Introduction
Dermatophytosis is a prevalent health issue in developing countries, affecting all age groups and different parts of the body. It estimated that between approximately 30% and 70% of adults are asymptomatic carriers of dermatophytes, while at least 10% of people develop dermatophytosis during their lifetime (41, 28). One causative agent of the disease is Microsporum canis, a zoophilic dermatophyte primarily afflicting dogs and cats (39). This zoonotic fungus can be transmitted through direct contact, making it one of the main species of keratinocytic and keratinolytic filamentous fungi that causes superficial infections in humans worldwide, by reason of the close relationships many humans have with pets (17). Associated pathologies include multifocal alopaecia and desquamation or tinea, marking M. canis infections as a significant public health issue (7). Conventional treatments include the use of topical and oral antifungal pharmacological agents such as amphotericin B, griseofulvin, terbinafine, itraconazole, flucytosine and fluconazole, either alone or in combination (1, 21). However, errors in use, including application, excessive or inadequate use and treatment interruption, have led to the occurrence of fungal resistance and adverse health effects such as nephrotoxicity, hepatotoxicity and neurotoxicity (21, 33, 27). In this context, natural products such as plant extracts are considered potential alternatives for developing new antifungal strategies or even for improving the existing ones (21). Various studies have explored the use of plant extracts as phytotherapeutic alternatives for the treatment of dermatophyte infections (7). Artemisia ludoviciana and Cordia boissieri are plants of considerable ethnopharmacological importance. Despite this, evidence regarding their antimycotic activity against M. canis remains elusive. Artemisia ludoviciana extracts have shown antiprotozoal, antioxidant, ixodicidal, antimicrobial and anti-inflammatory activity (34, 14, 30, 15). Similarly, C. boissieri has exhibited antimicrobial, antifungal, antioxidant, cytotoxic and ixodicidal properties (35, 38). Therefore, this study aimed to evaluate the antifungal activity of extracts from C. boissieri and A. ludoviciana against M. canis, and the extracts’ cytotoxicity to human epithelial cells, with the objective of identifying their therapeutic potential against M. canis infections.
Material and Methods
Plant collection
The aerial parts of A. ludoviciana were collected from Jaumave, Tamaulipas, Mexico (23°24′22.36, −99°22′50.59) and from C. boissieri in Guadalupe, Nuevo León, México (25°71′53.09, −100°22′52.36). Plant identification was performed by García-Ponce et al. (15) in connection with earlier research.
Extraction and characterisation of extracts
Methanolic extracts were obtained using a Soxhlet extractor, by placing 70 g of plant material and 700 mL of methanol in the apparatus for 48 h. The liquid was removed from the obtained product using a rotary evaporator (Laborota 4003-control; Heidolph Instruments, Schwabach, Germany) at 30°C and under reduced pressure. The resultant extract was dried at 25°C and was stored at 4°C. Phytochemical screening, as per the methods described by Verde-Star et al. (37), identified the presence of sterols and triterpenes, sesquiterpene lactones, coumarins, saponins, flavonoids, alkaloids, aromatic compounds, carbonyls and phenol oxides in the extracts.
Antifungal activity
Microsporum canis (American Type Culture Collection 11621) colonies were inoculated on oatmeal agar and incubated for 10 d at 32°C. Subsequently, a concentrated suspension of conidia was obtained by washing the colonies with 10 mL of sterile distilled water. The suspension was adjusted to a concentration of 3 × 104 colony-forming units/mL. A modified microdilution method was used, described in the Clinical and Laboratory Standards Institute protocol M38-A (11). A working solution of 20,000 µg/mL of extract diluted in methanol was prepared and stored at 6°C until use. Dilutions were made in 96-well microplates of the working solution (312.5 to 10,000 µg/mL), using Mueller–Hinton broth (Sigma-Aldrich, St. Louis, MO, USA) and phenol red as a growth indicator. A colour change to red signalled the growth of the dermatophyte through the release of alkaline metabolites. Subsequently, 100 µL of adjusted conidial suspension was added. A conventional fungicide, Imidazole (Sigma-Aldrich), was used as a positive control (1,000 µL/mL) and methanol served as a vehicle control. Regarding the growth control, 100 µL of each strain was inoculated into 100 µL of medium. The obtained solutions were incubated for 120 h at 32°C. Antifungal activity was determined by visual observation to obtain the minimum inhibitory concentration (MIC), which was the concentration that inhibited 80% of fungal growth relative to the growth control. In addition, the minimum fungicide concentration (MFC) was determined as the concentration that managed to inhibit 100% of fungal growth.
Cytotoxic evaluation
To assess cytotoxicity, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed using a healthy human keratinocyte cell line (HaCaT). Cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% bovine foetal serum, penicillin (100 U/mL) and streptomycin (100 µg/mL) and incubated in a sterile atmosphere with 4% CO2 for 5 d at 37°C until confluence was reached. After that, cells were cultured in 96-well microplates at a density of 5 × 103 cells/well. The cells were treated with extracts at the MIC and MFC obtained from the antifungal activity assays and with the extracts at a lower concentration diluted in dimethyl sulfoxide (DMSO) for 24 h. Growth control cells were given 100 µL of DMEM. After that, cells were washed with phosphate-buffered saline and 150 μL of MTT reagent at a concentration of 0.4 mg/mL. The cells were incubated for 3 h, the medium was removed and 100 μL of DMSO was added. The assay was performed in triplicate. Absorbance was measured by spectrophotometry at 560 nm. Additionally, trypan blue cell staining was performed using the same cell line and treatments. A cell quantity of 5 × 103 was cultured in each well of 96-well microchambers. The cells were incubated for 24 h in a sterile atmosphere with 4% CO2 at 37°C and then exposed to different concentrations of the methanolic extracts for 24 h. Finally, a cell count was performed adding trypan blue dye and using a hemacytometer.
Statistical analysis
A randomised block design statistical model was employed, and mean comparisons were performed using Tukey’s test. In all analyses, P-values ≤ 0.05 were considered statistically significant. Data were analysed using Minitab 17 software (Minitab, State College, PA, USA).
Discussion
In recent years, extensive research has been dedicated to exploring the therapeutic effects of plant extracts, particular emphasis falling on research regarding the study and exploitation of their antibacterial and antifungal activity. In this study, a partial phytochemical characterisation of methanolic extracts from A. ludoviciana and C. boissieri was performed. The analysis revealed that the C. boissieri extract contained sterols, triterpenes, saponins and flavonoids, which was consistent with the results reported by Alves et al. (4), who emphasised the abundance of these compounds in plants of the Cordia genus. Extracts obtained from this plant additionally exerted antibacterial (26), antioxidant (38) and antifungal activity against pulmonary mycosis– inducing pathogens (3). Extracts of A. ludoviciana were found to contain sterols, triterpenes, saponins, and flavonoids similarly to extracts of C. boissieri, additionally sesquiterpene lactones and coumarins were noted as present.
These results corroborated findings previously reported by several authors (22, 32, 8). Extracts from A. ludoviciana were previously associated with a spectrum of pharmacological activities, such as antioomycete (12), antinociceptive (5), hypoglycaemic (6), antioxidant (19), antimicrobial, cytotoxic, anti-inflammatory (14), antiparasitic (34) and antifungal (23) effects. Our research specifically evaluated the antimycotic activity of A. ludoviciana and C. boissieri methanolic extracts against the M. canis ATCC-11621 strain, using imidazole as a control, revealing MIC values of 2,500 and 1,250 µg/mL and MFC values of 5,000 and 2,500 µg/mL, respectively. Previous studies indicated that A. ludoviciana extracts inhibited Microsporum cookei and Microsporum gypseum, with halos of 10–15 mm assessed by a disc diffusion method (26). Similarly, Damián-Badillo et al. (13) reported inhibitory effects of flower, leaf and root extracts of A. ludoviciana against strains of Candida albicans, Colletotrichum lindemuthianum, Mucor circinelloides, Saccharomyces cerevisiae and Sporothrix schenckii. Moreover, C. boissieri hydroalcoholic extracts exhibited antifungal activity with a lower MIC value of 1,000 µg/mL against Candida albicans, Aspergillus fumigatus and Coccidioides immitis strains; and an even lower one of 125 µg/mL against Histoplasma capsulatum (3). A C. boissieri flower methanolic extract was also evaluated against species of the Candida genus and was found to be effective at a MIC value of 125 µg/mL against Candida glabrata (35). Our results show that the evaluated A. ludoviciana and C. boissieri extracts had antifungal activity against M. canis ATCC-11621, which may be considered to be directly dependent on the secondary metabolites contained in the extracts. Some studies suggest that tannins, flavonoids, triterpenes, sterols, phenolic compounds, terpenes and monoterpenes can be highly active antimicrobial agents (24, 10, 31). Other studies propose that some dermatophytes, including M. canis, are sensitive to terpenes and monoterpenes, particularly those from Euphorbia tirucalli L. (18, 9). It is worth noting that the extracts’ antifungal activity against M. canis may be attributed to the presence of sterols, terpenes and sesquiterpene lactones, compounds identified through the phytochemical screening of the extracts. However, it is essential to conduct phytochemical composition studies to identify and quantify the compounds present in the extracts used in this work. This step will enable us to attribute the observed biological activity to the major compounds found in the extracts. Additionally, it will aid in identifying any possible synergistic or individual effects they may exhibit.
To assess the safety profile of the methanolic extracts, cytotoxicity tests were conducted on a human keratinocyte cell line (HaCaT) using the MTT assay and trypan blue dye. The MIC and MFC of both extracts were tested in these assays, assessing the cell viability percentage. No statistically significant differences were observed when compared to the cell growth control, demonstrating that these extracts did not inhibit cell growth. While cytotoxic studies on A. ludoviciana and C. boissieri methanolic extracts have not previously been reported for this cell line, literature on related plant extracts from the Artemisia genus, specifically A. princeps, indicated cytoprotective effects, aiding the survival of HaCaT cells exposed to ultraviolet B radiation and reducing radiation-induced DNA damage (20). Other beneficial effects on human keratinocyte cells have also been described: Yang et al. (42) noted that ethanolic extracts from A. apiacea reduced proinflammatory chemokines in HaCaT cells in vitro; and Wang et al. (40) discussed the hydrating and antioxidant activity of an A. argyi extract in this cell line. In the case of plants from the Cordia genus, extracts from the species C. myxa were evaluated in the human L929 fibroblast cell line, and were reported to exert no antiproliferative effect (2). Similarly, Geller et al. (16) observed a C. americana extract at a concentration of 1 g/mL to increase cell proliferation and migration in albino Swiss 3t3 mice fibroblasts by 19.8% in a scratch assay. When loaded onto electrospun fibres applied in wound healing, C. myxa and C. curassavica extracts potentiated human fibroblast proliferation and significantly improved in vivo wound healing (36, 29). These findings support our results in cytotoxicity assays, which showed that methanolic extracts of A. ludoviciana and C. boissieri did not exhibit toxicity in HaCaT cells at the evaluated concentrations.
Profil Orcid
