Hydroalcoholic Extract of Myrcia bella Loaded into a Microemulsion System: A Study of Antifungal and Mutagenic Potential

• Abstract
• Introduction
• Results and Discussion
• Material and Methods
• Chemicals and culture media
• Plant material and extraction
• HPLC coupled with photodiode array detector analysis
• Nanostructured lipid system
• Infrared spectroscopy
• In vitro antifungal activity
• Mutagenicity assay
• In vitro release assay
• Contributorsʼ Statement
• References

Abstract

Myrcia bella is a medicinal plant used for the treatment of diabetes, hemorrhages, and hypertension in Brazilian folk medicine. Considering that plant extracts are attractive sources of new drugs, the aim of the present study was to verify the influence of incorporating 70% hydroalcoholic of M. bella leaves in nanostructured lipid systems on the mutagenic and antifungal activities of the extract. In this work, we evaluated the antifungal potential of M. bella loaded on the microemulsion against Candida sp for minimum inhibitory concentration, using the microdilution technique. The system was composed of polyoxyethylene 20 cetyl ether and soybean phosphatidylcholine (10%), grape seed oil, cholesterol (10%: proportion 5/1), and purified water (80%). To investigate the mutagenic activity, the Ames test was used with the Salmonella Typhimurium tester strains. M. bella, either incorporated or free, showed an important antifungal effect against all tested strains. Moreover, the incorporation surprisingly inhibited the mutagenicity presented by the extract. The present study attests the antimicrobial properties of M. bella extract, contributing to the search for new natural products with biological activities and suggesting caution in its use for medicinal purposes. In addition, the results emphasize the importance of the use of nanotechnology associated with natural products as a strategy for the control of infections caused mainly by the genus Candida sp.

Introduction

The rapid emergence of multidrug-resistant pathogenic fungi and the better-publicized threat of antibiotic-resistant bacteria together pose a considerable threat to human disease control [1]. In view of the importance of this global public health problem with clinical and economic consequences, several organizations, including the European Commission, US Centers for Disease Control and Prevention, and World Health Organization, among others, recognize the relevance of studying the emergence of microbial resistance and the development of surveillance and control systems [2].

The global mortality rate for fungal diseases, for example, now exceeds that of malaria or breast cancer and is comparable to those of tuberculosis and HIV. Recent estimates indicate that more than 300 million people are affected by serious fungal diseases worldwide, resulting in 1.6 million deaths annually [2], [3].

Research focused on this concern seeks therapeutic alternatives in natural products that are endowed with immense structural and chemical diversity. In addition to popular use, medicinal plants play a very important role in modern medicine, significantly contributing to the development of new therapeutic strategies through their secondary metabolites [4].

Species of the genus Myrcia have been used in folk medicine as astringents, diuretics, and antihemorrhagics in the treatment of hypertension, ulcers, and diabetes [5], [6]. Myrcia is one of the largest genera of the family Myrtaceae with 753 species and is widely distributed throughout the Brazilian territory [5]. The Myrtaceae family is represented by around 5500 accepted species, classified in 144 genera distributed especially in the forests of Southeast Asia, Australia, and South America [7]. It is one of the most representative species of the Brazilian flora, presenting significant potential and economic interest to Brazil. Among its species are medicinal plants and ornamental plants, producers of wood and edible fruits [5], [6], [7].

Myrcia bella Cambess is a common and important species in many savannah fragments (Cerrado) distributed in different biomes and in all 5 regions of Brazil [5]. Vareda et al. [8] confirmed that the extracts of M. bella leaves have hypoglycemic and hypolipidemic activities, acting in the reduction of glycemia, possibly due to the uptake and storage of glucose by the liver. Serpeloni et al. [9] verified the cytotoxic, antioxidant, and antimutagenic activity by the micronucleic test of hydroalcoholic extract of M. bella in normal and tumoral gastric cells, and the results showed that high concentrations of extract induce cytotoxicity and cell death by necrosis. Also, its antioxidant activity may be partly responsible for the antimutagenic effects observed and the protective effects against gastrointestinal disturbances previously described for this plant species. Recently, Santos et al. [10] also demonstrated its promising antioxidant activity, in addition to antiproliferative and antimicrobial activities against Escherichia coli. According to Saldanha et al. [11], this species contains phenolic acids, such as caffeic acid, ethyl gallate, gallic acid, and quinic acid, as well as glycosylated flavonoids and acylates mainly derived from quercetin and myricetin. Santos et al. [10] also found ellagic acid as a constituent.

Given the above, the biological studies performed with M. bella extract are promising and stimulate the search for alternatives that may improve its potential pharmacological parameters. Therefore, the search for tools such as pharmaceutical nanotechnology shows great impact and importance. Thus, the objective of this study is the evaluation of the mutagenic effect and antifungal activity of the 70% EtOH extract of M. bella leaves in microemulsions, in order to promote the innovation to create a new generation of treatments, as well as present a promising response to antimicrobial resistance.

Results and Discussion

The chemical characterization of the 70% EtOH leaves extract of M. bella via HPLC-PDA ([Fig. 1 a]) revealed the presence of peaks with UV spectra typical of phenolic acids derivatives with bands at 210 – 278 nm ([Fig. 1 b]) as well as flavonoids with bands at 240 – 285 and 350 – 380 nm ([Fig. 1 c]). These results confirm the presence of the main constituents of 70% EtOH extract of M. bella leaves as described in Saldanha et al. [11], which identified phenolic acids derivatives of gallic, caffeic, and quinic acids and flavonoid glycosides derivatives of quercetin, myricetin, and kaempferol.

The analysis carried out by Saldanha et al. [12] of 271 samples of M. bella harvested across 7 regions during the dry and rainy season by an LC-HRMS-based metabolomics approach revealed the existence of 3 M. bella chemotypes with variation in flavonoid and tannin content mainly linked to soil conditions. Specimens from the populations of the Goias and São Paulo regions, represented by the Parque Nacional das Emas, Jardim Botânico Municipal de Bauru (collection location of M. bella of the present study), and Pratânia, were clustered together and revealed the presence mainly of 2′,3,4′,6,7-pentahydroxyflavan, 3′-O-galloylprocyanidin B5, 2″-O-galloylisoquercitrin, quercetin 3-O-[3,4,5-Trihydroxybenzoyl-(→ 3)-α-L-rhamnopyranoside], and 4′,5,6,7,8-pentahydroxyisoflavone8-methyl ether, 7-O-βD-glucopyranoside.

The formulations were developed having, as difference, the presence (NLS1) or absence (NLS2) of GSO. The phase diagram was determined to characterize the NLS1 suitably. NLS2 was prepared according to Bonifácio et al. [13]. GSO provided a more aqueous and transparent system, while without oil, the system became opaque and viscous. Vegetable oils such as corn, cotton, orange, triglycerides, and esters of fatty acids (isopropyl myristate, ethyl oleate, etc.) are often used as oily components to develop “biocompatible microemulsions” [14], [15].

[Fig. 2] shows the points obtained with different structural characteristics according to the variation of the oil and water ratios, and the circled point in the phase diagram represents the composition of the formulation used to incorporate the M. bella extract in the present study.

According to the analyses of NLS1, 39% of the points were classified as a viscous and opaque system (VOS), followed by 31% as a viscous semi-transparent system (VSTS), 11% as a transparent viscous system (TVS), 8% with phase separation (PS), and, finally, a transparent liquid system (TLS) and an opaque liquid system (OLS) with 6% each ([Fig. 2]).

The more viscous formulation (NLS2) became interesting given the promising results obtained after the treatments against the different species of Candida because, due to this characteristic, it can allow for higher mucosal adhesiveness.

Both NLS1 and NLS2 were selected to obtain an oil-in-water system when diluted with aqueous buffer, consisting of 10% of the oil phase, 10% surfactant, and 80% aqueous phase.

As a surfactant, a mixture of Brij 58 and soy phosphatidylcholine was used. Brij 58 is a nonionic surfactant with hydrophilic-hydrophobic equilibrium [15]. Nonionic surfactants offer a considerable advantage in that they do not need a co-reagent for the formation of microemulsions [16].

[Table 1] shows the values of the means and standard deviations of the hydrodynamic diameters, PDI, and ZP for NLSs without the extract and for the M. bella-incorporated extract.

The values of the mean hydrodynamic diameter of the droplets varied between 42.7 ± 3.23 and 170.8 ± 2.18 nm, the ideal range for NLS, microemulsion type [17] (10 – 250 nm). The PDI values ranged from 0.224 ± 0.017 to 0.391 ± 0.021; this indicates uniformity in the samples (i.e., the lower the PDI value, the greater the homogeneity) [18]. Thus, the formulations were uniform, with NLS1 + MB being more homogeneous than M. bella in NLS2 since the values are smaller when compared.

The value of ZP is directly involved in the physical stability of the droplets, which can change due to the variations that occur at the interface with the dispersed medium, that is, in the dissociation of functional groups on the droplet surface or in the adsorption of possible ionic species present in the aqueous medium [19]. The values obtained for the formulations did not differ statistically from the systems without the extract. The observed negative value means that the droplets have an external surface with the predominance of negative charges, probably coming from the components of the formulations, such as soy phosphatidylcholine, which has free ester groups, as well as CHO, which in its structure has a free hydroxyl [OH−] [20].

All of these parameters were evaluated over a period of 3 mo with fortnightly readings, in order to monitor the stability of the samples. The results showed that there were no statistically significant differences in the assessments, thus demonstrating a good stability of the developed systems ([Fig. 3]).

The infrared (IR) spectra of NLS1 shows the association of the GSO and the surfactant, indicating summative peaks as well as peak displacements, which suggests the interaction between the components of the system. In NLS1, it is possible to visualize the peak in the region of 2800 – 2900 cm⁻¹ referring to the groups of CH₂ of the GSO, and in 1636 cm⁻¹ referring to the OH of the surfactant. Interestingly, the OH peak of the GSO was shifted from its original position of 1375 cm⁻¹ to 1351 cm⁻¹, suggesting a chemical interaction between the oil and the surfactant, possibly via hydrogen bonding. The chemical structure of GSO is rich in OH and COOC, and the terminal group of the surfactant is an OH group. The OH groups from GSO are capable of interacting with the OH from the surfactant through hydrogen bonds, resulting in the classical shift of the OH peaks in IR spectrum, shown in the spectrum of NLS. The large peak in the GSO spectrum at 1164 cm⁻¹ was not identified in the NLS1 spectra, and it can also be indicative of interactions among the system components, suppressing the vibration of the C = O group in that region of the spectrum ([Fig. 4]).

[Fig. 5] shows the spectrum of NLS1, M. bella, and NLS1 + M. bella. All the samples show the chemical groups CH₂, present in the GSO, indicated by the vibration of the bands in the region of 2800 – 2900 cm⁻¹ and OH vibration at about 3300 cm⁻¹, from the water in the aqueous systems and also from the OH of the chemical groups of the extract and system components.

Comparing the incorporated and unincorporated extract, the M. bella spectrum presents bands in the region of 1196 cm⁻¹ and 1326 cm⁻¹ corresponding to the C – H and OH binding vibrations, respectively, from its aromatic structures. These peaks completely disappeared in NLSs, suggesting the possibility of interaction of the components of the extract with the systems ([Fig. 6]).

The results of the analysis of the structural characterization of the microemulsions developed in the present study suggest an oil-in-water microemulsion, that is, oil-in-water droplets stabilized by an interfacial surfactant film.

[Table 2] presents the MIC values observed through the evaluation of the antifungal activity of the M. bella extract and its formulations against yeast: C. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis.

The results showed varying degrees of resistance and sensitivity of the Candida species after treatment with the different samples, being that C. glabrata was the species most sensitive to the inhibitory activity of the extract and its formulations.

When incorporated, the formulations with M. bella presented similar results to those of the free extract. The antifungal action of the extract was higher only after incorporation in NLS1 against C. glabrata and in NLS2 against C. tropicalis, probably due to the presence of CHO in the formulations, which facilitates the permeability of the extract due to the strong interaction between ergosterol present in the fungal plasma membrane.

Considering the medicinal importance of the microorganisms tested, the results of this study are considered very promising in the perspective of the discovery of new drugs from plant sources.

Among the non-albicans species, C. glabrata is considered to be the second most involved species in the vulvovaginal candidiasis [21], [22], [23]. For a long time, this species was considered a nonpathogenic yeast of the normal flora of healthy individuals and generally not associated with severe infections. However, the use of immunosuppressants associated with treatment with broad-spectrum antibiotics has resulted in a higher number of systemic and mucosal infections caused by this species [24].

Some extracts of Myrtaceae have already been analyzed as antimicrobial agents, mainly due to their chemical constitution rich in phenolic compounds. However, despite the popular use of M. bella, there is no data on the antimicrobial effect. Thus, the interest of this plant is justifiable because of its potential medicinal value. Some phenolics found in plants may be used in alternative therapies against conventionally resistant infections or as novel antiseptic agents [10].

Saldanha et al. [11] conducted a phytochemical study with M. bella extract that revealed the presence of nonglycosylated and glycoside flavonoids, such as kaempferol, quercetin, and myricetin, with the flavonoid-O-glycosides derivatives from quercetin being the major compound. It is known that flavonoids are synthesized by plants in response to microbial infection, thus explaining the in vitro activity of these substances against a wide range of microorganisms. Its activity is probably due to its ability to complex with extracellular and soluble proteins, and the lipophilic flavonoids can also break down the microbial membrane [25].

In addition to the interest in the effectiveness of herbal extract against various diseases, safety is also an important factor in traditional medicine. There is a growing assumption that natural products are safe. However, scientific studies have proven that various phytochemicals can be genotoxic and carcinogenic when consumed excessively [26], [27].

[Tables 3] and [4] show the mean and standard deviation of the number of (His+) revertants/plate and the MI after treatment with M. bella extract and its formulations, respectively.

In the present study, the extract of M. bella induced mutations of the base-pair substitution type (TA100 strain) and, at a much higher rate, frameshift mutations (TA98 strain) ([Table 3]).

When incorporated, the mutagenicity of the extract was significantly reduced by formulations, indicating an interaction of the extract with the components of the NLSs, preventing it from damaging bacterial DNA, in experiments with and without metabolic activation, in all tested strains and concentrations ([Table 4]).

These results may be related to the reservoir profile of the NLSs, which becomes more evident when the results of the evaluated surfactant alone are analyzed. The surfactant was mutagenic in the TA100 (with and without S9) and the TA102 (without S9) strains, and toxic in the TA98 (with and without S9) and the TA102 (with S9) strains. However, when in NLSs, it did not induce any mutagenic effect.

Studies have shown that the internal phase can constitute a dimensionally restricted microenvironment, with properties being able to bind or associate molecules with different polarities [28], forming microemulsions with reservoir properties.

The results of the in vitro release assays showed that the incorporation of the extract interferes significantly in its release process, producing significant inhibition of the release rate compared to the free extract, which suggests the retention of M. bella in the NLSs. The study showed that the extract of M. bella obtained a release of approximately 50% in 48 h. However, the formulations were not able to release the constituents of the extract through the dialysis membrane with the same efficiency and were probably retained in the pharmaceutical forms ([Fig. 7]). This fact is in agreement with the results of the IR spectroscopy, which indicate the possibility of interaction of the extracts with the components of the systems, justifying the decrease of the release.

In sum, in this study, we demonstrated the antifungal potential of the 70% EtOH extract of M. bella leaves free and in the formulations. Moreover, the incorporation inhibited the mutagenicity presented by the extract when evaluated by the Ames test in the Salmonella Typhimurium strains. Given the observed results, the present study supports the antimicrobial properties of the M. bella extract, contributing in the search of new natural products with biological activities and suggesting caution of its use for medicinal purposes.

Material and Methods

Chemicals and culture media

DMSO, NADP, D-glucose-6-phosphate disodium salt, magnesium chloride, L-histidine monohydrate, D-biotin, NPD, SA, MMC, 2-AA, 2-AF, amphotericin B, fluconazole, TTC, RPMI, CHO, and Brij 58 were purchased from Sigma Chemical Co. Oxoid nutrient broth No. 2 and Bacto Agar were used as bacterial media for mutagenicity assays. Mueller-Hinton agar, D-glucose, magnesium sulfate, citric acid monohydrate, anhydrous dibasic potassium phosphate, sodium ammonium phosphate, monobasic sodium phosphate, dibasic sodium phosphate, and sodium chloride were purchased from Merck. Sabouraud dextrose agar and chloramphenicol were purchased from Difco-Becton. SPC was obtained from Lipoid GMBH and GSO from Audaz Farmacopéia. DMEM was purchased from Gibco-Invitrogen and FBS from Nutricell.

Plant material and extraction

Leaf samples of M. bella Cambess were collected from the Municipal Botanical Garden of Bauru-SP, Brazil (22°20′30″ S and 49°00′30″ W) in November 2018. The plant specimen was identified by Prof. Dr. Anne L. Dokkedal, using macroscopic and microscopic methods, and a representative voucher specimen was deposited at the Herbarium of the São Paulo State University UNBA (UNESP) under voucher code 5508. The leaves were hot air-dried (60 °C), milled in a knife mill, and percolated at room temperature using EtOH : H₂O (7 : 3, v/v) with yielding of 23% as detailed by Saldanha et al. [11]. The obtained extract (70% EtOH) was chemically characterized by HPLC-PDA.

HPLC coupled with photodiode array detector analysis

The chemical characterization of the obtained extract was obtained using HPLC PU-2089S Plus (Jasco) coupled to a photodiode array detector (PAD) MD-2015 Plus (Jasco) and automatic injector AS-2055 (Jasco). The chromatographic separations were performed using a Luna C₁₈ column (250 × 4.6 mm, i. d.) with a particle size of 5 mm (Phenomenex) maintained at 35 °C, managed by Jasco ChromPass software. The eluents were: A (MeOH + 0.1% Formic acid) and B (H₂O + 0.1% Formic acid.). The gradient condition was: 5 – 45% of A in B in 190 min. The injected volume of the samples was 10 µL solution. The UV-vis spectra were recorded between 200 and 600 nm, and the chromatographic profile was registered at 254 nm. Compounds were dereplicated through retention time and UV spectrum detailed in Saldanha et al. [11], [12].

Nanostructured lipid system

M. bella extract was incorporated into 2 formulations, both containing as S, Brij 58 and SPC mixture in the ratio of 2 : 1, and as aqueous phase, PBS pH 7.4. What differs them from each other is that the first one possesses GSO and CHO in the ratio of 5 : 1 as oil phase (NLS1) and the other only CHO (NLS2). The latter was produced according to Bonifácio et al. [13].

The phase diagram was constructed by fixing the proportion of S and oily phase and titrating it with the aqueous phase, followed by visual inspection of the result of mixing the components. The mixtures were sonicated using a rod sonicator (QSonica Q500) with a potency of 500 watts, amplitude of 20%, batch mode, for 10 min with an interval of 30 s every 1 min, in an ice bath, during the process of sonication. The regions of the systems were visually classified as a viscous system, as a liquid that was optically transparent or opaque, or as phase separation, delimiting the different regions of the phase diagram.

For incorporation of extract in 1 mL of NLS1, 0.004 g of the plant extract was weighed and solubilized in the GSO, followed by low sonication, and the PBS, S, and CHO were added. Already in NLS2, the extract was, initially, solubilized in the aqueous phase followed by low sonication, and then the S and CHO were added. After the addition of all constituents, both were sonicated in a discontinuous mode for 10 min with an interval of 30 s every 1 min to facilitate incorporation of the nanostructured material into the lipid system.

The characterization of NLSs with and without M. bella extract was done through the analysis of the average droplet diameter, PDI, and ZP through DLS using the Zetasizer Nano NS equipment (Malvern Instruments). Samples were oriented in the analysis chamber so that the laser beam could cross throughout dispersion. The temperature of the system was maintained at 20 °C, and the laser wavelength was 532 nm.

Infrared spectroscopy

Infrared (IR) spectra of NLSs were obtained using a FTIR Cary 630 Agilent operating in ATR mode, from 4000 to 600 cm⁻¹ with a resolution of 4 cm⁻¹.

In vitro antifungal activity

The microbiological samples of Candida albicans ATCC-10231, C. glabrata ATCC-2001, C. krusei ATCC-6258, C. parapsilosis ATCC-22019, and C. tropicalis ATCC-750 were obtained from the American Type Culture Collection (ATCC). MIC was determined using the microdilution technique, according to the standard reference method M27-A3 [29], [30].

Yeasts were cultured at 37 °C for 48 h in SDB, adjusted to a final density of 2.5 × 10³ CFU/mL, and used as the inoculum.

M. bella extract was dissolved in 20% DMSO and RPMI for an initial concentration of the extract of 4000 µg/mL. Two-fold serial dilution was performed to obtain concentrations ranging from 1.95 µg/mL to 1000 µg/mL. For each concentration, 100 µL/well was added to a 96-well microplate containing 80 µL/well of RPMI-1640, and the standardized yeast inoculum. The positive controls were amphotericin B (from 8 at 0.062 µg/mL) and fluconazole (from 64 at 0.5 µg/mL); 20% DMSO was used as negative/solvent control, as well as NLSs without extract.

The microplates were again incubated at 37 °C for 48 h, and after that, the mixture from each well was spiked into a sabouraud agar plate to observe the presence or absence of fungal growth. The MIC of the samples was determined after the addition (30 µL) of 2% of TCC and incubation at 37 °C for 2 h. Yeast growth changes the colorless TCC to red color. The samples were processed in triplicate.

Mutagenicity assay

For the evaluation of mutagenicity, the Ames test was performed according to the preincubation methodology developed by Maron and Ames [31]. Five different concentrations of the incorporated and unincorporated extract were evaluated. M. bella was taken up at a concentration of 5000 µg/mL and diluted in phosphate buffer when toxicity was observed in the preliminary tests. Toxicity was evidenced by a reduction in the number of His + revertants or as background growth on the minimal agar test plates.

The Salmonella Typhimurium tester strains (TA98, TA100, and TA102) were kindly provided by Dr. B. N. Ames, and the experiments were performed with and without metabolic activation. The metabolic activation mixture (S9 fraction), prepared from livers of Sprague-Dawley rats treated with the polychlorinated biphenyl mixture Aroclor 1254 (500 mg/kg), was purchased from Molecular Toxicology Inc. and freshly prepared before each test. The metabolic activation system consisted of 4% S9 fraction, 1% 0.4 M MgCL₂, 1% 1.65 M KCl, 0.5% 1 M D-glucose-6-phosphate disodium and 4% 0.1 M NADP, 50% 0.2 M phosphate buffer, and 39.5% sterile distilled water.

To perform the test, 0.5 mL of 0.2 M phosphate buffer or 0.5 mL of 4% S9 mixture and 0.1 mL of bacterial culture were added to the concentrations of the extract and then incubated at 37 °C for 20 min. Then, 2 mL of top agar were added and the mixture poured on to a plate containing minimal agar. The plates were incubated at 37 °C for 48 h, and the His + revertant colonies were counted manually. The assay was performed in triplicate.

The standard mutagens used as positive controls in experiments without the S9 mix were NPD (10 µg/plate) for TA98, SA (1.25 µg/plate) for TA100 and MMC (0.5 µg/plate) for TA102. 2-AA (1.25 µg/plate) was used with TA98 and TA100 and 2-AF (1.25 µg/plate) with TA102 in the experiments with metabolic activation. DMSO and NLSs without extract (100 µL/plate) were used as solvent controls. Also, negative control (without any treatment) that corresponds to the rate of spontaneous reversion of each strain also was performed, as well as GSO and S alone (2.4 µL/plate).

The results were analyzed with the Salanal statistical software package, adopting the Bernstein et al. [32] model. The data (revertants/plate) were assessed by analysis of variance (ANOVA), followed by linear regression. The MI was also calculated for each concentration tested, this being the average number of revertants per plate with the test compound divided by the average number of revertants per plate with the negative control. A sample was considered mutagenic when a dose-response relationship was detected, and a 2-fold increase in the number of mutants (MI ≥ 2) was observed with at least 1 concentration [33].

In vitro release assay

The release process was carried out by placing 0.5 mL of the sample into dialysis tubes (permeability at 14 KDa). The tubes were immersed in beakers containing 10 mL of solvent, sealed to avoid evaporation, and incubated at 37 °C under stirring at 150 rpm on an orbital shaker. The solvent for the free extract was PBS containing 5% DMSO, and the solvent for the incorporated extract was PBS. At time intervals of 0.05, 0.15, 0.30, 1, 2, 4, 8, 24, and 48 h, 10 mL of the solvent was withdrawn and stored in glass tubes under refrigeration. The solvent was replaced at each sampling. The absorbance was monitored by UV-Vis at 350 nm to compare the release time of the pure extract with the incorporated extract.

Contributorsʼ Statement

Conception and design of the study: G. D. Marena, F. A. Resende, M. Chorilli, T. M. Bauab, F. R. Pavan, E. Trovatti, W. R. Lustti; data collection: G. D. Marena, L. Girotto, L. L. Saldanha, M. A. S. Ramos, R. A. De Grandis, P. B. Silva; statistical analysis: G. D. Marena, L. Girotto, L. L. Saldanha, M. A. S. Ramos, R. A. De Grandis, P. B. Silva; analysis and interpretation of the data: G. D. Marena, L. Girotto, L. L. Saldanha, M. A. S. Ramos, R. A. De Grandis, P. B. Silva, A. L. Dokkedal, M. Chorilli, T. M. Bauab, F. R. Pavan, E. Trovatti, W. R. Lustti, F. A. Resende; produced, characterized and supplied M. bella extract: L. L. Saldanha, A. L. Dokkedal; resources: F. A. Resende, A. L. Dokkedal, M. Chorilli, T. M. Bauab, F. R. Pavan, E. Trovatti, W. R. Lustti; wrote the paper: G. D. Marena, F. A. Resende; critical revision of the manuscript: G. D. Marena, L. Girotto, L. L. Saldanha, M. A. S. Ramos, R. A. De Grandis, P. B. Silva, A. L. Dokkedal, M. Chorilli, T. M. Bauab, F. R. Pavan, E. Trovatti, W. R. Lustti, F. A. Resende.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2016/08559-5, 2017/16278-9, 2017/18782-6, 2018/12590-0), National Council for Scientific and Technological Development (CNPq): Research Grant 423371/2018-5, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazilian state and federal agencies.

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• 12 Saldanha LL, Allard PM, Afzan A, Melo FPSR, Marcourt L, Queiroz EF, Vilegas W, Furlan CM, Dokkedal AL, Wolfender JL. Metabolomics of Myrcia bella populations in Brazilian savanna reveals strong influence of environmental factors on its specialized metabolism. Molecules 2020; 25: 2954
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• 19 Obeidat WM, Schwabe K, Müller RH, Keck CM. Preservation of nanostructured lipid carriers (NLC). Eur J Pharm Biopharm 2010; 76: 56-67
  CrossrefPubMedSearch in Google Scholar
• 20 Silva PB, Freitas ES, Bernegossi J, Gonçalez ML, Sato MR, Leite CQF, Pavan FR, Chorilli M. Nanotechnology-based drug delivery systems for treatment of tuberculosis–a review. J Biomed Nanotechnol 2016; 12: 241-260
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• 21 Kucharíková S, Tournu H, Lagrou K, van Dijck P, Bujdáková H. Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin. J Med Microbiol 2011; 60: 1261-1269
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• 22 Lim CSY, Rosli R, Seow HF, Chong PP. Candida and invasive candidiasis: back to basics. Eur J Clin Microbiol Infect Dis 2012; 31: 21-31
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• 23 Merenstein D, Hu H, Wang C, Hamilton P, Blackmon M, Chen H, Calderone R, Li D. Colonization by Candida species of the oral and vaginal mucosa in HIV-infected and noninfected women. AIDS Res Hum Retroviruses 2013; 29: 30-34
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• 24 Moretti ML, Trabasso P, Lyra L, Fagnani R, Resende MR, De Oliveira Cardoso LG, Schreiber AZ. Is the incidence of candidemia caused by Candida glabrata increasing in Brazil? Five-year surveillance of Candida bloodstream infection in a university reference hospital in southeast Brazil. Med Mycol 2013; 51: 225-230
  CrossrefPubMedSearch in Google Scholar
• 25 Carneiro NS, Alves CCF, Alves JM, Egea MB, Martins CHG, Silva TS, Bretanha LC, Balleste MP, Micke GA, Silveira EV, Miranda MLD. Chemical composition, antioxidant and antibacterial activities of essential oils from leaves and flowers of Eugenia klotzschiana Berg (Myrtaceae). An Acad Bras Cienc 2017; 89: 1907-1915
  CrossrefPubMedSearch in Google Scholar
• 26 Caparroz-Assef SM, Grespan R, Freire Batista RC, Bersani-Amado FA, Baroni S, Araujo Dantas J, Cuman RKN, Bersani-Amado CA. Toxicity studies of Cordia salicifolia extract. Acta Sci Health Sci 2005; 27: 41-44
  PubMedSearch in Google Scholar
• 27 Ahmed AS, Elgorashi EE, Moodley N, McGaw LJ, Naidoo V, Eloff JN. The antimicrobial, antioxidative, anti-inflammatory activity and cytotoxicity of different fractions of four South African Bauhinia species used traditionally to treat diarrhoea. J Ethnopharmacol 2012; 143: 826-839
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• 28 Oliveira AG, Scarpa MV, Correa MA, Rodrigues Cera LF, Formariz TP. Microemulsões: Estrutura e aplicações como sistema de liberação de fármacos. Quim Nova 2004; 27: 131-138
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• 29 Clinical & Laboratory Standards Institute (CLSI). M27-A3: Reference Method for Broth Dilution antifungal Susceptibility Testing of Yeasts; Approved Standard – Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2008
  Search in Google Scholar
• 30 Duarte MCT, Figueira GM, Sartoratto A, Rehder VLG, Delarmelina C. Anti-Candida activity of Brazilian medicinal plants. J Ethnopharmacol 2005; 97: 305-311
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• 31 Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res 1983; 113: 173-215
  CrossrefPubMedSearch in Google Scholar
• 32 Bernstein L, Kaldor J, McCann J, Pike MC. An empirical approach to the statistical analysis of mutagenesis data from the Salmonella test. Mutat Res 1982; 97: 267-281
  CrossrefPubMedSearch in Google Scholar
• 33 Resende FA, Vilegas W, Santos LC, Varanda EA. Mutagenicity of flavonoids assayed by bacterial reverse mutation (Ames) test. Molecules 2012; 17: 5255-5268
  CrossrefPubMedSearch in Google Scholar

Correspondence

Publication History

Received: 26 August 2020

Accepted after revision: 23 November 2020

Article published online:
28 January 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 21 Kucharíková S, Tournu H, Lagrou K, van Dijck P, Bujdáková H. Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin. J Med Microbiol 2011; 60: 1261-1269
  CrossrefPubMedSearch in Google Scholar
• 22 Lim CSY, Rosli R, Seow HF, Chong PP. Candida and invasive candidiasis: back to basics. Eur J Clin Microbiol Infect Dis 2012; 31: 21-31
  CrossrefPubMedSearch in Google Scholar
• 23 Merenstein D, Hu H, Wang C, Hamilton P, Blackmon M, Chen H, Calderone R, Li D. Colonization by Candida species of the oral and vaginal mucosa in HIV-infected and noninfected women. AIDS Res Hum Retroviruses 2013; 29: 30-34
  CrossrefPubMedSearch in Google Scholar
• 24 Moretti ML, Trabasso P, Lyra L, Fagnani R, Resende MR, De Oliveira Cardoso LG, Schreiber AZ. Is the incidence of candidemia caused by Candida glabrata increasing in Brazil? Five-year surveillance of Candida bloodstream infection in a university reference hospital in southeast Brazil. Med Mycol 2013; 51: 225-230
  CrossrefPubMedSearch in Google Scholar
• 25 Carneiro NS, Alves CCF, Alves JM, Egea MB, Martins CHG, Silva TS, Bretanha LC, Balleste MP, Micke GA, Silveira EV, Miranda MLD. Chemical composition, antioxidant and antibacterial activities of essential oils from leaves and flowers of Eugenia klotzschiana Berg (Myrtaceae). An Acad Bras Cienc 2017; 89: 1907-1915
  CrossrefPubMedSearch in Google Scholar
• 26 Caparroz-Assef SM, Grespan R, Freire Batista RC, Bersani-Amado FA, Baroni S, Araujo Dantas J, Cuman RKN, Bersani-Amado CA. Toxicity studies of Cordia salicifolia extract. Acta Sci Health Sci 2005; 27: 41-44
  PubMedSearch in Google Scholar
• 27 Ahmed AS, Elgorashi EE, Moodley N, McGaw LJ, Naidoo V, Eloff JN. The antimicrobial, antioxidative, anti-inflammatory activity and cytotoxicity of different fractions of four South African Bauhinia species used traditionally to treat diarrhoea. J Ethnopharmacol 2012; 143: 826-839
  CrossrefPubMedSearch in Google Scholar
• 28 Oliveira AG, Scarpa MV, Correa MA, Rodrigues Cera LF, Formariz TP. Microemulsões: Estrutura e aplicações como sistema de liberação de fármacos. Quim Nova 2004; 27: 131-138
  CrossrefPubMedSearch in Google Scholar
• 29 Clinical & Laboratory Standards Institute (CLSI). M27-A3: Reference Method for Broth Dilution antifungal Susceptibility Testing of Yeasts; Approved Standard – Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2008
  Search in Google Scholar
• 30 Duarte MCT, Figueira GM, Sartoratto A, Rehder VLG, Delarmelina C. Anti-Candida activity of Brazilian medicinal plants. J Ethnopharmacol 2005; 97: 305-311
  CrossrefPubMedSearch in Google Scholar
• 31 Maron DM, Ames BN. Revised methods for the Salmonella mutagenicity test. Mutat Res 1983; 113: 173-215
  CrossrefPubMedSearch in Google Scholar
• 32 Bernstein L, Kaldor J, McCann J, Pike MC. An empirical approach to the statistical analysis of mutagenesis data from the Salmonella test. Mutat Res 1982; 97: 267-281
  CrossrefPubMedSearch in Google Scholar
• 33 Resende FA, Vilegas W, Santos LC, Varanda EA. Mutagenicity of flavonoids assayed by bacterial reverse mutation (Ames) test. Molecules 2012; 17: 5255-5268
  CrossrefPubMedSearch in Google Scholar