UPLC-MS-ESI-QTOF Analysis and Antifungal Activity of the Spondias tuberosa Arruda Leaf and Root Hydroalcoholic Extracts

The aim of this study was to identify and evaluate the chemical compositions and effects of the S. tuberosa leaf and root hydroalcoholic extracts (HELST and HERST) against different strains of Candida. Chemical analysis was performed by Ultra-Performance Liquid Chromatography Coupled to Quadrupole/Time of Flight System (UPLC-MS-ESI-QTOF). The Inhibitory Concentration of 50% of the growth (IC50) as well as the intrinsic and combined action of the extracts with the antifungal fluconazole (FCZ) were determined by the microdilution method while the minimum fungicidal concentrations (MFCs) and the effect on fungal morphological transitions were analyzed by subculture and in humid chambers, respectively. From the preliminary phytochemical analysis, the phenols and flavonoids were the most abundant. The intrinsic IC50 values for HELST ranged from 5716.3 to 7805.8 µg/mL and from 6175.4 to 51070.9 µg/mL for the HERST, whereas the combination of the extracts with fluconazole presented IC50 values from 2.65 to 278.41 µg/mL. The MFC of the extracts, individually, for all the tested strains was ≥16384 µg/mL. When fluconazole was combined with each extract, the MFC against CA URM 5974 was reduced (HELST: 2048 and HERST: 4096 µg/mL). Synergism was observed against standard C. albicans (CA) and C. tropicalis (CT) strains and with the root extract against the CT isolate. The leaf extract inhibited the morphological transition of all strains while the root extract inhibited only CT strains.


Introduction
Candida species, especially C. albicans, are commonly found on human mucosal surfaces but are becoming an important progressive invasive pathogen due to their increased prevalence in immunocompromised patients and the increased use of antibiotics [1].
Infections caused by the Candida genus are associated with a high morbidity and mortality rate, where these species are responsible for superficial and systemic candidiasis, with the latter being a serious problem for health systems and patients [2]. Several Candida species are polymorphic and able to transition between different morphological conditions involving yeast, hyphal, and pseudohyphal forms [3].
The inappropriate use of antifungal drugs contributes to the increase in microbial resistance, thus an understanding the mechanisms of antifungal drugs, the cellular molecular mechanisms involved in the antifungal resistance process, is sought [4]. Given the few commercially available antifungals and their varied side effects, a need to produce new and more effective antifungal agents with fewer adverse effects exists [5].
Studies addressing the use of plants have been growing in Brazil as well as worldwide, increasing the importance and knowledge of their chemical components [6]. In this sense, natural products and their chemical diversity are seen as options for potentially active therapeutic sources, which can be a means of discovering new drugs [7].
Using ethnobiology as a guiding strategy for bioactivity research, the objective of this study was to identify the chemical composition of the S. tuberosa leaf and root hydroalcoholic extracts and their antifungal effect, both the intrinsic and combined effect with conventional fluconazole, as well as their action on morphological transition, a Candida spp. virulence factor.

Results
The leaf and root extract yields were, respectively, 0.74% and 0.635%. Preliminary phytochemical analysis of the extracts revealed the presence of several metabolite classes, such as: Alkaloids, steroids, phenols, flavonoids, triterpenoids, and xanthones (Table 1). It is noteworthy that the presence of phenols and flavonoids prevailed in all extracts. Among the secondary metabolites investigated, coumarins, flavanonols, and tannins were not detected. Table 1. Preliminary phytochemical analysis of extracts.

Special Metabolite Classes (SMC) SMC 1 SMC 2 SMC 3 SMC 4 SCM 5 SMC 6 SMC 7 SMC 8 SMC 9 SMC 10
HELST In UPLC-MS-ESI-QTOF, identification of the compounds was based on their molecular ion mass, retention time, fragmentation pattern, and data available in the literature, as shown in Tables 2 and 3, according to the order of elution, molecular formula, error, and major fragments (MS2).  [16,21] [M-H] --Reference Ion on negative ion mode. Table 4 shows the IC 50 values of the extracts, fluconazole, and their combined action, where the concentration in which the natural product or the drug could reduce the microorganismal population by 50% can be seen. The HESTL and HESTR values were not clinically significant while fluconazole achieved a 50% reduction in the fungal population, whereas when associated with leaf extract, there was an increase in drug action. With root extract, the effect was similar to fluconazole for C. albicans strains, and for CT INCQS 40042, there was a decrease in the effect of the drug, while for CT URM there was an improvement in fluconazole action.  The action of the extracts and fluconazole on the cell viability curve can be seen in Figure 1. The extracts presented no relevant action against the tested strains while fluconazole presented a more effective action against CA URM 5974 (32 µg/mL), and against other strains when its concentration was increased. inhibition at the HCE concentration (8192 µ g/mL) and a decrease in hyphae and pseudohyphae growth at the HCE/4 concentration (2048 µ g/mL). The HCE/16 concentration (512 µ g/mL) presented similar results to the growth control.
Generally, the HELST obtained better results in all the performed tests, which is interesting from the point of view of the conservation of the species, as since the leaves are more accessible, the collection is practical and less harmful to the plant.  The MFC of the extracts against all tested strains was ≥16,384 µg/mL, as well as the fluconazole MFC against C. tropicalis, thus observing a fungistatic effect. The FCZ effect was termed fungicidal at a concentration of 8192 µg/mL (Table 5) against standard and isolated C. albicans strains. An MFC of 2048 and 4096 µg/mL, respectively, was observed against the CA isolate in the fluconazole combination with the HELST and HERST extracts while the MFC ranged from 8192 to ≥16,384 µg/mL against the other tested strains. When combined with the antifungal agent fluconazole, the HELST potentiated the effect of the drug, where a synergistic effect against CA INCQS 40006 (8 to 256 µg/mL) and CT INCQS 40042 (8 to 512 µg/mL) was observed. On the other hand, the cell viability curve for CA URM 5974 and CT URM 4262 isolates was similar to that of fluconazole ( Figure 2).

Discussion
Phytochemical studies using the S. tuberosa leaf methanolic and ethyl acetate extracts also revealed the presence of phenols, flavonoids, flavones, triterpenes, and steroid compounds, similar to those found in the present study analysis; however, these also found cinnamic derivatives, saponins, and leucoanthocyanidins, which differ from the present results [14,24]. The S. monbin ethanolic extract revealed the presence of polyphenols and flavonoids [25]. The authors of [9] found chlorogenic acid in S. tuberosa leaf hydroethanolic extract by HPLC analysis, with the same compound being identified in the root hydroalcoholic extract of the same species.
The authors of [26] inhibited C. albicans growth with S. tuberosa leaf hexanic extract using a different methodology, obtaining an IC50 of 2.0 mg/mL, a result that is different to those of the present Generally, the HELST obtained better results in all the performed tests, which is interesting from the point of view of the conservation of the species, as since the leaves are more accessible, the collection is practical and less harmful to the plant.

Discussion
Phytochemical studies using the S. tuberosa leaf methanolic and ethyl acetate extracts also revealed the presence of phenols, flavonoids, flavones, triterpenes, and steroid compounds, similar to those found in the present study analysis; however, these also found cinnamic derivatives, saponins, and leucoanthocyanidins, which differ from the present results [14,24]. The S. monbin ethanolic extract revealed the presence of polyphenols and flavonoids [25]. The authors of [9] found chlorogenic acid in S. tuberosa leaf hydroethanolic extract by HPLC analysis, with the same compound being identified in the root hydroalcoholic extract of the same species.
The authors of [26] inhibited C. albicans growth with S. tuberosa leaf hexanic extract using a different methodology, obtaining an IC 50 of 2.0 mg/mL, a result that is different to those of the present study; however, the authors did not detect a fungicidal effect, a result similar to that of this study. S. tuberosa bark ethanolic extract did not inhibit growth of the C. albicans, C. glabrata, C. krusei, or C. tropicalis strains with the disc diffusion technique [27], whose effect may be explained by the fact that the hexane extract have a lower polarity and the extracted constituents are different from those obtained with the extracts in this study [28].
Some of the compounds identified in the S. tuberosa leaf and root extracts are reported in the literature for their antifungal activity. The author of [29] reports the antifungal activity of mangiferin. An inhibition in filament growth was observed with the C. albicans (ATCC 90028 and MTCC 186) strains and clinical isolates (CA1, CA2, CA3, and CA4) in a study addressing the synergistic association of quinic acid with undecanoic acid on Candida spp. morphological transition. However, only a moderate inhibition was observed for C. tropicalis (MTCC 184) strains and isolates (CT1, CT2, and CT3) [30]. The authors of [31] tested several compounds using the disc diffusion method where one of them was narigenin, a compound present in HERST, which demonstrated activity against C. albicans.
The authors of [30] showed that quinic acid, which was identified in the HELST, did not inhibit the growth of Candida strains at varying concentrations (12.5-800 µg mL −1 ). In a study on the antifungal activity of chlorogenic acid, the yeasts Trichosporon beigelii (KCTC 7707), Malassezia furfur (KCTC 7744), and C. albicans (TIMM 1768) were susceptible to the compound, with MIC values between 40 and 80 µg/mL [32]. Chlorogenic acid achieved a partial reduction in the mycelial growth of some pathogenic plant fungi [33]. The chlorogenic acid effect on C. albicans dimorphism was investigated, where this was able to inhibit and destroy hyphae. The authors attribute the action of this compound, which affects yeast cells, to it damaging their membranes and disrupting their membrane potential [32].
The use of different Spondias monbin L. parts in combination with fluconazole inhibited the growth of C. albicans ATCC 90029 and C. krusei, where the author associated this result with the compounds present in the plant, which may have increased the effect of the drug [34]. The S. monbin leaf extract inhibited the Saccharomyces cerevisiae NCPF 3178 yeast strain (7 to 7.5 mm) in the disk diffusion method and presented a minimum inhibitory concentration (MIC) of 250 µg/mL [35] by microdilution. The authors of [25] tested S. monbin ethanolic extract against C. albicans ATCC 10231, C. guilliermondii ATCC 6260 and C. krusei ATCC 34135 using a similar technique to that applied in this study, obtaining MIC values >1000 µg/mL −1 .
Other species that are also from the Anacardiaceae family have been investigated for their activity against fungi. A study with Anacardium occidentale L. and Mangifera indica L. leaves against C. albicans obtained an MIC of 1250 µg/mL and MFC of 1250 and 2500 µg/mL, respectively [36]. The A. occidentale ethanolic extract from the plant's flowers, leaves, and stem bark were investigated using the disc diffusion technique, where the flower extract inhibited different C. albicans and C. tropicalis (1 mm) strains, while the leaf and stem bark extracts inhibited C. albicans 37 (1 mm) and fluconazole inhibited the C. albicans and C. tropicalis strains (1 to 2 mm) [37].
The diversity of chemical classes that are present in an extract are defined as complex heterogeneous mixtures, each of which has different biological or pharmacological activities and collectively contribute to the bioactivity of the extract as a whole [38,39], thus the moderate activity demonstrated by the S. tuberosa leaf and root hydroalcoholic extracts against species from the Candida genus may have occurred due to the synergistic action of the chemical constituents present in the extracts.

Botanical Material Collection and Identification
The botanical material was collected with consent from the Biodiversity Authorization and Information System SISBIO-number 64293-1, in the community of Lameiro (07 • , 15 03.1 S and 39 • , 23 48.3 W of Greenwich), in the municipality of Crato, southern Ceará, Brazil. Specimens from seven individuals were collected from 8:00 to 9:30 in the morning. A sample of the collected material was deposited in the Herbarium Dárdano de Andrade Lima (HCDAL) of the Regional University of Cariri-URCA with the herbarium number 13.728, identified by Professor Ana Cleide Alcantara Morais Mendonça as being the species Spondias tuberosa Arruda.

Extract Acquisition
For the extract preparations, healthy young roots and leaves collected from the upper part of the plant were used. The hydroalcoholic extract (tincture or alcohol) was prepared in 70% alcohol (ethanol) at 500 g of fresh leaves or 400 g of dried root for every 2.652 L of alcohol/water proportion [40] with modifications.
Extract drying was performed by spray drying using the Mini-spray dryer MSDi 1.0 (Labmaq do Brasil) using a 1.2-mm spray nozzle under the following operating conditions: (a) Flow control: 500 mL/h; (b) inlet temperature: 130 ± 2 • C; (c) outlet temperature: 74 ± 2 • C; (d) atomization air flow: 45 L/min; and (e) blower flow: 1.95 m 3 /min. The spray drying process consists of a product that is in the liquid state changing to a solid state in powder form by passing through a heated medium under continuous operation [41].

Preliminary Phytochemical Analysis
The extracts were subjected to preliminary phytochemical analysis based on qualitative methods proposed by [42]. In these experiments, characterization of the main metabolite classes was performed by chemical reactions, with the addition of specific reagents resulting in color changes and/or precipitate formations characteristic of the following metabolite classes: Alkaloids, steroids, phenols, flavonoids, tannins, and triterpenoids. All experiments were performed in triplicates and the secondary metabolite classes present in the extracts were classified as present (+) or absent (-).

Compound Identification by Ultra-Performance Liquid Chromatography Coupled to Quadrupole/Time of Flight System (UPLC-MS-ESI-QTOF)
Identification of the compounds present in the extracts was performed in an Acquity ® UPLC system coupled to a Quadrupole/Time of Flight (QTOF) system (Waters Corporation, Milford, MA, USA), provided by the Chemical and Natural Products Laboratory, Embrapa Tropical Agroindustry (Fortaleza, Ceará). Chromatographic runs were performed with a Waters Acquity ® UPLC BEH column (150 × 2.1 mm; 1.7 µm), a fixed temperature of 40 • C, mobile phases with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B), gradient ranging from 2% to 95% B (15 min), 0.4 mL/min flow rate, and 5 µL injection volume. The ESImode was obtained in the 110-1180 Da range with a fixed source temperature of 120 • C, 350 • C desolvation temperature, 500 L/h desolvation gas flow, 0.5 V extraction cone, and 2.6 kV capillary voltage. Leucine encephalin was used as the lock mass. The MSE (high energy mass spectrometry) acquisition mode was used. The instrument was controlled by the Masslynx  The Sabouraud Dextrose Agar (SDA) solid medium, prepared according to the manufacturer's instructions, and the doubly concentrated Sabouraud Dextrose Broth (SDB) liquid medium, purchased from HIMEDIA ® , were used. The Potato Dextrose Agar (PDA) medium, purchased from Difco ® , depleted by dilution and added to agar was used for the fungal morphology analysis. The media were solubilized with distilled water and autoclaved at 121 • C for 15 min.
All strains were initially kept in refrigerated (8 • C) slanted SDA at the Cariri Applied Mycology Laboratory-LMAC of the Regional University of Cariri-URCA. For microdilution assays, strains were initially cultivated on SDA media at 37 • C for 24 h. Cell suspensions were then prepared in tubes containing 4 mL of sterile saline solution (0.9% NaCl) and their turbidity was compared and adjusted according to the MacFarland 0.5 scale [43].
Dimethyl sulfoxide (DMSO-Vetec) was used for the dilution of the extracts and the antifungal fluconazole 150 mg (prati-donaduzzi), diluted in water, was used as the commercial drug reference in the tests. For the initial extract solution preparations, 0.15 g were weighed and solubilized in 1 mL of DMSO. The extracts and fluconazole were then further diluted in sterile distilled water to obtain the desired test concentration (16,384 µg/mL).

IC 50 Determination and Cell Viability Curve
This assay was performed using the broth microdilution technique in 96-well plates. Each well was filled with the medium and inoculum, followed by the extracts and fluconazole, before being individually microdiluted (81,92 to 8 µg/mL). The last well was reserved as a growth control, all tests were performed in quadruplicate [44], with changes in concentration. Natural product and fluconazole dilution controls (with 0.9% sodium chloride solution instead of inoculum) and sterility controls were also performed. The plates were incubated at 37 • C for 24 h, then read on an ELISA spectrophotometer (Kasuaki-DR-200Bs-BI) at a wavelength of 450 nm. Results from the absorbance were used to construct the cell viability curve; from the mean cell viability curve, the IC 50 values of the extracts and fluconazole were calculated [45].

Minimum Fungicidal Concentration (MFC) Determination
For this test, a sterile rod was introduced into each well of the microdilution test plate (except for the sterility control), where after homogenization of the medium contained in the well, a petri dish with SDA was sub-cultured by transferring a small aliquot of the test solution (medium + inoculum + natural product). The plates were incubated at 37 • C for 24 h and checked for Candida colony growth or lack thereof [46], with modifications. The MFC was defined as the lowest natural product concentration capable of inhibiting the growth of fungal colonies.

Evaluation of the Modifying Effect of Fluconazole Action
The combination of the extracts with the reference drug (fluconazole) was performed to verify whether the antifungal action was modified by the presence of the extract in the medium. Solutions containing the extracts were tested at a sub-inhibitory concentration (MFC/16), according to the methodology used by [47], with changes in concentrations. If the extracts potentiated the action of the antifungal, the observed effect was considered to be synergistic; however, if it impaired its action, it was considered to be an antagonistic effect. Fluconazole was microdiluted at a 1:1 proportion up to the penultimate well. Sterility controls were also performed. All tests were performed in quadruplicate. The plates were incubated at 37 • C for 24 h. The readings were performed in an ELISA spectrophotometry device (Kasuaki-DR-200Bs-BI) [45].

Effect of the Extracts and Fluconazole on Candida Morphological Transition
Microculture chambers were used to verify changes in fungal morphology by the extracts and fluconazole through the reduction or prevention of hyphae development. The extracts and fluconazole were individually evaluated at different concentrations (HCE-8192 µg/mL, HCE/4-2048 µg/mL, and HCE/16-512 µg/mL, where HCE is the highest concentration evaluated by microdilution).
The tests were performed according to [48,49], with modifications: Two parallel striae were made in the solid medium (PDA), which were subsequently covered with a sterile cover slip. For comparative purposes, growth controls as well as fluconazole antifungal controls were performed. The chambers were incubated for 24 h (37 • C), inspected, and recorded by an optical microscope (AXIO IMAGER M2-3525001980-ZEISS-Germany) using a 20X objective. The Zen 2.0 software was used to measure hyphae and pseudohyphae extensions, where images were taken from each complete stria, with five random images being selected from these, according to the concentration. The values were used for statistical analysis [50].

Statistical Analysis
A two-way ANOVA was applied to each sample, comparing the values for each extract concentration, with Bonferroni's post hoc test, in which p < 0.05 and p < 0.0001 were considered significant while p > 0.05 was not significant. IC 50 values were obtained by nonlinear regression with unknown interpolation of standard curves obtained from fungal growth values as a function of the extract concentration, expressed in µg/mL. Statistical analysis was performed using the Graphpad Prism software, version 6.0.
Measurement of the complete striae borders, as well as of the regions where hyphal growth was observed, was performed for virulence analysis. Then, measurements of all hyphal filaments identified in five randomly selected regions from each striae and concentration were performed. Hyphal filament length was averaged and analyzed by ANOVA followed by Bonferroni's correction for multiple comparisons according to the product concentration [50].

Conclusions
Preliminary phytochemical analysis of the extracts detected the presence of phenolic and flavonoid compounds in both extracts. The activity of the extracts against the tested strains was not clinically relevant since direct contact growth inhibition only began at high concentrations. In the fluconazole modulatory assay, the extracts presented synergistic effects with some concentrations. The natural product inhibited hyphal and pseudohyphal growth in the fungal morphological transition assay, depending on the tested concentrations.
Studies on the modulatory and morphological activity of S. tuberosa extracts are rare, with the present study being the first to report on these activities, as well as to report on the analysis of S. tuberosa extracts by high performance liquid chromatography. Thus, research addressing the mechanisms of action of these extracts and the bioactivity of their chemical constituents is necessary to better explore the pharmacological potential of the species.