Cytoprotective effect of Fridericia chica (Bonpl.) L.G. Lohmann extract associated with geranylgeraniol enriched-fraction from Bixa orellana L. on epithelial cells treated with bisphosphonate

Abstract Bisphosphonates are drugs used to treat bone disorders. The chronic use of bisphosphonates is associated with the occurrence of medication-related osteonecrosis of the jaw (MRONJ). Previous data reported the positive effects of Geranylgeraniol on different cell types treated with Bisphosphonates. Foregoing work done by our research group demonstrated the wound healing capacity of Fridericia chica (Bonpl.) L.G.Lohmann standardized ethanol extract. Herein in vitro cytoprotective synergistic effect of the association of F. chica extract associated with an enriched geranylgeraniol fraction on keratinocytes exposed to zoledronic acid is reported. An association of F. chica at 1 and 5 µg/mL with geranylgeraniol at 15 µg/mL, increased cell viability by 73.5% and 71.1%, respectively. This treatment did not increase tumor cells viability; whereas the clonogenic potential assessment showed that, the association with F. chica (5 µg/mL) reversed the effects of zoledronic acid on the cells. This study provides data for a potential treatment for MRONJ. Graphical Abstract


Introduction
Approximately 65% of the drugs used in cancer therapy are isolated or somehow inspired from natural sources, whilst being important for various diseases (Newman and Cragg 2020). Medicinal plant research has provided an array of molecules with unique mechanisms of action that are leads for the pharmaceutical industry (Pye et al. 2017;Rosa 2020).
Among the main species listed in the National List of Medicinal Plants of Interest to the Brazilian Health System (RENISUS), F. chica is a potential therapeutic agent for wound healing (Minist erio do Meio Ambiente 2021).
Our research group demonstrated F. chica standardized extract healing activity potential with fibroblasts and collagen experimental models (Jorge et al. 2008). Further studies demonstrated an increase in collagen production in the wound area in tendon repair (Aro et al. 2013). Thereafter 2.5% F. chica extract Natrosol gel for Phase I and II clinical trials for volunteers with head and neck cancer treated with radiotherapy demonstrate that the product produced significant improvement of wounds when compared to standard laser treatment (Queiroz et al. 2018).
In addition to healing issues, some diseases lead to loss of bone tissue, such as malignant hypercalcaemia, Paget's disease, bone metastasis, multiple myeloma, and advanced breast cancer. In all these situations that represent high morbidity, bisphosphonates (BPs) are an important class of drugs used to treat these skeletal complications (Gdowski et al. 2017;Macedo et al. 2017;Svensson et al. 2017). Despite the component's tissue-related benefits in different bone pathologies, BP has straight relationship with medication-related osteonecrosis of the jaw (MRONJ), whose incidence is 12%, deranging feeding and speaking in patients with this disorder (AAOMS 2014;Zafar et al. 2014;Koneski et al. 2018;Fliefel et al. 2019).
Based on the assumption that promoting epithelial healing could help in the treatment of MRONJ (Scheper et al. 2010), the standardized F. chica extract was used as a local agent to induce fibroblasts and collagen production, with results indicating a cytoprotective effect (Zago et al. 2020).
Previous studies reported benefits of Geranylgeraniol on improving cell viability and proliferation in tissue regeneration, thus overcoming Bisphosphonate-induced osteonecrosis of the jaw (Zafar et al. 2016;Dequigiovani et al. 2017;Koneski et al. 2018;Fliefel et al. 2019). This substance is a byproduct of the production of bixina, a dye obtained from annatto seeds by molecular distillation of Bixa orellana L. (Carvalho 2015).
Herein the cytoprotective effects of the association of F. chica standardized extract with an enriched geranylgeraniol fraction obtained from Bixa orellana tested with in vitro models is reported providing data for the development of an adjuvant herbal medicine for the management of bisphosphonate-induced osteonecrosis wounds.

Results and discussion
This study aimed to assess the combination of F. chica extract and geranylgeraniol obtained from Bixa orellana L. through in vitro experiments, with evaluations of the cytoprotective activity via cell viability, clonogenic potential, and antiproliferative activity (Tables S2, S3, and Figure S1, respectively).
In the cell viability assay, the cells treated with ZA (zoledronic acid) combined with two concentrations of F. chica presented significant results when compared to the control, while the fraction enriched with geranylgeraniol presented better results of cell viability when tested at the highest concentration (Table S2).
The results also showed that extracts association presented higher cell viability results when compared to individual treatments, which allows the identification of a potential synergistic effect between the studied extracts, that shall be further investigates with in vivo experimental models.
Results reinforced the healing properties previously reported for both F. chica and geranylgeraniol, in addition to the negative effects of nitrogen-containing bisphosphonates when in contact with cells that participate in the healing process (Jorge et al. 2008;Aro et al. 2013;Zafar et al. 2016;Koneski et al. 2018;Zago et al. 2020). They also demonstrated the benefits attained by combination of active compounds with the same therapeutic effect (Basting et al. 2019; Table S2).
The antiproliferative data showed that ZA reduced all cell lines (except for NCI-H460) and non-tumor cell lines (HaCaT) tested, in agreement with Li et al. (2012), who identified the antiproliferative activity of ZA in tumor cells.
However, a cytostatic effect was observed only for 786-0 and HaCaT cell lines, i.e., inhibition of cell proliferation above 50%, in agreement with the studies conducted by Pabst et al. (2012), who investigated the effects of ZA on keratinocytes, demonstrating the compound's harmful impact on these cells ( Figure S1).
The main advantage of the clonogenic assay used in this study is the ability to measure the potential of cells to maintain their reproductive integrity after an extended period of exposure; for example, approcimately one to two weeks (Franken et al. 2006).
The surviving fraction (SF), defined as the number of cells that survive in the clonogenic test, showed that the extracts treated only with ZA, enhanced the negative effects of the drug. When the cells were treated with the association of ZA þ AC5 þ GG15, the effects of ZA in the long term, were reversed whereas the combination of ZA þ AC1 þ GG15 was able to reverse the effects of ZA in the short term (cell viability test), nevertheless in the long term, this association did not show the same effect.
Therefore, the association with AC at the highest concentration tested showed the ability to reverse the effects of ZA on cells, which may have relationship with the extract sediment inside the cells with extended release, ensuring the cell preservation effects (Table S3).
The results found herein agree with data obtained by Romani et al. (2009) who determined the ability of geranylgeraniol to reverse the antiproliferative action of ZA in liver tumor cells, showing that the mechanism of action of geranylgeraniol has relationship with prenylation of proteins that are part of the mevalonate pathway.

Conclusion
The associations of ZA þ AC1 þ GG15 and ZA þ AC5 þ GG15 exposed to the cell viability assay suggest a synergistic effect of F. chica extract with Geranylgeraniol. In the antiproliferative activity assay, none of the evaluated treatments presented a significant proliferative or antiproliferative effect on tumor cells tested, ensuring safety parameters. The association of ZA þ AC5 þ GG15 showed the highest colony formation potential, being feasible for preservation of cells exposed to zoledronic acid. Therefore, herein data for the development of an adjuvant herbal medicine for the management of bisphosphonate-induced osteonecrosis wounds is reported.

Disclosure statement
No potential conflict of interest was reported by the authors.