Evaluation of the antimicrobial potential of extracts Myrciaria cauliflora in strains of Staphylococcus aureus ATCC and Staphylococcus aureus BLAC

Avaliação do potencial antimicrobiano de extratos Myrciaria cauliflora em cepas de Staphylococcus aureus ATCC e Staphylococcus aureus BLAC

Abstract

Bacteria of medical interest have increasingly acquired resistance to available antimicrobials over the years. This resistance makes these microorganisms a challenge for conventional medicine treatments, and new research is focusing on herbal medicine to fight these pathogens. The present work aimed to evaluate the cytotoxic activity of Myrciaria cauliflora and Genipa americana extracts, considering an in vitro bacterial inhibition, using a modified antibiogram technique, the extracts were studied in strains of Staphylococcus aureus ATCC 29213 and Staphylococcus aureus BLAC, where inhibition was observed in the two strains studied by the extract. This study observed that the M. cauliflora bark extract has antimicrobial potential when tested in vitro on S. aureus ATCC 29213 and S. aureus BLAC strains, indicating it is a potential herbal medicine for infections by this pathogen.

Keywords:
extract; antibacterial; Myrciaria cauliflora; Staphylococcus aureus; herbal medicine

Resumo

As bactérias de interesse médico vêm cada vez mais, ao longo dos anos, adquirindo resistência a antimicrobianos disponíveis. Essa resistência torna esses microrganismos um desafio para o tratamento da medicina convencional, e novas pesquisas se apoiam na fitoterapia para conseguir combater esses patógenos. O presente trabalho teve como objetivo avaliar a atividade citotóxica do extrato da casca de Myrciaria cauliflora e Genipa americana e avaliar a inibição bacteriana in vitro, utilizando a técnica de antibiograma modificado. Foram testados extratos em cepas de Staphylococcus aureus ATCC 29213 e Staphylococcus aureus BLAC, nas quais se observou a inibição nas duas cepas estudadas. Neste estudo observou-se que o extrato da casca de M. cauliflora possui potencial antimicrobiano quando testado in vitro em cepas de S. aureus ATCC 29213 e S. aureus BLAC, indicando-o como um potencial fitoterápico para infecções causadas por esse patógeno.

Palavras-chave:
extrato; antibacteriano; Myrciaria cauliflora; Staphylococcus aureus; fitoterapia

1. Introduction

The constant increase in the number of microorganisms resistant to antimicrobials has been intensifying daily, becoming a public health problem in recent years (Salam et al., 2023a). According to the World Health Organization (WHO), infections with resistant bacteria could lead to around 10 million deaths by 2050 (Giono-cerezo et al., 2020).

Studies show that the genus Staphylococcus is one of the main groups of microorganisms highlighted, as it has a high multi-resistance character, making it essential to develop measures to control this pathogen (Torres, 2020).

Staphylococcus aureus is a pathogenic microorganism with several mechanisms of adaptation to an extensive collection of available medications; this resistance results from changes in its genetic material (Lakhundi and Zhang, 2018). Research demonstrated that 45.1% of the S. aureus strains analyzed had the mecA gene, a change in the genetic material that causes a mutation in the cell membrane wall, the target of beta-lactam drugs. This change in the molecular bases makes this pathogen an aggravating factor for cardiovascular and immunological diseases (Pournajaf et al., 2014).

Looking for new ways to combat bacterial infections of medical interest, several works are being carried out with plants seeking therapeutic effects (Cardoso et al., 2019; Tatiya-aphiradee et al., 2019). Work carried out with plant essential oils demonstrates an excellent response against the growth of S. aureus (in vitro tests) (Nascimento et al., 2020).

A plant that has been extensively studied is Myrciaria cauliflora, characteristic of the Brazilian cerrado; this species is popularly known as “jabuticaba”. M. cauliflora has been shown to have several therapeutic effects, such as anti-inflammatory activity (Zhao et al., 2019), vasodilator (Andrade et al., 2016), and oxidative stress suppressor (Hsu et al., 2016).

Bacterial inhibition of methicillin-resistant S. aureus (MRSA) was observed in vivo in a study that used Myrciaria cauliflora extract in conjunction with the photoactivation technique with blue LED light. In an animal model, the administration of photoactivated M. cauliflora extract led to an increase in TNF-α and neutrophils and, consequently, a decrease in bacterial load (Santos et al., 2019).

Another species that has been widely studied is Genipa americana, with the fresh fruit having notable levels of total carbohydrates, soluble solids, phenolic components, acidity, vitamin C, and excellent antioxidant activity. The parameters analyzed suggest that the consumption of the fruit is of great importance and nutritive value (Pacheco et al., 2015). Studies indicate that the hydroalcoholic extract of G. americana bark demonstrated antimicrobial activity against Pseudomonas aeruginosa (Lakhundi and Zhang, 2018).

Understanding the need to develop new ways to combat resistant microorganisms and observing the phytotherapeutic and antibiotic activity of M. cauliflora and Genipa americana when applied to strains of different bacteria of medical interest, the present study aimed to evaluate the antimicrobial activity of the jaboticaba bark extract on strains of S. aureus ATCC and S. aureus resistant to beta-lactams.

2. Materials and Methods

2.1. Collection and preparation of Genipa americana and Myrciaria cauliflora extracts

The ripe fruits of the Genipa americana species were collected in June 2016 in the rural area of the city of Guapó, Goiás. The fruits were sanitized, their skins removed, and their pulp was frozen. Using a blender, the pulp was crushed to prepare the standardized liquid extract, and characterization was carried out.

The fruits of M. cauliflora were provided by Vinícola Jabuticabal in Hidrolândia, Goiás, and its skin was used. The separation was done manually; after collecting the waste, the plant material was dried at 40 °C in an oven with air circulation. Then, the sample was ground in a number 100 tamis knife mill, and the resulting powder of plant material was adequately packed in plastic bags and stored at a temperature of -20 °C.

2.2. Obtaining RAW cells

RAW 264.7 cells, macrophages (American Type Culture Collection - ATCC), were cultured in RPMI 1640 (Sigma Chemical Co. St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Cripion, São Paulo, Brazil), inactivated at 56 °C for 30 min, 2 mM of L-glutamine (Sigma Chemical Co), 50µM of 2 mercaptoethanol (Sigma-Aldrich®), 10U/mL of penicillin and 100 µg mL-1 of streptomycin (Sigma-Aldrich®), 10 mM of Herpes (Sigma-Aldrich®). 2x10⁵ cells were cultured in 2 mL of complete RPMI per well in 6-well culture plates (Costar, New York, USA), incubated at 5% CO₂ and 37 °C.

2.3. Assessment of the cytotoxicity of the extract from the fruits of Genipa americana and Myrciaria cauliflora

For each concentrated alcoholic extract obtained, RAW cells were added to verify their cytotoxicity (Taia et al., 2023). The experiment is based on the Tetrazolium dye uptake assay, mitochondrial metabolic activity (MTT), which has as its principle the absorption of [3-(4,5-dimethylthiazol-2-yl) -2,5 diphenyltetrazolium] bromide salt by viable cells, which reduce, inside the mitochondria, to a product called formazan through the enzyme succinate dehydrogenase and form insoluble purple crystals (Mosmann, 1983).

Raw 264.7 cells (1x10⁵ cells/mL) were seeded, in triplicates, in 96-wells culture plates (Corning-Costar - Kennebunk, ME, EUA) in RPMI 1640 medium and exposed to five concentrations from 5 to 60 µg/mL (5, 10, 20, 40 and 60 µg/mL) of the extracts studied. After 48 hours of incubation at 37 °C and 5% CO₂, 5 µL of MTT was added to each well and incubated for another 3 hours. This process carried out readings in an ELISA reader (Spectrophotometer) (Biolisa Reader, Bioclin, Belo Horizonte, MG) with a 450 and 630 nm double filter.

Cells not treated with the extracts were considered a 100% cell viability parameter. Cytotoxicity was calculated using the Equation 1:

$$\%Viability=\left(AverageofAbsorbanceofeachextractconcentration–controlbasal/Absorbancemeanofcontrols–controlbasal\right)x100$$ (1)

Cell viability was expressed, when applicable, in IC₅₀ (a concentration that inhibited cell growth by 50% compared to the untreated group). The results were analyzed with calculations and expressed in graphs with the help of the statistical tool GraphPad Prism version 7.0.

2.4. Application of the Allium cepa test to evaluate the cytotoxicity, genotoxicity, and mutagenicity of Myrciaria cauliflora fruit extract

To evaluate cytotoxicity, genotoxicity, and mutagenicity, 12 Allium cepa (onion) bulbs were used, purchased from a local market, and grown in a cylindrical container. After 48 hours, the bulbs were treated with different concentrations of the product (5%, 10%, and 20%) for 24 hours, together with a negative control composed of drinking water. Three exposures were performed for each treatment (including the negative control). After 24 hours of exposure, part of the roots were collected and fixed in carnoy solution (3:1 methanol and concentrated acetic acid) for at least 24 hours.

To prepare the slides, the roots were subjected to acid hydrolysis in 1N HCl for 5 minutes, followed by immersion in distilled water for 10 minutes and then in 2% acetic orcein dye, finishing with the preparation of the slide by cutting off the meristematic portions of the roots followed by crushing via a coverslip on a microscope slide. 2000 cells were analyzed per exposure (replicates), totaling 6000 cells per treatment and negative control, recording the number of cells in interphase and cell division for cytotoxic effect analysis, in addition to counting micronuclei and nuclear changes in the different phases of mitosis.

Analysis under an optical microscope allowed the counting of cells in the meristematic portion of Allium cepa roots, a portion with a high mitotic index. Thus, providing the analysis of the parameters: cytotoxicity, which consists of comparing the number of dividing cells in contrast to the number of cells in interphase; genotoxicity, which consists of DNA damage observed in dividing cells in this method; and mutagenicity, which refers to changes in DNA that have been fixed, observed in the form of micronuclei in interphase cells.

The genotoxicity and mutagenicity analysis were performed in an adjusted manner since the obtained count of micronuclei in interphase cells and nuclear changes in mitotic cells were reduced. Thus, the two mentioned parameters were calculated, combining their frequencies and assigning the variable as total nuclear changes.

The data obtained were analyzed using the IBM SPSS (Statistical Package for the Social Sciences) software version 22.0.0.0, and subjected to the Chi-square (χ2) statistical test and proportion comparison analysis through the Z test with values adjusted by the Bonferroni method. All statistical tests were applied at a significance level of 5%.

2.5. Fingerprint of liquid extract from M. cauliflora bark

The ses were conducted on a Waters® model HPLC Alliance® chromatograph with e2695 separation module, 2998 diode array detector (DAD), and Empower 2.0 data processing system. Chromatographic separations were conducted on a reversed-phase Zorbax Eclipse (solvent A) and 0.05% formic acid in ultrapure water (Mili-Q®) (pH=3.15) (solvent B) at a flow rate of 1 mL/min. The following mobile phase gradient was applied: 0%-5% A (0-5 min), 5%-10% A (5-15 min), 10%-15% A (15-25 min), 15%-20% A (25-35 min), and 5 min isocratic 20% A (35-40 min). The injection volume was 10 μL, and the temperature was 35 °C. The mobile phase was previously filtered through a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millex®) and degassed in an ultrasonic bath (USC 1800A, 40 kHz, Uniques).

For the analyses, 1 mL of the extract was diluted in 5 mL HPLC grade methanol.The standards (Sigma Aldrich) were prepared at a concentration of 0.1 mg/mL in HPLC-grade methanol. It was evaluated: quercetin, rutin, p-coumaric acid, catechin, epicatechin, chlorogenic acid, kaempferol, rosmarinic acid, hesperidin, ellagic acid, gallic acid, and caffeic acid. The wavelengths used for detection were 254, 327, and 366 nm. The extract identified compounds by comparing the retention times and the ultraviolet absorption spectrum (190 to 400 nm) of the peaks obtained. The solutions were previously filtered through a 0.45 μm Millex® membrane.

2.6. In silico biological evaluation of gallic acid

The structure of gallic acid was drawn using the ACD/ChemSketch version. 12.01 through selecting pre-defined structures in the program: benzene group and carboxyl radical. Carbon, hydrogen, and oxygen atoms have been selected and added to the structure in the atom toolbar. The molecule was adjusted using the “clean structure” function and saved in the MDL Molfile (.mol) format.

2.7. In silico prediction of gallic acid bioactivity

The PASS (Prediction of Activity Spectra for Substances) server (Lagunin et al., 2010) was used to predict the antimicrobial activity and the possible mechanisms of action involved for gallic acid. The values ​​that define the probability of presence (Pa) and absence (Pi) for each particular activity range from 0.000 to 1.000 and only activities with Pa>Pi were considered.

2.8. Bacterial susceptibility test against extracts on Mueller Hinton agar

The agar well diffusion technique was chosen to carry out bacterial susceptibility tests against extracts on Mueller Hinton agar, as studies have demonstrated its greater sensitivity when carrying out tests with hydroalcoholic plant extracts.

2.9. Technical microbiology procedures

Preparation of Mueller-Hinton Agar, Cled, MacConkey, Mannitol Salt

In a flat-bottom flask graduated to 1000 mL, 22.8 grams of OXOID Mueller-Hinton Agar,, were added and dissolved in 600 mL of deionized water. The flask was sealed with anti-bacteriological cotton sealed with tape suitable for autoclaving, and placed in the autoclave for 15 minutes. The agar was plated in petri dishes measuring 150 mm in diameter and 15 mm in depth. Immediately after cooling the agar, the plates were placed in a UV oven for 30 minutes.

In a 500 mL flat bottom flask graduated to, 10.3 g of OXOID MacConkey Agar were added and dissolved in 200 mL of deionized water; the flask was sealed with anti-bacteriological cotton and autoclave tape, then placed in an autoclave for 15 minutes. The agar was plated into petri dishes measuring 90 mm in diameter and 15 mm in depth. Immediately after cooling the agar, the plates were placed in a UV oven for 30 minutes.

In a 500 mL graduated flat-bottom flask, 7.6 grams of OXOID Mannitol Salt Agar, lot 052818507, were added and dissolved in 200 mL of deionized water. The flask was sealed with anti-bacteriological cotton and tape suitable for autoclave and autoclave for 15 minutes. The agar was plated in petri dishes measuring 90 mm in diameter and 15 mm in depth. Immediately after cooling the agar, the plates were placed in a UV oven for 30 minutes.

2.10. Preparation of S. aureus, E. coli and K. pneumoniae strains

Strains of E. coli ATCC 25923, K. pneumoniae ATCC 700603, S. aureus ATCC 29213, and a clinical isolate of S. aureus BLAC were used, stored, and made available by the microbiology sector of the Clinical Analysis Laboratory at PUC Goiás.

With a sterile loop, the colonies were transferred to a selective medium. E. coli and K. pneumoniae were inoculated in MacConkey media and S. aureus in Mannitol Agar. The plates were incubated for 24 hours in a bacteriological incubator at 37 °C to verify bacterial proliferation.

The isolated colonies of each strain studied were collected with a sterile swab, a bacterial suspension reaching a turbidity of 0.5 on the MacFarland scale. In a bacteriological oven at 37 °C, the strains of K. pneumoniae ATCC 700603, E. coli ATCC 25923, Staphylococcus aureus ATCC 29213, and Staphylococcus aureus BLAC, which were duly purified, were inoculated, using sterile swabs, and in different Petri dishes (150 × 15 mm) containing Mueller-Hinton agar). Inoculation was done in two positions covering the entire plate. Soon after, circular cavities were made with hematology tubes with diameters of 10 mm. Each cavity was identified adequately for the application of Genipa americana and Myrciaria cauliflora extracts, in three different volumes (50 µL, 100 µL, and 200 µL). This procedure was performed 4 times on other days, each in triplicate for each extract used.

3. Results

3.1. Assessment of the cytotoxicity and genotoxicity of the bark extract of Myrciaria cauliflora and Genipa americana

To begin studies with biomedical devices in laboratory animals, these devices must indicate biocompatibility by in vitro tests, demonstrating that they do not have cellular toxicity. These in vitro tests suggest that these study objects, mainly for clinical use, will not be toxic for future human applications. Although the in vitro test does not represent the actual situation in vivo, it is cataloged by ISO 10993-1 as initial research using cell culture, as it generates initial results that demonstrate the interaction of the studied material with the biological body.

In 98-well plates, RAW 264.7 cells were subjected to contact with Myrciaria cauliflora bark extract at concentrations of 2, 6, 10, 20, and 30 µg/mL, and the readings were subsequently analyzed by spectrophotometry.

The IC50 was observed through data generated from absorbance analyses, having a value of 20.0 µg/mL. Cell decay was observed at a concentration of 10 µg/mL, showing 58% cell viability, and at concentrations of 20 and 30 µg/mL, showing 45% cell viability, with stability observed from this point on the graph (Figure 1).

Figure 1
Variation in cell viability (%) in concentrations (µg/mL) of Myrciaria cauliflora bark extract (µg/mL).

The cytotoxicity of Genipa americana fruit extract at concentrations of 5, 10, 20, 40, and 60 µg/mL incubated with RAW 267.7 cells. The cells were not viable with any volume or concentration of the extract used in the experimentz; therefore, it was impossible to calculate the IC₅₀ (Figure 2).

Figure 2
Variation in cell viability (%) in concentrations (µg/mL) of Genipa americana extract (µg/mL).

3.2. Antimicrobial activity

The antimicrobial activity of the Myrciaria cauliflora bark extract was observed in the three volumes applied against Staphylococcus aureus: 50 µL presenting an inhibition halo of 1 mm, 100 µL with an inhibition halo of 6 mm, and 200 µL presenting the most prominent inhibition, both in the ATCC and BLAC strains, both wells of the two strains had a diameter of 10 mm. No inhibition was observed against Klebsiella pneumoniae ATCC 700603 and Escherichia coli ATCC 25923.

The antimicrobial activity of the extracts against the selected microorganisms was verified. In the Genipa americana extract, there was no inhibition, both in the ATCC strains of Staphylococcus aureus and in the resistant strain of Staphylococcus aureus BLAC, and it was not possible to observe any inhibition halo formed by the extracts used (Table 1).

Table 1
Diameter of bacterial inhibition by extracts of Myrciaria cauliflora and Genipa americana at different concentrations.

S. aureus strains	Myrciaria cauliflora			Genipa americana
	50 µl	100 µl	200 µl	50 µl	100 µl	200 µl
Staphylococcus	02 mm	06 mm	10 mm	-	-	-
aureus ATCC 29213
Staphylococcus aureus BLAC	02 mm	06 mm	10 mm	-	-	-

• Antimicrobial activity of the extracts studied in volumes of 50 µl, 100 µl, and 200 µl on Staphylococcus aureus ATCC 29213, Staphylococcus aureus BLAC.

3.3. Analysis of cytotoxic, genotoxic, and mutagenic potential

The analysis inferred that the treatments tested showed a significantly (p < 0.0001) lower number of dividing cells than the negative control, in which 5% of the cells counted were in mitosis. The treatments of 5%, 10%, and 20% did not differ, as demonstrated by the Z test, demonstrating a percentage of dividing cells between 1.8 and 2.0%. Table 2 shows the results obtained, including the categorization of treatments according to proportion patterns, received in the analysis of the adjusted Z test according to the Bonferroni method (Bland and Altman, 1995; Sokal and Rohlf, 2012).

Table 2
Number of cells observed in division per treatment and their respective mitotic indices (MI)

Number of cells in division recorded
Exposure time	Negative control	5%	10%	20%	p(χ2)
24 hours	302a (5.03)	118b (1.97)	116b (1.93)	110b (1.83)	<0.0001

• ^a,b
  Assignments of treatments and negative control were done according to post-hoc analysis using the Z test with data adjusted according to the Bonferroni method.

The frequencies of nuclear changes observed between treatments were close, with no significant differences (p > 0.05). In this way, treatments are categorized in the same proportion pattern when considering the parameters mentioned above. Different types of nuclear changes were observed, considering genotoxic and mutagenic indicators. However, this constitutes a basal rate. Table 3 shows the results obtained.

Table 3
Frequency of total nuclear changes in onion root meristematic cells exposed to different treatments of Myrciaria cauliflora bark extract and the negative control.

Frequency of total nuclear alterations
Exposure time	Negative control	5%	10%	20%	p(χ2)
24 hours	3a (0.1%)	13b (0.2%)	8b (0.1%)	10b (0.2%)	0.1

• ^a,b
  Assignments of treatments and negative control according to post-hoc analysis using the Z test with data adjusted according to the Bonferroni method.

3.4. Fingerprint analysis of M. cauliflora bark extract

After the results of bacterial inhibition, the M. cauliflora bark extract, which had the most relevant results, was selected for further analysis. An analysis was carried out using the fingerprint of the M. cauliflora bark extract to verify the compounds as components of the extract. The data obtained from the HPLC fingerprint analysis revealed that only gallic acid was identified in the extract, with a similar retention time (7.032 min in the extract and 6.906 min in the standard) and with the same ultraviolet absorbance spectrum. Furthermore, the presence of gallic acid indicates the presence of other phenolic compounds (gallotannins) in the extract (Figures 3 and 4).

Figure 3
Graph demonstrating the gallic acid peak in M. cauliflora extract on HPLC.

Figure 4
Chemical structure of gallic acid, drawn using the ACD/ChemSketch v program. 12.01.

3.5. In silico prediction of gallic acid bioactivity

The PASS server (Lagunin et al., 2010) was used to predict the antimicrobial activity and the possible mechanisms of action involved in gallic acid. The compound was designed in the ACD/ChemSketch Freeware Version program (Advanced Chemistry Development, Inc.), saved in MDL Molfile (.mol) format, and submitted for analysis on the server. The approach used by PASS is based on the principle that the activity of a molecule is closely related to its chemical structure. Thus, by comparing the structure of the analyzed molecule with the structures of molecules with biological activity already described in the literature, it is possible to estimate the potential activities of the molecule of interest. The values ​​that define the probability of presence (Pa) and absence (Pi) for each particular activity range from 0.000 to 1.000. Therefore, only activities with Pa>Pi were considered for the predicted activities (Lagunin et al., 2010).

The spectrum of biological activity represents the therapeutic and toxic effects and their mechanisms of action observed for a particular chemical compound, describing its biological properties according to its chemical characteristics (Musiol et al., 2007). Several computational programs, such as PASS, are based on the structure-activity relationship of bioactive molecules already known in the literature to estimate the potential activities of the molecule of interest, thus proposing the spectrum of bioactivity and a better understanding of the mechanisms involved.

The prediction of biological activities suggested for gallic acid revealed a wide variety of pharmacological activities, which have already been verified in scientific studies, including anti-inflammatory (Bensaad et al., 2017; Dludla et al., 2018) and antimutagenic (Abdelwahed et al., 2007; Kaur et al., 2015) antinociceptive (Kumar and Jain, 2014), antioxidant (Kongpichitchoke et al., 2016; Velderrain-Rodríguez et al., 2018), and antimicrobial activity, such as antiprotozoal activity (Antwi et al., 2019), antifungal (Alves et al., 2014b) and antibacterial (Borges et al., 2013).

The activities related to antimicrobial action predicted for gallic acid from the bioactivity spectrum are presented in Table 3. The suggested activities represent critical approaches to mechanisms of antimicrobial action associated with the inhibition of microbial growth (inhibition of cell wall biosynthesis) and facilitation of the action of antibiotics (inhibition of the efflux pump and induction of membrane permeability) until cell death (cytostatic activity).

Farrag et al. (2019) found that the membrane permeability-inducing activity of gallic acid facilitated the passage of antibiotics through the membrane of antimicrobial-resistant Gram-negative bacteria and verified the inhibitory activity of the efflux pump of antimicrobial-resistant S. aureus strains. Furthermore, several antimicrobial drugs act through mechanisms described in Table 4, such as cell wall synthesis, inhibition of beta-lactamases, inhibition of the efflux pump, and cytostatic activity (Gajdács, 2019).

Table 4
Predicted antimicrobial activity for gallic acid using the PASS program.

Pa	Pi	Activity
0.645	0.005	Nitrate reductase inhibitor
0.516	0.023	Cytostatic
0.496	0.016	Membrane permeability inducer
0.418	0.026	Anti-bacterial
0.414	0.004	Cell wall biosynthesis inhibitor
0.266	0.008	Beta-lactamase inhibitor
0.177	0.004	Bacterial efflux pump inhibitor

• Pa are the values that define the probability of presence and Pi the probability of absence.

3.6. Inhibition of bacterial growth against Gallic Acid

To verify whether the antimicrobial potential presented was specific to gallic acid or whether it was from the Myrciaria cauliflora bark extract, another bacterial susceptibility test was carried out with gallic acid against strains of Staphylococcus aureus ATCC 29213 and a clinical isolate of Staphylococcus aureus BLAC. No inhibition halos were observed. However, inhibitory activity was observed again when these same strains were exposed to Myrciaria cauliflora extract (Table 5).

Table 5
Diameter of bacterial inhibition by Myrciaria cauliflora extract and Gallic acid.

Myrciaria cauliflora				Gallic acid
	50 µl	100 µl	200 µl	50 µl	100 µl	200 µl
Staphylococcus aureus ATCC 29213	02 mm	06 mm	10 mm	-	-	-
Clinical isolate of Staphylococcus aureus BLAC	02 mm	06 mm	10 mm	-	-	-

• Inhibitory action of Myrciaria cauliflora and gallic acid on S. aureus ATCC 29213 and S. aureus BLAC.

4. Discussion

After the launch of the National Policy on Medicinal Plants and Phytotherapeutics, it is possible to notice a large increase in the number of scientific publications with detailed data on the use of herbal medicines to meet the safety criteria to ensure a more correct and safe application. RDC nº 26/14 includes, in its scope, conduct for the appropriate use of herbal medicine studies, bringing together criteria for good manufacturing practices, validation of analytical methods, quality control, stability, and pharmacodynamics (Oshiro et al., 2016).

The Brazilian Cerrado is the biome that involves one of the greatest variability of living beings (Duarte and Leite, 2020). Several species from the cerrado have phytochemical characteristics that are used by pharmaceutical industries (Pereira-Filho and Castro, 2019). There are also studies by our group that have already observed antimicrobial activities of extracts derived from the cerrado, such as S. mombin, which did not interfere with cell metabolism (Taia et al., 2023), as seen in this study in the extracts evaluated.

Studies with an ethanolic extract from Genipa americana leaf showed cytotoxic activity against Artemia salinas larvae (Bezerra and Morais, 2017); results in the present work did not verify the viability of RAW cells in any volume or concentration studied. When analyzing the antimicrobial potential of the Genipa americana extract, no development of an inhibition halo was observed in any volume of the extract administered to any bacteria studied. These results agree with studies by Gonçalves et al. (2005) that did not verify antibacterial activity under the extract of Genipa American.

The toxicity results in this research showed little lethality for RAW cells when observing the Myrciaria cauliflora extract, as at concentrations of 20 to 30µg/mL, no changes in the viability of RAW cells were observed. There are few studies related to the toxicity of the Myrciaria cauliflora species however an in vivo test carried out by Alves et al. (2014a) showed that the jaboticaba bark extract was cytotoxic to Spodoptera frugiperda, causing an increase in mortality of the species' larvae, according to the author, larval mortality may be related to the presence of phenolic compounds in the extract that may reduce insect survival.

Previous studies identified the antimicrobial activity of Myrciaria cauliflora extract on bacteria and fungi of dental importance (Diniz et al., 2010; Macedo-Costa et al., 2009). Another study demonstrated the antimicrobial activity of jaboticaba bark extract on Gram-negative and Gram-positive bacteria (Albuquerque et al., 2020); these in vitro antimicrobial assays corroborate the results of the present study, which showed bacterial inhibition in S. aureus ATCC and clinical isolate of S. aureus BLAC. According to Oliveira et al. (2018), the bioactive compounds that inhibit bacterial growth are phenolic compounds, mainly cyanidin chloride, catechin, and epicatechin, which have inhibitory activity mainly on S. aureus. These phenolic compounds are thought to disrupt bacterial cell membranes and interfere with essential enzyme systems, inhibiting bacterial growth and survival. Furthermore, the presence of these compounds in jaboticaba bark extract suggests a dual mechanism of action that targets cell wall integrity and curtails metabolic processes within the bacteria. Future research should explore the synergistic effects of these compounds with conventional antibiotics, potentially enhancing their efficacy and reducing the required doses of synthetic antibiotics. Such approaches could also mitigate the development of antibiotic resistance, a growing concern in clinical settings worldwide (Górniak et al., 2019).

The inhibition of bacterial growth in agar is qualified by measuring in millimeters the diameter of the inhibition halo formed around the disc containing the antimicrobial (Salam et al., 2023b). In the case of this work, the inhibition halo was measured around the wells where the extract volumes were applied, discarding the well diameter and measuring only the area of ​​inhibition. With an extract from the bark of Myrciaria cauliflora, S. aureus ATCC 29213, and clinical isolate of S. aureus BLAC had the exact halo measurement, with a 10 mm halo for the volume of 200 µL in all repetitions of the method.

The cytotoxic effect on onion root meristematic cells was observed in the present study, with a reduced number of cells undergoing cell division (mitosis) when exposed to fractions between 5 and 20% of Myrciaria cauliflora bark extract for at least 24 hours. However, other effects have also been observed by the scientific community, as in the case of research by Silva et al. (2016), who reported the protective effect of Myrciaria cauliflora seed extract on the Genomic DNA of mouse bone marrow cells. When considering the parameters of genotoxicity and mutagenicity, the study cited does not contradict the present study, which, although it did not find a protective effect, did not show genomic or mutagenic damage.

Franscescon et al. (2018) reported the prevention of cytotoxicity and genotoxicity of Plinia peruviana (Poir.) Govaerts in copper-induced Allium cepa root cells. Thus, the results obtained in the present study are contrasting. However, it should be noted that aspects such as the pharmacogen used, exposure time, variety of species, and concentration, among other components, may justify differences in results between studies, which are still scarce when considering analyses of cytotoxicity, genotoxicity, and mutagenicity of cells, meristematics of onion root (Allium cepa).

Farag et al. (2015) conducted a structure-activity relationship study to identify desirable molecular characteristics of gallic acid and analogs for activity against Ralstonia solanacearum. It was observed that, concerning the effect of methylation, the analogs with free phenolic groups presented lower MIC values ​​than the methylated analog. On the other hand, the positions of the hydroxyl (-OH) and carboxyl (-COOH) groups did not significantly influence the MIC values.

The bioactivity prediction for gallic acid highlights the antimicrobial potential of this compound and the possibility of showing promise in experimental tests. Numerous studies have reported that gallic acid exhibits significant antibacterial properties against various pathogens, suggesting its role as an effective antimicrobial agent when used with other compounds (Keyvani-Ghamsari et al., 2023). In this way, the virtual screening stage served as the basis for antimicrobial tests on the Myrciaria cauliflora extract. Although gallic acid did not show activity alone, it may have contributed to the antibacterial activity of the extract. This synergy could be attributed to other bioactive constituents in the extract, which may enhance the overall effect and provide a more potent antimicrobial profile. Further investigations into the interactions between gallic acid and other compounds in Myrciaria cauliflora are essential to understanding the playful mechanisms and optimizing the extract's antimicrobial efficacy in potential therapeutic applications (Araujo et al., 2013; Senes et al., 2021).

When evaluating the antimicrobial activity of the extracts, it was noted that the M. cauliflora bark extract revealed better results with antibacterial activity on S. aureus ATCC 29213 and clinical isolate of S. aureus BLAC. However, the potential to inhibit bacterial growth of jabuticaba extract can only be observed when the extract is applied. The presence of the compound gallic acid, which is a component of the extract, can influence the inhibition of bacterial growth. Still, this compound alone cannot inhibit the growth of S. aureus in vitro. This analysis suggests that other compounds contained in the M. cauliflora extract are essential for the proposed pharmacological action. New studies are needed to identify the main compounds that together act to inhibit bacteria. Further studies are required to verify the adaptation and resistance mechanisms of S. aureus BLAC and how jabuticaba extract acts on this microorganism.

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Publication Dates

• Publication in this collection
  07 Feb 2025
• Date of issue
  2024

History

• Received
  23 May 2024
• Accepted
  18 Nov 2024