Phenolic profile, antioxidant and antifungal activity of extracts from four medicinal plants of the Anacardiaceae family

The aim of this study was to evaluate the chemical profile and the antioxidant and antifungal activities of the ethanol extracts of four Anacardiaceae species, also including the computational study of the mechanism of action of phenolic compounds against the Trichophyton rubrum aspartate semialdehyde dehydrogenase enzyme. For this, the contents of phenols and flavonoids in the extracts were determined. The antifungal activity of the extracts was evaluated by microdilution in broth on T. rubrum fungi and the antioxidant capacity was analyzed by measuring the scavenging effect of the extracts against the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical. For the study of molecular docking, computer simulations of the interaction between enzyme and ligands were performed using the AutoDock Vina Code. The phenolic and flavonoid contents ranged from 227.47 ± 18.62 to 493.44 ± 13.89 gallic acid equivalent (EAG), 14.86 ± 0.22 to 86.07 ± 1.67 quercetin equivalent (EQ), respectively. Antioxidant activity ranged from 3.73 ± 0.12 to 11.21 ± 0.49 μg/mL, especially for Spondias mombim. The ethanol extracts showed antifungal activity against most strains of T. rubrum. The compounds rutin, ellagic acid, quercetin, isoquercitrin and mangiferin present in the species, showed higher affinity for the enzyme aspartate semialdehyde dehydrogenase in relation to fluconazole, the antifungal standard. These results provide evidence that the ethanol extracts of Anacardiaceae can be potential sources of new biotechnological products, acting as natural antioxidant and antifungal agents.


Introduction
Dermatophytoses are among the most recurrent human infections in the world, and a variety of antifungal drugs are used to treat this condition. However, these infections are becoming increasingly resistant to the different existing treatments, especially when they are caused by species of Microsporum and Trichophyton (Akroum, 2018). Given this problem, the development of new safe antifungal agents for the management of these pathogens represents not only an obstacle, but also an urgent need for new drugs (Espino et al., 2019).
Oxidative stress is a biological condition that leads to the formation of free radicals. These molecules are extremely harmful to living organisms, and thus have received greater attention due to their undesirable effects on health. To combat the harmful actions of free radicals, antioxidants are useful tools in reducing the risk of some chronic and acute diseases associated with the redox state of the human body. In this context, studies with medicinal plants are gaining more and more importance in the pharmaceutical, food and cosmetic industries, since the antioxidant properties of plant extracts have been attributed to their phenolic content (Bardacki et al., 2019;Penido et al., 2017).
Natural compounds derived from plants provide a promising research possibility, representing an alternative to synthetic drugs, due to their significant pharmacological and toxicological properties. In addition, natural products are considered potential new drugs against drug-resistant pathogens, among other conditions in the treatment of inflammatory and tumoral diseases (Ziani et al., 2020). Bioactive compounds present in plants, particularly in their extracts, are mainly secondary (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i8.17421 3 metabolites. Among them, phenolic compounds and alkaloids are the most relevant groups. In addition, these compounds have been reported for their therapeutic activities acting as antimicrobial, anti-inflammatory, antifungal and cardioprotective (Nascimento et al., 2021;Espino et al., 2019;Rodrigues et al., 2019;Sales et al., 2021).
Brazil is one of the countries with the huge biodiversity of plants existing in different biomes, and that these plants can present therapeutic potential. However, several plants from the phytogeographic domain present in Northeastern Brazil have been well investigated, regarding their pharmacological properties, although studies have shown that plants in this region are a rich source of biotechnological compounds (Luna et al., 2020;Araújo et al., 2014). The Cocó State Park, located in the city of Fortaleza-CE, occupies an environmental preservation area of 1,571.29 hectares. Its flora consists mostly of mangroves, also including decorative and medicinal plants as the representative species of the Anacardiaceae family (Morais et al., 2021). This family is represented by about 75 genera and more than 700 species distributed in tropical and subtropical regions of the world, which is composed of species of trees or shrubs, used as ornamentals and also for medicinal purposes (Madikizela, Aderogba, Van Staden, 2013). Among the species of Anacardiaceae, Anacardium occidentale L., popularly known as cashew tree, Schinopsis brasiliensis Engl., whose popular name is baraúna, Spondias mombin L. popularly known as cajá, and Mangifera indica L. (mango tree), stand out. These species are traditionally used in the treatment of allergies, diarrhea, mycoses and diabetes, also acting as an antitumor, antidepressant, antimicrobial, anti-inflammatory and anxiolytic (Nivedha et al., 2020;Santos, Amaral, Silva, 2018;Sampaio et al., 2018). In addition to their pharmacological properties, these species are also widely studied due to their antimicrobial and antioxidant properties, which are attributed to the presence of anacardic acid, mangiferin and other compounds, such as tatrols and tannins, phenolic compounds derived from galoyl and ellagic acid compounds and chlorogenic acid Cabral et al., 2016;Singh et al., 2015;Agedah, Bawo, Nyananyo, 2010).
Thus, the objective of this work was to quantify and characterize the phenolic compounds of the ethanol extracts of four species of Anacardiaceae, evaluate the antioxidant and antifungal activities against species of the genus T. rubrum, as well as the computational study of the mechanism of action against the enzyme crystal structure of aspartate semialdehyde dehydrogenase.

Plant material
The collection of plant material was carried out at Cocó State Park in Fortaleza-CE, between june and december 2018.
All samples were packed in plastic bags and transported to the Natural Products Chemistry Laboratory of the State University of Ceará (UECE) where the exsiccates were prepared. The botanical identification was carried out at the Herbarium Prisco Bezerra of the Federal University of Ceará -UFC, and then deposited under the exsiccate numbers shown in Table 1. Source: Herbarium Prisco Bezerra -UFC.

Preparation of ethanol extracts
To prepare the extracts, 50g of dry plant material (leaves, branches and stem bark) was ground and immersed in 200 mL of ethanol solvent for 10 days, and then the extract was filtered and concentrated in a rotary evaporator at 60°C and, after this process, it was placed in a water bath (40°C) until the total elimination of the solvent, obtaining the ethanol extract of the leaves (EEF), the ethanol extract of the branches (EEG) and the ethanol extract of the stem bark (EECC) (Matos, 2009).

Quantification of total phenols
The total phenolic contents of the extracts were determined by the Folin-Ciocateau spectrophotometric method using gallic acid as a reference standard (Sousa et al., 2007). Briefly, 100 μl of each extract with 500 μl of Folin Ciocalteau reagent was added to each volumetric flask for 30 seconds. Then, 6 mL of distilled water and 2 mL of 15% sodium carbonate Na2CO3 were added to each flask and the mixtures were stirred again for another 1 minute, completing the volume of 10 mL with distilled H2O. The balloons were left to rest in the absence of light for 2 hours. The blank was conducted under the same conditions. After resting time, the absorbance spectra were recorded in a UV-Vis spectrophotometer at 750 nm. The test was performed in triplicate and the same procedure was used to obtain the standard calibration curve (y= 0.127x + 0.011, coefficient of determination R2=0.995) obtained by diluting gallic acid in concentrations from 0 to 4 μg. mL-1. The results were expressed in mg of gallic acid equivalent per gram of extract (EAG.g-1), based on a standard curve prepared with gallic acid.

Quantification of total flavonoids
The total flavonoid content was analyzed by the colorimetric aluminum chloride method (Funari & Ferro, 2006).
Briefly, in each volumetric flask, 2 mL of the sample was added with 1 mL of 2.5% aqueous solution of aluminum chloride AlCl3, making up the volume of 25 mL with ethanol. For the blank, the same procedure was performed, substituting only the sample for ethanol. After the balloons were left to rest in the absence of light for 30 minutes, the absorbance reading was performed in a UV-Vis spectrophotometer at a wavelength of 425 nm. All determinations were made in triplicate and the same procedure was used to obtain the standard calibration curve (y= 0.067x + 0.011, coefficient of determination R2=0.999) obtained by diluting the isolated quercetin in concentrations from 0 to 14 μg. mL-1. Flavonoid content was expressed in mg of quercetin equivalent per gram of extract (EQ/g), based on a standard curve prepared with quercetin.

Antioxidant activity -2,2-diphenyl-1-picrylhydrazyl (DPPH) methodology
The evaluation of the antioxidant capacity of the extracts of Anacardiaceae was carried out using the DPPH free radicals capture method (2.2-diphenyl-1-pricril-hydrazyl) described by Yepez et al. (2002). Each extract was weighed and diluted in order to obtain serial dilutions from the stock solution at a concentration of 10000 ppm (15 mg of the extract solubilized in 1.5 mL of methanol). The ethanolic solutions obtained from the stock solution were diluted at concentrations of 5000, 1000, 500, 100, 50, 10 and 5 ppm. Once diluted, an aliquot of 0.1 ml of each dilution was placed in test tubes, together with 3.9 ml of the DPPH solution. Afterwards, the tubes were kept in the absence of light for 1 hour, this procedure was performed in triplicate. After the incubation period had elapsed, the absorbance of the samples was read in a UV-Vis spectrophotometer (Genesys 10S UV-Vis Thermo Scientic) at wavelengths of 515 nm and quercetin was used as a positive control.
The percentage of DPPH free radical inhibition by the extracts at different concentrations was calculated by expressing the scanning index in percentage (IV%): IV% = (DPPH Abs -Sample Abs / DPPH Abs) x 100. The effective concentration to inhibit 50% of the DPPH free radical (CE50) was obtained with the aid of the Excel 2019 software, where, from the values of the final concentrations of the sample and the scanning index (IV%), scatter plots were generated whose equations of the straight lines were used for to obtain the mean and standard deviation values. For comparison purposes, a calibration curve was constructed with different percentages of the flavonoid quercetin, which has high antioxidant activity in DPPH free radicals.

In vitro antifungal assay
In the assay to verify the minimum inhibitory concentration (MIC) of the ethanol extracts of the studied species, strains of T. rubrum obtained from the mycothek of the Federal University of Pernambuco were used. For this assay, the broth microdilution method was used, according to the methodology described by Neves et al. (2019) with the standards of the Clinical Laboratory Standards Insitute protocol (CLSI, 2008).
The broth microdilution method was performed in 96-well microdilution plates, in which growth and sterility control in the wells were included in each strain tested. Initially, 100 μL of RPMI medium was added to all wells and 10 mg/mL of extracts diluted in DMSO was added to all wells of the first column to, in sequence, make the serial dilutions. Finally, 100 μl of the inoculum was added to the plate wells. As a control, ketoconazole was used for dermatophytes.
For susceptibility analysis, the ethanol extracts were tested at concentrations ranging from 0.002 to 2.5 mg/mL. Plates were covered with parafilm and incubated at 37ºC and visual reading was performed after five days for T. rubrum. All tests were performed in duplicate and MIC was defined as the lowest concentration of extract capable of inhibiting 100% of the visible growth of the fungus (Fontenelle et al., 2007). Results were determined by visualization as recommended by CLSI. The minimum fungicidal concentration (MFC) was determined by subculturing 100 µL of the turbidity-free well solution in potato agar at 28˚C (Fontenelle et al., 2007).

Molecular docking with the enzyme aspartate semialdehyde dehydrogenase
The structure of the enzyme aspartate semialdehyde dehydrogenase from T. rubrum was obtained from the Protein Data Bank database (https://www.rcsb.org/), deposited with the PDB 4ZIC code generated from X-Ray diffraction with resolution 2.55 Å, classified as oxidoreductase, organism T. rubrum BMU01672 .
For analysis were selected phenolic compounds isolated from the studied Anacradiaceae species, which showed better atifungal activity (mangiferin, ellagic acid, isoquercitrin, quercetin, rutin and gallic acid). The bibliographic search was carried out in indexed databases of portals and periodicals (CAPES, Scielo and Google Scholar) without date restrictions. The chemical structures of the phenolic compounds for molecular docking were obtained from the PubChem repository (https://pubchem.ncbi.nlm.nih.gov/), in which the structure of mangiferin is deposited with CID5281647, ellagic acid CID5281855, isoquercitrin CID5280804, quercetin CID5280343, rutin CID5280805 and gallic acid CID370. The chemical structures were optimized using the Avogadro code (Hanwell et al., 2012), applying the MMFF94 force field (Nachbar & Halgren, 2000).
After the preparation of the ligands and the receptor, molecular docking was performed using the AutoDock Vina program (version 1.1.2), using 3-way multithreading and Lamarkian Genetic Algorithm (Trott & Olson, 2010). The analysis of results was performed using the programs Discovery Studio Visualizer (Biovia, 2020) and UCSF Chimera (Pettersen et al., 2004).

Statistical analysis
The statistical analysis was performed using the Graph Pad Prism v5.01 program, where the data were submitted to the one-way test of analysis of variance (ANOVA) to determine the statistical differences followed by the multiple comparison between pairs by the Tukey test, considering significant values of p <0.05.

Quantification of total phenols and flavonoids
Phenolic compounds are bioactive structures, and for this reason, they are among the main antioxidants and free radical scavengers present in plants, acting to interrupt chain reactions caused by free radicals due to their ability to donate hydrogen atoms (Torres et al., 2018;Penido et al., 2017). The quantitative estimate of total phenols and flavonoids show statistical differences between most of the ethanol extracts of the different studied plant species (Table 2). Among the species of Anacardiaceae surveyed, the total phenol content ranged from 227.47 to 493.44 EAG/g, while the flavonoid levels showed high variability among the species tested, from 12.28 to 86.07 EQ/g. High levels of total phenols and flavonoids were detected all species in this study. Comparison with literature data is difficult, since few studies have been carried out previously with the same species and, even when the species were the same, they originated from other regions. Another problem is the use of different

Antioxidant activity
The antioxidant potential of the ethanol extracts of four species of Anacaradiaceae was quantified as the concentration needed to inhibit 50% of the DPPH radical (EC50) ( Table 2). This method has been widely used to determine the antioxidant activities of food and vegetable components due to its speed, sensitivity and simple execution (Andrade et al., 2018). The results show that EC50 values ranged from 3.73 ± 0.12 to 11.21 ± 0.49 µg/mL. S. mombin extract was the best DPPH inhibitor, followed by A. occidentale, M. indica and S. brasiliensis. The EECC A. occidentale and the S. mombin EEF did not show statistical differences. The variation observed in EC50 values among the species studied may be associated with differences in the contents of phenolic compounds and other constituents present in the samples (Rahimmalek et al., 2020). Similar to our results, previous studies revealed the antioxidant potential of Anacardiaceae species, attributing this activity to the presence of phenolic compounds Cabral et al., 2016;Abdullah et al., 2015;Ajileye et al., 2015). The antioxidant activity performed by plant extracts may be due to their secondary metabolites, including flavonoids and other phenolic compounds. The scavenging of free radicals by these metabolites is especially attributed to their oxidationreduction capacity, acting directly in the absorption and neutralization of free radicals. This antioxidant protection will directly interfere with the formation of lesions and loss of cell integrity (Mendes et al., 2021). Table 3 shows the results of the antifungal activity of plant extracts on T. rubrum strains. Holetz et al. (2002) classified the antimicrobial efficacy of plant extracts as good (MIC <100μg/mL), moderate (MIC, 100-500μg/mL), weak (MIC, 500-1000μg/mL) and inactive (MIC > 1000μg/mL). It is observed that the extract with the most expressive antifungal activity against all the tested fungi was the EECC of A. occidentale at a concentration of 39 μg/mL. It should be noted that the S. mombin EEF presented MIC ranging from 39 to 78 μg/mL against dermatophytes, demonstrating good inhibitory activity.

Antifungal activity
For the EEF of M. indica it was found good activity against the species of T. rubrum (5908 and 6213) with MIC ranging from 78 to 39 μg/mL, respectively. However, T. rubrum strains (6753 and 6205) were not inhibited by the M. indica extract. Similar activity was observed for S. brasiliensis EEG, which showed moderate antifungal activity with a MIC of 150 μg/mL against T. rubrum (6753, 6205 and 5908) and good activity against T. rubrum 6213 with a MIC of 39 μg/mL. This study confirmed that the studied Anacardiaceae species have antifungal properties against dermatophytes. Thus, the results confirm the ethnopharmacological use of these plants as antimicrobial agents. In line with our results, previous studies using different parts of the species studied here, as well as isolated compounds, extracts, antifungal methods and other fungal species, also revealed inhibition potential (Disegha & Akani, 2017;Temitope et al., 2017;Kannan et al., 2009).
All extracts showed considerable amounts of phenolic compounds, which may be involved in the antifungal activity observed in this study. Research has shown that the antifungal potential of plant extracts is strongly influenced by the quantitative and qualitative variations of their phenolic compounds, including flavonoids. As for the antifungal action mechanism demonstrated by these phenolic substances, there are the disruption of the cell membrane, inhibition of cell division, the development of hyphae through targets in specific genes, as well as interference in metabolic pathways and/or inducing apoptosis by disturbing redox homeostasis (Lagrouh, Dakka, Bakri & 2017;Mohamed et al., 2016).

Molecular docking
Studies using ethanol extracts prepared from flowers, leaves and stem bark of A. occidentale identified the presence of gallic acid in all samples (Silva et al., 2016). Quercetin, on the other hand, was reported from extracts from the leaves of this species. This flavonoid was effective against different strains of T. rubrum, reducing the ergosterol content in these microorganisms (Bitencourt et al., 2013;Ayra et al., 1989). Rutin has also been identified in A. occidentale leaves (Chotphruethipong, Benjakul, Kijroongrojana, 2017). This compound has been shown to be effective against different bacteria and also in the treatment of arthritis caused by Candida albicans (Oliveira et al., 2016).
Tests carried out with the pulp of M. indica showed the presence of some phenolic compounds, including gallic acid (Palafox-Carlos, Yahia & González-Aguilar, 2012). Mangiferin was also identified in this species, which showed antifungal activity against filamentous fungi (Singh et al., 2012). Gallic acid is reported to inhibit the growth of many fungal species such as C. albicans and T. rubrum in vitro and in vivo assays. For T. rubrum species, gallic acid also decreases the ergosterol content, inhibiting the enzymes sterol 14α-desmethylase and squalenopoxidase (Li et al., 2017).
For S. mombin, studies with the hydroethanolic extract of the leaves of this species allowed the identification and quantification of ellagic acid (Cabral et al., 2016) and isoquercitrin . Studies have shown that ellagic acid has a broad spectrum of antifungal activity in vitro, being the best activity observed against T. rubrum. As for in vivo trials, topical administration of ellagic acid was shown to significantly increase the cure rate in a guinea pig infection model of T.
rubrum (Li, Guo & Aibai, 2015). Isoquercitrin is a flavonoid that acts as a potent antifungal agent, targeting the cell membrane, causing permeabilization and depolarization of this structure (Yun et al., 2015). Fernandes et al. 2015 also observed gallic acid in the hydroalcoholic extract of S. brasiliensis stem bark.
In this study, some phenolic compounds presented in A. occidentale, M. indica, S. mombin and S. brasiliensis, as above described, were chosen to evaluate their molecular docking with the enzyme aspartate semialdehyde dehydrogenase from T. rubrum. Among them, the phenolic compounds mangiferin, ellagic acid, isoquercitrin, rutin, quercetin and gallic acid, which stand out for their antifungal activity (Figure 1). The computational study will enable to compare the antifungal potential of these phenolic compounds against T. rubrum. Source: Authors.
In the molecular docking with the enzyme aspartate semialdehyde dehydrogenase from T. rubrum, after simulations, it was possible to observe that all ligands presented ideal RMSD values (Table 4), less than 2 Å (Yusuf et al., 2008). Regarding the binding energy of the formed complexes, it was possible to observe that all phenolic compounds under study showed high affinity for the enzyme, being more favorable to mangiferin (-9.9 kcal/mol), ellagic acid (-9.6 kcal/mol), isoquercitrin (-9.4 kcal/mol), quercetin (-9.1 kcal/mol), rutin (-8.5 kcal/mol) and gallic acid (-7.6 kcal/mol). Five ligands were more favorable to interact with the protein target than fluconazole, and gallic acid was similar to this reference antifungal ( Figure 2). The formed complexes showed interactions varying between 1.92 Å and 5.73 Å in distance (Table 5), and it is possible to identify as the most relevant bonds of the mangiferin complex, five strong-intensity hydrogen bonds (Imberty et al., 1991) with the ASN9A residues, GLU278A, ARG297A, ARG312A, SER618B and two medium intensities with ARG312A and LYS598B. In the case of ellagic acid, the most prominent bonds were three hydrophobic interactions with ALA162A, GLN282A, LYS598B and two strong hydrogen bonds with GLN282A and ARG312A. With the compound isoquercitrin it was possible to observe two hydrophobic interactions with LYS598B, GLN623B and five strong hydrogen bonds with residues SER277A, GLU278A, GLN282A, TYR284A and LEU621B. Hidrogen bond classification: 2,5 Å < d < 3,1 Å = Strong interaction; 3,1 Å < d < 3,55 Å = Average interaction; d > 3,55 Å = Weak interaction. Source: Imberty et al. (1991).
The quercetin complex had five hydrogen bonds, four of which were of strong intensity with ALA11A, GLU278A, ARG297A, ARG312A and one of medium intensity with SER277A. Quercetin also showed a π -Stacking interaction with PHE281A and a π -Cation with ARG312A. Regarding the rutin complex, three hydrophobic interactions with PRO670B,

Conclusion
The findings research show that the species studied presented relevant levels of phenolic compounds, as well as an antioxidant and antifungal effect. As for molecular docking, the compounds rutin, isoquercitrin, quercetin, ellagic acid and mangiferin, showed high affinity for the enzyme aspartate semialdehyde dehydrogenase from T. rubrum, thus demonstrating that several phenolic compounds from Anacardiaceae species are important natural products that inhibit fungal development.
Thus, the selected medicinal species are promising sources of natural products that can be used in further studies to discover new therapeutic compounds against fungal infections. However, future research will be needed to elucidate the antioxidant and antifungal mechanisms in vivo, as well as the bioavailability and metabolic pathways involved.