Antibacterial and antioxidant potential of Spondias tuberosa Arruda ( Anacardiaceae ) extracts

Antimicrobial resistance and the consequent inefficiency of antibiotics are the main problems faced by medicine. In view of this, numerous researches have been aimed at looking for new agents with antibacterial activity, among them natural products. Thus, this study aims to evaluate the antioxidant activity of aqueous and hydroalcoholic extracts of the leaves and roots of Spondias tuberosa by Thin Layer Chromatography (TLC), as well as to verify the antibacterial action of the extracts alone and in combination with commercial antibiotics to evaluate its potential in action of change of antibiotics. The extracts showed polar and nonpolar phenolic substances with antioxidant action. The Minimum Inhibitory Concentration (MIC) of the hydroalcoholic extracts of the leaves and roots was 1,024 μg/mL compared to the Staphylococcus aureus 25923 strain, whereas with the other strains, the products showed an MIC ≥ 2,048 µg/mL. The effect of combining extracts with amikacin, ampicillin and norfloxacin against the multidrug-resistant bacteria Escherichia coli 06, Staphylococcus aureus 10 and Pseudomonas aeruginosa 24 resulted in synergistic effects with aminoglycoside alone, achieving up to 75 % reduction in the MIC of the antibiotic. In view of the results obtained, it can be concluded that the extracts of S. tuberosa presented polar and nonpolar phenolic substances, in the antibacterial activity it can positively modify the effect of the aminoglycoside antibiotic against multi-resistant bacteria, but future studies are necessary to discover the mechanism of action of such an effect.


Introduction
Antimicrobial resistance represents a serious threat to global public health and food security, affecting anyone, of any age, in any country, being responsible for a longer stay in hospitals, high medical costs and increased mortality Vitalini & Varoni, 2020).
This mechanism is mainly due to the inappropriate use of antibiotics, which leads to the selection of resistant microorganisms. This resistance can be developed, acquired or even transported through the bacterial gene recombination, being increasingly virulent (Lima et al., 2019).
Such mechanisms mainly affect nosocomial microorganisms, such as the Gramnegative bacteria Escherichia coli and Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus .
In this context, natural products have a wide source of active substances, containing complex mixtures of several distinct constituents that can be synergistically active once administered. In addition, plant extracts and phytochemicals may be important in therapy helping to improve and the effectiveness of conventional antimicrobials, reducing their adverse effects and reversing resistance to numerous drugs (Aboody & Mickymaray, 2020; Bezerra et al., 2019;Costa et al., 2020).
In this case, the use of natural products based on their therapeutic use appears as a viable alternative, since they are culturally accepted, have a low cost and high availability (Bezerra et al., 2017). Thus, ethnopharmacological studies assist in the selection of plants with possible active principles, as they are based on popular knowledge and the medicinal use of plants in traditional practices (Albuquerque & Hazanaki, 2006). Such traditional uses and knowledge are present in several regions of the world, and one that can stand out is the Northeast of Brazil, due to its history, since before 1988 the local populations had no access to a public health system. As a result, communities used plants from the Caatinga, a type of seasonally dry tropical forest that has high biological diversity, to treat diseases (Magalhães et al., 2019).
Based on these premises, the hypothesis is raised that the species S. tuberosa has biological activity against pathogenic microorganisms and has antioxidant action. Thus, this work sought to evaluate the antioxidant activity of the aqueous and hydroalcoholic extracts of the leaves and roots of S. tuberosa Arruda (umbu), as well as to verify the antibacterial action of the extracts alone and in combination with conventional antibiotics to evaluate their potential in the action of antibiotics change.

Botanical Material Collection
The leaves and roots of Spondias tuberosa were collected under the consent of the Biodiversity Authorization and Information System -SISBIO with number 64293-1, in the Lameiro community (07º15'03.1" south latitude and 39º23'48.3" west longitude Greenwich), in the municipality of Crato, Ceará, Brazil, were collected in June 2018 from 7 individuals, from 8:00 am to 9:30 am. Of the material collected, a copy was deposited at the Herbário Caririense Dárdano de Andrade Lima (HCDAL) of the Regional University of Cariri -URCA with a voucher of #13,728, being identified by M.ª Ana Cleide Alcântara Morais Mendonça.

Preparation of Extracts
The aqueous extract was acquired through an infusion process, in which the leaves and roots were crushed to increase their contact surface. Subsequently, 1 L of distilled water at 100 ºC was added to each 132.2 g of plant part, after which the container was closed, where it was kept at rest for 15 minutes, for later filtration. While the hydroalcoholic extract (tincture) was prepared by maceration in 70 % ethanol in a proportion of 500 g of fresh leaves and 400 g of dried roots for each 2.652 L of ethanol (70 %). The mixture was stored in a dark place for Research, Society and Development, v. 9, n. 12, e12791210845, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10845 7 The drying of the extracts was performed using the spray drying technique (spray drying) using the Mini-spray dryer MSDi 1.0 equipment (Labmaq do Brasil), using a 1.2 mm spray nozzle, under the following operational conditions: a) flow control: 500 mL/h; b) inlet temperature: 130 ± 2 ºC; c) outlet temperature: 74 ± 2 ºC; d) atomization air flow: 45 L/min; e) blower flow: 1.95 m 3 /min. The spray drying process consists of changing a product that is in a liquid to a solid state in powder form, through its passage in a heated medium, in a continuous operation (Masters, 1991).

Antioxidant Activity by Thin Layer Chromatography (TLC)
The trial was carried out based on methodologies proposed by Formagio et al., (2014) & Hidalgo, Nunomura & Nunomura (2016), with adaptations. The plant extracts were analyzed, in triplicate, by TLC using quercetin and gallic acid as positive comparison standards (1 mg/mL in methanol). As an adaptation of the method, chromatographic plates were prepared, using glass plates (10 x 5 x 0.3 cm) as support, and as a stationary phase the mixture of plaster and corn starch (1:1) (Collins, 2010;Pereira, 2010). 20 μL of each sample were applied to the plates, together with the positive controls and the elution system: chloroform/ethanol (9:1) was used. After drying at room temperature (30 ± 2 ºC), the plates

Bacterial Strains and Inoculum Preparation
The strains used in the tests were the standard strains of bacteria Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 9027, and multi-resistant strains Escherichia coli 06, Staphylococcus aureus 10 and Pseudomonas aeruginosa 24 (Table 1). The bacteria were grown on solid slopes on Heart Infusion Agar and maintained at 37 °C for 24 h. The inoculants were derived from this solid medium using test Research, Society and Development, v. 9, n. 12, e12791210845, 2020 (CC BY 4.

Culture Mediums
In the tests for antibacterial evaluation, the solid medium Heart Infusion Agar (HIA), which was prepared according to the manufacturer's instructions and the liquid medium Brain Heart Infusion (BHI) at 10 %. The media were solubilized with distilled water and sterilized in an autoclave at 121 °C for 15 minutes.

Preparation of Extracts and Drugs
Was weighed 20 mg of extract and diluted in 1 mL of dimethylsulphoxide (DMSO, Merck, Darmstadt, Germany), then diluted in 8,765 µL of distilled water to obtain a concentration of 2,048 µg/mL and reduce the concentration DMSO, which was used in the tests. The antibiotics amikacin (aminoglycoside), norfloxacin (fluoroquinolone) and ampicillin (beta-lactam) (SIGMA Chemical Co., St. Louis, USA) were used at a concentration of 1,024 μg/mL in the experiments.

Determination of Minimum Inhibitory Concentration (MIC)
The MIC of the extracts was determined using the broth microdilution technique, using 96-well plates and in triplicate. Eppendorfs® were prepared, each containing approximately 1,350 μL of 10 % BHI and 150 μL of the bacterial suspension (corresponding Research, Society and Development, v. 9, n. 12, e12791210845, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10845 9 to 10 % of the solution). The plate was filled in the numerical direction by adding 100 μL of this solution to each well, and then serial microdilution was performed in a 1:1 ratio, varying in concentrations from 1,024 to 0.5 μg/mL. The plates were taken to the incubator for 24 hours at 37 ºC (Javadpour et al., 1996). The determination of bacterial MIC was made using the addition of 20 μL of resazurin in each well and observation after 1 hour. The MIC was verified through the color change caused by resazurin, where the change from blue to pink indicates bacterial growth and the unchanged blue color indicates growth inhibition, verifying the lowest concentration of the product capable of inhibiting bacterial growth. In all tests, the last wells were not microdiluted, as they are used to control bacterial growth.

Evaluation of the Antibiotic Action Modifying Effect
To verify whether the extracts would modify the action of antibiotics against the tested resistant strains, the method proposed by Coutinho et al., (2008). The extracts were tested in sub-inhibitory concentration (MIC/8). Eppendorfs® containing 10 % BHI, 150 μL of the bacterial suspension and the volume corresponding to the MIC/8 of the tested extracts were prepared. For the control of antibiotics, eppendorfs® tubes were prepared with 1.5 mL of solution containing 1,350 μL BHI (10 %) and 150 μL of microorganism suspension. The plate was filled in the numerical direction by adding 100 μL of this solution to each well.
Then, 100 μL of the antibiotics were added, proceeding in series microdilution, in a proportion of 1:1 until the penultimate well. The plates were taken to the incubator for 24 hours at 37 ºC. Antibiotic concentrations ranged from 512 μg/mL to 0.5 μg/mL. All tests were performed in triplicate and the reading was done through the colorimetric variation of resazurin as previously described.

Results and Discussion
Thin layer chromatography of the extracts demonstrated the presence of phenolic substances with varying polarities, the most polar being located at the bottom of the chromatoplate ( Figure 1A), as they showed dark blue color when reacted with FeCl3, as well as the positive quercetin controls (Spot 1 in Figure 1A) and gallic acid (Spot 2 in Figure 1A).
The yellow-colored bands in Figure 1B indicate DPPH radical scavenging sites, as well as the positive controls quercetin (Spot 1 in Figure 1B) and gallic acid (Spot 2 in Figure 1B).
Analyzing the two plates of the two figures, it can be seen that the studied extracts presented polar and nonpolar phenolic substances with antioxidant action. HELST and 6: HERST; Elution: chloroform/ethanol (9:1). Source: The Author. Development, v. 9, n. 12, e12791210845, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i12.10845 11 The results presented are indicative of the presence of phenolic compounds with antioxidant action, because as pointed out by Formagio et al., (2014) & Hidalgo, Nunomura & Nunomura (2016), plant extracts that have blue colored substances revealed with FeCl3 and yellow colors revealed with DPPH in chromatoplates are phenolic substances and with antioxidant action. This antioxidant effect was also found by Uchôa et al., (2015), who, when evaluating the effect on the reduction of DPPH free radicals, demonstrated that the methanol extract from the leaves is capable of reducing the amount of radicals in up to 50 % in concentrations of 500 µg/mL. In addition to the leaves, the barks of the species have constituents with antioxidant action (Araújo et al., 2012).
The assessment of the intrinsic antibacterial activity of the extracts showed that the MIC of the HELST and HERST was 1,024 μg/mL compared to the S. aureus 25923 strain, whereas with the other strains, the products showed an MIC of ≥ 2,048 μg/mL. These results indicate that the extracts of S. tuberosa used do not have antibacterial activity in concentrations of clinical relevance, considering that this concentration, in plasma values, can trigger toxic effects in the human organism (Houghton et al., 2007).
The study by Cristofoli et al., (2018) also showed no significant effects against E. coli, P. aeruginosa and S. aureus with the aqueous extract of the leaves of Spondias mombin L.
using the agar diffusion method. Jaiswal et al., (2019), in the same sense, corroborate the present research, since the aqueous and ethanolic extracts of the leaves of Spondias mangifera Willd. were not effective against Gram-positive and Gram-negative bacteria.
In contraposition, Silva et al., (2012) showed that the methanolic extract of the leaves of S. tuberosa had MIC of 125 μg/mL and zone of inhibition of 20.1 mm against P. aeruginosa, however, it is noteworthy that different solvents have distinct selectivity, resulting in unequal bioactivities with a same vegetable, due to the difference of the extracted components (Costa & Hoscheid, 2018).
Although the isolated extracts did not have a significant action on bacterial strains, when associated with amikacin, these products potentiated the action of antibiotics against multi-resistant nosocomial bacteria (Figure 2). These are the greatest reductions in MIC of amikacin: in S. aureus 10, MIC was reduced from 161.27 to 40.32 μg/mL by the HERST ( Figure 2A); in P. aeruginosa 24 the AELST and AERST reduced the MIC from 64 to 32 μg/mL ( Figure 2B); and in E. coli the HELST decreased the MIC from 256 to 101.59 μg/mL ( Figure 2C).
In addition, the extracts also demonstrated indifferent or antagonistic effects when combined with antibiotics, especially with ampicillin and norfloxacin, not interfering or reducing their activities against the tested multidrug-resistant strains.
The antagonistic effects presented may have been due to the antioxidant substances present in the extracts, and can then be explained by the capture of these radicals by antioxidant compounds, intercepting functional oxygen and developing stable radicals, so that the action of the antibiotic is reduced (Mello & Santos, 2017;Andrade et al., 2019). In the same way as Temitope et al., (2017) demonstrated that the association between aqueous, ethanolic and ethyl acetate extracts from leaves, bark and stem of S. mombin with ofloxacin (fluoroquinolone) resulted in significant zones of inhibition against E. coli ATCC 25922 (up to 31 mm), S. aureus ATCC 29213 (up to 35 mm) and P. aeruginosa ATCC 25619 (up to 29 mm).
In the same way as Temitope et al., (2017) demonstrated that the association between aqueous, ethanolic and ethyl acetate extracts from leaves, bark and stem of S. mombin with ofloxacin (fluoroquinolone) resulted in significant zones of inhibition of E. coli ATCC 25922 (up to 31 mm), S. aureus ATCC 29213 (up to 35 mm) and P. aeruginosa ATCC 25619 (up to 29 mm).
The synergistic effects mentioned above with antibiotics of the beta-lactam and fluoroquinolone classes can be explained because in these studies the extracts were used in concentrations ranging from 6,250 to 200,000 μg/mL, 24 to 781 times higher than the present study (256 μg/mL), without necessarily potentiating the drugs by inhibiting resistance, but high toxicity of the extracts themselves on microorganisms (Zhou et al, 2013;Leja et al., 2019).

Conclusion
In view of the results obtained, it can be concluded that the extracts of S. tuberosa presented polar and nonpolar phenolic substances with antioxidant action. They have no efficacy in antibacterial activity by direct action, however, they positively modify the effect of the aminoglycoside antibiotic against multi-resistant bacteria. It is necessary to elucidate the mechanism by which this synergism occurs in order to understand its pharmacological potential.