that impair the development of suitable dosage forms. In order to overcome the AmB insolubility, several lipid carriers such as microemulsions have been developed. In this context, the bullfrog oil stands out as an eligible oily phase component, since its cholesterol composition may favor the AmB incorporation. Thus, the aim of this study was to develop a microemulsion based on bullfrog oil containing AmB. Moreover, its thermal stability, antifungal activity, and cytotoxicity in vitro were evaluated. The microemulsion formulation was produced using the pseudo-ternary phase diagram (PTPD) approach and the AmB was incorporated based on the pH variation technique. The antifungal activity was evaluated by determination of minimal inhibitory concentration (MIC) against different species of Candida spp. and Trichosporon asahii. The bullfrog oil microemulsion, stabilized with 16.8% of a surfactant blend, presented an average droplet size of 26.50 ± 0.14 nm and a polydispersity index of 0.167 ± 0.006. This system was able to entrap AmB up to 2 mg mL-1. The use of bullfrog oil as oily phase allowed an improvement of the thermal stability of the system. The MIC assay results revealed a growth inhibition for different strains of Candida spp. and were able to enhance the activity of AmB against T. asahii. The microemulsion was also able to reduce the AmB toxicity. Finally, the developed microemulsion showed to be a suitable system to incorporate AmB, improving the system's thermal stability, increasing the antifungal activity, and reducing the toxicity of this drug.

Thermo-Oxidative Stability Evaluation of Bullfrog (Rana catesbeiana Shaw) Oil.

Bullfrog oil (BO), a natural product obtained from recycling of adipose tissue from the amphibian Rana catesbeiana Shaw, has been recently evaluated as a therapeutic activity ingredient. This work aimed to evaluate the long-term and accelerated thermal oxidative stabilities of this product, which is a promising raw material for emulsion technology development. BO was extracted from amphibian adipose tissue at 70 °C with a yield of 60% ± 0.9%. Its main fatty acid compounds were oleic (30.0%) and eicosapentaenoic (17.6%) acids. Using titration techniques, BO showed peroxide, acid, iodine and saponification indices of 1.92 mEq·O2/kg, 2.95 mg·KOH/g oil, 104.2 g I2/100 g oil and 171.2 mg·KOH/g oil, respectively. In order to improve the accelerated oxidative stability of BO, synthetic antioxidants butylhydroxytoluene (BHT) and buthylhydroxyanisole (BHA) were used. The addition of BHT increased the oxidation induction time compared to the pure oil, or the oil containing BHA. From the results, the best oil-antioxidant mixture and concentration to increase the oxidative stability and allow the oil to be a stable raw material for formulation purposes was derived.

New Trends on Antineoplastic Therapy Research: Bullfrog (Rana catesbeiana Shaw) Oil Nanostructured Systems.

Bullfrog oil is a natural product extracted from the Rana catesbeiana Shaw adipose tissue and used in folk medicine for the treatment of several diseases. The aim of this study was to evaluate the extraction process of bullfrog oil, to develop a suitable topical nanoemulsion and to evaluate its efficacy against melanoma cells. The oil samples were obtained by hot and organic solvent extraction processes and were characterized by titration techniques and gas chromatography mass spectrometry (GC-MS). The required hydrophile-lipophile balance and the pseudo-ternary phase diagram (PTPD) were assessed to determine the emulsification ability of the bullfrog oil. The anti-tumoral activity of the samples was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for normal fibroblast (3T3) and melanoma (B16F10) cell lines. Both extraction methods produced yielded around 60% and the oil was mainly composed of unsaturated compounds (around 60%). The bullfrog oil nanoemulsion obtained from PTPD presented a droplet size of about 390 nm and polydispersity = 0.05 and a zeta potential of about -25 mV. Both the bullfrog oil itself and its topical nanoemulsion did not show cytotoxicity in 3T3 linage. However, these systems showed growth inhibition in B16F10 cells. Finally, the bullfrog oil presented itself as a candidate for the development of pharmaceutical products free from cytotoxicity and effective for antineoplastic therapy.

Chemical Characterization and Antimicrobial Activity Evaluation of Natural Oil Nanostructured Emulsions.

The aim of this work was to investigate the antimicrobial activity of nanostructured emulsions based on copaiba (Copaifera langsdorffii) resin-oil, copaiba essential oil, and bullfrog (Rana catesbeiana Shaw) oil against fungi and bacteria related to skin diseases. Firstly, the essential oil was extracted from copaiba resin-oil and these oils, along with bullfrog oil, were characterized by gas chromatography combined with mass spectrometry (GC-MS). Secondly, nanostructured emulsion systems were produced and characterized. The antimicrobial susceptibility assay was performed, followed by the Minimum Inhibitory Concentration (MIC) determination, the bioautography assay, and the antibiofilm determination. Strains of the genera Staphylococcus, Pseudomonas, and Candida were used. The CG-MS analysis was able to identify the components of copaiba resin-oil, copaiba essential oil, and bullfrog oil. The MIC assay in association with the bioautography revealed that some esters of palmitic and oleic acids, a-curcumene, a-himachalene, isothujol, and α-fenchene--probably inhibited some strains. The nanostructured emulsions based on copaiba resin-oil and essential oil improved the antimicrobial activity of the pure oils, especially against Staphylococcus and Candida, resistant to azoles. The bullfrog oil nanostructured emulsion showed a lower antimicrobial effect when compared to the copaiba samples. However, bullfrog oil-based nanostructured emulsion showed a significant antibiofilm activity (p < 0.05). Given the significant antimicrobial and antibiofilm activities of the evaluated oils, it may be concluded that nanostructured emulsions based on copaiba and bullfrog oils are promising candidates for the treatment of infections and also may be used to incorporate other antimicrobial drugs.