In Vitro Antifungal Antibacterial Activity of Partitions from Euphorbia tirucalli L.

: We determined the antifungal and antimicrobial sensitivity of Euphorbia tirucalli extracts in vitro. Antifungal and antibacterial activity was determined based on the M38-A and M26-A proto-cols, respectively. The methanolic and ethanolic partitions demonstrated antidermatophytic activity against Trichophyton rubrum (MIC 125 µ g/mL for ethanol and MIC 125 µ g/mL for methanol) and T. interdigitalis (MIC 500 µ g/mL for ethanol; 125 µ g/mL for methanol). These partitions also showed antibacterial activity—the ethanolic partition had an MIC of 1.56 ± 0.02 mg/mL against methicillin-resistant Staphylococcus aureus (clinical isolate), 6.25 ± 0.04 mg/mL against Staphylococcus aureus BAA-44, 3.13 ± 0.13 mg/mL against Pseudomonas aeruginosa 27853, and 3.13 ± 0.15 mg/mL against Escherichia coli ATCC 25922; the methanolic partition showed an MIC of 1.56 ± 0.02 mg/mL against P. aeruginosa 27853 and 1.56 ± 0.043 mg/mL against E. coli ATCC 25922. These partitions show promise as antimicrobial agents or adjuvants in the treatment of infections caused by these microorganisms.


Introduction
In the last three decades the pharmaceutical industry has seen a renewed interest in natural products and their possible applications in the search for new and more efficient drugs. Plants are an important source for drug production; in effect, numerous studies have confirmed the biological potential of phytochemical compounds against human diseases, with antimicrobial [1,2], antiviral [3], antioxidant [2,4], and antidiabetic properties [5], among others. It is worth mentioning that about 30% of the drugs used in industrialized countries have been synthesized from plant products [6]. Within the wide range of plants with pharmacological potential, the Euphorbia genus is among the most used in traditional medicine in many parts of the world, and reports of its antimicrobial activity has aroused interest in the scientific community [7]. Euphorbia tirucalli is a native plant of southern Africa, widely used in Indian and Brazilian ethnomedicine in the treatment of skin conditions such as excrescences, nodules, abscesses, warts, epithelioma, sarcoma, skin tumors, and leprosy, among others [8]. The alcoholic extracts of the stem bark and leaves of E. tirucalli have antimicrobial activity against P. aeruginosa, Candida tropicalis, C. albicans, and Aspergillus niger. Stem extracts with acetone, hexane, methanol, chloroform, and petroleum are reported to have antibacterial activity against E. coli and Bacillus megaterium [9], whereas the aqueous extract of aerial parts eliminates superoxide anions and scavenges hydroxyl radicals [10]. Based on these properties, it is necessary to continue researching the pharmacological properties of E. tirucalli. The number of human diseases caused by microorganisms has increased, which is mainly attributed to a deficiency in immune function, along with the low bioavailability of antifungals and antibiotics [11]. According to the WHO, fungal mycoses have a global prevalence of 20% to 25% for the general population; 5% to 10% of these cases are caused by dermatophytes, with Trichophyton rubrum and T. interdigitalis being the most frequent species. The incidence of chronic dermatophytosis has sparked interest in seeking alternative solutions to combat these diseases [12][13][14]. Another pressing issue is presented by nosocomial infections caused by organisms acquired during stays in health centers, which are an important cause of morbidity and mortality in hospitals and incur an increase in medication costs. Global prevalence is relatively high, reaching 10% of the population; more people die each year from nosocomial diseases caused by multidrug-resistant bacteria than from homicides and traffic accidents [15]. These infections are commonly caused by Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, which cause conditions in the urinary and digestive tracts (diarrhea), toxic shock syndrome, pneumonia, and septicemia [16][17][18].
Due to the continued use of medicinal plants, it is necessary to continue using valid experimental models to demonstrate their therapeutic properties. We evaluated the in vitro antifungal activity of different partitions of extracts obtained from E. tirucalli against clinical isolates of dermatophytes (T. rubrum and T. mentagrophytes), as well as against bacterial strains responsible for nosocomial infections (S. aureus, P. aeruginosa, and E. coli).

Microorganisms
Isolates of the fungi Trichophyton rubrum and T. interdigitalis, as well as the bacterial strains Escherichia coli 25922, Staphylococcus aureus BAA-44, Staphylococcus aureus clinical isolate, and Pseudomonas aeruginosa 27853, were used. All strains were obtained from the microorganism collection from the Laboratory of Analytical Chemistry of the College of Biological Sciences of the Universidad Autónoma de Nuevo León (UANL).

Collection of Plant Material
The E. tirucalli (E.t) collection was carried out in northern San Nicolás de los Garza, in Nuevo León, México. The samples were taxonomically classified in the Botany Department of the College of Biological Sciences in UANL, and were registered in the herbarium under folio number: 029755. The plant material was repeatedly washed with distilled water and dried at 40 • C with a white light lamp (150 W). Subsequently, the material was mashed using a manual grinder.

Preparation of Extracts
First, 40 g of dried and ground plant material was subjected to continuous extraction using Soxhlet equipment with hexane, chloroform, ethanol, and methanol (CTR Scientifics). Partitions were recovered and solvents were removed under reduced pressure (Yamato rotary evaporator model RE200). The plant material was brought to dryness in a stove oven at a temperature no higher than 40 • C (BTC-9100, TERLAB, Zapopan, México). Once solvent-free, all four partitions (hexane: E.t-Hex-Part; chloroform: E.t-Clo-Part; ethanol: E.t-EtOH-Part; methanol: E.t-MeOH-Part) were stored at ambient temperature until use ( Figure 1).
The yield was obtained by the rule of three, considering the initial plant material (40 g) as 100%. The yield was obtained by the rule of three, considering the initial plant material (40 g) as 100%.

Preparation of Treatments
Plant treatment stocks (20 mg/mL with corresponding solvent) were prepared and homogenized by ultrasound (Ney ultrasonic cleaner 19 h) for 20 min. For antibacterial activity, initial solutions of 25 mg/mL were prepared via the same procedure.

Preparation of Inoculate
For the dermatophyte strains: Strains were initially activated on potato dextrose agar (PDA; CTR Scientifics, Monterrey, México), for 21 days at 28 °C (Quincy lab, Model 12-140 Bench Top Incubator). Subsequently, fungal colonies were covered with 10 mL of sterile distilled water, and their surfaces were gently scraped with a sterile loop. Later, the obtained mixtures were filtered with a sterile gauze to separate the conidia from the hyphae and agar particles. Before performing biological tests, conidia suspensions were adjusted to 1-3 × 10 3 CFU/mL concentrations after being counted with a hemocytometer (CTR Scientifics, Monterrey, México).

Determination of the Antidermatophytic Activity of the E. tirucalli L. Partitions
In vitro antifungal sensitivity was determined through modification of the microdilution method described in the M38-A protocol by the Clinical for Laboratory Standards Institute, 2008 [20], using clotrimazole (Sigma-Aldrich, St. Louis, MI, USA) as a control. Partition solutions were diluted from 20,000 µg/mL to 8000 µg/mL to carry out

Preparation of Treatments
Plant treatment stocks (20 mg/mL with corresponding solvent) were prepared and homogenized by ultrasound (Ney ultrasonic cleaner 19 h) for 20 min. For antibacterial activity, initial solutions of 25 mg/mL were prepared via the same procedure.

Preparation of Inoculate
For the dermatophyte strains: Strains were initially activated on potato dextrose agar (PDA; CTR Scientifics, Monterrey, México), for 21 days at 28 • C (Quincy lab, Model 12-140 Bench Top Incubator, Waltham, MA, USA). Subsequently, fungal colonies were covered with 10 mL of sterile distilled water, and their surfaces were gently scraped with a sterile loop. Later, the obtained mixtures were filtered with a sterile gauze to separate the conidia from the hyphae and agar particles. Before performing biological tests, conidia suspensions were adjusted to 1-3 × 10 3 CFU/mL concentrations after being counted with a hemocytometer (CTR Scientifics, Monterrey, México).  In vitro antifungal sensitivity was determined through modification of the microdilution method described in the M38-A protocol by the Clinical for Laboratory Standards Institute, 2008 [20], using clotrimazole (Sigma-Aldrich, St. Louis, MI, USA) as a control. Partition solutions were diluted from 20,000 µg/mL to 8000 µg/mL to carry out subsequent dilutions. With respect to the positive controls, 1000 mg/mL dilutions were obtained from stock solutions (1000 µg/mL).
Initially, 100 mL of MH (Müller-Hinton) medium was prepared, and 0.02 mg of phenol red was added before sterilization (MH-Ph). Subsequently, the biological testing was carried out through serial dilutions in flat-bottomed 96-well microplates, using MH-Ph broth as a diluent from an initial partition concentration of 8000 µg/mL. To the MH-Ph-Part broth initially placed in the microplate, 100 mL of conidia solution was added in order to obtain final concentrations of 0.0076-2000 µg/mL. With respect to the control with clotrimazole (CLSI, 2008), final concentrations of 0.0001-25 µg/mL were obtained. Dilutions without inoculum were used as blanks; methanol dilutions were used as negative controls. For growth control, 100 µL of each strain was inoculated in 100 µL of medium. The obtained mixtures were incubated at 28 • C for 120 h (Figure 2). subsequent dilutions. With respect to the positive controls, 1000 mg/mL dilutions were obtained from stock solutions (1000 µg/mL).
Initially, 100 mL of MH (Müller-Hinton) medium was prepared, and 0.02 mg of phenol red was added before sterilization (MH-Ph). Subsequently, the biological testing was carried out through serial dilutions in flat-bottomed 96-well microplates, using MH-Ph broth as a diluent from an initial partition concentration of 8000 µg/mL. To the MH-Ph-Part broth initially placed in the microplate, 100 mL of conidia solution was added in order to obtain final concentrations of 0.0076-2000 µg/mL. With respect to the control with clotrimazole (CLSI, 2008), final concentrations of 0.0001-25 µg/mL were obtained. Dilutions without inoculum were used as blanks; methanol dilutions were used as negative controls. For growth control, 100 µL of each strain was inoculated in 100 µL of medium. The obtained mixtures were incubated at 28 °C for 120 h (Figure 2). Antifungal activity was determined using the minimum inhibitory concentration (MIC), according to the CLSI as the concentration capable of inhibiting the fungal growth (% H) at 80% compared to the control growth. Revelation was determined with a microplate ELx800 universal microtiter plate reader (BioTek, Santa Clara, CA, USA) at 550 λ, and the inhibition percentage (% H) was determined using the following formula % ⁄ 100

Antibacterial Activity of E. tirucalli L. Partitions
MIC values for plant extracts were determined according to the M26-A protocol (CLSI, 1999) [21]. The inocula were prepared from 18 h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. Partitions were first diluted to the highest concentration, and then serial 2-fold dilutions were carried out in a concentration range from 25-0.781 mg/mL (final concentrations: 12.5-0.391 mg/mL). MIC values against bacteria were determined based on a micro-well dilution method. Microbial growth was determined by absorbance values at 540 nm using an ELx800 universal microtiter plate reader (BioTek, CA, USA). The MIC was defined as the lowest compound concentration to inhibit microorganism growth. In order to reveal whether a partition had bacteriostatic or bactericidal activity, a sample was removed from the MIC and inoculated in Müller-Hinton agar. Growth in the medium was considered bacteriostatic, whereas no growth was considered bactericidal. All assays were carried out in triplicate. MIC values for plant extracts were determined according to the M26-A protocol (CLSI, 1999) [21]. The inocula were prepared from 18 h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. Partitions were first diluted to the highest concentration, and then serial 2-fold dilutions were carried out in a concentration range from 25-0.781 mg/mL (final concentrations: 12.5-0.391 mg/mL). MIC values against bacteria were determined based on a micro-well dilution method. Microbial growth was determined by absorbance values at 540 nm using an ELx800 universal microtiter plate reader (BioTek, Santa Clara, CA, USA). The MIC was defined as the lowest compound concentration to inhibit microorganism growth. In order to reveal whether a partition had bacteriostatic or bactericidal activity, a sample was removed from the MIC and inoculated in Müller-Hinton agar. Growth in the medium was considered bacteriostatic, whereas no growth was considered bactericidal. All assays were carried out in triplicate.

Percentage of Extraction Yield
Successive extractions were carried out from 40 g of dry plant material. The partition yield for hexane was 4.63%; for chloroform, 3.028%; for ethanol, 7.775%, and for methanol, 12.423% (Table 1).

Antifungal Activity
The methanolic and ethanolic partitions had inhibitory effects on the growth of clinical dermatophyte isolates. The two dermatophytic strains had an MIC of 125 µg/mL for the methanolic partition, whereas for ethanolic partitions the MIC for T. rubrum was 125 µg/mL, while for T. interdigitalis it was 500 µg/mL ( Table 2). No inhibitory effect was observed on dermatophyte growth in solvent controls (methanol and ethanol); therefore, the solvents do not contribute to the partitions' activity. Moreover, using a blank in the formula to evaluate the inhibition eliminated the absorbance effect of the extract. The same MICs were obtained in both the ethanolic and methanolic partitions against T. rubrum, whereas a highly significant difference was found between the MICs of T. rubrum and T. mentagrophytes (Table 2). Qualitatively, changes in the color of culture medium to fuchsia red indicated the growth of fungus, whereas slight changes in color showed a low inhibition of fungal growth.

Antibacterial Activity
Both partitions showed growth inhibition of both Gram-positive and -negative strains, but only the ethanolic partition had activity against MRSA strains (ATCC and Clinical Isolates). For the methanolic partition, the bacteriostatic activity is shown in Table 3. The ethanolic partitions demonstrated bactericidal activity against P. aeruginosa ATCC 27853 (3.125 ± 0.129) and S. aureus BAA-44, (6.25 ± 0.038 mg/mL). These results are interesting because these bacteria are usually common etiological agent for nosocomial diseases.

Discussion
The methanolic and ethanolic partitions obtained in this study showed both bactericidal and bacteriostatic activity against antibiotic-resistant microorganisms; the ethanolic partition showed a greater bactericidal potential against P. aeruginosa ATCC 27853 (3.125 mg/mL). This compares to the agar plating method used by Kumar et al. 2010 [10], who found that a concentration of 2.5 mg/mL inhibited approximately 40% of growth. For S. aureus, our methods showed 100% inhibition, whereas Kumar et al. (2010) [10] showed an inhibition of 30.76% at a concentration of 2.5 mg/mL compared to its inhibition control (streptomycin). It should be noted that the strain used in our work is considered multidrugresistant, and did not present any resistance; however, at 6.25 mg/mL it already had a 100% inhibition, which translates into a bactericidal effect. In relation to the methanolic partitions, our results show greater inhibitory potential at concentrations below 2.5 mg/mL. The differences between studies suggest that when partitioning from nonpolar solvents to methanol, the metabolites present behave either as bacteriostatic or bactericidal, because they are separated by their polarity gradients.
The in vitro sensitivity of partitions obtained from E. tirucalli against clinical isolates of dermatophytes and multidrug-resistant bacteria were determined using clotrimazole as a control. The E.t-MeOH-Part and E.t-EtOH-Part were found to have antifungal activity against dermatophyte clinical isolates (T. rubrum and T. interdigitalis) and multidrugresistant bacterial strains; T. rubrum showed an MIC of 125 µg/mL with the E.t-EtOH-Part, while T. interdigitalis had an MIC of 500 µg/mL. Both dermatophytic strains had an MIC of 125 µg/mL with the E.t-MeOH-Part (Table 2). It should be noted that these results are similar to those obtained by Kumar et al. in 2010, who introduced a methanolic extract of E. tirucalli to strains of clinical importance such as Candida albicans, C. tropicalis, Aspergillus niger, A. fumigatus, A. flavus, and Fusarium oxysporum [10]. Our MICs obtained with E.t-MeOH-Part were similar to those obtained by Parekh and Chanda (2008) for different strains of Candida [22]. However, the slight improvement in MIC that we found could be related to the partitions obtained, which could favor the solubility of secondary metabolites, thereby increasing the contact surface on microorganisms. According to Savjani et al. (2012), the phenomenon of creating dilutions from a stock solution with the purpose of obtaining a homogeneous system is one of the important parameters used to achieve the desired concentration for a strong pharmacological response [23]. Furthermore, secondary metabolites such as tannins and other phenolic compounds are classified as highly active antimicrobial compounds [24]. Extract partitions from E. tirucalli L. revealed the presence of tannins, flavonoids, triterpenes, and sterols, which are biologically active against various microbial agents responsible for human diseases [24][25][26].
Although the two partitions had different minimum fungicide concentrations, they presented equal MICs (125 µg/mL) against T. rubrum (Table 2). This is related to the small differences in their phytochemical profiles, given that the E.t-EtOH-Part showed the presence of tannins, flavonoids, triterpenes, and sterols, whereas the phytochemical profile of the E.t-MeOH-Part exhibited the presence of tannins, flavonoids, sterols, and sesquiterpenes, in addition to those previously mentioned. According to the results obtained by Miron et al. (2014) [27], the antifungal activity of these partitions may be related to the presence of terpenes. Dermatophytes (e.g., T. rubrum, T. mentagrophytes, Microspurum gypseum, and M. canis) are considerably sensible to terpenes (i.e., monoterpenes). Qasim and Rasool [28,29] found that the terpenes taraxerano and cycloeuphordenol are present in the aerial parts of E. tirucalli L. Because the main difference between the E.t-EtOH-Part and the E.t-MeOH-Part is the presence of sesquiterpenes in the latter, the sensitivity shown by T. interdigitalis may be related to this compound. Duong et al. (2019) reported the presence of sesquiterpenes in E. tirucalli L.; these compounds have shown antibiotic activity against different organisms, and this may be the case for T. interdigitalis [30]. In a previous study, the methanolic partition of E. tirucalli L. showed antimicrobial activity against B. subtilis, E. coli, E. faecalis, and C. albicans [31]. Partitions obtained from E. tirucalli L. also contain flavonoids and tannins-compounds generally recognized for their antioxidant and antimi-crobial potential, as well as their role in collagen synthesis [32,33]. It is possible that synergy against dermatophytic strains occurs between the compounds present in the partitions [34].

Conclusions
In the present study, ethanolic and methanolic partitions of E. tirucalli L. showed antimicrobial potential against clinical isolates of dermatophytes (T. rubrum and T. interdigitalis) and bacteria (E. coli 25922, S. aureus BAA44, S. aureus MRSA, and P. aeruginosa 27853). We found that the ethanolic partition had the highest biological activity against bacteria, whereas the methanolic partition was most effective against dermatophytes. These biological activities were related to the phytochemical profiles of each of the partitions. The secondary metabolites of E. tirucalli are promising antimicrobial agents that could be used in the treatment of the diseases caused by these microorganisms. Although the pharmacological potential of these partitions was evidenced, it is worth mentioning that even though Soxhlet extraction offers numerous advantages, the use of high temperatures in this method increases the possibilities of thermal degradation of compounds such as polyphenols and alkaloids, generally known for their antimicrobial potential. Therefore, in the future we will try to evaluate the same biological tests using partitions of E. tirucalli obtained via other extraction methods that use more tolerable temperatures.