The biocompatibility of Achyrocline satureioides plant extract over human gingival fibroblasts

Objective: Achyrocline satureioides “A.satureioides” is a subshrub, widely distributed in South America because of its medicinal properties. Although it is widely used in folk medicine, there is still no approval for its therapeutic use, and its biocompatibility is little explored. This study aimed to evaluate its cytotoxicity and genotoxicity over human gingival fibroblasts (FMM-1). Methodology: Ten different concentration of the glycolic extract of A.satureioides were tested for 5min and 24h of contact with the cells to evaluate its cytotoxicity using the MTT colorimetric assay and to evaluate its genotoxicity using the micronucleus assay. Data were analyzed with one-way ANOVA and Tukey test with a significance level (α=0.05). Results: All tested concentrations of the extract presented cell viability more than 70% and has no significant difference of the control group after 5min and 24h. However, for 5min the 100 mg/mL was cytotoxic and for 24h the 1.56 mg/mL stimulated cell proliferation. For genotoxicity analysis, only the concentration 6.25 mg/mL showed results similar to the control of cell culture in the micronucleus count after 5min and 24h. Conclusions: The glycolic extract of A.satureioides doesn’t have cytotoxic and genotoxic effects in concentrations up to 6.25 mg/mL, but in high concentrations it is considered genotoxic. Research, Society and Development, v. 10, n. 1, e37610111902, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i1.11902 2


Introduction
Phytotherapy was used in dentistry and more specifically in endodontics since the last century (Groppo et al., 2008) due to its antimicrobial, anti-inflammatory and antifungal actions and because it is considered biocompatible (Guandalini Cunha et al., 2020;De La Chapa, Singha, Lee, & Gonzales, 2018;Marcia C Valera et al., 2016;Viegas et al., 2020) in addition to other techniques like photodynamic therapy and passive ultrasonic irrigation (Abu Hasna, Ferrari, & Talge Carvalho, 2019; Abu Hasna, Khoury, et al., 2020;Abu Hasna, Pereira Da Silva, et al., 2020). This use was encouraged by the World Health Organization (WHO); however, the agency establishes specific safety requirements including traditional use certificate and toxicity analyzes of the extracts (Organization, 2019).
Even A. satureioides extracts are widely used in folk medicine (Polydoro et al., 2004). The literature reported divergent results about its biocompatibility by few in vitro and in vivo studies (Calvo et al., 2006;Polydoro et al., 2004;Rivera, Gervaz, Sere, & Dajas, 2004;M C Sabini et al., 2013). To the best of our knowledge there is not studies evaluated the biocompatibility of A. satureioides extracts over the oral cells.
Therefore, this study aimed to evaluate the cytotoxic and genotoxic effects of A. satureoides extracts over human gingival fibroblasts (FMM-1) in periods of 5 min and 24 h of contact by MTT and micronucleus assays respectively, to assist in the future development of dental materials like intracanal medications, endodontic irrigants, mouthwashes and toothpastes.

Extract and cells lineage
The glycolic extract of A. satureiroides was used at a concentration of 200 mg / mL (20%) diluted in propyleneglycol (Mapric -São Paulo, SP).

Cytotoxicity analysis by MTT assay
In 96-well plates, 200 µL of DMEM + 10% SFB medium containing 2 x 10 4 viable cells were added. These plates were incubated at 37 °C with 5% CO2 for 24 h. Then, the cells were exposed to 200 µL / well of 10 different concentrations of A. satureioides extract (0.195, 0.390, 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50 and 100 mg / mL), n = 12 for each group, and incubated for a period of 5 min and 24 at 37ºC with 5% CO2. Culture medium cells were used as a positive control group without any extract.
The plates were washed with phosphate buffered saline (PBS -Cultilab, Campinas, SP, Brazil) and the MTT solution (100 µL/well) was added to the 96-well plate. The plates were incubated at 37 °C with 5% CO2 for 1 h. Later, the MTT solution was discarded and 100 µL / well of dimethyl sulfoxide (DMSO -Sigma) was added and the plated incubated again fot 10 min. Lastly, the plates were shaken for 10 min and read by a multi-well spectrophotometer (Bio-Tek, Winooski, Vermont, USA) at 570 nm. The obtained optical densities (OD) were converted into a percentage of cell viability using the following formula: % Viability = (OD of treated group x 100) / average OD of the control group (Memari Trava et al., 2020).

Genotoxicity analysis by micronucleus assay
In 24-well plates, FMM-1 were grown in DMEM (LGC Biotechnology) medium supplemented with 10% fetal bovine serum (SFB) (Invitrogen) where 2 x 10 4 cells / well were plated and incubated at 37 °C with 5% CO2 for 24 h. Then, the cells were exposed to A. satureioides extracts diluted in DMEM + 10% SFB and to ethyl methane sulfanate (EMS); 5 mM as a positive control group for 24 h.
Later, the cells were fixed with 4% formaldehyde. Then, 500 µL of PBS and 1 drop of Fluoroshield solution with DAPI (Sigma-Aldrich) were added to the wells, which were photographed with a digital camera (Sony F828 digital, CyberShot, 8.0 megapixels) coupled with an inverted light microscope. At least 10 photos per well were taken and the number of micronuclei was determined to be 2,000 cells / well. DNA structures contained in the cytoplasm separated from the main nucleus were identified as micronuclei, with an area less than 1/3 of the main nucleus area. Cells in mitosis and that exhibited nuclear fragmentation by apoptosis were not considered in the count Sousa et al., 2020).

Statistical analysis
The data were statistically analyzed using the one-way ANOVA and Tukey tests, with a significance level of 5% by GraphPad Prism 6 (La Jolla, CA, USA).

MTT assay
In the 5 min contact period, all the tested concentration of the extract presented cell viability more than 70% and has no significant difference of the control group. Conversely, the 100 mg/ mL of the extract presented 65.9% of cell viability and had significant difference of the control group (p <0.05) ( Figure. 1). In the 24 h contact period, all the tested concentrations showed cell viability greater than 70% and has no significant difference of the control group except the concentration of 1.56 mg / mL that significantly stimulated cell proliferation compared to the control group (p <0.05) ( Figure. 2).

Genotoxicity assay
In the 5 minutes contact period, the micronucleus count increased according to the extract concentration, with the highest concentration analyzed (100 mg / mL) averaging 13.5 micronuclei. Only the concentration of 6.25 mg / mL of the extract showed similar results to the control ( Figure. 3). As well, in the 24 h contact period, the concentration 6.25 mg / mL also showed results similar to the control of cell culture in the micronucleus count ( Figure. 4).

Discussion
Diverse plant extracts were used in dentistry principally as endodontic irrigant or intracanal medication (Horiba, Maekawa, Ito, Matsumoto, & Nakamura, 1991;Maekawa et al., 2015;Qunqiang & Farnham, 1989;Marcia C Valera et al., 2016) or even associated with other intracanal medication (Marcia Carneiro Valera et al., 2015) and this may be related to the fact of the increased resistance of micro-organisms against antibiotics overtime (Jungermann et al., 2011).
The aim of this study was to introduce the A. satureioides extract to be used like others (Ghonmode et al., 2013) as an effective substance over the endodontic and oral pathogens. For this, the cytotoxicity of the extract was evaluated over the human gingival fibroblasts (FMM-1). In this study, it was founded that the different concentrations of the extract were biocompatible and had no significant difference of the control group (without extract) when applied for 5 minutes or 24 hours, except of the 100 mg / mL concentration when applied for 5 minutes. Even more, the concentration of 1.56 mg / mL that significantly stimulated cell proliferation compared to the control group when applied for 24 hours.
To the best of our knowledge, there is no studies in the literature evaluated the effect of A. satureioides extract over oral cells. The study of Guss et al. (2017) evaluated this effect over mice fibroblasts and macrophage cell lines; however, it did not define the fibroblast cell line exactly, in this study, it was concluded that the extract show no toxicity to fibroblasts agreeing with the results of the present study (Guss et al., 2017). Even more, the extract has no toxic effect over the human peripheral blood mononuclear cells (M C Sabini et al., 2013). It was also verified that only high concentrations of cold extract of A. satureioides induced cytotoxicity, corroborating the current study, when the concentration of 100 mg / mL of glycolic extract of A. satureioides promoted viability of FMM-1 below 70% (65.9%), demonstrating that higher concentrations of the extract are cytotoxic (María Carola Sabini et al., 2012). Low toxicity was also identified by Salgueiro et al. (2016), the authors used extracts obtained by water infusion and tested its cytotoxic over human lymphocytes and it presented low toxicity (Salgueiro et al., 2016).
In this study as well, the genotoxicity of the extract was evaluated, and it was founded that, the major the extract concentration, the major the genotoxicity, being its dose-dependent genotoxic potential. Only the concentration of 6.25 mg / mL of the extract showed similar results to the control group in both 5 min and 24 h contact period.
In the literature, similar results were obtained, Sabini et al. (2013) found that the cold aqueous A. satureioides extract did not show genotoxicity in vitro against Vero cells in the concentration of 10 to 50 μg / mL, but in high concentrations it was genotoxic by the micronucleus assay (M C Sabini et al., 2013) and in a previous study, it was concluded the absence of genotoxicity of cold aqueous extract of A. satureioides by Allium test. These findings show the existence of a limit between safe and toxic concentrations that need to be further studied in order to guarantee safety for clinical use.
Beside the extract concentration, another point to be emphasized is the extract temperature, it seems that the hot aqueous extract of A. satureioides is more cytotoxic and genotoxic over cells than cold one and this may be related to high concentrations of flavonoids including luteolin, quercetin, and 3-O-methylquercetin (Cariddi et al., 2015). In this study, the extract was used at room temperature.
Then, the results obtained in the present study suggest the use of the A. satureioides at low concentration (below 6,25 mg / mL) as it presents lower cytotoxicity and genotoxicity and preferably to use the cold or room-temperature extract when used as a component of dental products like mouthwashes, toothpastes, or as endodontic irrigant and intracanal medication bringing benefits to the population.

Conclusion
It may be concluded that: • The glycolic extract of A. satureioides (≤ 50 mg / mL) was not cytotoxic to gingival fibroblasts, in the periods of 5 min and 24 h of contact.
• The concentration of 6.25 mg / mL of the glycolic extract of A. satureioides did not induce an increase in micronucleus production in the two periods tested, with no genotoxicity.
• The concentration of 6.25 mg / mL of the glycolic extract of A. satureioides is indicated to be used as component of mouthwashes, toothpastes, or as endodontic irrigant and intracanal medication bringing benefits to the population.