Antifungal Activity, Phytochemical Characterization and Thermal Profile of Anadenanthera colubrina (Vell.) Brenan

Objective: To investigate the antifungal potential of A. colubrina , and its phytochemical characteristics, thermal profile and toxicity. Material and Methods: To assess potential antifungal activity, the technique of microdilution was used with the determination of the Minimum Inhibitory Concentration and Minimum Fungicidal Concentration, using standard species of Candida and recent clinical isolates of Candida albicans . Analyses of action of the extract were performed on the wall and cell morphology of C. albicans , of the interactive effect between the plant extract and nystatin on C. albicans through the checkerboard method, and of growth kinetics. The phytochemical screening was determined by spectrophotometry. The thermal profile was traced with the determination of thermogravimetric curves (TG) and differential scanning calorimetry (DSC). The toxicity was evaluated by the method of hemolysis. Results: The extract of A. colubrina showed a fungistatic potential against all bacteria tested and it acted by modifying the cellular morphology of C. albicans . There was a synergism between nystatin and the plant extract (FIC=0.375), and 53.18% of total polyphenols were determined. The TG curve showed the occurrence of three steps of thermal decomposition. None of the tested concentrations became the effective cytotoxic concentration. Conclusion: Further studies should be conducted to understand the efficacy and the mechanisms of action involved in the antifungal activity of the plant extract of A. colubrina in order to produce a new drug for the treatment of oral candidiasis.


Introduction
Oral candidiasis is an opportunistic infection that has a high occurrence rate and that is frequently associated with Candida albicans [1].The predisposing factors for its development can be classified into local factors such as use of prosthesis, reduced salivary flow and sugar-rich diet, and systemic factors such as advanced age, endocrine disorders, immunosuppression, use of broadspectrum antibiotics and nutritional deficiencies [2].
Among the most appropriate drugs used for the treatment of oral candidiasis, miconazole, from the group of azoles, and nystatin, from the group of polyenics, stand out [3].Despite its efficacy, miconazole can trigger undesired drug interactions, reducing drug absorption and increasing the risk of adverse effects through inhibition of human cytochrome P450 [4,5].Nystatin, because it binds directly to ergosterol and interacts with a component of the human membrane (cholesterol), may lead to side effects [6,7].Thus, there is a demand for new antifungals with different structural classes that can act selectively on new cellular targets with fewer side effects [8].
In this perspective, the antifungal activity of medicinal plants, aiming to develop new drugs, has been investigated.
Among plants used by the population for medicinal purposes is the Anadenanthera colubrina (Vell.)Brenan, used to treat diarrhea, cough, bronchitis, the flu, inflammation and tissue damage, allergy, rash, constipation, gastritis, among other uses [9][10][11].An analgesic action of parts of this plant was identified.It is a result of its antinoceptive effects mediated by central and peripheral mechanisms [12] and with a potential healing and tissue repair action [13].Regarding antimicrobial activity, activity against P. aeruginosa [14], Staphylococcus aureus [15] and Candida albicans [16] was observed.This study aims to investigate the activity antifungal, phytochemical and thermic profil of the hydroalcoholic extract of the bark of Anadenanthera colubrina (Vell.)Brenan.

Plant Extract Collection
Barks from Anadenanthera colubrina (Vell.)Brenan were used.They were collected in the region of the Brazilian semi-arid (7º22'25" S, 35°59'32" W) with the guidance of a botanist.The testimony specimen is deposited in the collection of the Herbarium Manuel de Arruda Câmara (ACAM) of the State University of Paraíba, Campina Grande, Paraiba, under registration nº 667/ACAM.The material was dehydrated in an air circulating oven at 40°C, subsequently ground and then immersed in ethyl alcohol 80% (10g/25mL) for 48 hours at room temperature.The resulting mixture was filtered and the filtration residues were immersed again twice in the solvent.

Determination of Minimum Inhibitory Concentration (MIC)
Minimum Inhibitory Concentration (MIC) was determined by broth microdilution technique using 96-well plates (Alamar Tecno Científica Ltda., Diadema, SP, Brazil) in accordance with the provisions of the document M27A2 [17].The inoculum with a 24-hour growth was standardized in a spectrophotometer, corresponding to 2.5 x 10 3 CFU/mL.To each well of the microtiter plate 100 µL of broth culture medium Sabouraud dextrose, 100 µL of plant extract, whose concentration ranged from 15.62 to 2,000 μg/mL, and 100 µL of the inoculum were placed (final concentration: 5 x 10 6 UFC/mL).The viability control of the tested yeast strain and the sensitivity control to nystatin of the strain were made.The plates were incubated for 24 hours at 37°C.After the incubation period, 10 μL of the aqueous solution of resazurin 0.01% were added to each well to perform a MIC reading.
Viable microorganisms were reduced from a blue color to a pink color.The experiment was performed in triplicate on three different occasions.

Determination of the Minimum Fungicidal Concentration (MFC)
After determining the MIC, the concentration corresponding to the inhibitory and to higher concentrations, as well as the controls, were subcultured on Sabouraud dextrose agar plates.After 24 hours of incubation at 37°C, MFCs' readings were made, being the MFC the lowest extract concentration in which a visible growth of the subculture was prevented.

Action of the Extract on the Cell Wall of Candida albicans
The action of the extract on the cell wall was analyzed by the microdilution in broth technique, with MIC determination, in the presence of sorbitol (0.8 M).Microtiter plates containing 96 wells, with the bottom in a "U" shape, were used (Alamar Tecno Científica Ltda., Diadema, SP, Brazil).In each well of the plate, 100 µL of broth culture medium Sabouraud Dextrose, previously added to sorbitol with molecular weight of 132,17g (Vetec Fine Chemicals Ltda., Rio de Janeiro, RJ, Brazil), and 100 mL of the extract in concentrations from 32 to 0.25 mg/mL were added.Then, 10 μL of the microbial inoculum were added at a concentration of 2.5 x 10 3 CFU/mL in each well.The microplates were seeded and incubated at 37°C for 48 hours, then added to an aqueous resazurin 0.01% solution to perform the MIC reading.The experiment was performed in triplicate.

Cell Morphology Analysis of Candida albicans
The cellular morphology of C. albicans treated with the plant extract was analyzed by scanning electron microscopy (SEM).Aliquots of fungal strains treated with the plant extract and nystatin, and antifungal free strains in culture medium, were centrifuged at 3,500 rpm for 15 minutes to perform deposition of organic material.The supernatant was removed and the samples were fixed with a solution of 2.5% glutaraldehyde and 0.1 M cacodylate buffer.There was a new centrifugation and a subsequent wash with Caco Buffer 0.1M.After removing the buffer, the fungal material was inserted into a glass coverslip to be processed.The processing consisted of three washes with B. Caco 0.1M for 10 minutes, post-fixation at a 1:1 proportion + Osmium 2% + B. Caco 0.1M for 30 minutes to enable a greater material contrast upon reading, two washes with B. Caco 0.1M for 10 minutes and two washes with distilled water for 10 minutes.Dehydration was also performed with a wash in acetone at concentrations of 30%, 50%, 70%, 90% and 100%.The material was dried in a Critical Point (CPD 030 BAL-TEC) for 2 hours, and then the assembly in stubbs was made (carbon tape and silver ink).The stubbs act as electron conductors.Finally, the gold plating was performed for 30 minutes in a sputtering system (Leica EM SCD 500) and then it was analyzed by scanning electron microscope (Quanta 200 FEG, Fei Company, Oregon, USA).

Checkerboard Method
The combined effect of the two substances (nystatin and plant extract of A. colubrina) was determined from the microdilution-checkerboard method for derivation of the Fractional Inhibitory Concentration index (FIC index).The microbial inoculum was standardized in a spectrophotometer to obtain a 2.5 x 10 3 CFU/mL concentration.The extract and the nystatin were tested considering the MIC.Initially, 100μL of the Sabouraud dextrose agar broth culture medium was added to sterilized microplate wells containing 96 wells with a "U" shape (Alamar Tecno Científica Ltda., Diadema, SP, Brazil) Then, 50μL of each substance in concentrations MIC/8, MIC/4, MIC/2, MIC, MICx2, MICx4 and MICx8 were added to the microplate, and finally the inoculum was added.The extract was placed on horizontal lines and the nystatin on vertical lines.The microplates were incubated at 37°C for 48 hours and fungal growth was evidenced by the use of an aqueous 0.01% resazurin solution.The experiment was performed in triplicate.
The FIC index was calculated by the sum of the FIC A + FIC B , where A represents the plant extract and B the nystatin.The FIC A is calculated by the MIC A combined (extract+nystatin)/MIC A alone, while the FIC B = MIC B combined (extract+nystatin)/MIC B alone.This index was interpreted as follows: synergism (<0.5), additivity (0.5-1.0), indifference (>1 and <4) or antagonism (>4) [18].

Growth Kinetics
The study of the interference of the plant extract, associated or not to nystatin, on the growth of C. albicans was conducted through the viable cell count method.Initially, 0.5 mL of yeast suspension was inoculated to 4.5 mL of Sabouraud dextrose broth containing different concentrations of the extract (MIC/8; MIC/4, MIC), nystatin (MIC/8; MIC/4, MIC) and two associations of substances (MIC/8 of extract + MIC/4 of nystatin and MIC/8 of nystatin + MIC/4 of extract).At 0 min and 30 min intervals, 1, 2, 3, 4, 5, 6, 12 and 24 hours after incubation, an aliquot of 10μL of this inoculum was evenly inoculated on Petri plates containing Sabouraud dextrose agar culture medium.In addition, the control experiment was carried out with free antifungal growth.
The inoculated plates were incubated at 35°C for 48 hours.After the incubation period, the counting of the number of viable cells was made.It was expressed as CFU/mL and presented in the form of a microbial kill curve.Data were analyzed and processed using SPSS software version 20.0.The Friedman and Kruskal-Wallis tests were made with a 5% significance level.

Phytochemistry Prospecting
To determine the total polyphenol content, the colorimetric method was used.It uses the Folin-Ciocalteu reagent [19]. 1 mL of the aqueous extract solution was added to 1 mL of the Folin-Ciocalteu 1N reagent and, after a 2-minute rest, 2 mL of an aqueous solution of Na2CO3 at 20% (p/v) was added, then allowing it to rest for another 10 minutes.Then, the absorbance reading at 757nm was made in a spectrophotometer (Shimadzu ® UV mini -1240), against a blank composed of distilled water, Folin-Ciocalteu reagent and 20% of a solution of Na2CO3.The calibration curve was obtained from gallic acid solutions at concentrations of 1, 3, 6, 9, 12, 15, 20, 25, 30, 35, and 40 mg/mL.The concentration of polyphenols was measured in equivalent milligrams of gallic acid.
The determination of the total flavonoid content was performed by adding 5 mL of each extract solution (in methanol) to the same volume of a solution (in methanol) of AlCl2 at 2% (p/v) [20].The mixture rested 10 minutes before the absorbance reading at 415 nm against a blank consisting of an AlCl3 solution was made.For this determination, a calibration curve obtained from quercetin solutions at concentrations of 2, 4, 6, 8, 10, 13, 16, 19, 22, 26, 28 and 30 μg/ml was made.
The concentration of flavonoids was expressed in equivalent milligrams of quercetin.
The content of condensed tannins was quantified by adding 0.5 mL of the sample plant extract to 3 mL of a vanillin solution (4% p/v in methanol).Then, 1.5 mL of concentrated HCl (37%) was added [21].The reaction occurred in test tubes soaked in water at about 22°C.The reading was taken at 500nm against a blank consisting of the vanillin solution, HCl and a solution of ethanol 50% (v/v) in water.The calibration curve for this assay was made using catechin solutions at concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL.The concentration of tannins was expressed as equivalent milligrams of catechin.All analyzes were performed in triplicate.

Thermal Profile
Thermogravimetric curves (TG) were obtained with a simultaneous analyzer Model SDT Q600 (TA Instruments) using alumina crucibles containing samples with 8±0.1 mg, in a nitrogen atmosphere, under a flow of 50 mL.min - .The experiments were conducted between temperature ranges from 25 to 900°C at a heating rate of 10°C min -1 .For the calibration of the equipment, a standard calcium oxalate monohydrate was used.DSC curves were obtained from a calorimeter Model DSC Q20 (TA Instruments) using alumina crucibles containing samples with 2±0.1 mg, in a nitrogen atmosphere, under a flow of 50 mL.min - .The experiments were conducted between temperature ranges from 25 to 400°C at a heating rate of 10°C min -1 .For the calibration of the equipment, an Indium standard (m.p. = 156.6°C)was used.

Toxicity
The toxicity of A. colubrina extract was analyzed by the hemolysis method.A red 4% blood cell suspension was prepared in a 0.9% saline solution.Then, 1 mL of this suspension was distributed in test tubes and homogenized with 1 mL of the diluted extract in different concentrations in order to obtain 0.25, 0.5, 1, 2, 4, 8, 16 and 32 mg/mL.After 1 hour, the samples were centrifuged at 3,000 rpm for 10 minutes and the reading was visually performed, taking into consideration the amount of erythrocytes lysed.The hemolysis visualization was classified as -(0% hemolysis) + (25% hemolysis), ++ (50% hemolysis), +++ (75% hemolysis), and ++++ (100% hemolysis) [22].This reading was made in a spectrophotometer with a 540nm wavelength (Shimadzu ® UV mini -1240), using as a blank a 5% saline solution to confirm the results of the visual reading.Two negative controls were used, one being the suspension of 4% red blood cells and the other the diluted plant extract.As a positive control, a Turk Liquid hemolizing solution was used.

Results
The extract of A. colubrina inhibited the growth of all strains of tested Candida (Table 1) and produced a slight change in the morphology of C. albicans ATCC 18804 (Figure 1).The MIC of the extract of this species increased from 1 mg/mL to 8 mg/mL in the presence of sorbitol.The interactive effect of the extract of A. colubrina and nystatin showed a synergistic activity, with a FIC equal to 0.375 (Table 2) and a reduction of the number of CFU/mL (p <0.05) in the first 6 hours (Figures 2, 3 and 4).The TG curve of the extract of A. colubrina showed the occurrence of three stages of thermal decomposition and the DSC curves of the extract showed that the thermal processes occurred in temperature ranges from 52.37 to 195,52ºC (Figure 5).In the hemolysis assay, the extract of A. colubrina showed a toxicity equivalent to 25% when compared to the positive control (Table 3).

Discussion
The hydroalcoholic extract of the bark of A. colubrina has an antifungal activity for different species of Candida, whose potential ranged from moderate to strong [23].It is worth noting that other authors consider different parameters to assess the antimicrobial potential of medicinal plants, which is considered moderate or strong when the MIC is less than 500 mg/mL or 100 mg/mL, respectively [24].These data are relevant especially when discussing sustainability and production of new pharmaceutical compounds.The extract of A. colubrina presented a fungistatic profile and was not fungicidal against species of Candida, which deserves attention when considering the balance and maintenance of oral microbiota, where the substance controls the growth of the microorganism without removing it completely from the surface where it commonly resides [25].
It is possible that the inhibition of growth of Candida species produced by the extract of A.
colubrina has occurred due to its action on the cell wall, since its MIC increased in the presence of an osmotic shield, sorbitol.Slight alterations in the cell wall were observed in photomicrographs by scanning electron microscopy, especially represented by the loss of the typical rough aspect of the cell without treatment [16].The action of the extract on the cell wall has a positive clinical significance, for it is a structure that mammalian cells do not have [7,26] which favors selectivity and therefore more security to the host.Although the results suggest the A. colubrina as a new source for the development of anti-fungal drugs with greater specificity, there is a great need for more research on its mechanism of action.
Interaction tests of the extract of A. colubrina and nystatin indicated a synergistic effect, probably by acting simultaneously on different cellular targets.Nystatin acts on the membrane of the fungal cell [4,5] and A. colubrina extract acts possibly on the cell wall.Different mechanisms may be involved in synergistic activity between two agents.Some authors [27,28] described four of them: One of the simplest and most known protocols for determination of antimicrobial interaction is the checkerboard test, which provides a two-dimensional array of different concentrations of analyzed substances.This test allows the calculation of the Fractional Inhibitory Concentration Index (FIC index) [18].The checkerboard test and the microbial kill curve are indicated for this evaluation in vitro [29].The synergistic effect between A. colubrina extract and nystatin, even at low concentrations, was maintained over time with a reduction in the number of CFU/mL in relation to the control.
The synergism between antifungal agents may increase the fungal clearance rate, shorten the duration of the therapy, prevent the emergence of drug resistance, broaden the spectrum of activity, and diminish the toxicity associated with the drug, allowing the use of lower doses of antifungal agents [30].In this study, it was observed that the contact time of the substances with cells positively favored such reduction in the first 6 hours.The time factor is important in the prospecting of antifungals, for the prolonged use of antifungal agents and longer dosing regimens appear to contribute to the development of microbial resistance [31].
In the phytochemical prospecting, total polyphenols, tannins and flavonoids were identified.
Their results are in agreement with the findings of other authors [32,33].The high content of phenolic compounds found in the A. colubrina extract may be responsible for its antifungal potential.
These compounds are capable of causing metabolic instability in C. albicans and they destroy the enzymatic activity of proteasomes, thereby contributing to a reduction in the growth rate of microorganisms, as well as biofilm formation and maturation [34].The tannins are part of the group of phenolic compounds and have an antimicrobial activity due to the ability to precipitate proteins [35], leading to inactivation of adhesins, enzymes or the transport of proteins in the cell envelope, interfering with solute transportation or decreasing the availability of metal ions essential to the metabolism of microorganisms [36,37].To a lesser extent, flavonoids have been identified, which also compose the group of phenolic compounds [38], and they may also be associated with the antifungal action of A. colubrina extract [38][39][40].
In thermogravimetric analysis, the first step of TG occurred at temperature ranges from 39.17 to 141,26ºC, representing a mass loss of the sample of approximately 5.97%.This step was attributed to water loss and certain volatile products of the sample.This moisture content is in accordance with the criteria of the Brazilian Pharmacopoeia, which establishes for the control of plant material a limit of 14% as the maximum acceptable amount of moisture.The TG is an effective technique to determine the moisture content of the plant and, with these results, it is possible to establish physical parameters for an efficient drying and proper preservation of the material [41].
The second stage occurred within a temperature range from 141,26 to 229,17ºC, having a mass loss of 9.13%, corresponding to the first step of extract decomposition.The most significant mass loss of the extract was observed between the temperatures ranging from 229,17ºC and 657,39ºC, with loss of 37.44%.This event is probably related to the metabolites present in the plant, already identified as tannins and flavonoids.
In the DSC curve, an endothermic peak was observed at 69,96ºC, which may be related to the vaporization of the sample, with loss of water and volatile constituents.Decomposition processes start over 195,52ºC, which are possibly related to the initial decomposition of secondary metabolites present in the sample as flavonoids, tannins and other [41].This high temperature for the beginning of compounds decomposition present in the plant suggests a good stability of the plant extract.The data obtained by thermal analysis are directly related to the final quality of a pharmaceutical product, either as to the therapeutic efficacy of the product or as to its stability throughout the period of validity.TGs are used to measure mass variation according to temperature in a controlled atmosphere under a heating program.The DSC is used to measure the heat flow difference between a substance and a reference material in function of a heating or cooling program.However, some difficulties are encountered in obtaining reproducible peaks of plant extracts in DSC curves.This is because these extracts are a mixture of substances that interact in the vegetable matrix.In addition, the degradation products are often formed in different concentrations due to various factors such as the shape and imperfections of the particles, loss of gaseous products and the heating rate the sample.
In addition, impurities present in plant extracts have a direct effect on the width of peaks obtained in the endothermic process [41,42].
None of the tested concentrations became an effective 50% cytotoxic concentration (EC50), i.e., able to hemolyze 50% of a 4% suspension of erythrocytes in the hemolysis assay [43].Thus, it was possible to determine the Selectivity Index (SI) of the plant extract, obtained by a IC50/MIC ratio greater than 32.The higher the value of SI, the more active it is against the microorganism and less toxic to the host [44].The results indicate that A. colubrina can be considered a promising source for the development of new drugs.However, other studies in vitro should be conducted to assess biocompatibility and the mechanism of action of the A. colubrina extract, mimicking the biological conditions of the oral mucosa, as well as in vivo tests to prove its effectiveness aiming to produce a new drug for the treatment of oral candidiasis.
Given the limitations of this study, oral cavity condition differs from in vitro status, further studies in vitro and in vivo should be conducted to understand the efficacy and the mechanisms of action involved in the antifungal activity of plant extract of A. colubrina in order to produce a new drug for the treatment of oral candidiasis.

Conclusion
The hydroalcoholic extract from the bark of Anadenanthera colubrina (Vell.)Brenan showed a fungistatic activity against Candida species and a synergistic interaction when combined with nystatin.It exhibited a high concentration of total polyphenols, a good thermal stability and an absence of toxicity to red blood cells.
The final three liquid phases were concentrated on a rotary evaporator in vacuum at 39°C, subsequently lyophilized and kept under refrigeration.Antimicrobial Activity Microorganisms The microorganisms used were Candida albicans (ATCC 18804), Candida krusei (ATCC 34135), Candida glabrata (ATCC 15545), Candida guillermondii (ATCC 6260), Candida parapsilosis (ATCC 22019), Candida tropicalis (ATCC 13803), all provided by the Reference Materials Laboratory of National Institute for Health Quality Control (Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil), and fresh isolates of Candida albicans (LM 11, LM 520 LM 70, LM 410) were provided by the Mycology Laboratory of the Federal University of Paraíba.

Figure 2 .
Figure 2. Kinetics of C. albicans growth (ATCC 18804) under the activity of the test products in MIC.

Figure 3 .Figure 4 .
Figure 3. Kinetics of C. albicans (ATCC 18804) growth under the activity of the test products in MIC/4.

Figure 5 .
Figure 5. TG and DSC curves.10°C min -1 heating rate.Temperature ranges from 25 to 900°C for TG, and from 25 to 400°C for DSC.

( 1 )
inhibition of different stages in intracellular fungal biochemical pathways, essential to cell survival; (2) increased penetration of the antifungal agent provided by the action of another antifungal on fungal cell membrane; (3) inhibition of carrier proteinases; and (4) inhibition of different cellular targets simultaneously.