Ethyl acetate fractions of Myrciaria floribunda, Ocotea pulchella, and Ocotea notata exhibit promising in vitro activity against Sporothrix brasiliensis isolates with low susceptibility to itraconazole

Abstract

Sporothrix brasiliensis with low susceptibility isolates were described from the Brazilian zoonotic sporotrichosis hyperendemics. The aim of this work was to evaluate distinct fractions of Ocotea pulchella, Ocotea notata, Myrciaria floribunda, and Hypericum brasiliense plant extracts against itraconazole-sensitive and low susceptibility S. brasiliensis isolates. Crude extracts were tested against clinical isolates and the ATCC MYA4823 to determine the minimum inhibitory concentrations (MICs) and fungicidal or fungistatic activities (MFC). A high MICs and MFCs amplitude (1 – > 128 µg/mL) were obtained for seven extracts. The highest antimicrobial activities against sensitive S. brasiliensis were displayed by the ethyl acetate extracts of O. notata (MIC = 2–128 μg/mL) and M. floribunda (MIC = 1–8 μg/mL). A fungicidal effect was observed for all fraction extracts. Ocotea spp. and M. floribunda ethyl acetate extracts provide promising profiles against itraconazole-sensitive or low susceptibility S. brasiliensis. Future studies will determine if these extracts can contribute as alternative therapies to this neglected zoonosis.

Introduction

Sporotrichosis is a traumatically implanted subcutaneous mycosis that affects animals and humans worldwide [1]. In Brazil, a three-decade epidemics due to zoonotic transmission resulted in a serious public health neglected mycosis, in which diseased cat play a central role [2, 3]. In this country, Sporothrix brasiliensis is considered to be the most virulent species [4] responsible for the majority of cases, with reports of isolates showing diminished susceptibility to antifungals [5, 6]. A considerable rate of S. brasiliensis coinfected felines displaying distinct drug susceptibility profiles was also described [7]. Furthermore, zoonotic transmission of this species in neighboring countries, such as Argentina, highlights its importance as a potential Public Health issue in South America [8].

Itraconazole is the drug of choice for both human and feline sporotrichosis [9, 10]. However, treating feline mycosis is still a challenge due to the limited number of antifungal agents, their adverse effects, and ideal drug dosage availability. In addition, it is a costly and prolonged therapy, involving the isolation of the patient, which altogether contributes to treatment interruption and animal abandonment [10]. Likewise, there is a pressing need for new therapeutic alternatives for cat sporotrichosis as an important measure of controlling this hyperendemic zoonosis.

The Brazilian flora is well known to be composed of different plant genera, whose extracts are described as having therapeutic properties [11, 12]. Crude extracts and essential oils are alternatives considered of great interest due to various biological activities, including antimicrobial activity against fungi of medical and veterinary importance, such as Sporothrix schenckii and S. brasiliensis [13, 14].

Ocotea pulchella, Ocotea notata, Hypericum brasiliense, and Myrciaria floribunda were previously described as containing distinct biological properties such as anti-inflammatory [15], antiproliferative [16], insecticide [17] as well as antimicrobial activities such as antiviral [18, 19] and antibacterial [11, 20, 21], including antifungal properties against Cladosporium cucumerinum, a fungal plant pathogen [22], and also against human yeast pathogenic species of the genus Candida [21].

To our knowledge, the present study is the first to evaluate the potential antifungal properties of these seven crude extracts of three different plant genera (Ocotea, Myrciaria, and Hypericum) against S. brasiliensis. The aim of the present work was to evaluate distinct fractions of Ocotea pulchella, Ocotea notata, Myrciaria floribunda, and Hypericum brasiliense extracts for their potency against Sporothrix brasiliensis itraconazole “sensitive” and “low susceptibility” clinical isolates.

Material and methods

Ethical statement

The study was approved by and conducted according to the norms of the Ethics Committee on Animal Use by the Fluminense Federal University, Rio de Janeiro / BR (CEUA-UFF, protocol number 208/2012; December 13th, 2012 and protocol number 7561040518; June 14th, 2018). The authorization for academic purposes collection, nº 13,659–13, was issued by SisBio (Chico Mendes Institute for Biodiversity Conservation, Ministry of Environment). All plant species were registered under nº A0D648D in the National System for the Management of Genetic Heritage and Traditional Knowledge (SISGEN).

Clinical epidemiological data

S. brasiliensis isolates were obtained from the lesions of six domestic cats with sporotrichosis, clinically classified as responsive or unresponsive to treatment with itraconazole, all residing in the hyperendemic region of Rio de Janeiro, Brazil. Treatment varied from 2 to 60 months, and, as expected, S. brasiliensis showed MICs compatible with reduced sensitivity to itraconazole or “non-Wild Type” phenotype, whenever obtained from unresponsive cats. Figure 1 shows the clinical-epidemiological data referring to the diseased felines from which the S. brasiliensis isolates were obtained. It is important to emphasize that more than one episode of sporotrichosis relapse was reported for two domestic felines: the carriers of the WT3 and NWT1 isolates. In addition, the cat carrying the NWT1 was submitted to tail amputation and currently suffers from a new episode of sporotrichosis in the tail stump. Overall, itraconazole treatment ranged from 2 to more than 120 months, in a dosage of 100 mg/cat/day.

Fig. 1.

Clinical-epidemiological and laboratorial data of domestic cats infected by the six S. brasiliensis isolates investigated in the present study

Fungal isolates

Clinical S. brasiliensis isolates were later genotyped as Sporothrix brasiliensis by a species-specific PCR, as previously described [3]. Briefly, the isolate selection occurred among those from the Center for Microrganisms’ Investigation fungal (CIM) collection, based on the previous itraconazole in vitro results, following the criteria proposed by Espinel-Ingroff (2017) [23], as Wild Type (MIC ≤ 2 µg/mL) or non-Wild Type (MIC ≥ 4 µg/mL). These were further designated as “WT1,” “WT2,” “WT3” and “NWT1,” “NWT2,” “NWT3,” respectively, and reference strain, S. brasiliensis ATCC MYA-4823 (Sbra) was included in all experiments.

Growth conditions

To ensure standardized growing conditions, the clinical isolates were maintained by cryopreservation in its yeast phase at − 20 °C in the CIM-UFF fungus collection until its reactivation for the conduction of the experiments. For reactivation, cryotubes were defrosted with subsequent replication on Sabouraud agar 2% dextrose (Becton, Dickinson, and Company—BD, NJ, USA) and incubated at room temperature (25 °C) for 5 days for microbial growth in the form of conidia.

Crude extracts

Seven different plant fraction extracts obtained from Ocotea pulchella, Ocotea notata, Hypericum brasiliense, and Myrciaria floribunda were tested for potential antifungal activity against clinical isolates and standard strain of S. brasiliensis (Sbra; Table 1). These crude extracts were provided by the Natural Products Technology Laboratory (LTPN), Faculty of Pharmacy, Fluminense Federal University. O. notata, O. pulchella, and M. floribunda were collected in the Restinga de Jurubatiba National Park (PNRJ), in the municipality of Carapebus (22° 13′ 53″ S, 41° 35′ 46″ W), the northern area of Rio de Janeiro state, Brazil. Hypericum brasiliense was collected in the municipality of Trajano de Moraes (22° 12 ′ 17″ S, 43° 11′ 35″ W), Mountain Region of the same state.

Table 1.

Taxonomic information on the crude extracts investigated and the respective diluents and deposit in the herbarium

Scientific name	Family	Extracts/fractions	Herbarium deposit
Ocotea pulchella	Lauraceae	Ethanol; Dichloromethane; Ethyl acetate	RFFP 14,500
Ocotea notata	Lauraceae	Ethyl acetate	RB 2300
Hypericum brasiliense	Hypericaceae	Hexane	RB 4837
Myrciaria floribunda	Myrtaceae	Dichloromethane; Ethyl acetate	RFFP 13,789

RFFP: Herbarium of the Faculty of Teacher Education, (State University of Rio de Janeiro, campus São Gonçalo/RJ, responsible: Dra. Ana Angélica Monteiro de Barros; RB: Herbarium of the Botanical Garden of Rio de Janeiro/RJ, responsible: Dra Rafaela Campostrini Forzza

Leaves of O. notata, O. pulchella, M. floribunda, and the Hypericum brasiliense whole plant were separately dried at 45 °C in an air circulation oven, subjected to grinding, and then extracted by maceration until depletion using 5 L of 96% ethanol. After filtration, the solvents were evaporated under vacuum in a rotary evaporator, obtaining the crude ethanol extracts that were then lyophilized. Finally, each crude extract was resuspended and partitioned in solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, and butanol) to obtain the respective fraction extracts.

Antifungal susceptibility assays

Susceptibility testing was performed according to the standardized broth technique described by the CLSI in documents M38-A2 and M27-A3 for yeast-like cells and conidia [24, 25]. The antifungal used as the experimental control was itraconazole (Sigma-Aldrich, MO, USA).

Minimum fungicidal concentrations (MFCs) were obtained from subcultures on Petri dishes, including Sabouraud agar 2% dextrose (SDA; Becton, Dickinson, and Company—BD, Franklin Lakes, NJ, USA), filamentous phase, and brain heart infusion (BHI; Becton, Dickinson, and Company—BD, Franklin Lakes, NJ, USA), yeast phase, with 30 μL of the MIC cell suspension. In plates, they were incubated at 25 °C for 5 days (conidia) and at 37 °C for 7 days (yeast). After reading the number of colonies, the MFC was established as the lowest fraction extract concentration capable of eliminating 99.9% of the fungal growth [26]. A plant fraction extract presented a “'fungicide” effect whenever its MFC was equal or up to 4 × greater than its MIC, whereas the “'fungistatic” effect was determined whenever this value was greater than 4 the MIC, as previously referred [6, 7].

Results

A wide variation was observed for both MICs and MFCs (1 – > 128 µg/mL) obtained for the seven crude extracts (four plant species) against S. brasiliensis saprophytic and yeast forms (Table 2). Overall, M. floribunda^ea, O. pulchella^e, and O. pulchella^d extracts showed different MICs and MFCs, with lower values against the yeast forms of the S. brasiliensis isolates, either WT or NWT. The highest antimicrobial activities against the two morphotypes of the NWT S. brasiliensis were displayed by the ethyl acetate extracts of O. notata^ea (MIC = 2 – 128 μg/mL) and M. floribunda^ea (MIC = 1 – 8 μg/mL). All fraction extracts exhibited fungicidal profiles. MIC and MFC ranged from 16 to > 128 µg/mL against H. brasiliense, regardless of the morphotype. The geometric means (GMs) obtained by the filamentous form of WT/NWT isolates were higher than those against the parasitic form of S. brasiliensis, as shown in Table 3.

Table 2.

Comparison of the in vitro susceptibility evaluation (µg/mL) of the six isolated Sporothrix brasiliensis (conidial and yeast form) planktonic cells to crude extract

Azole/crude extracts		ATCC/clinical isolates
		S. bra		WT 1		WT 2		WT 3		NWT 1		NWT 2		NWT 3
		C	Y	C	Y	C	Y	C	Y	C	Y	C	Y	C	Y
Itraconazole	MIC	1	2	16	1	16	4	16	1	32	32	16	16	16	8
	MFC	8	16	64	8	32	32	64	8	64	> 128	64	128	32	128
O. pulchella ea	MIC	> 128	16	> 128	32	> 128	16	64	64	> 128	> 128	64	16	128	2
	MFC	> 128	16	> 128	32	> 128	32	64	32	> 128	> 128	64	8	128	1
O. pulchella e	MIC	4	1	> 128	32	> 128	1	64	1	> 128	> 128	64	2	64	32
	MFC	4	1	> 128	16	> 128	2	32	1	> 128	> 128	32	2	64	32
O. pulchella d	MIC	128	> 128	64	16	64	8	128	128	64	128	32	> 128	64	8
	MFC	128	> 128	64	16	64	8	128	128	64	128	32	> 128	64	16
M. floribunda d	MIC	32	> 128	> 128	> 128	128	4	2	> 128	16	16	16	128	16	32
	MFC	32	> 128	> 128	> 128	128	8	2	> 128	16	32	8	128	16	32
M. floribundaea	MIC	16	32	128	> 128	> 128	128	8	128	8	4	2	1	4	8
	MFC	16	32	> 128	> 128	> 128	128	8	128	8	4	2	1	4	8
O. notata ea	MIC	128	64	> 128	8	> 128	16	8	64	2	2	64	64	128	16
	MFC	128	64	> 128	8	> 128	8	16	64	4	4	64	64	128	16
H. brasiliense h	MIC	128	128	128	> 128	> 128	64	16	128	16	> 128	128	64	64	128
	MFC	128	128	128	> 128	> 128	64	16	128	32	> 128	128	64	64	128

Sbra: Sporothrix brasiliensis (ATCC MYA-4823), MIC: minimum inhibitory concentration, MFC: minimum fungicidal concentration, C: conidia, Y: yeast

^eaFraction ethyl acetate

^eFraction ethanol

^dFraction dichloromethane

^hFraction hexane

Table 3.

Geometric means generated from the in vitro susceptibility test (µg/mL) of the six isolated Sporothrix brasiliensis (yeast form and conidial form) planktonic cells to crude extracts

Azole/crude extracts		ATCC/clinical isolates
		S. bra (C)	S. bra (Y)	WT (C)	NWT (C)	WT (Y)	NWT (Y)
				GM	GM	GM	GM
Itraconazole	MIC	1	2	16	21.3	2	18,7
	MFC	8	16	53.3	53.3	16	128
O. pulchella ea	MIC	> 128	16	106.7	106.7	37.3	73
	MFC	> 128	16	106.7	106.7	32	45.7
O. pulchella e	MIC	4	1	106.7	106.7	11.3	54
	MFC	4	1	96	74.7	6.3	54
O. pulchella d	MIC	128	> 128	85.3	53.3	50.7	88
	MFC	128	> 128	85.3	53.3	50.7	90.7
M. floribunda d	MIC	32	> 128	86	16	86.7	58.3
	MFC	32	> 128	86	13.3	88	64
M. floribundaea	MIC	16	32	88	4.7	128	4.3
	MFC	16	32	88	4.7	128	4.3
O. notata ea	MIC	128	64	88	64.7	29.3	27.3
	MFC	128	64	90.7	65.3	26.7	28
H. brasiliense h	MIC	128	128	90.7	64.3	106.7	106.7
	MFC	128	128	90.7	74.7	106.7	106.7

Sbra: Sporothrix brasiliensis (ATCC MYA-4823), MIC: minimum inhibitory concentration, MFC: minimum fungicidal concentration, GM: geometric mean, C: conidia, Y: yeast

^eaFraction ethyl acetate

^eFraction ethanol

^dFraction dichloromethane

^hFraction hexane

Discussion

The Ocotea extracts provided MICs and MFCs which can be interpreted as promising values and worthy of future research investment for sporotrichosis treatment. Besides, the O. notata ethyl acetate fraction extract was particularly interesting against the parasitic form of S. brasiliensis. This could be of great importance on diminishing fungal load in cat’s lesions [27], mainly if new antifungal formulations are to be considered [28], in order to promptly reduce zoonotic transmission. Previously, other species from this genus displayed antifungal properties, such as Ocotea glomerata and Ocotea quixos against Candida krusei [29] and Candida albicans [30], respectively. Yamaguchi and coworkers (2011) evaluated the in vitro potential of the ethanol extract from Ocotea odorifera leaves on Candida parapsilosis with a MIC of 1.5 µg/mL [31] while Ocotea macrophylla inhibited the opportunistic filamentous fungi Fusarium oxysporum at 250 µg/L [30].

Myrciaria floribunda (H. West ex Willd.) O. Berg, widely distributed from North to South of Brazil, is a native plant species of the Atlantic Rain Forest [32]. In the current study, the ethyl acetate fraction of M. floribunda was the most effective for in vitro killing the yeasts of S. brasiliensis NWTs isolates. M. floribunda essential oil antifungal profile was demonstrated before against C. albicans contrarily to the observed for the methanol extract against C. albicans and C. krusei [21]. Its chemical profile description revealed several triterpenoids similar to betulinic acid, such as 2α,6α,30-trihydroxybetulinic acid, betulinic aldehyde, 6α-hydroxybetulinic, and the flavonoids catechin, quercetrin, and mirycitrin. In addition, Tietbohl (2014) [32] isolated betulinic acid and myricetin 3-O-galactoside, from the M. floribunda ethyl acetate extract. Furthermore, Tietbohl and coworkers (2017) [16] described the total growth inhibition (TGI) of M. floribunda ethyl acetate extract from leaves in immortalized human skin keratinocytes (HaCat) as 213.6 µg/mL. In this context, the concentrations for S. brasiliensis inhibition by the ethyl acetate fraction extract from M. floribunda (4.3 – 128 µg/mL) are lower than the TGI described by Tietbohl et al. (2017) [16] suggesting the safety of the extract to normal cells for both itraconazole S. brasiliensis clinical and standard strains. Further investigation will be able to prove if the high contents of flavonoids and triterpenoids from the ethyl acetate extract of M. floribunda leaves are responsible for the anti-S. brasiliensis in vitro activity.

Although displaying a sensitivity profile compatible with a potential antifungal candidate, the hexane fraction of H. brasiliense showed the highest MIC and MFC values (16 – > 128 µg/mL). Hypericum spp. is composed by several metabolites, such as ɣ-pyrone and xanthones, phloroglucinols as japonicine A, isouliginosin B and uliginosin B [33], hyperbrasilol B and C, flavonoids as kaempferol, quercetin, quercitrin and isoquercitrin, hyperoside, and luteolin [11, 34]. These extracts have been previously capable of antibacterial and antifungal activities against Bacillus subtilis, Staphylococcus aureus, the phytopathogen Cladosporium cucumerinum [22] and Candida albicans [21].

Few reports described the antifungal properties of plant origin compounds against Sporothrix spp., mainly concentrated in essential oils [13]. Cleff and collaborators (2008) published promising in vitro results of the essential oil of Origanum vulgare against S. schenckii from diseased domestic cats with MICs of 250 to 500 µg/mL. Another in vitro study with extracts of Origanum manjorana (marjoram) and Rosmarinus officinalis (rosemary) against S. brasiliensis from animals residing in the Brazilian South Region revealed MICs around 40 mg/mL⁻¹ [35]. In the present study, Lauraceae, Hypericaceae, and Myrtaceae plant families’ fractions extracts exhibited anti-S. brasiliensis in vitro inhibition. However, the ethyl acetate fractions of Ocotea spp. and Myrciaria floribunda showed the most promising antifungal profiles against S. brasiliensis. More importantly, MICs and MFCs were even lower for the yeast forms of the itraconazole low susceptibility strains of S. brasiliensis. An important issue to be addressed is the toxicological profile of the studied plant extracts, a limitation we acknowledge. As a matter of fact, ongoing studies of our group (unpublished data) have shown that Myrciaria floribunda and Ocotea pulchella did not show evidence of cytotoxicity either to cancer cells or normal fibroblasts in vitro cultures within the concentrations tested in the present investigation (up to 1000 µg mL⁻¹).

Altogether, these results encourage new research efforts on natural compounds as important alternatives for feline sporotrichosis treatment. One hypothesis that is presented is the possibility that the antifungal activity is related to the presence of plant metabolite substances, such as xanthones and flavonoids, as previously described against C. albicans [36, 37] and Cryptococcus neoformans [38].

Conclusions

M. floribunda and Ocotea spp. ethyl acetate extracts are capable of high antimicrobial activity against itraconazole-sensitive and low susceptibility S. brasiliensis clinical isolates. Future studies, including in vivo experiments, will be able to confirm that these fraction extracts can contribute as future alternative therapies to control and prevent sporotrichosis, a severe public health concern.

Author contribution

L.C.S.V.S.: conceptualization, biological investigation, data curation, writing original draft. N.F.R.: conceptualization, biological investigation, data curation, writing original draft. L.M.A.: biological investigation, writing the original draft. S.R.L.S.S.: biological investigation, writing the original draft. B.A.P., B.K.R.: supervision, manuscript reviewing and editing. RCSS: biological investigation, writing the original draft. F.P.M., B.K.R.: biological investigation, writing the original draft. H.C.C.: validation, supervision, manuscript reviewing and editing. R.L.D.M.: visualization, investigation, manuscript reviewing and editing. L.R.: visualization, supervision, manuscript reviewing and editing. A.R.S.B.: visualization, supervision, manuscript reviewing and editing, project administration.

Funding

This study was supported by grants from the Brazilian Agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (PIBIC-CNPq-UFF, Brazil); Fundação de Amparo à Pesquisa no Estado do Rio de Janeiro, Brazil (FAPERJ-E-26/103.198/2011; E-26/010.001882/2014), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil (CAPES)—Financial Code 001, Rede Micologia RJ and PROEX-MEC. A.R.S.B, R.L.D.M. and L.M.R. are research fellows of CNPq (PQ-CNPq).

Data Availability

All data generated or analyzed during this study were included in this published article. Further details on particular patients are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare no competing interests.