ANTIFUNGAL POTENTIAL OF PLANT SPECIES FROM BRAZILIAN CAATINGA AGAINST DERMATOPHYTES.

Trichophyton rubrum and Trichophyton mentagrophytes complex, or Trichophyton spp. are the main etiologic agents of dermatophytosis, whose treatment is limited by the high cost of antifungal treatments, their various side effects, and the emergence of resistance amongst these species. This study evaluated the in vitro antidermatophytic activity of 23 crude extracts from nine plant species of semiarid vegetation (caatinga) found in Brazil. The extracts were tested at concentrations ranging from 1.95 to 1,000.0 mg/mL by broth microdilution assay against the reference strains T. rubrum ATCC 28189 and T. mentagrophytes ATCC 11481, and 33 clinical isolates of dermatophytes. All plants showed a fungicidal effect against both fungal species, with MIC/MFC values of the active extracts ranging from 15.6 to 250.0 µg/mL. Selected extracts of Eugenia uniflora (AcE), Libidibia ferrea (AE), and Persea americana (AcE) also exhibited a fungicidal effect against all clinical isolates of T. rubrum and T. mentagrophytes complex. This is the first report of the antifungal activity of Schinus terebinthifolius, Piptadenia colubrina, Parapiptadenia rigida, Mimosa ophthalmocentra, and Persea americana against both dermatophyte species.

INTRODUCTION

Trichophyton rubrum and Trichophyton mentagrophytescomplex, or Trichophyton spp. are the main etiologic agents of dermatophytosis in many parts of the world, including Brazil1 , 2 , 3. Overall, this contagious infection, commonly referred to as ringworm or tinea, is restricted to the outermost layers of the epidermis and its appendages, resulting in either a mild or intense inflammatory reaction, and in many cases, it is long lasting and difficult to treat1. Topical antifungal agents, mainly azoles or allylamines, are currently used for the treatment of most dermatophytoses. In some cases, such as infections of the nail and hair, a systemic treatment is required4 , 5 , 6. However, therapeutic efficacy can be limited by antifungal side effects and/or resistance, patient non-adherence or therapy discontinuation, the cost of medication, and drug interactions7 , 8. Antifungal drugs have a limited number of cellular targets such as ergosterol and the enzymes involved in its synthesis, nucleic acids and the cell wall synthesis, and the formation of microtubules9. Studies on compounds with potential antifungal action are important, not only for the treatment of dermatophytosis, but also to lead to the discovery of new cellular targets for the treatment of fungal infections.

Natural products have contributed significantly in healthcare since ancient times, when they have been extensively used in folk medicine for the treatment of various diseases10. Indeed, the antimicrobial activity of extracts and essential oils from native plants, including their effects on dermatophytes, has been reported worldwide6 , 11 , 12 , 13 , 14 , 15. Importantly, they exhibit a large chemical diversity and biological activity, representing an alternative easily obtainable for the treatment of various diseases.

In this study, the in vitro antifungal activity of different extracts obtained from nine plant species of semiarid vegetation (caatinga) of Northeast Brazil were investigated againstT. rubrum and T. mentagrophytes complex.

MATERIAL AND METHODS

Microorganisms: T. rubrum ATCC 28189, T. mentagrophytes ATCC 11481, and 33 clinical isolates of T. rubrum (n = 24) and T. mentagrophytes complex (n = 9) were used in this study. The clinical isolates were recovered from nail and skin infections, which were obtained from the fungal collection of the Medical Mycology Laboratory of the Universidade Estadual de Maringá.

Plant material: Nine plant species of the semiarid vegetation (caatinga) of Northeast Brazil (seven native and two cultivated) were selected for this study. The voucher specimens were deposited and identified at the herbaria of the Universidade Federal de Pernambuco (UFPE), Universidade Federal do Rio Grande do Norte (UFRN), and Instituto Agronômico de Pernambuco(IPA) (Table 1).

Table 1

Plant species from Brazilian caatinga: tradicional use and in vitro antifungal activity of extracts from selected plant parts

Species (family) (voucher number)	Local name	Origin	Part tested	T. rubrum* ATCC 28189	T. mentagrophytes* ATCC 11481	Folk medicine use
				MICand MFC (µg/mL)	MIC and MFC (µg/mL)
				AE	EE	AcE	AE	EE	AcE
Eugenia uniflora Linn (Myrtaceae) (11763 UFRN)	Pitanga	N	Leaves	125.0	125.0	62.5	125.0	125.0	31.3	Throat complaints 11
Schinus terebinthifolius Raddi (Anacardiaceae) (8758 IPA)	Aroeira	N	Stem bark	1000.0	62.5	NA	1000.0	62.5	NA	Injury, inflamation of internal organs, gastritis, ulcer 19
Piptadenia colubrina Vell. Benth (Mimosaceae) (38384 IPA)	Angico	N	Stem bark	500.0	125.0	250.0	500.0	125.0	250.0	Bronchitis, gastritis, pneumonia, colds 38
Parapiptadenia rigida Benth. Brenan (Fabaceae) (83115 UFPE)	Angico vermelho	N	Stem bark	250.0	250.0	15.6	250.0	250.0	15.6	Asthma, bronchitis 19
Libidibia ferrea Mart. (Caesalpiniaceae) (88145 IPA)	Pau-ferro	N	Stem bark	62.5	62.5	62.5	31.3	31.3	31.3	Blow, throat complaints, bronchitis, anemia, swelling, back pain, injury, labyrinthitis, renal problems, inflammation, stress, fatigue 19
Psidium guajava Linn. (Myrtaceae) (8214 UFRN)	Goiaba	C	Leaves	125.0	62.5	125.0	125.0	62.5	125.0	Stomach ache, dysentery, digestive problems, headache, inflammation, gingivitis, throat complaints, leukorrhea and skin diseases 11 , 19
Mimosa ophthalmocentra Mart. ex Benth (Mimosaceae) (83114 UFPE)	Jurema vermelha	N	Stem bark	125.0	125.0	NA	125.0	125.0	NA	Bronchitis, cough 19
Mimosa tenuiflora Wild. Poir (Mimosaceae) (83113 UFPE)	Jurema preta	N	Stem bark	62.5	NA	62.5	62.5	NA	62.5	Injury, inflammation, fever 19
Persea americana Mill. (Lauraceae) (89420 IPA)	Abacate	C	Leaves	NA	31.3	31.3	NA	31.3	31.3	Renal problems 19

The activity of the extracts was classified as follows: MIC ≤ 75.0 µg/mL, classified as strong activity; 75.0 < MIC ≤ 150.0 µg/mL, moderate activity; 150.0 < MIC ≤ 250.0 µg/mL, weak activity, and MIC > 250.0 µg/mL, inactive18. Crude extract: AE - aqueous, EE: ethanol:water and AcE: acetone:water. N: Native; C: Cultivated. *Terbinafine (MIC/MFC) ≤ 0.004 µg/mL. NA: Not analyzed.

Preparation of crude extracts: The plant parts were dried at 45oC, until a constant weight was achieved, and ground into powder using a Willey mill (Adamo(r) ). The extract solutions were obtained by reflux using water (aqueous extract, AE), ethanol: water (1:1, v/v, ethanolic extract, EE), or acetone: water (1:1, v/v, acetone extract, AcE) as previously described16 (Table 1). Stock solutions (2,000.0 mg/mL) of crude extracts were prepared in water containing 10% dimethylsulfoxide [DMSO, v/v (Sigma-Aldrich, USA)] and twofold serial dilutions were prepared in growth medium (1,000.0 -1.95 mg/mL), and added once in each assay. The final concentration of DMSO in the assays did not exceed 1% (v/v).

In vitro antifungal assay: The minimum inhibitory concentrations (MIC) of crude extracts against dermatophytes were determined using the standard broth microdilution assay17, except for the conidia counts that were performed in a Neubauer chamber. The conidial suspensions were adjusted to obtain a final concentration ranging from 2.5 × 103 to 5 × 103 CFU/mL. Terbinafine was used as a quality control. Wells containing 1% DMSO (v/v) and wells without fungal cells in each plate served as growth and sterility controls, respectively. For determination of minimal fungicidal concentrations (MFC), the contents from the wells showing no growth were transferred to Sabouraud dextrose agar plates and incubated at 25 oC for seven days. Selected extracts (according to MIC values and quantity available for testing) were also assayed against 33 isolates of T. rubrum (n = 24) and T. mentagrophytes complex (n = 9). All experiments were performed in duplicate on two different occasions.

The antifungal activity of the extracts was ranked according to MIC values using the criteria established by SCORZONI etal.18: MIC ≤ 75.0 µg/mL, classified as strong activity; 75.0 < MIC ≤ 150.0 µg/mL, moderate activity; 150.0 < MIC ≤ 250.0 µg/mL, weak activity, and MIC > 250.0 µg/mL, inactive.

RESULTS AND DISCUSSION

The Brazilian caatinga is one of the richest biomes in terms of biodiversity, harboring several native or cultivated plant species. Many plant species of this region are used in folk medicine or as commercial herbal to treat different conditions. However, few ethnobotanical and ethnopharmacological studies have been conducted on these medicinal plants19. Moreover, antifungal activity against dermatophytes has not yet been studied for many of these plant species. Previous results by our research group have shown that the nine plant species (seven native and two cultivated) selected from this biome exhibited growth-inhibitory activity against variousCandida species16. In this study, the antifungal activity of these plants was evaluated against T. rubrum and T. mentagrophytes complex.

First, T. rubrum ATCC 28189 and T. mentagrophytesATCC 11481 reference strains were used for the initial screening of 23 crude extracts obtained from the plant species of the Brazilian caatingaand results are reported in Table 1. The presence of 1% DMSO (v/v) did not affect fungal growth. Overall, all plants showed a fungicidal effect (MIC and MFC values of each extract were exactly at the same concentration) against either species, with MIC/MFC values of the active extracts ranging from 15.6 to 250.0 µg/mL. The highest values of MIC/MFC (1,000.0 µg/mL and 500.0 µg/mL, against both species) were observed for AE of Schinus terebinthifolius and for AE of Piptadenia colubrina,and these extracts were considered inactive according to the criteria of SCORZONIet al.18. The lowest MIC/MFC values (15.6 µg/mL), against both species, were obtained for AcE ofParapiptadenia rigida. No differences in MIC/MFC values were observed between the solvents used for preparing the crude extracts ofLibidibia ferrea, Persea americana, Mimosa tenuiflora, and Mimosa ophthalmocentra, and except for the last species, all crude extracts displayed strong antifungal activity. For the other plants, there was a two- to 16-fold decrease in MIC/MFC values depending on the solvent used in extract preparations. Indeed, the type of solvent used during extraction can affect both the yield and the number and type of phytochemicals obtained. The aqueous mixtures of ethanol or acetone have been used to extract mainly soluble polyphenols from plant material20. Polyphenols comprise a large family of compounds found in several plant species, which have a chemical structure consisting of at least one phenolic ring and which display various biological properties, including antifungal activity21. A high content of condensed tannins was detected in AcE of stem bark of P. rigida 22, which may be responsible for the antidermatophytic activity reported previously21 and in the present study. However, the presence of other phytochemical groups cannot be ruled out.

Currently, there are still few reports describing the antifungal activity of extracts or substances derived from plant species against dermatophytes6 , 11 , 12 , 13 , 14 , 15. Among the plant species analyzed here, most have shown antifungal activity againstCandida and Cryptococcus species11 , 16 , 23 , 24 , 25 , 26 , 27 , 28. More precisely, most extracts analyzed in this study were active against four Candida species (C. albicans, C. dubliniensis, C. glabrata, and C. krusei) with a MIC range of 15.62 to 500.0 mg/mL. However, in contrast to our results, MFC values were at least four times greater than the MIC values16. Regarding dermatophytes, only the antifungal activity of E. uniflora, L. ferrea, M. tenuiflora, and P. guajava had been previously described. An inhibitory effect on dermatophytes growth was observed for essential oils29 and EE from leaves of E. uniflora cultivated in the Brazilian cerrado14. In the latter study, the authors reported MIC values of EE ranging from 500.0 to 1,000.0 mg/mL for T. rubrumand T. mentagrophytes 14, which differs from our results (125.0 mg/mL for both species). Chemical analyses of E. uniflora leaves revealed the presence of terpenes, mainly oxygenated sesquiterpenes, which may be responsible for the antifungal activity21 , 25 , 29. By using an agar well-diffusion method, LIMA et al.30 reported that 2,500.0 mg/mL of AE and EE of stem bark fromM. tenuiflora inhibited the growth of clinical isolates of four dermatophyte species (T. rubrum, T. mentagrophytes, Microscopum canis and, Epidermophyton floccosum). The antidermatophytic activity of M. tenuiflora may be attributed to tannins21, which represent the major class of compounds in stem bark of this plant31. In the study conducted by DUTTA et al.32, tinctures from leaves and stem bark of P. guajava (which mimics the popular use) at concentrations ranging from 5 to 15% exhibited a fungicidal effect on dermatophytes. SUWANMANEE et al.33 reported the antidermatophytic activity of AE of leaves from P. guajavacultivated in Thailand, with MIC of 2,670.0 mg/mL, and 3,330.0 mg/mL for T. rubrum and T. mentagrophytes, respectively, which also differ from our results (125.0 mg/mL for both species). Several compounds have been detected in the leaves of P. guajava 34, and the antibacterial and antifungal activities may be due to the content of polyphenols, such as flavonoids, and hydrolysable and condensed tannins21 , 22 , 35 , 36 ,. Condensed tannins have also been detected in the aqueous extract of L. ferrea 22, which in this study showed strong activity against both species of dermatophytes. By using the agar well-diffusion assay, LIMA et al.30 found that 1,250.0 mg/mL AE and EE of L. ferreainhibited the growth of clinical isolates of T. rubrum, T. mentagrophytes, M. canis, and E. floccosum. Finally, SCHMOURLO et al. 28 evaluated the AE obtained by decoction of aerial parts of S. terebinthifolius on the growth of T. rubrum, by using an agar well-diffusion (1,000.0 mg/mL) and broth microdilution (10-6 mg/mL - 1,000.0 mg/mL) methods, and the results showed no inhibitory effect for this extract. Similarly, no antidermatophytic activity was detected for the AE from the stem bark of this plant in the present study. On the other hand, the EE of bark showed strong antifungal activity with MIC/MFC of 62.5 mg/mL for both fungal species. The differences observed between the results described in the literature and the present study may be due to the conditions of plant cultivation, such as soil type and climate, which affect the production of active compounds37, extraction systems for plant compounds20, and the antifungal susceptibility testing, which can affect the MIC values of the extracts18.

According to the MIC/MFC values obtained with dermatophyte reference strains and the availability of plant extracts, E. uniflora, L. ferrea, and P. americana were also tested in clinical isolates, which were all susceptible to terbinafine (MIC ≤ 0.004 µg/mL, Table 2). Selected extracts showed a fungicidal effect (MIC=MFC) against all clinical isolates of T. rubrum and T. mentagrophytes complex, corroborating the results obtained with the reference strains. MIC/MFC values of extracts against isolates of T. rubrum ranged from 7.8 to 250.0 µg/mL for E. uniflora (AcE), 7.8 to 62.5 µg/mL for L. ferrea (AE), and 15.6 to 62.5 µg/mL for P. americana (AcE). For T. mentagrophytes complex, the MIC/MFC values of these extracts ranged from 7.8 to 62.5 µg/mL for E. uniflora, 15.6 to 62.5 µg/mL for L. ferrea, and 7.8 to 62.5 µg/mL for P. americana. Table 2 shows the MIC50 and MIC90 of extracts of these three plant species, for 50 and 90% growth-inhibition of all isolates, respectively. Overall, a slight increase (twofold) in the MIC90 values was observed for all extracts against both fungal species. However, except for AcE of E. uniflora, which exhibited moderate activity against most clinical isolates (MIC90 of 125.0 µg/mL), the AE of L. ferrea(MIC90 of 62.5 µg/mL) and AcE of P. americana(MIC90 of 62.5 µg/mL) showed strong activity against most isolates.

Table 2

MIC50 and MIC90 of extracts ofLibidibia ferrea, Persea americanaand Eugenia uniflora against 33 clinical isolates of dermatophytes.

Isolates	N	Libidibia ferrea (AE)	Persea americana (AcE)	Eugenia uniflora (AcE)
		MIC50	MIC90	MIC50	MIC90	MIC50	MIC90
T. rubrum	24	31.3	62.5	31.3	62.5	62.5	125.0
T. mentagrophytes complex	9	31.3	62.5	62.5	62.5	31.3	62.5

N - number of isolates; MIC50 and MIC90: concentration (µg/mL) of each extract that inhibited growth by 50 and 90% of all isolates, respectively.

In conclusion, all the caatinga plants studied showed a fungicidal effect against T. rubrum and T. mentagrophytescomplex. Except for E. uniflora,L. ferrea, M. tenuiflora, and P. guajava, the antifungal activity of S. terebinthifolius, P. colubrina, P. rigida, M. ophthalmocentra, and P. americana against both fungal species was described for the first time. These results are useful as a preliminary step towards further antidermatophytic-guided studies of plant species from the Braziliancaatinga.