Effect of Stryphnodendron adstringens (Mart.) Coville extract in aerobic and anaerobic microorganisms and mammalian cells

Stryphnodendron adstringens (Mart.) Coville is a medicinal plant known for its anti-inflammatory and antimicrobial properties. This study evaluated some biological activities of extract from S. adstringens. Antimicrobial activity was checked in planktonic cultures and monomicrobial biofilms on aerobic, and anaerobic dental microorganisms. Analyzes of cytotoxicity using MTT assay, and genotoxicity by micronucleus test were performed in human keratinocytes (HACAT), murine macrophages (RAW 264.7), and murine fibroblasts (L929). The anti-inflammatory effect was checked in RAW 264.7 stimulated by lipopolysaccharide (LPS) from Escherichia coli, and treated with the plant extract. The levels of cytokines, and nitric oxide (NO) were measured by ELISA, and Griess method, respectively. Data were analyzed by ANOVA, followed by Tukey's, or Kruskal-Wallis, and Dunns tests (P ≤ 0.05). Biofilms of anaerobic bacteria were very susceptible to the plant extract. Effective concentrations showed cell viability > 50%, except 25 mg/mL for HACAT after 24 h of exposure. The extract of S. adstringens was not genotoxic for RAW 264.7. LPS associated with extract increased the production of all cytokines, except TNF-α. However, the plant extract decreased the production of NO. In conclusion, the extract of S. adstringens affected biofilm of anaerobic bacteria using non-cytotoxic concentrations for RAW 264.7, L929, and HACAT cells.


Introduction
The search for new antimicrobial drugs has become increasingly important due, mainly, to the resistance that microorganisms develop after a while under the action of these drugs. The study of medicinal plants can be an excellent alternative because they have a rich constitution of active biomolecules. Stryphnodendron adstringens (Mart.) Coville is a medicinal plant rich in tannins belonging to the Fabaceae family. Native to southeastern and central-western Brazil, it produces fleshy, light-brown fruits with many seeds (Lorenzi H & Matos FJA, 2002;Maroni BC, 2006). Empirically, S. adstringens has been used as an antiseptic and antimicrobial as well as for treatments of diabetes, inflammation, gastritis, liver disease, pain, diarrhea, and bleeding (Costa et al., 2013;De Santana et al., 2016). Scientifically, some studies demonstrate that the aqueous extract of S. adstringens has antioxidant and anticancer properties (da Silva Baldivia et al., 2018), antifungal, including action against fluconazole-resistant isolates (de Freitas et al., 2018), pronounced anti-inflammatory activity (Henriques et al., 2016), and antimicrobial action (Costa et al., 2010;Ferreira, Palmeira, Souza, Almeida, Figueiredo, Pequeno, Arruda, 2010). This fact is important because several oral diseases have a dysbiotic microbial biofilm as the main etiological factor.
Biofilms are three-dimensional structures in which several species of microorganisms are surrounded by an extracellular matrix that hinders the action of the immune system and antimicrobial agents. The species that make up the oral biofilm change as the maturity of the structure increases and may also vary between individuals. (Karched et al., 2015;Pathan, 2017).
There are many species capable of forming biofilms, such as Candida albicans, a yeast that can also form germ tubes and has adhesion proteins (Ganguly & Mitchell, 2011). It is the main species responsible for causing oral candidosis. It can grow in anaerobiosis, being able to contribute to periodontal and endodontic infections (Bernal-Treviño et al., 2018;Razina et al., 2017). Enterococcus faecalis is an optional anaerobic bacterium that can invade dentinal tubules, initiate biofilm, and is associated with secondary endodontic infections (Tennert, Fuhrmann, Wittmer, Karygianni, Altenburger, Pelz, 2014;Wilson et al., 2015). Pseudomonas aeruginosa is an opportunistic aerobic pathogen that causes different acute and chronic infections.
One of the main causes of nosocomial infections, and also associated with persistent root canal infections (Crone et al., 2020;Pourhajibagher et al., 2017). Staphylococcus aureus, considered an important opportunistic nosocomial pathogen is an optional anaerobic bacterium that frequently colonizes the nostrils without causing problems (Mehraj J, Witte W, Akmatov MK, Layer F, 2016). Streptococcus mutans is an optional anaerobic microorganism prevalent in the caries process. Important in the biofilm initiation process, it is capable of synthesizing extracellular polymers (Krzyściak, Jurczak, Kościelniak, Bystrowska B, 2014).
Obligate anaerobic bacteria can also form a biofilm, such as Fusobacterium nucleatum, an important bacterium for the maturation of a biofilm, and also is correlated with cases of gingivitis and chronic periodontitis (Karched et al., 2015;Okuda et al., 2012). Parvimonas micra is an anaerobic. and proteolytic bacterium found in the gingival sulcus. Currently, the frequency of case reports in the literature correlating P. micra with spondylodiscitis (spinal infection) has been extremely high and some patients had a dental problem before spinal infection (Durovic et al., 2020;van Duijvenbode et al., 2018). Porphyromonas endodontalis is a pathogen isolated from periodontal pockets, periapical lesions, endodontic infections and biofilm on the back of the tongue of patients with chronic periodontitis. (Pérez-Chaparro et al., 2018;Rôças & Siqueira, 2018). Porphyromonas gingivalis is considered the main etiologic agent of periodontal diseases (Bostanci & Belibasakis, 2012;Tamai et al., 2011).
In addition, microorganisms can also generate significant inflammatory processes in their hosts, due to their presence in the organism or only their constituents, such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), for example. Oral infections caused by the microorganisms described above can cause acute or chronic inflammatory processes requiring pharmacological intervention to minimize additional tissue damage (Headland & Norling, 2015). Also, to an active substance being effective, it needs to be safe. There are many tests to show how much an organism tolerates a substance. Among them, in vitro tests using cell, and tissue cultures preferably mammals. The cytotoxicity tests allow evaluating the biological response of cells to a particular substance, through specific aspects of metabolism, morphological, or even cell growth and viability.
Genotoxicity tests demonstrate effects on DNA, gene mutations, and chromosomal aberrations. Some studies have shown that S. adstringens maintains cell viability when analyzed on mammalian cell culture (de Oliveira et al., 2013;Henriques et al., 2016;Ishida et al., 2006), and has a protective effect on cellular genetic material (Costa et al., 2010;Pellenz et al., 2018).
There is a lack of studies in the literature that evaluated the antimicrobial activity of S. adstringens extract on such microorganisms of dental interest, particularly on anaerobic bacteria. It is also scarce studies with analysis of cytotoxicity to find a concentration that is effective and without toxic effects and that can be applied in the dental clinic in the future.
The purpose of this study was evaluated the biological activities of extract from S. adstringens (Mart.) Coville, such as antimicrobial effect on planktonic culture, and biofilms of C. albicans, E. faecalis, P. aeruginosa, S. aureus, S. mutans, F. nucleatum, P. endodontalis, P. gingivalis, and P. micra; cytotoxicity and genotoxicity in mammalian cells (murine macrophages, murine fibroblasts, and human keratinocytes); and anti-inflammatory action in LPS-stimulated murine macrophages.

Extract
The glycolic extract of S. adstringens (Mart.) Coville was prepared from the stem bark of the plant and obtained commercially at 200 mg/mL of propylene glycol (Mapric, São Paulo, Brazil).

Antimicrobial activity in planktonic cultures
The Minimum Inhibitory Concentration (MIC) was determined by the broth microdilution method, according to the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute, 2002, 2003, 2007, as described in documents M27-A2 for yeasts, M7-A6 for aerobic microorganisms, and M11-A7 for anaerobic microorganisms. The microorganisms were prepared in sterile saline solution (NaCl 0.9%) with turbidity adjusted to 0.5 of the McFarland scale, from a 24 h culture for aerobic microorganisms at 37 °C. The anaerobic bacteria were grown in Brucella broth supplemented at 37 °C for 48 h, before preparation in saline solution. In 96-well plates, 100 µL of broth was added in 10 wells, and 100 µL of the extract was added only in the first well of the microplate, from which a two-fold serial dilution was performed, totaling 10 different concentrations. For this, Mueller Hinton broth (Himedia) for bacteria, and RPMI 1640 medium (with glutamine, without bicarbonate and with red phenol indicator) (Himedia) buffered with 3-(N-morpholino) propane sulfonicpropane sulfonic acid (MOPS -Sigma-Aldrich) at pH 7.0, for C. albicans were used. For aerobic bacteria and yeast, 100 µL of inoculum was added to the wells and the microplates were incubated at 37 °C for 24 h. For anaerobic bacteria, 10 μL of standardized inoculum was added to the wells, and the microplates were incubated in anaerobiosis for 48 h. The MIC was determined as the lowest concentration of extract capable of inhibiting microbial growth was observed with the naked eye.
To determine the Minimal Microbicide Concentration (MMC) of the plant extract on aerobic bacteria and yeast, the contents of the wells (100 µL) of the MIC, as well as the adjacent wells were seeded on BHI agar for the bacteria, and Sabouraud-dextrose (Himedia) for yeast. After 48 h of incubation, MMC was determined on the plate with no colonial growth.
Regarding the anaerobic bacteria, the well contents were homogenized and 20 µL of each concentration were seeded in supplemented Brucella agar, and incubated under anaerobic condition for 48 h.

Antimicrobial activity in monomicrobial biofilms
Initially, the action of the plant extract was verified in monomicrobial biofilms of C. albicans, E. faecalis, P. aeruginosa, S. aureus, and S. mutans (De Oliveira et al., 2014). For biofilm formation, each microbial suspension was standardized at 10 7 Colony-Forming Units per milliliter (CFU/mL) in saline and added to microtiter plates (200 µL/well). The microplates were incubated (37 °C) under agitation (75 rpm) for 90 min. The supernatant was discarded and culture medium was added, being YNB broth for C. albicans, and BHI broth for bacteria. Incubation (37 °C) was performed for 48 h, with the replacement of the culture medium after 24 h. The biofilms were exposed to the plant extract for 5 min or 24 h, using effective concentrations. Sterile saline was used as a negative control. Ten replicates were performed in each experimental group.
Posteriorly, 200 µL of MTT solution (3-(4,5-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) was added to the wells and the microplates were incubated at 37 ºC for 1 h in the dark. Afterward, the MTT solution was removed and 200 µL of dimethylsulfoxide (DMSO -Sigma-Aldrich) was added to the wells. After incubation of 10 min, the microplates were stirred for another 10 min, and proceeded to readon a spectrophotometer (Biotek ELx808) at 570 nm. The Optical Densities (OD) were converted into the viability percentage of the biofilm.
To evaluate the action of the plant extract on biofilms of F. nucleatum, P. micra, P. endodontalis, and P. gingivalis, 100 μL/well of microbial suspension (1.5 x 10 8 CFU/mL), and 100 μL/well of supplemented Brucella broth were added in microplates. The plates were then incubated at 37 °C in anaerobiosis for seven days with medium replacement every 48 h.
After, the biofilms were exposed to the plant extract for 5 min or 24 h. Supplemented Brucella broth and 0.1% chlorhexidine were used as controls. These solutions were discarded, the biofilms were washed with sterile saline and disintegrated with an ultrasonic homogenizer (Sonoplus HD 2200 -Bandelin Electronic GmbH & Co. KG, Berlin, Germany), with 25% energy for 30 s. The suspension was diluted, and 20 μL seeded on Brucella agar. After 48 h of incubation, colonies were counted and given their concentration in CFU/mL.
The culture medium was changed every 48 h. After subconfluency, the cells were used in the tests. The Trypan blue exclusion test (0.4%, Sigma-Aldrich) was performed to count the cells.

Cytotoxicity analysis
In 96-well microplates (Kasvi), 200 μl of DMEM containing 4 x 10 4 viable cells were added to the wells. After 24 h of incubation, 200 μL/well of the extract were added on the cells at different concentrations, pure DMEM was used as control (n = 8/group). The supernatants were discarded and cell viability checked by MTT assay (as mentioned before), after 4 h of incubation.

Genotoxicity analysis
The micronucleus (MN) test was performed according to the Organization for Economic Co-operation and Development (Organization for Economic Co-operation and Development (OECD)., 2014).
For the test, 5 x 10 5 viable cells/well were grown in 24-well plates for 24 h. The cells were then exposed at different extract concentrations diluted in DMEM, 5 μM ethyl methanesulfonate (EMS), was used as a positive control, or pure DMEM was used as a negative control for 24 h. Next, the cells were incubated with cytochalasin B (Sigma-Aldrich) at 6 μg/mL for 24 h.
Then, the cells were fixed with methanol for 10 min and stained with 300 nM DAPI diluted in PBS for 10 min. Cells were washed with PBS and observed under fluorescence microscopy, where 2000 cells/well per concentration (n = 3) were evaluated. MN were identified as DNA structures contained in the cytoplasm separated from the main nucleus, surrounded by a nuclear membrane, and including an area smaller than 1/3 of the area of the main nucleus. Cells with less than 5 MN were counted.

Anti-inflammatory activity
In 24-well plates, RAW 264.7 were cultured at 5 x 10 5 cells/well for 24 h. After incubation, culture medium with different concentrations of extract from S. adstringens was added. At the same time, LPS from E. coli (Sigma) at 1 μg/mL was added to the wells. Groups without LPS were used as controls. After 24 h of incubation, the supernatants were collected and stored at -20 °C for quantification of pro-inflammatory (TNF-α, IL-1β, IL-6, and IL-17), and anti-inflammatory (IL-10) cytokines by ELISA (R & D Systems, Minneapolis, United States of America) according to the manufacturer's instructions.

Production of nitric oxide (NO)
The production of NO by RAW 24.7 was determined by the concentration of nitrite detected by the Griess reagent.
For this, 100 μL of cell supernatant, and 100 μL of Griess reagent were added in 96-well plates. After 10 min, the absorbance of the wells was read on a microplate spectrophotometer at 570 nm. To calculate the nitrite concentration, a standard curve of nitrite from 23 to 0.72 pg/mL was used.

Statistical analysis
The results were presented in mean values (± standard deviation) and data distribution was assessed by Shapiro-Wilk.
The data that presented normal distribution were analyzed by ANOVA complemented by Tukey's test, with significance of 5% (P ≤ 0.05). The results that did not present normal distribution were analyzed by Kruskal-Wallis and Dunns tests (P ≤ 0.05).

Antimicrobial activity
Regarding the antimicrobial activity of extract from S. adstringens on aerobic microorganisms, it was possible to observe action only on P. aeruginosa. There was no growth inhibition for the other microorganisms. In the microdilution tests, the highest concentration analyzed was 50 mg/mL and in the tests of the antibiofilm action of the extract, higher concentrations (50, 100, and 200 mg/mL) were used, and only the biofilm of P. aeruginosa suffered a significative reduction, however, no statistical difference (p > 0.05) was found.
Regarding anaerobic microorganisms, it was not possible to determine the MIC due to the turbidity of extract from S. adstringens, and the color of the medium (data not shown). Our study showed an MMC of 50 mg/mL for F. nucleatum, and > 100 mg/mL for other species. Concerning monomicrobial biofilms from anaerobic bacteria, a significant susceptibility to the plant extract was verified, with a minimum reduction of 50% (F. nucleatum, 5 min), as shown in Figure 1.

Cytotoxicity
Ten concentrations were evaluated of bark extract from S. adstringens in RAW 264.7, L929, and HACAT. Different responses were observed ( Figure 2); however, the effective concentrations for anaerobic bacteria showed cell viability >50% in mammalian cells, generally.

Genotoxicity
The genotoxicity of two concentrations (50 and 100 mg/mL) of extract from S. adstringens was tested in HACAT, RAW 264.7, and L929 (Figure 3). These concentrations were not genotoxic for murine macrophages. At the concentration of 100 mg/mL, the extract was not cytotoxic to murine fibroblasts. Human keratinocytes were less sensitive to the genotoxic agent used (EMS) and all treatments were statistically similar making the results inconclusive.

Anti-inflammatory activity
In the evaluation of anti-inflammatory activity, it was possible to observe that the extract only increased the release of IL-1β and IL-17 at 50 mg/mL in non-LPS-stimulated macrophages. On the other hand, when the extract was associated with LPS stimulation, all the cytokines were increased, except TNF-α, which was reduced in the concentration of 100 mg/mL but was similar to the positive control with 50 mg/mL (Figure 4).

Production of NO
The ability to inhibit the production of nitric oxide (NO) can contribute to the performance of the extract's antiinflammatory activity. The average values of nitrite (pg/mL), referring to the production of nitric oxide, obtained in each experimental group, in comparison with the control groups, are shown in Table 1. It was observed that in the groups treated with S. adstringens extract at 25 mg/mL (+ LPS), and 50 mg/mL (+ LPS) there was a significant reduction in the production of NO in relation to the group with LPS and without extract (P < 0.05). The groups treated with S. adstringens extract at 100 mg/mL group (+ LPS) promoted higher levels of nitric oxide, which was similar to the positive control (LPS without extract).
The negative control group (without LPS stimulation) had the lowest level of nitrite, being statistically different from the other groups (P < 0.05).

Discussion
In the present study, the plant extract was not able to inhibit the growth of the aerobic microorganisms analyzed, except P. aeruginosa. Likewise, only the monomicrobial biofilm of P. aeruginosa was significantly decreased. There are no reports in the literature regarding the antimicrobial activity of this plant extract on E. faecalis, and P. aeruginosa regarding the antibiofilm action on the other strains tested in the present study.
To check the antimicrobial action of some fractions from extract of S. adstringens, the agar diffusion test, and microdilution were used on ATCC strains of C. albicans, S. mutans, S. aureus, and Aggregatibacter actinomycetemcomitans.
The vegetable compound showed antimicrobial activity at different levels for all strains tested (Pereira et al., 2011). albicans, S. aureus, S. mutans, E. faecalis, P. gingivalis, F. nucleatum, and P. micra among clinical isolates and ATCC strains.
All the evaluated extracts effectively inhibited the obligate anaerobic bacteria (Hickl et al., 2018).
Also, the inhibitory potential of Olea europaea extract has been found in periodontal pathogens, such as P. gingivalis, F. nucleatum, and Prevotella intermedia . Some compounds from O. europaea, and Pistacia lentiscus have also demonstrated antimicrobial action on several oral pathogens. These molecules were highly effective against Streptococci spp., P. gingivalis, F. nucleatum, and P. micra. However, the extracts were ineffective for P. intermedia (Karygianni et al., 2019). Melaleuca alternifolia oil has also shown antimicrobial activity against P. gingivalis and P.
Concerning anaerobic biofilm studies, 24 h contact to the S. adstringens extract on biofilms of F. nucleatum, and P.
micra promoted a reduction of 100% at all analyzed concentrations, differing statistically from the chlorhexidine 0.12% that promoted a percentage low reduction. This substance is considered a gold standard anti-plaque agent and in the present study, the extract of S. adstringens acted better than chlorhexidine. Regarding 5 min contact to the S. adstringens extract, all evaluated concentrations acted similarly to 0.12% chlorhexidine in the reduction P. gingivalis, P. endodontalis, and P. micra infections related to P. micra such as spondylodiscitis (infection in the spine). A case series reported in 2020 showed that of the six cases, five P. micra is the only pathogen isolated from spine infection (Durovic et al., 2020). Analyzing alternative therapeutic agents that are effective against infections caused by these microorganisms is very important and in the present study, the plant extract was very efficient against this pathogen.
Regarding cytotoxicity analyzes, in the present study, the effective concentrations for anaerobic bacteria showed cell viability >50%. tannins from the S. adstringens (Mart.) Coville bark were not very cytotoxic for murine macrophage (J774G8) cells and green monkey kidney cells (Vero), but the cutoff point of this study was 50% (Ishida et al., 2006). The extract of the bark of S. adstringens (Mart.) Coville was tested on RAW 264.7, and was found cell viability of 65% using a concentration of 12.5 mg/mL and > 70% for concentrations of 50, 25, and 3.13 mg/mL (de Oliveira et al., 2013). Another study with an extract from the S. adstringens bark demonstrated cell viability > 90% for the human monocytes (THP-1) cells (ATCC TIB-202) (Henriques et al., 2016). The cytotoxic effect of the extract from leaves S. adstringens (Mart.) Coville was tested in two human breast cancer cell lines (MCF-7 and MDA-MB-435), and also on the primary culture of rat marrow cells. The study concluded that the extract was cytotoxic with a 50% cutoff point (Sabino et al., 2018).
Concerning genotoxicity analyzes, in the present study, the highest concentrations tested were not genotoxic for RAW 264.7 and L929. The effect of the S. adstringens hydroalcoholic extract has also been evaluated for cell viability, DNA damage, and apoptosis induction in keratinocytes (HaCaT), and fibroblast (HFF-1) cell lines. The results have suggested that the extract may have a genoprotective and anti-apoptotic effects for human keratinocytes and fibroblasts (Pellenz et al., 2018).
The S. adstringens ethanolic extract was used at 50, 100, and 200 mg/kg, and was found that the two higher concentrations reduced the genotoxicity caused by mitomycin C (Silva- de-Andrade et al., 2006). Another study demonstrated that the proanthocyanidins rich fraction of the stem bark of S. adstringens was not genotoxic at 750, 1500, and 2000 mg/kg and also conferred protection to the cellular genetic material of the cyclophosphamide-treated groups at 750 mg/mL (Costa et al., 2010).
Besides, was verified the antigenotoxic activity of aqueous and ethanolic extracts of S. adstringens leaves on genotoxicity caused by cyclophosphamide (Filho et al., 2011). On the other hand, the aqueous solution of S. adstringens was genotoxic at 30 mg/mL for erythrocytes of mice treated with mitomycin C (Chaves DS, Lemes SR, Montes de Sousa MA, da Rocha Souza EGA, Alves de Araújo L, Mrué F, 2017). The plant product showed relative genotoxicity when evaluated by SOS-Inductest (Vilar et al., 2010).
About anti-inflammatory activity, it has already been reported that there is the increased IL-1β in RAW 264.7 treated with 50 mg/mL of extract from S. adstringens, but in this study, the same concentration reduced the release of TNF-α which did not occur in the present study (de Oliveira et al., 2013). Another study demonstrated that there is a reduction of TNF-α using human monocytes (THP-1) and the extract of S. adstringens (Mart.) Coville at concentrations of 250, 125, and 62.5 μg/mL (Henriques et al., 2016). It was also observed that the extracts did not increase the production of TNF-α in cells not stimulated with LPS. No studies on the action of S. adstringens on other cytokines have been found in the literature.
To the production of NO, there are no reports in the literature on the action of S. adstringens for the production of NO.
This shows the importance of the current study because we found that S. adstringens may be a potential NO inhibitor. Some natural extracts and their phytochemicals, such as Apios americana (Seo et al., 2018), Inula Japonica (Jin et al., 2016), and the propolis ethanolic extract (Onbas et al., 2016) have demonstrated potent NO inhibitor effect when evaluated on LPS-stimulated RAW 264.7 cells.

Conclusion
In conclusion, extract of S. adstringens (Mart.) Coville was effective for monomicrobial biofilms of anaerobic bacteria at low non-cytotoxic concentrations for RAW 264.7, L929, and HACAT cells. This study has elucidated important biological activities of S. adstringens extract however, for further analysis, the authors believe to be necessary additional in vitro trials and the near future, start in vivo analyzes in invertebrate models that allow to obtain preliminary results such as the toxicity of the plant extract in vivo.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant number 15/08776-3.