1 Volume 24 2025 e255546 Original Research Braz J Oral Sci. 2025;24:e255546http://dx.doi.org/10.20396/bjos.v24i00.8675546 1 Universidad Autónoma de Nuevo León, Facultad de Odontología/ Laboratorio de Microbiología Oral. Monterrey, Nuevo León, México. 2 Universidad Autónoma de Nuevo León, Centro de Investigación y Desarrollo en Ciencias de la Salud. Monterrey, Nuevo León, México. Corresponding author: Myriam Angélica de la Garza-Ramos Universidad Autónoma de Nuevo León, Facultad de Odontología/ Laboratorio de Microbiología Oral. Calle Dr.Eduardo Aguirre Pequeño y Silao S/N, Col. Mitras Centro, CP. 64460, Monterrey, Nuevo Leon, Mexico. Tel.: +52 81 8329 4000 ext. 3168. E-mail: myriam.garzarm@uanl.edu.mx Editor: Dr. Altair A. Del Bel Cury Received: January 19, 2024 Accepted: September 17, 2024 Antifungal and wound healing potential of calendula officinalis for dental use: an in vitro study Nadxiely Ixchel Ricardez-García1 , Myriam Angélica de la Garza-Ramos1* , Arturo Abraham Cienfuegos-Sarmiento2 , Guillermo Cano-Verdugo1 Aim: To demonstrate the antifungal and healing properties of Calendula officinalis. Methods: We included four assays: inhibition halo, germicidal test, cytotoxicity test, and scratch test. Results: The extract at 100% and 50% concentrations was effective, and both presentations (Calendula officinalis extract and Calendula officinalis infusion) exhibited no cytotoxic effects. Furthermore, the infusion demonstrated a high healing effect without affecting the morphology of the cell monolayer and promoted fibroblastic proliferation. The extract did not show the expected healing results. Conclusions: Calendula officinalis holds considerable potential for further research across various biological processes, highlighting its promise as a subject for continued investigation. Keywords: Antifungal agents. Calendula. Dentistry. Phytotherapy. Wound healing. https://orcid.org/0009-0003-1970-1261 https://orcid.org/0000-0003-0792-3695 https://orcid.org/0009-0009-7376-1904 https://orcid.org/0000-0002-4905-1939 2 Ricardez-García et al. Braz J Oral Sci. 2025;24:e255546 Introduction In dentistry, some therapeutic substances have side effects. For example, chlor- hexidine gluconate 0.12% (CHX) can cause staining of teeth, alteration or irritation of taste, and alteration of the oral mucosa1. Currently, the practice of herbal medi- cine has gained significant momentum in the therapeutic use of medicinal plants, either as replacements for or in combination with pharmaceutical medicines2. In dentistry, homeopathic remedies such as Calendula officinalis L. (C. officinalis) and its petals have been proposed due to their anti-inflammatory, antipyretic, antitumor, and healing effects3,4. Calendula officinalis, a well-known therapeutic herb used for millennia, belongs to the Kingdom Plantae and the Asteraceae family5,6. Traditionally, it has been utilized as an anti-inflammatory, immunostimulant, diaphoretic, analgesic, antiseptic, and in the treatment of open and lacerating wounds with bleeding7. It has been demonstrated that C. officinalis possesses pharmacological proper- ties relevant to dentistry, including its antimicrobial property for controlling bac- terial growth in dental biofilm8, analgesic effect (as its use has been reported among tribal peoples who use C. officinalis as an analgesic)7, anti-inflammatory action promoting hemostasis5,9, significant benefits in controlling the progression of periodontal disease and tissue regeneration10, healing properties8,11,12, promo- tion of wound healing13, antifungal effect against several strains of Candida spp.6, and antiviral effect by suppressing herpes simplex virus replication and exhibiting anti-HIV activity6,7. The main purpose of this research was to determine the properties of the infusion and extract of C. officinalis for use in dentistry as an alternative method for mouth- wash. The focus was on demonstrating the antifungal and wound healing properties of C. officinalis fluid extract and flower infusion. The rationale behind this study is the need to find therapeutic alternatives in dentistry that are not only effective but also reduce the side effects associated with conventional treatments. Methods This was an in vitro study included three control groups. To assess the antifungal and wound healing potential of Calendula officinalis (C. officinalis) for dental applications, we utilized a hydroethanolic extract of C. officinalis, sourced as a fluid extract from an herbalist shop in Monterrey, Mexico. Following this, dried C. officinalis flowers were purchased from an herbalist shop, also located in Monterrey. For preparing the C. officinalis infusion, 7 grams of dried flowers were weighed, and 100 mL of sterilized water was boiled and poured over the flowers in a glass container. After steeping for 15 minutes, the infusion was filtered through a fine sieve to remove flower residues. The resulting infusion was then transferred to a sterilized glass bottle and tightly sealed for storage. The in vitro study utilized drugs including Chlorhexidine Gluconate 0.12%, an orally administered antiseptic commonly used in dentistry, and Nystatin, also administered 3 Ricardez-García et al. Braz J Oral Sci. 2025;24:e255546 orally and frequently used to treat oral candidiasis. These drugs were sourced from a drugstore in Monterrey, Mexico. No conflicts of interest were reported by the authors in the development of this study. Candida albicans strains (reference ACCT 90029) were obtained from the Research and Development Center in Health Sciences at the Universidad Autónoma de Nuevo León (Monterrey, Mexico). The inoculum was prepared by adding the Candida albi- cans strain to YPD broth as a culture medium and adjusting it to a turbidity of 0.5 on the McFarland scale to ensure proper suspension. Subsequently, this inoculum was plated on Petri dishes. The FGH cell line was obtained from the Research and Development Center in Health Sciences at the Universidad Autónoma de Nuevo León (Monterrey, Mexico), and cul- tured in plates with DMEM medium supplemented with 10% fetal bovine serum (FBS) and a mixture of antibiotics and antifungals. The culture was maintained at 37°C in an atmosphere of 5% CO2. The trypan blue method was used to evaluate cell viability. After obtaining the C. officinalis infusion, we performed five trials: sensidisc antimicro- bial test, antimicrobial assay MIC, germicidal assay, MTT gingival fibroblast cytotoxic- ity assay, and wound healing assay gingival fibroblast. Sensidisc antimicrobial test The diffusion method on antimicrobial susceptibility test discs was employed to determine the antifungal activity of the test solutions against Candida albicans. An inoculum of Candida albicans (50 microliters) previously prepared was cultured and evenly spread on YPD agar plates. Three discs were loaded with respective mouthwash preparations of C. officinalis fluid extract at different concentrations: 25% (25 g/100 mL), 50% (50 mL of fluid extract in 50 mL of sterilized water), and 100% (C. officinalis fluid extract per se). Additionally, one disc was loaded with C. officinalis infusion (7 g/100 mL), one disc served as a positive control with CHX, and another disc served as a negative control with nystatin (a common drug for treating oral candidiasis). The tests were repeated three times to minimize errors. The plates were then incubated at 37°C for 24 hours. Finally, the inhibition halos were measured using a millimeter ruler. The antimicrobial susceptibility test was conducted in accordance with Standard NMX-BB-012-1974, “Multidiscs for Antimi- crobial Susceptibility Testing”14. Antimicrobial Assay MIC The aim of this test was to determine the minimum inhibitory concentration (MIC) of Candida albicans by incubating each sample for 24 hours at 37°C. A 1:100 dilu- tion was prepared by combining 1 mL of the activated strain with 99 mL of pure YPD broth. Ten tubes were prepared with varying concentrations of C. officinalis extract, ranging from 15 mg/mL in tube 1 to 100 mg/mL in tube 10. The absence of growth, indicated by translucency compared to positive chlorhexidine and negative pure YPD controls, in the tube with the lowest extract concentration, was designated as the MIC. The incubation was maintained at 37°C throughout the experiment, 4 Ricardez-García et al. Braz J Oral Sci. 2025;24:e255546 with absorbance readings of the culture taken every 30 minutes at 595 nm using a microplate reader. The antimicrobial MIC broth dilution test followed the NCCLS standard: Perfor- mance Standards for Antimicrobial Susceptibility Testing, document M7 Methods for Dilution Antimicrobial Susceptibility Testing. Germicidal Assay For the performance of this test, the species Candida albicans was used as the organ- ism of interest. The dilution procedure was followed by adding 1 mL of the inoculum of the microorganisms to the six dilutions previously prepared with a phosphate buffer solution. Each dilution was then placed in Petri dishes with gentle swirling to ensure homogeneous distribution, and the medium was allowed to solidify. After 24 hours of incubation at a constant temperature of 37°C, the colony-forming units (CFU) present in each of the Petri dishes used in the experiment were counted. The assay followed the NMX-BB-040-SCFI-1999 standard: General Methods of Analysis: “Determination of Antimicrobial Activity in Germicidal Products”15. MTT Gingival Fibroblast Cytotoxicity Assay For this study, a gingival fibroblast cell suspension at a concentration of 1x10^5 cells/mL was prepared in a 96-well culture plate. After a 24-hour incubation period, the culture medium was removed, and 100 μL of C. officinalis infusion dilu- tions in DMEM were added: C. officinalis infusion dilutions/DMEM; infusion per se, 90g/10mL, 80g/20mL, and so on up to a concentration of 10g/90mL. A negative control (CHX) and a positive control (DMEM) were also included. After a further 24 hours of incubation, the contents of each well were removed, and cell viability was assessed by measuring the reduction of 50 μL/well of MTT solution (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium) by metabolically active cells. Cell quantification was performed using a microplate reader, and the results were recorded in the corresponding tables. For the cytotoxicity assay, the protocol by Martinez-Redríguez et al.16 (2020) was followed. Wound Healing Assay Gingival Fibroblast To conduct this study, a gingival fibroblast cell suspension was prepared to achieve 90% confluence, forming a confluent monolayer. A 200-microliter (p200) pipette tip was used to create a straight line both horizontally and vertically in the cell mono- layer, simulating an experimental “wound.” The fibroblast cell culture was treated with the infusion at its non-toxic dose of 10 g/90 mL C. officinalis infusion dilution, while a positive control group was treated with DMEM culture medium. Each treat- ment was performed in triplicate to ensure robustness of the results. To document the wound evolution in the cells, images were captured at specific time intervals: 0 hours, 6 hours, 24 hours, 30 hours, and 48 hours, using an inverted fluorescence motorized microscope. 5 Ricardez-García et al. Braz J Oral Sci. 2025;24:e255546 Chemical composition of C. officinalis C. officinalis, commonly known as marigold, contains a diverse array of chemical compounds that contribute to its therapeutic properties. The primary constituents present in the plant are listed in Table 1. Table 1. Phytochemical analysis results of C. officinalis flowers and leaves, (+) Present, (-) Absent Metabolite/Test Chemical Test Result Alkaloids Dragendorff - Carbohydrates Molisch + Coumarins Baljet Test + Double Bonds Beayer Test + Flavonoids Shinoda + Phenolic Hydroxyl Groups Ferric Chloride + Sterols and Triterpenes Liebermann-Burchard - Xanthophylls Xanthophylls test - Statistical analysis Each study was accompanied by a specific statistical analysis. In the antimicrobial susceptibility test, inhibition halo values were compared using a single halo measure- ment taken 24 hours post disk placement, adhering to Standard NMX-BB-012-1974, “Multidiscs for Antimicrobial Susceptibility Testing”14. The antimicrobial MIC broth dilution test was conducted following the NCCLS standard: Performance Standards for Antimicrobial Susceptibility Testing, document M7 Methods for Dilution Antimicro- bial Susceptibility Testing. In the germicidal assay, the initial plate readings were averaged to calculate via- ble cell counts, and the percentage reduction was determined following the NMX-BB-040-SCFI-1999: General Methods of Analysis: “Determination of Antimicro- bial Activity in Germicidal Products”15. For the cytotoxicity assay, culture plates were read on a microplate reader at 570 nm, and the cytotoxicity was calculated using the formula from Martinez-Rodríguez et al.16 (2020). The wound healing assay data were processed using SPSS v23, with normality tests, Kruskal-Wallis (K-W), and Mann-Whit- ney U tests conducted to identify significant differences between groups, setting the significance level at p ≤ 0.05. Results Sensidisc antimicrobial test The antimicrobial susceptibility test discs applied to Candida albicans indicate that a 5 mm inhibition halo is observed when using 100% C. officinalis extract. When using 50% C. officinalis extract, the inhibition halo measures 4 mm. For 25% C. officinalis extract, the inhibition halo is the same as that of C. officinalis. Additionally, the C. officinalis flower infu- 6 Ricardez-García et al. Braz J Oral Sci. 2025;24:e255546 sion results in a 1 mm inhibition halo. On the other hand, the positive control (CHX) shows a 5 mm inhibition halo, while the negative control (Nystatin) has a 1 mm inhibition halo. 0 1 2 3 4 5 6 In hi bi tio n H al o Treatment SENSIDISC ASSAY 1 5 4 3 5 1 Infusio n Extr act 100% Extr act 50% Extr act 25% Chlorhexid ine Nys tatin Source: Created by the Author Figure 1. Bar chart. Halo of inhibition. The results of the susceptibility test are observed. Antimicrobial assay MIC The test tubes were incubated for a duration of 24 hours. Upon completion of this incu- bation period, turbidity was observed in tube 1, containing a concentration of 15 mg/mL, indicating the presence of microbial growth. Conversely, from tube 2 through tube 10, the contents remained translucent, suggesting that no appreciable microbial growth had occurred. These results indicate that the minimum inhibitory concentration (MIC) is located within tube 2, which contained a concentration of 20 mg/mL. The minimum inhibitory concentration (MIC) of the positive control, chlorhexidine (CHX), was observed in tube 2, which contained a concentration of 10 mg/mL. Table 2. MIC Antimicrobial Assay. The results of various concentrations of C. officinalis applied to Candida albicans are shown. Tube No. Extract concentration mg/mL Result N° 1 15 mg/mL Presence of bacterial growth N° 2 20 mg/mL Bacterial growth absence N° 3 30 mg/mL Bacterial growth absence N° 4 40 mg/mL Bacterial growth absence N° 5 50 mg/mL Bacterial growth absence N° 6 60 mg/mL Bacterial growth absence N° 7 70 mg/mL Bacterial growth absence N° 8 80 mg/mL Bacterial growth absence N° 9 90 mg/mL Bacterial growth absence N° 10 100 mg/mL Bacterial growth absence 7 Ricardez-García et al. Braz J Oral Sci. 2025;24:e255546 Germicidal Assay Table 3 demonstrates effective germicidal activity, indicating the sensitivity of Candida albicans to a 50% extract of C. officinalis, along with the cell count. Table 3. Germicidal Test. The percentage of the germicidal effect of C. officinalis applied to Candida albicans is shown in this table. Test Microorganism Treatment application Initial C.V C.V Final Percentage reduction ACCT 90029: Candida albicans 24hrs 105 x 108 CFU/mL <10 CFU/mL 99.999% C.V. Viable UFC Colony Forming Units. MTT fibroblast cytotoxicity assay Cell cytotoxicity test showed no negative effects on viable cells during a 24-hour incu- bation period with the C. officinalis extract, unlike the positive control group. However, the negative control group exhibited a high level of cytotoxicity after 24 hours. There- fore, the C. officinalis extract had a cytotoxicity percentage of 62%. Regarding the C. officinalis infusion, the 90/10 dilution (DMEM/infusion) was deter- mined to be a non-toxic dose as it resulted in an increase in cell number after 24 hours of incubation compared to the positive control. Conversely, the nega- tive control group showed significant cytotoxicity during the same period. By using the formula, the cytotoxicity percentage for the C. officinalis infusion was calculated to be -23%. Scratch assay gingival fibroblast The data show that in the control group, the initial value (0 hours) is 514.760 μm and remains constant until 30 hours. At 48 hours, the value decreases to 205.01 μm, indi- cating a significant decrease from the initial value (p < 0.015). In the experimental group, the initial value (0 hours) is 545.945 μm and shows an increase (p > 0.085) compared to the reference value. At 6 hours, the value is 478.885 μm, which represents a decrease compared to 0 hours but is still higher than the reference value, indicating that the healing effect of the C. officinalis infusion is most effective at 6 hours. Subsequently, at 24 hours, the value decreases signifi- cantly (p < 0.015) compared to 0 hours, and this trend continues in the following hours (30 and 48 hours). 8 Ricardez-García et al. Braz J Oral Sci. 2025;24:e255546 0 200 400 600 800 0h Re ad in g Period Descriptive Analysis of the Wound Healing Assay Control Infusión 48h30h24h6h Source: Created by the Author. Figure 2. The graph shows significant differences between the treated groups and the control, determined by the descriptive analysis, at a level of P < 0.05. Dots represent the mean ± S.E.M. of the groups, demonstrating the greatest healing effect at 6 hours of application. Discussion The aim of this project was to explore and scientifically validate the use of C. officina- lis, renowned for its multiple therapeutic benefits, in dental practice. The main results obtained indicated that the antimicrobial susceptibility test discs applied to Candida albicans showed a greater halo of inhibition compared to the control group. In addi- tion, it was found that the infusion and extract of C. officinalis did not present cytotox- icity in gingival fibroblast cells after 24 hours of application. Finally, a significant effect on cell migration was observed 6 hours after the treatment application. The antimicrobial susceptibility test by Vinola et al.3 (2021) showed that C. officinalis has antimicrobial and antifungal activity against E. faecalis and C. albicans3; however, chlorhexidine is more potent. Bermúdez Hoyos et al.17 (2016) determined that the cream with the addition of 40% garlic extract and 20% Calendula showed the best results, with a reduction of 99% for P. aeruginosa, 98.8% for S. aureus, and 99.9% for T. rubrum. Cardona Rivero et al.18 (2019) determined the minimum inhibitory con- centration (MIC) of the 70% ethanolic extract of C. officinalis against Escherichia coli using the tube dilution method, presenting an MIC of 25.0 mg/mL. This result is similar to that obtained in this study, as the extract of C. officinalis showed an MIC of 20 mg/mL. Verma et al.6 (2018) concluded that C. officinalis has antifungal activity against Candida albicans as well as against other fungal pathogens. Regard- ing our cytotoxicity study, it was found that both presentations of C. officinalis had a lower cytotoxic effect compared to Acuña Piña and Sánchez Flórez19 (2023). Cell via- bility for both oregano oil, calendula oil, and turmeric oil did not present a statistically significant difference, indicating that the implementation of these oils does not affect fibroblast cell activity. On the other hand, Fronza et al.20 (2009) obtained similar results to our in vitro study on wound healing assay, demonstrating that both extracts of C. officinalis stimulated the proliferation and migration of fibroblasts at low concentrations; for example, 10 μg/mL enhanced cell numbers by 64.35% and 70.53%, respectively. Similarly, 9 Ricardez-García et al. Braz J Oral Sci. 2025;24:e255546 Madrid Ahumada et al.21 (2010) demonstrated that the major proliferative effect was achieved by the ethanolic extract of C. officinalis in concentrations of 750 and 500 μg/mL at 12 hours. C. officinalis contains several chemical compounds that act on Candida albicans. These compounds affect motor and cytoskeletal proteins, as well as the cytoskele- ton, lipid transport, and synthesis, thereby compromising the function and integrity of the cell membrane. The primary strengths of this study are found in the economic and accessible nature of C. officinalis, making it readily available to the general population. Unlike many commonly used dental medications, C. officinalis is characterized by the absence of adverse side effects, providing a safer alternative for patients. An innovative approach to dental practice is introduced through the incorporation of sustainable, natural prod- ucts. The potential for C. officinalis to replace chemical-based treatments not only allows for the reduction of healthcare costs but also aligns with the growing demand for eco-friendly and health-conscious medical practices, thereby significantly contrib- uting to the advancement of dental medicine. A notable limitation of our study is the use of a single type of fibroblast cell, which may affect the generalizability of our findings to other oral structures. Additionally, financial constraints limited the scope of our research. Therefore, further studies are essential to evaluate the therapeutic applications of C. officinalis across a broader range of oral tissues, including oral mucosal and labial cells. We acknowledge these limitations and strongly encourage future research to address these gaps. The primary application of C. officinalis in dental practice is its incorporation into daily oral care routines as a natural alternative to conventional products. Its proven antimi- crobial and antifungal properties, especially against Candida albicans, make it effec- tive for maintaining oral hygiene and preventing infections. Additionally, its non-cyto- toxic nature ensures safety for oral tissues, while its promotion of cell migration aids in wound healing and tissue regeneration. Integrating C. officinalis into mouthwashes, toothpaste, and topical gels can enhance patient health and well-being by reducing dependence on synthetic chemicals. In conclusion, significant antifungal activity was demonstrated by the extract of C. officinalis. The minimum inhibitory concentration (MIC) of the C. officinalis extract was determined to be 20 mg/mL. Both forms of presentation exhibited no harmful effects on cells. The infusion of the flower extract revealed a noticeable healing effect, particularly six hours after application, without compromising the structure of the cell layer and promoting the growth of fibroblasts. These results suggest that C. officinalis holds substantial potential for further research in various biological processes, high- lighting its promise as a subject for continued investigation. Acknowledgements The authors are grateful for the support provided by the Oral Microbiology Laboratory and the Undergraduate Research Office of the Faculty of Dentistry at the Universidad Autónoma de Nuevo León. 10 Ricardez-García et al. Braz J Oral Sci. 2025;24:e255546 Conflict of Interest The authors have no conflict of interest to disclose. Data availability Datasets related to this article will be available to the corresponding author upon request. Author Contribution Nadxiely Ixchel Ricardez-Garcia: Conceptualization, Methodology, Research, Writing- original draft, Acquisition of funds. Myriam Angélica de la Garza-Ramos: Supervision, Validation, Resources, Visualization, Project Management, Formal Anal- ysis, Conceptualization. Arturo A. Cienfuegos-Sarmiento: Resources, Methodology, Conceptualization. Guillermo Cano-Verdugo: Writing- revision and editing, Visualiza- tion, Formal analysis. We declare that all authors actively participated in two distinct criteria related to authorship. References 1. Meccatti VM, Ribeiro MCM, Oliveira LD. The benefits of phytotherapy in Dentistry. Res Soc Dev. 2022 Mar;11(3): e46611327050. Portuguese. doi: 10.33448/rsd-v11i3.27050. 2. Quispe Poccohuanca LJ. [Antibacterial efficacy of traditional toothpaste vs. phytotherapy toothpaste against Streptococcus mutans in vitro] [thesis]. Lima, Peru: Universidad Privada Norbert Wiener; 2021 [cited 2022 Jul 5]. Available from: https://hdl.handle.net/20.500.13053/5848. Spanish. 3. Vinola SM, Sekar M, Renganathan SK, Dhiraviam S. Comparative evaluation of C. officinalis and 2% chlorhexidine against Enterococcus faecalis and Candida albicans. J Interdiscip Dent. 2021;11(3):119-23. doi: 10.4103/jid.jid_28_21. 4. Ukiya M, Akihisa T, Yasukawa K, Tokuda H, Suzuki T, Kimura Y. Anti-inflammatory, anti-tumor- promoting, and cytotoxic activities of constituents of marigold (Calendula officinalis) flowers. J Nat Prod. 2006 Dec;69(12):1692-6. doi: 10.1021/np068016b. 5. Patil K, Sanjay CJ, Doggalli N, Devi KRR, Harshitha N. A Review of C. officinalis-Magic in Science. J Clin Diagn Res. 2022 Feb;16(2):23-7. doi: 10.7860/JCDR/2022/52195/16024. 6. Verma P, Raina R, Agarwal S, Kour H. Phytochemical ingredients and Pharmacological potential of C. officinalis Linn. Pharm Biomed Res. 2018;4(2):1-17. doi: 10.18502/pbr.v4i2.214. 7. Ashwlayan VD, Kumar A, Verma M, garg VK, Gupta SK. Therapeutic Potential of C. officinalis. Pharm Pharmacol Int J. 2018;6(2):149-55. doi: 10.15406/ppij.2018.06.00171. 8. Jácome EVM, Macedo DS, Ferreira FD, Diógenes RFP, Alves ADD, Lima Álvaro MP. [Phytotherapy in dental pre and post-surgical treatments]. Rev Fitos. 2022 Mar;16(1):83-92. Portuguese. doi: 10.32712/2446-4775.2022.1136. 9. Bohneberger G, Machado MA, Debiasi MM, Dirschnabel AJ, Ramos GO. [Phytotherapy in dentistry, when can we use them?]. Braz J Health Rev. 2019;2(4):3504-17. Poortuguse. doi: 10.34119/bjhrv2n4-114. 10. Escalante Casco JC, Moreno Tercero I. [Use of phytotherapy as an alternative treatment of Natural and Traditional Medicine by dentists in two cities of Nicaragua] [undergraduate final work]. León: Universidad Nacional Autonoma de Nicaragua, Leon, Facultad de Odontologia; 11 Ricardez-García et al. Braz J Oral Sci. 2025;24:e255546 2020 [cited 2022 Apr 5]. Available from: http://riul.unanleon.edu.ni:8080/jspui/ handle/123456789/9078. Spanish. 11. Givol O, Kornhaber R, Visentin D, Cleary M, Haik J, Harats M. A systematic review of Calendula officinalis extract for wound healing. Wound Repair Regen. 2019 Sep;27(5):548-61. doi: 10.1111/wrr.12737. 12. Roveroni-Favaretto LH, Lodi KB, Almeida JD. Topical Calendula officinalis L. successfully treated exfoliative cheilitis: a case report. Cases J. 2009 Nov;2:9077. doi: 10.1186/1757-1626-2-9077. 13. Silva JFM, Nascimento GNL do N, Ferreira EMS, Pimenta RSP. Diálogos sobre fitoterapia. EDUFT. 2020 Dec[cited 2022 Sep 2];1(9) Lv9. Available from: https://sistemas.uft.edu.br/periodicos/index. php/editora/article/view/9246. 14. Diario Oficial de la Federación. México. 2008 Apr 14[cited 2024 Jun 29]. Available from: https://www.dof.gob.mx/nota_detalle.php?codigo=5032938&fecha=14/04/2008. Spanish. 15. Diario Oficial de la Federación. México. 1999 Nov 3[cited 2024 Jun 29]. Available from: https://www.dof.gob.mx/nota_detalle.php?codigo=4955916&fecha=03/11/1999&print=true. Spanish. 16. Martínez-Rodríguez MA, Madla-Cruz E, Urrutia-Baca VH, de la Garza-Ramos MA, González-González VA, Garza-Navarro MA. Influence of polysaccharides’ molecular structure on the antibacterial activity and cytotoxicity of green synthesized composites based on silver nanoparticles and carboxymethyl-cellulose. Nanomaterials (Basel). 2020 Jun;10(6):1164. doi: 10.3390/nano10061164. 17. Bermúdez Hoyos M, López Naranjo LM, Zabala González DA. [Development of a master preparation based on garlic (Allium sativum) and calendula (C. officinalis) and evaluation of its antimicrobial and antifungal activity]. Cienc Tecnol Innov Salud. 2016 Oct;1:6-11. Spanish. 18. Cardona Rivero A, Medina Cardoso F, Santillan Palomino N. [Evaluation of the in vitro antibacterial activity of ethanolic extracts of “Roque” (Colletia spinosissima) and “Calendula” (C. officinalis) against Staphylococcus aureus, Escherichia coli and formulation of an antibacterial liquid soap] [thesis]. Cuesco, Perú: Universidad Nacional de San Antonio Abad Del Cusco. Facultad de Ciencias de la Salud. Escuela Profesional de Farmacia y Bioquímica; 2019. Available from: http://hdl.handle.net/20.500.12918/4641. Spanish. 19. Acuña Piña LD, Sánchez Flórez V. [Evaluation of cell viability in bacterial cellulose hydrogels impregnated with essential oils of calendula, turmeric, and oregano with potential application in dressings for second-degree burns] [thesis]. Universidad Autónoma de Bucaramanga, Facultad de Ingeniería; 2023. [cited 2023 Nov 20] Available from: http://hdl.handle.net/20.500.12749/20263. Spanish. 20. Fronza M, Heinzmann B, Hamburger M, Laufer S, Merfort I. Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts. J Ethnopharmacol. 2009 Dec 10;126(3):463-7. doi: 10.1016/j.jep.2009.09.014. 21. Madrid Ahumada MA, Mahecha Donato LC, Oviedo Peñaloza VA, Chaves Clavijo M, Roa Molina NS, García Robayo DA, et al. [Effect of Calendula officinalis on the proliferation of human gingival fibroblast]. Univ Odonto. 2010;29(63):107-12. Spanish.