Anti-Sporothrix spp. activity of medicinal plants

ABSTRACT

Cases of sporotrichosis in humans and animals without satisfactory clinical response have increased, a warning sign of strains resistant to conventional antifungal agents. The urgent search for alternative therapies was an incentive for research on medicinal plants with anti-Sporothrix spp. properties. A bibliographic survey was performed based on scientific papers about in vitro and in vivo antifungal activity of essential oils and extracts of plants in differents solvents against the fungal of the Sporothrix schenckii complex. The study methodology consisted of a literature review in Google Scholar, Science Direct, Pubmed, Bireme and Springer link with papers from 1986 to 2015. We found 141 species of plants that were investigated, of which 100 species were concentrated in 39 botanical families that had confirmed anti-Sporothrix activity. Combretaceae, Asteraceae and Lamiaceae represented the botanical families with the greatest number of plants species with antifungal potential, using different methodologies. However, there are few studies with medicinal plants in experimental infection in animals that prove their activity in the treatment of sporotrichosis. It reinforces the need for further research related to standardization of in vitro methodologies and in vivo studies related to safety and to toxicity potential of these plants with anti-Sporothrix spp. activity.

Uniterms: Sporothrix schenckii complex; Antifungals; Combretaceae/pharmacognosy; Asteraceae/pharmacognosy; Lamiaceae/pharmacognosy; Medicinal plants/antifungal activity

RESUMO

Casos de esporotricose em humanos e animais sem resposta clínica satisfatória têm aumentado, sinal de alarme para o surgimento de cepas resistentes aos antifúngicos convencionais. A urgente busca por alternativas terapêuticas tem incentivado as pesquisas em plantas medicinais com atividade anti-Sporothrix spp. Um levantamento bibliográfico foi realizado com base em artigos científicos sobre a atividade antifúngica in vitro e in vivo de óleos essenciais e extratos de plantas preparados em diferentes solventes contra o complexo Sporothrix schenckii. A metodologia do estudo consistiu em uma revisão bibliográfica em Google Scholar, Science Direct, Pubmed, Bireme e Springer link com artigos desde 1986 até 2015. Foram encontradas 141 espécies de plantas já investigadas, das quais 100 espécies concentradas em 39 famílias botânicas apresentaram atividade anti-Sporothrix spp. confirmada. Combretaceae, Asteraceae e Lamiaceae representaram as famílias botânicas com maior número de espécies vegetais com potencial antifúngico, empregando diferentes metodologias. Entretanto, há poucos estudos com plantas medicinais em infecção experimental animal comprovando sua atividade no tratamento da esporotricose. Reforça-se a necessidade de mais pesquisas relacionadas à padronização de metodologias in vitro e a estudos in vivo relacionados à segurança e potencial tóxico dessas plantas com atividade anti-Sporothrix spp.

Unitermos: Sporothrix schenckii; Antifúngicos; Combretaceae/farmacognosia; Asteraceae/farmacognosia; Lamiaceae/farmacognosia; Plantas medicinais/atividade antifúngica

INTRODUCTION

Among the diseases with zoonotic potential, sporotrichosis is recognized as an emerging mycosis with worldwide occurrence that tends to increase exponentially in the number of cases in domestic animals and humans and is considered a neglected epidemic in the state of Rio de Janeiro, Brazil (Schubach, Barros, Wanke, 2008; Barros et al., 2010; Silva et al., 2012; Rodrigues et al., 2013). This disease is the most common subcutaneous mycosis in the Americas, especially in Brazil, Mexico, Colombia and Peru, and also occurs in South Africa, India and Japan (Carrada-Bravo, Olvera-Macías, 2013). The sources of environmental fungus infection include soil, decomposed vegetation, tree stems, and other elements. The infection may also occur through bites and scratches of cats that may carry Sporothrix spp. conidia and, through inoculation, transmit the fungal agent into the dermis (Larsson, 2011; Romeo, Criseo, 2013). Although the treatment with antifungal drugs is effective, in vitro studies acknowleded the emergence of strains of Sporothrix spp. resistant to antifungals (Meinerz et al., 2007; Marimon et al., 2008; Rodrigues et al., 2013; Stopiglia et al., 2014).

There is a continuing and urgent need to discover new antifungal compounds and to understand their mechanisms of action (Kontoyiannis, Lewis, 2002; Cleff et al., 2008; Gaitán et al. 2011). It is known that the use of plants and derivatives for medicinal purposes is an ancient practice used worldwide, primarily in Eastern cultures, and is utilized by up to 80% of the population of developing countries and has been increasingly used in the West, according to data from the World Health Organization in the 1990's (Veiga Jr., Pinto, 2005).

Faced with resistant cases of sporotrichosis, the search for alternative therapies in their treatment has increasingly been conducted by several international researchers, whose papers reveal the potential anti-Sporothrix spp. of certain medicinal plants (Rojas et al., 2003; Masoko, Picard, Eloff, 2007; Cleff et al., 2008; Suleiman et al., 2009; Johann et al., 2011; Joshi et al., 2011; Stopiglia et al., 2011). However, there is a lack of standardization of tests with medicinal plants and this makes the comparison of antifungal activity of the plants difficult. Thus, this review aimed for a new approach of sporotrichosis and grouped the studies in the medicinal plants with anti-Sporothrix spp. activity according to the methodology for antifungal evaluation. A bibliographic survey was performed based on scientific papers researched in Google Scholar, Science Direct, Pubmed, Bireme and Springer link and papers from 1986 to 2015 were evaluated.

SPOROTRICHOSIS AND ITS CONVENTIONAL THERAPY

Chronic or subacute, the clinical manifestations of sporotrichosis occur in dependence of the host immune status, commonly resulting in limited damage to the dermis, with or without involvement of the lymphatic system in humans and animals, which can produce subcutaneous nodules, and may also produce manifestations in the respiratory system, such as dyspnea, nasal discharge and sneezing (Schubach, Barros, Wanke, 2008; Barros et al., 2010; Madrid et al., 2012).

The conventional therapy for sporotrichosis includes the use of antifungal agents such as potassium iodide, itraconazole, ketoconazole, amphotericin B and terbinafine, among others, as well as the adoption of local thermal therapy and the surgical excision of the lesion (Meinerz et al., 2008; Gremião et al., 2009; Honse et al., 2010; Pereira et al., 2011; Gremião et al., 2011; Reis et al., 2012). The preferred antifungal therapy for cat and dogs with sporotrichosis has been itraconazole administered daily orally at the dose of 10 to 40 mg kg^-1 for a minimum period of three months and up to one year with no apparent adverse effects (Madrid et al., 2010; Larsson, 2011; Rossi et al., 2013). However, poor clinical response has been observed in cats with sporotrichosis (Gremião et al., 2011).

The daily use of potassium iodide in capsules at dose of 2.5 to 20 mg kg^-1 has provided clinical cure, although side effects may occur, as appetite loss and clinical signs of hepatotoxicity (Reis et al., 2012). The association of subcutaneous amphotericin B with oral itraconazole is also an alternative in cats with sporotrichosis refractory to itraconazole (Gremião et al., 2011). Furthermore, the association of itraconazole with (1-3) β-glucan promoted the case resolution after four weekly applications of glucan in a canine with refractory sporotrichosis to itraconazole by Sporothrix brasiliensis and it may be considered a promising alternative in cases of resistance to conventional therapy (Guterres et al., 2014). In humans, the recommended the daily dosage of 100 up to 400 mg kg^-1 of itraconazole has been succesfully used in a study with 645 patients, which the recovery rate was 94.6% (Barros et al., 2010).

Experimental models in rats showed better activity of terbinafine (250 mg kg^-1) compared to itraconazole (100 mg kg^-1) in treatment of systemic sporotrichosis (Meinerz et al., 2008), but in the cutaneous sporotrichosis, terbinafine (20 e 30 mg kg^-1) has shown little efficacy while itraconazole (10 mg kg^-1) showed good results (Antunes et al., 2009). In human isolates of S. schenckii, S. brasiliensis and S. globosa, terbinafine had better in vitro antifungal activity, followed by ketoconazole and fluconazole (Stopiglia et al., 2014).

The therapeutic protocol adopted must be associated with clinical monitoring of the patient, especially in felines because they are sensitive to many medications (Nobre et al., 2002; Madrid et al., 2012). Toxic effects has been cited in cats and dogs treated with azoles, such as anorexia, emesis and hepatotoxic signs, as well as lethal cases in cats treated with saturated sodium solutions or 20% potassium iodide (Larsson, 2011).

The difficulties in obtaining therapeutical success can be associated with treatment time and the high cost of drugs that has been discouraging to the animals´ owners, who often abandon the therapy. These factors coupled with the indiscriminate use of antifungal drugs contributed to the problem of antifungal resistance (Schubach et al., 2004; Marimon et al., 2008; Rodrigues et al., 2013). Unfortunately, in vitro studies showed the emergence of Sporothrix spp. resistant to antifungals, including itraconazole, which is used as the preferred therapy in sporotrichosis (Marimon et al., 2008; Rodrigues et al., 2013; Stopiglia et al., 2014).

The difficulties observed in the therapeutical cure in human and animals show that the current situation calls for new therapeutic alternatives and/or new complements to conventional antifungal therapy. It is know that medicinal plants present antimicrobial properties which have been increasingly reported in international studies and it is expected that plant extracts have different target sites than used by currently available antimicrobials in being effective against drug-resistant microbial pathogens (Gibbons, 2003), including resistant strains of Sporothrix spp.

Currently, the agent of sporotrichosis has been reclassified and related to several species of Sporothrix schenckii complex, such as S. brasiliensis, S. globosa, S. mexicana, S. albicans, S. inflata, S. schenckii var. luriei and S. schenckii var. schenckii; which are identified only by molecular biology techniques (Marimon et al., 2007). However, the studies on medicinal plants found in this review were only conducted with isolates of S. schenckii because this agent was, at the time, the only species identified in cases of sporotrichosis, since the genre had not yet been classified as Sporothrix schenckii complex.

PLANTS WITH ANTI- SPOROTHRIX SPP. POTENTIAL

Among the first reports of the search of plants with possible antifungal activity against S. schenckii, Minami and Oliveira (1986) studied alcoholic extract at 30% of Bidens pilosa (Compositae), a plant distributed in America, Asia and Africa, and they found no inhibitory antifungal activity. Fortunately, studies in the years following detected satisfactory in vitro activity of other plant species tested in different extracts types, concentrations and methodologies (Sinha, Gulati, 1990; Apisariyakul, Vanittanakom, Buddhasukh, 1995; Verástegui et al., 1996; Saikia et al., 2001; Rojas et al., 2003; Masoko, Picard, Eloff, 2005; Luqman et al., 2007; Damián-Badillo et al., 2008).

According to the queried data, 141 species of plants have been subjected to in vitro tests against S. schenckii, and anti-Sporothrix spp. activity was conferred in 100 species belonging to 39 botanical families, in which were highlighted the families of Combretaceae (31% - 31/100) (Masoko, Picard, Eloff, 2005; 2007; Suleiman et al., 2009), Asteraceae (11% - 11/100) (Rojas et al., 2003; Damián-Badillo et al., 2008; Salomão et al., 2008; Stopiglia et al., 2011) and Lamiaceae (7% - 07/100) (Sinha, Gulati, 1990; Luqman et al., 2007; Fernández, 2005; Cleff et al., 2008; Couto et al., 2015), which have the largest number of species with confirmed activity of interest. From the in vitro studies revised, a total of 233 extract products showed anti-Sporothrix spp. activity. The botanical information according to the consulted references is described in Table I.

TABLE I
Medicinal plants with confirmed anti-Sporothrix spp. activity and their taxonomical and anatomical identifications, extract types and the country where the study was conducted by the consulted references

Botanical Family	Plant Species	Plant Parts	Extract	Country	Reference
Agavaceae	Agave lecheguilla	Root	E	Mexico	Verástegui et al.,1996; 2008
		Leaves	E	Mexico	Verastegui et al., 2008
Agavaceae	Agave lophantha	Leaves	E	Mexico	Verastegui et al., 2008
Agavaceae	Agave picta	Leaves	E	Mexico	Verastegui et al., 2008
Agavaceae	Agave scabra	Leaves	E	Mexico	Verastegui et al., 2008
Anacardiaceae	Schinus terebinthifolius	Leaves, Stems	Di, E, EA, He	Brazil	Johann et al., 2007
Anacardiaceae	Loxostylis alata	Leaves	Ac, Aq, Bu, Ch, CT Di, He, Ch, M	South Africa, Nigeria	Suleiman et al., 2009; Suleiman, Naidoo, Eloff, 2012
Anacardiaceae	Protorhus longifolia	Leaves	Ac, Di, He, M	South Africa, Nigeria	Suleiman et al., 2009
Apiaceae	Cuminum cyminum	Seed	Aq, EO, Ha, M	India	Chaudhary, Husain, Ali, 2014
Araucariaceae	Araucaria spp.	Resin	E	Brazil	Salomão et al., 2008
Asteraceae	Artemisia ludoviciana	Leaves, Root, Flowers	Aq, EA, MC	Mexico	Damián-Badillo et al., 2008
Asteraceae	Heliopsis longipes	Leaves, Root, Flowers	Aq, EA, MC	Mexico	Damián-Badillo et al., 2008
Asteraceae	Tagetes lucida	Leaves, Root, Flowers	Aq, EA, MC	Mexico	Damián-Badillo et al., 2008
Asteraceae	Baccharis dracunculifolia	Resin	E	Brazil	Salomão et al., 2008
Asteraceae	Ophryosporus peruvianus	Leaves, Stems	E	Peru	Rojas et al., 2003
Asteraceae	Senecio culcitioides	Aerial Parts	E	Peru	Rojas et al., 2003
Asteraceae	Pterocaulon balansae	Aerial Parts	M	Brazil	Stopiglia et al., 2011
Asteraceae	Pterocaulon cordobense	Aerial Parts	M	Brazil	Stopiglia et al., 2011
Asteraceae	Pterocaulon lanatum	Aerial Parts	M	Brazil	Stopiglia et al., 2011
Asteraceae	Pterocaulon lorentzii	Aerial Parts	M	Brazil	Stopiglia et al., 2011
Asteraceae	Pterocaulon polystachyum	Aerial Parts	M	Brazil	Stopiglia et al., 2011
Burseraceae	Commiphora harveyi	Leaves	Ac, Di, He, M	South Africa, Nigeria	Suleiman et al., 2009
Caesalpiniaceae	Senna alata	Leaves	E	Guatemala	Fernández, 2005
Caesalpiniaceae	Hymenaea courbaril	Leaves	E	Guatemala	Sánchez, 2009
Campanulaceae	Lobelia pyramidalis	Aerial Parts	EO	Índia	Joshi et al., 2011
Chloranthaceae	Hedyosmum mexicanum	Leaves	E	Guatemala	Castellanos, 2007
Clusiaseae	Hypericum uliginosum	Grass	E	Guatemala	Fernández, 2005
Combretaceae	Terminalia brachystemma	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2005
Combretaceae	Terminalia gazensis	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2005
Combretaceae	Terminalia mollis	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2005
Combretaceae	Terminalia prunioides	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2005
Combretaceae	Terminalia sambesiaca	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2005
Combretaceae	Terminalia sericea	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2005; 2010b; Masoko et al., 2010a;
Combretaceae	Combretum acutifolium	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum albopunctactum	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007; 2010b; Masoko et al., 2010a
Combretaceae	Combretum apiculatum	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum bracteosum	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum caffrum	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum celastroides spp. celastroides	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum celastroides spp. orientale	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum collinum spp. suluense	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum collinum spp. taborense	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum edwardsii	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum erythrophyllum	Leaves	Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum hereroense	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum imberbe	Leaves	Di	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum kraussii	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum microphyllum	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum moggii	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum molle	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum mossambicense	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum nelsonii	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007; 2010b; Masoko et al., 2010a;
Combretaceae	Combretum padoides	Leaves	Ac, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum paniculatum	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum petrophilum	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum woodii	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum zeyheri	Leaves	Ac, Di, He, M	South Africa	Masoko, Picard, Eloff, 2007
Combretaceae	Combretum vendae	Leaves	Ac, Di, He, M	South Africa, Nigeria	Suleiman et al., 2009
Compositae	Baccharis glutinosa	Leaves	E	Mexico	Verástegui et al.,1996
Cornaceae	Curtisia dentata	Leaves, Stems	Ac, Di, He, M	South Africa	Shai et al., 2008
Euphorbiaceae	Croton ruizianus	Leaves, Stems	E	Peru	Rojas et al., 2003
Fabaceae	Glycyrrhiza glabra	Root	E, EA	Índia	Fatima et al., 2009
Fabaceae	Diphysa robinioides	Leaves	E	Guatemala	Sánchez, 2009
Geraniaceae	Perlagonium graveolens	-	EO	Índia	Singh et al., 2012
Hydrophyllaceae	Wigandia urens	Leaves, Stems	E	Peru	Rojas et al., 2003
Kirkiaceae	Kirkia wilmsii	Leaves	Ac, Di, He, M	South Africa, Nigeria	Suleiman et al., 2009
Labiateae	Satureja macrostema	Leaves, Root	Aq, EA, MC	Mexico	Damián-Badillo et al., 2008
Lamiaceae	Origanum vulgare	Aerial Parts	EO	Brazil	Cleff, 2008; Couto et al., 2015
Lamiaceae	Salvia lavanduloides	Leaves, Flowers	E	Guatemala	Fernández, 2005
Lamiaceae	Rosmarinus officinalis	Leaves	EO	Índia	Luqman et al., 2007
Lamiaceae	Ocimum basilicum	-	EO	Índia	Sinha, Gulati, 1990
Lamiaceae	Ocimum canum (americanum)	-	EO	Índia	Sinha, Gulati, 1990
Lamiaceae	Ocimum gratissimum	-	EO	Índia	Sinha, Gulati, 1990
Lamiaceae	Ocimum sanctum	-	EO	Índia	Sinha, Gulati, 1990
Lauraceae	Cinnamomum zeylanicum	Cortex	He	Mexico	Márquez, 2010
Liliaceae	Agapanthus africanus	Root	E	India	Singh et al., 2008
Meliaceae	Khaya anthotheca	Leaves	Ac, Di, He, M	South Africa, Nigeria	Suleiman et al., 2009
Moraceae	Dorstenia contrajerva	Root	E	Guatemala	Castellanos, 2007
Myristicaceae	Iryanthera lancifolia	Stems	E	Peru	Rojas et al., 2003
Myrtaceae	Eucalyptus camaldulensis	Leaves	M	Mexico	Márquez, 2010
Myrtaceae	Eucalyptus citriodora	-	EO	India	Akhtar et al., 2014
Myrtaceae	Psidium guajava	Leaves	Ac, He	Mexico	Márquez, 2010
Myrtaceae	Syzygium aromaticum	Sprouts	He	Mexico	Márquez, 2010
Myrtaceae	Psidium acutangulum	Leaves	E	Peru	Wen et al., 2011
Ochnaceae	Ochna natalitia	Leaves	Ac, Di, He, M	South Africa, Nigeria	Suleiman et al., 2009
Onagraceae	Oenothera multicaulis	Aerial Parts, Root	E	Peru	Rojas et al., 2003
Piperaceae	Piper abutiloides	Aerial Parts	Ha	Brazil	Johann et al., 2009
Poaceae	Cymbopogon flexuosus	-	EO	Índia	Saikia et al., 2001
Poaceae	Cymbopogon martini	-	EO	Índia	Saikia et al., 2001
Poaceae	Cymbopogon winterianus	-	EO	Índia	Saikia et al., 2001
Polygalaceae	Polygala campestris	Whole Plant	Di	Brazil	Johann et al., 2011
Polygalaceae	Polygala paniculata	Whole Plant	E	Brazil	Johann et al., 2011
Polygalaceae	Polygala sabulosa	Whole Plant	Di, EA	Brazil	Johann et al., 2011
Polygonaceae	Rumex acetosa	Leaves, Stems, Flowers	E	Brazil	Johann et al., 2007
Rosaceae	Rubus urticaefolius	Leaves, Stems, Flowers	E, EA	Brazil	Johann et al., 2007
Smilacaceae	Smilax domingensis	Leaves, Stems, Root, Flowers	E	Guatemala	Fernández, 2005
Solanaceae	Cestrum auriculatum	Leaves	E	Peru	Rojas et al., 2003
Valerianaceae	Valeriana prionophylla	Root	E	Guatemala	Fernández, 2005
Verbenaceae	Lippia graveolens	Leaves	E, He	Guatemala	Fernández, 2005; Beteta, 2005
Zingiberaceae	Curcuma longa	Rhizomes	EO	Thailand	Apisariyakul, Vanittanakom, Buddhasukh, 1995
Zygophyllaceae	Larrea tridentata	Leaves	E	Mexico	Verástegui et al.,1996

• Ac, acetone; Aq, aqueous; Bu, butanol; Di, dichloromethane; Ch, chloroform; CT, carbon tetrachloride; E, ethanol; EA, ethyl acetate; EO, essential oil; Ha, hydroalcoholic; He, hexane; M, methanol; MC, methanol, chloroform, - Not informed by consulted reference.

IN VITRO SUSCEPTIBILITY TESTS

In the studies reviewed, the methodology utilized for research on plants were agar diffusion (Apisariyakul et al., 1995; Saikia et al., 2001; Rojas et al., 2003; Luqman et al., 2007; Damián-Badillo et al., 2008; Verástegui et al., 2008; Fatima et al., 2009; Márquez, 2010; Joshi et al., 2011), broth microdilution technique (Sinha, Gulati, 1990; Verástegui et al., 1996; Johann et al., 2007; Luqman et al., 2007; Cleff et al., 2008; Fatima et al., 2009; Johann et al., 2009, 2011; Joshi et al., 2011), bioautographic assay (Betina, 1973; Johann et al., 2007; 2011), serial microdilution at 24 and 48 hours (Masoko, Picard, Eloff, 2005; 2007; Suleiman et al., 2009) and agar cup method (Salomão et al., 2008).

In the various methodologies utilized in the references, variation was observed in the values of concentration tested in extracts, making it difficult to compare the results and, thus, the designation of those plants with high, medium and low anti-Sporothrix spp. activity was not performed. According to Fennel et al. (2004), the lack of standardized methods for antimicrobial tests with natural products results in variations of minimum inhibitory concentration values. In addition, according to them, the factors that influence the outcome of antimicrobial activity of a plant are the technique applied, the strain of microorganism used, the origin of the plant and the time of its collection, the type of preparation and the amount of extract tested, among others.

Agar Diffusion

The anti-Sporothrix spp. activity of medicinal plants by agar diffusion test have been described and, for most authors, the studies focused on human isolates (Apisariyakul et al., 1995; Saikia et al., 2001; Rojas et al., 2003; Damián-Badillo et al., 2008; Luqman et al., 2008; Verástegui et al., 2008; Fatima et al., 2009; Márquez, 2010; Joshi et al., 2011; Wen et al., 2011), according to Table II.

TABLE II
Results of agar diffusion test done in plants with antifungal activity against Sporothrix schenckii complex and its characteristics of solvent extract tested, botanical family, plant species and fungal strain origin according to reference that demonstrated values of inhibition zone

Botanical Family	Plant Species	Solvent Extract	Origin of Sporothrix spp.	Inhibition Zone*	Reference
Agavaceae	Agave lecheguilla	Ethanol	Human	10 mm	Verastegui et al., 2008
Agavaceae	Agave lophantha	Ethanol	Human	10 mm	Verastegui et al., 2008
Agavaceae	Agave picta	Ethanol	Human	16 mm	Verastegui et al., 2008
Agavaceae	Agave scabra	Ethanol	Human	9 mm	Verastegui et al., 2008
Asteraceae	Artemisia ludoviciana	Ethanol	Unspecified	1to 1.99 cm	Damián-Badillo et al., 2008
Asteraceae	Artemisia ludoviciana	Methanol	Unspecified	1 to 1.99 cm	Damián-Badillo et al., 2008
Asteraceae	Heliopsis longipes	Ethanol	Unspecified	1 to 1.99 cm	Damián-Badillo et al., 2008
Asteraceae	Ophryosporus peruvianus	Ethanol	Human	13 mm	Rojas et al., 2003
Asteraceae	Senecio culcitioides	Ethanol	Human	15 mm	Rojas et al., 2003
Asteraceae	Tagetes lucida	Methanol	Unspecified	1 to 1.99 cm	Damián-Badillo et al., 2008
Asteraceae	Tagetes lucida	Ethanol	Unspecified	1 to 1.99 cm	Damián-Badillo et al., 2008
Campanulaceae	Lobelia pyramidalis	Essential oil	MTCC† 1359	13 mm	Joshi et al., 2011
Euphorbiaceae	Croton ruizianus	Ethanol	Human	13 mm	Rojas et al., 2003
Fabaceae	Glycyrrhiza glabra	Ethanol	AIIMS◊	5 mm	Fatima et al., 2009
Fabaceae	Glycyrrhiza glabra	Acetyl acetate	AIIMS◊	7 mm	Fatima et al., 2009
Hydrophyllaceae	Wigandia urens	Ethanol	Human	13 mm	Rojas et al., 2003
Labiateae	Satureja macrostema	Ethanol	Unspecified	1 to 1.99 cm	Damián-Badillo et al., 2008
Labiateae	Satureja macrostema	Methanol	Unspecified	1 to 1.99 cm	Damián-Badillo et al., 2008
Lamiaceae	Rosmarinus officinalis	Essential oil	AIIMS◊	< 5 mm	Luqman et al., 2007
Lauraceae	Cinnamomum zeylanicum	Hexane	Unspecified	14 mm	Márquez, 2010
Myristicaceae	Iryanthera lancifolia	Ethanol	Human	13 mm	Rojas et al., 2003
Myrtaceae	Eucalyptus camaldulensis	Acetone	Unspecified	12 mm	Márquez, 2010
Myrtaceae	Eucalyptus citriodora	Essential oil	MTCC† 1359	6.67 to 27 mm	Akhtar et al., 2014
Myrtaceae	Psidium acutangulum	Ethanol	IHEM§15503	22 mm	Wen et al., 2011
Myrtaceae	Psidium guajava	Methanol	Unspecified	16 mm	Márquez, 2010
Myrtaceae	Syzygium aromaticum	Hexane	Unspecified	43 mm	Márquez, 2010
Onagraceae	Oenothera multicaulis	Ethanol	Human	15 to 19 mm	Rojas et al., 2003
Poaceae	Cymbopogon flexuosus	Essential oil	Human	35 mm	Saikia et al., 2001
Poaceae	Cymbopogon martini	Essential oil	Human	19 mm	Saikia et al., 2001
Poaceae	Cymbopogon winterianus	Essential oil	Human	12 mm	Saikia et al., 2001
Solanaceae	Cestrum auriculatum	Ethanol	Human	25 mm	Rojas et al., 2003
Zingiberaceae	Curcuma longa	Essential oil	Human	3 mm	Apisariyakul, 1995

• *The resuls were described according authors reference and only species plants with results of inhibition zone were considered; ^†Microbial Type Culture Collection and Gene Bank (Chandigarh, India); ^◊All India Institute of Medical Sciences (New Delhi, India); ^§Instutute of Hygiene and Epidemiology-Mycology Laboratory (Brussels, Belgium).

An Agar diffusion test consists of defying a microorganism against a biologically active substance in a solid medium culture, in which the absense of microbial growth is related to the size of the halo formed, demonstrating sensitivity of the strain to the extract (Pinto, Kaneko, Ohara, 2003). Considered one of the simplest and most trusted methods of sensitivity, this method is advantageous because of its ease of implementation and interpretation of results, as well as its use of inexpensive reagents and antibiotics without requiring special equipment. However, there is a deficiency of mechanization or automation to this method (CLSI, 2007). In regards to plant extracts, the difficulties with the test are the diffusion of the extract in the culture medium and pH of the substances used (Ostrosky et al., 2008), as well as the presence of contaminants such as heavy metals, drugs and other medicinal plants (Veiga Jr., Pinto, 2005).

In this methodology, methanol-chloroformic and ethyl acetate extracts of Satureja macrostema (Labiateae) and Tagetes lucida, Artemisia ludoviciana and Heliopsis longipes (Asteraceae) gave growth inhibition of S. schenckii in the zone ranging from 1.0 to 1.99 cm (Damián-Badillo et al., 2008). Better results were observed in 10% ethanolic extract of Agave lecheguilla, A. picta, A. scabra and A. lophantha (Agavae) between 9.0 and 16 mm at concentrations of 4.0 to 6.0 mg mL^-1 (Verástegui et al., 2008). Popularly known as licorice, ethanolic extract from the roots of Glycyrrhiza glabra (Fabaceae) also inhibited the growth of S. schenckii at 5 mm diameter, as well as the active constituent of this plant - glabridin - and the ethyl acetate fraction of root with 6 and 7 mm, respectively (Fatima et al., 2009).

In traditional Peruvian medicine, a study with 36 ethanolic extracts of 24 plants from Asteraceae family showed the inhibition of S. schenckii to Croton ruizianus, Iryanthera lancifolia, Oenothera multicaulis, Ophryosporus peruvianus, Senecio culcitioides and Wigandia urens that promoted halo of growth inhibition of 13 to 19 mm and the best anti-Sporothrix spp. activity was related to Cestrum auriculatum with halo of 25 mm, including against strains with no susceptibility to amphotericin B and fluconazole, indicating the importance of the search for new compounds for resistant isolates (Rojas et al., 2003).

In another similar study, ethanolic extracts of Calycophyllum spruceanum (Rubiaceae), Spondia mombin (Anacardiaceae) and Thevetia peruviana (Apocynaceae) did not show anti-Sporothrix spp. activity, but leaves of Psidium acutangulum (Myrtaceae) inhibited S. schenckii with an halo of 22 mm and the active chemical composition "3'-formil-2',4',6'-trihidroxidihidrochalcone" was individually tested against strains resistants to amphotericin B and fluconazole by broth microdilution technique and showed MIC of 32 µg ml^-1, being considered promising for therapeutic alternatives in sporotrichosis (Wen et al., 2011).

Popularly known as clove, extracts of hexane and acetone at 20% of Syzygium aromaticum (Myrtaceae) had greater anti-Sporothrix spp. activity with inhibition growth of 28 mm to 43 mm in the disc diffusion method on agar and the extracts of Cinnamomum zeylanicum (Lauraceae), Eucalyptus camaldulensis and Psidium guajava (Myrtaceae), popularly known as cinnamom, eucalyptus and guava, respectively, that inhibited S. schenkii in zones of 10 to 23 mm, but the most of the methanolic extracts of these tested plants did not show antifungal activity (Márquez, 2010). Still in Myrtaceae family, the ointment formulation of Eucalyptus citriodora Hook at 2% was more pronounced against S. schenckii with zone of growth inhibition of 27 mm, but cream formulation with essential oil and the proper essential oil of this plant had also fungistatic activity between 6.67 to 19.67 mm through agar disc diffusion (Akhtar et al., 2014).

In another study, Cymbogon winterianus (Poaceae) essential oil constituted a potential antifungal product (Pereira et al., 2011). S. schenckii was inhibited in the growth zone of 12 mm at dilution of 1/800 for C. winterianus, known as citronella, while C. martini and C. flexuosus inhibited with 19 mm and 35 mm, respectively, at dilution of 1/1600 (Saikia et al., 2001). In the Ocimum genus (Lamiaceae), the essential oils of O. sanctum, O. canum (O. americanum) and O. gratissimum was conferred activity between 8 and 14 mm of diameter (Sinha, Gulati, 1990). In other botanical species, S. schenckii has been inhibited by Lobelia pyramidalis (Campanulaceae), known as lobelia, with 13 mm of diameter (Joshi et al., 2011); Curcuma longa (Zingiberaceae), known as turmeric, with 3 mm (Apisariyakul et al., 1995), and Rosmarinus officinalis (Lamiaceae), known as rosemary, with 2.5 mm (Luqman et al., 2007). In commercially-available natural products from Perlagonium graveolens (Geraniaceae), Singh et al. (2012) showed that the compounds of terpineol and geranium oil had antifungal activity, while geraniol, phenyl ethyl alcohol and citronella acetate had not shown satisfactory activity.

In the methodology adapted by Brancato and Golding (1953), Beteta (2005) tested aqueous extracts and portions extracted with hexane, chloroform and ethyl acetate derived from the leaves of Lippia graveolens (Verbenaceae) and the flowers of Bourreria huanita (Boraginaceae) and showed that the antifungal activity was conferred only to L. graveolens whose hexanic fraction inhibited the yeast and filamentous phases when tested at a concentration of 0.5 mg mL^-1, while the ethanolic fraction inhibited only the filamentous phase at 0.25 mg ml^-1.

Ethanolic extracts of seeds of Phasoelus lunatus (Fabaceae) and leaves of Cassia grandis (Fabaceae), Diphysa robinioides (Fabaceae), Hymenaea courbaril (Caesalpiniaceae), Phasoelus vulgaris (Fabaceae), Senna occidentalis (Fabaceae) and Vicia faba (Fabaceae) were already tested and proved no activity against the yeast phase, but the filamentous phase showed sensitivity only to extracts of H. courbaril and D. robinioides tested at the concentration of 1 mg mL^-1 and the MIC value of 0.5 mg mL^-1 and 1 mg mL^-1, respectively (Sánchez, 2009).

In another study, yeast and filamentous phases of S. schenckii were inhibited by the ethanolic extracts from roots of Dorstenia contrajerva (Moraceae) and leaves of Hedyosmum mexicanum (Chloranthaceae), being the mycelial phase inhibited at MIC of 0.1 mg mL^-1 and 0.05 mg mL^-1, respectively, but the yeast phase was only inhibited by H. mexicanum at MIC of 0.1 mg mL^-1, however, the extracts from leaves of Baccharis triversis (Asteraceae), Lippia chiapasensis (Verbenaceae) and Ocimum micranthum (Lamiaceae) and the roots of Petiveria alliaceae (Phytolaccaceae) did not have activity against S. schenckii at maximum concentration of 0.2 mg mL^-1 for both fungal phases (Castellanos, 2007). According to these authors, the plants that did not show satisfactory activity may have active chemical compounds in other botanical portions or when tested in higher concentrations, which does not rule out the possibility of possessing potential antifungal activity.

In ethanolic extracts from twelve plants native to Guatemala at the concentration of 1 mg mL^-1 using agar diffusion adapted by Brancato and Golding (1953), the extracts of Hypericum uliginosum (Clusiaseae), Smilax domingensis (Smilacaceae), Salvia lavanduloides (Lamiaceae) and Senna alata (Caesalpiniaceae) inhibited S. schenckii in the yeast phase, while only the extracts of Lippia graveolens (Verbenaceae) and Valeriana prionophylla (Valerianaceae) showed activity for both phases of this dimorphic fungus. The other tested plants - Cornutia pyramidata (Verbenaceae), Quercus crispifolia (Fagaceae), Solanum americanum (Solanaceae), Sterculia apetala (Malvaceae), Tabebuia rosea (Bignoniaceae) and Tithonia diversifolia (Asteraceae) - did not exhibit activity against S. schenckii (Fernández, 2005).

According to Fernández (2005), the yeast phase would be more susceptible to the extracts for the lesser amount of ergosterol present in the membrane cell at this phase, leading to a greater susceptibility to permeability changes, as well as by the biosynthesis of ergosterol inhibition and other steroids by the active compounds present in the extracts. Another hypothesis of the antifungal mechanism would be the inhibition of its triglycerides and phospholipids biosynthesis or the inhibition of oxidative and peroxidative enzymatic activity that results in accumulation of toxic concentrations of hydrogen peroxide and leads to deterioration of subcellular organs and cell necrosis.

Broth Microdilution Method

Plants from different botanical families presented anti-Sporothrix spp. activity through tests of broth microdilution in standard strains isolated from humans and felines (Verástegui et al., 1996; Johann et al., 2007; Luqman et al., 2007; Cleff et al., 2008; Fatima et al., 2009; Johann et al., 2011; Joshi et al., 2011; Stopiglia et al., 2011). This method is based on the proportion of microbial growth in liquid medium challenged with the concentration of the measured substance using a 96-well microplate (CLSI, 2002; Pinto et al., 2003). Among the main advantages cited of this quantitative test are savings in space and reagents, generation of quantitative results through values of minimum inhibitory concentration and the use of pre-fabricated microdilution plates (CLSI, 2002). However, the cost of microdilution plates is a disadvantage, because the realization of a large number of antifungal tests may elevate costs (CLSI, 2002). Furthermore, Eloff (1998) observed compounds precipitation in some plants extracts and interference in the analysis by the high concentration of chlorophyll. In spite of these factors, this method is still considered advantageous for its high sensitivity and reproducibility in relation to other techniques (Eloff, 1998). The results of plants with anti-Sporothrix spp. activity confirmed by broth microdilution technique, as well as their botanical information are displayed in Table III.

TABLE III
Results of microdilution test of plants species with activy against strains of Sporothrix schenckii complex and their different values of minimal inhibitory concentration according to references and solvent extract with action

Botanical Family	Plant Species	Solvent Extract	Origin of Sporothrix spp.	Minimal Inhibitory Concentration	Reference
Agavaceae	Agave lecheguilla	Ethanol	Human	5.0 mg mL-1	Verástegui et al.,1996
Anacardiaceae	Schinus terebinthifolius	Ethanol, Hexane	ATCC♦ 22019	15 μg mL-1	Johann et al., 2007
Asteraceae	Pterocaulon balansae	Methanol	Human	625 μg mL-1	Stopiglia et al., 2011
Asteraceae	Pterocaulon cordobense	Methanol	Human	625 μg mL-1	Stopiglia et al., 2011
Asteraceae	Pterocaulon lanatum	Methanol	Human	625 μg mL-1	Stopiglia et al., 2011
Asteraceae	Pterocaulon lorentzii	Methanol	Human	625 μg mL-1	Stopiglia et al., 2011
Asteraceae	Pterocaulon polystachyum	Methanol	Human	156 μg mL-1	Stopiglia et al., 2011
Campanulaceae	Lobelia pyramidalis	Essential oil	MTCC† 1359	6.25 mg mL-1	Joshi et al., 2011
Compositae	Baccharis glutinosa	Ethanol	Human	12 mg mL-1	Verástegui et al.,1996
Fabaceae	Glycyrrhiza glabra	Ethanol	AIIMS◊	1 mg mL-1	Fatima et al., 2009
Lamiaceae	Origanum vulgare	Essential oil	Human; Feline	250 μL mL-1	Cleff et al., 2008
Lamiaceae	Origanum vulgare	Essential oil	Human; ATCC 1099-18; ATCC 5110; IPEC* 15383; IPEC 17943	216 to 1735 μg mL-1	Couto et al., 2015
Lamiaceae	Rosmarinus officinalis	Essential oil	AIIMS◊	11 mg mL-1	Luqman et al., 2007
Piperaceae	Piper abutiloides	Hydroalcoholic	ATCC♦ 20679	125 μg mL-1	Johann et al., 2009
Polygalaceae	Polygala campestris	Dichloromethane	ATCC♦ 20679	500 μg mL-1	Johann et al., 2011
Polygalaceae	Polygala paniculata	Ethanol	ATCC♦ 20679	1000 μg mL-1	Johann et al., 2011
Polygalaceae	Polygala sabulosa	Acetyl acetate	ATCC♦ 20679	30 μg mL-1	Johann et al., 2011
Polygonaceae	Rumex acetosa	Ethanol	ATCC♦ 22019	1000 μg mL-1	Johann et al., 2007
Rosaceae	Rubus urticaefolius	Acetyl acetate, Ethanol	ATCC♦ 22019	125 μg mL-1	Johann et al., 2007
Zygophyllaceae	Larrea tridentata	Ethanol	Human	16 mg mL-1	Verástegui et al.,1996

• ^♦
  American Type Culture Collection (Manassas, VA, USA); ^†Microbial Type Culture Collection and Gene Bank (Chandigarh, India); *Instituto de Pesquisa Clínica Evandro Chagas (Rio de Janeiro, Brazil); ^◊All India Institute of Medical Sciences (New Delhi, India).

Lamiaceae family is known with potential antifungal for controlling the growth of pathogenic fungi and the occurrence of mycosis (Souza et al., 2010). Rosemary oil (Rosmarinus officinalis) showed in vitro activity against S. schenckii at concentration of 11 mg mL^-1 (Luqman et al., 2007), as well oregano oil (Origanum vulgare), that inhibited S. schenckii concentrations between 250 to 500 μL mL^-1 (Cleff et al., 2008; 2010). In S. schenckii and S. brasiliensis,Couto et al. (2015) showed the antifungal potential of oregano oil, as well as in its major compound (γ-terpinene), which caused morphological alterations in hyphae and reduced the adhered conidia numbers.

From the plants used in traditional medicine in Brazil, Johann et al. (2011) exhibited that ethanolic and ethyl acetate extracts of Rubus urticaefolius (Rosaceae) inhibited the fungal growth at the concentration of 125 μg mL^-1, while Rumex acetosa (Polygonaceae) demonstrated activity at 1000 μg mL^-1. The same activity was observed in the essential oil of Lobelia pyramidalis (Campanulaceae), which inhibited the fungal growth at MIC of 6.25 mg mL^-1 (Joshi et al., 2011). Fatima et al. (2009) showed greater activity of ethanolic and hexanic extracts of Schinus terebinthifolius (Anacardiaceae) at 15 μg ml^-1, as well as the ethanolic extracts at 15% from roots of Glycyrriza glabra (Fabaceae) at 1 mg mL^-1 and glabridin and ethyl acetate fraction at 0.25 μg mL^-1. Other plants were tested on S. schenckii, such as Piper regnelii (Piperaceae), Herissantia crispa (Malvaceae), Baccharis dracunculifolia (Asteraceae), Inga dulcis (Leguminosae) and Alternanthera brasiliana (Amaranthaceae) and did not show antifungal activity (Johann et al., 2007). In the Chihuahuan region (encompassing parts of Northern Mexico and the Southwestern United States), fungistatic activity was observed in the ethanolic extract at 80% of the leaves from Baccharis glutinosa (Compositae) and Larrea tridentata (Zygophyllaceae) and roots of Agave lecheguilla (Agavaceae) at MIC of 12, 16 and 5 mg mL^-1, respectively (Verástegui et al., 1996).

In the Piperaceae family, Piper abutiloides presented MIC value of 125 μg mL^-1 when tested in form of hydroalcoholic extract at 80%, but hexane, dichloromethane, ethyl acetate and aqueous extractions did not show antifungal activity at the maximum tested concentration of 1000 μg mL^-1 (Johann et al., 2009). In the extract with antifungal activity, these authors isolated the compounds pseudodillapiol, eupomatenoid-6 and conocarpan, which provided fungistatic activity at MIC of 12.5 μg spot^-1, 25 μg spot^-1 and 50 μg spot^-1 respectivelly, when individually tested against S. schenckii.

In the Polygala genus (Polygalaceae family), the ethyl acetate and dichloromethane of P. sabulosa had the better activity at MIC of 30 μg mL^-1 and 250 μg mL^-1, respectively, and dichloromethanic and ethanolic extracts of P. campestris, P. sabulosa and P. paniculata has activity between 500 to 1000 μg mL^-1, but no activity was observed in extracts from P. cyparissias. The authors isolated the compounds "prenyloxycoumarin" and "1,2,3,4,5,6-hexanehexol" from P. sabulosa and these showed activity in the MIC value of 125 μg mL^-1 and 250 μg mL^-1, respectively (Johann et al., 2011).

Stopiglia et al. (2011) demonstrated in vitro efficacy of plants from the Pterocaulon genus (Asteraceae family) against 24 strains of S. schenckii, and the methanolic extract at 10% of P. polystachyum, P. lorentzii, P. lanatum, P. cordobense and P. balansae had inhibitory and fungicidal activity. Of these, the extract of P. polystachyum showed greater results with MIC ranged of 156 to 312 μg mL^-1 and MFC varied between 312 to 1250 μg mL^-1.

A variation of the broth microdilution technique, the Serial Microdilution Method is performed through the confirmation of the MIC value by the use of p-iodonitrotetrazolium violet (INT) in each well-test as a fungal growth indicator, indicated by reduction of the red color of INT formazan and evaluated within a period of 24 to 48 hours of incubation (Eloff, 1998; Masoko, Picard, Eloff, 2005). According to Table IV, acetonic, hexanic, dichloromethanic and methanolic extracts from leaves of six native species of trees in South Africa, belonging to the genus Terminalia (Combretaceae family) showed high activity in the MIC values of 0.02 to 0.64 mg mL^-1 (Masoko, Picard, Eloff, 2005), as well as extracts from 24 plants of Combretum genus (Combretaceae) (Masoko, Picard, Eloff, 2007).

TABLE IV
Results of Minimal Inhibitory Concentration (MIC) in the serial microdilution dilution method in different extracts of medicinal plants with activity against Sporothrix spp. in the period of 24 hours

Botanical Family	Plant Species	Solvent Extract	Origin of Sporothrix spp.	MIC at 24 hours*	Reference
Anacardiaceae	Loxostylis alata	Acetone	Unspecified	0.04 mg mL-1	Suleiman et al., 2009
		Methanol, Butano, Hexane, Chloroform, Carbon Tetrachloride, Water	Unspecified	0.2 to 1.88 mg mL-1	Suleiman et al., 2012
	Protorhus longifolia	Dichloromethane	Unspecified	0.84 mg mL-1	Suleiman et al., 2009
Burseraceae	Commiphora harveyi	Acetone	Unspecified	0.31 mg mL-1	Suleiman et al., 2009
Combretaceae	Combretum acutifolium; C. albopunctactum; C. apiculatum; C. edwardsii; C. hereroense; C. moggi; C. nelsonii; C. zeyheri	Acetone	Equine	0.02 to 0.04 mg mL-1	Masoko, Picard, Eloff, 2007
	C. apiculatum; C. edwardsii; C. moggi; C. molle; C. mossambicense; C. petrophilum	Methanol	Equine	0.02 to 0.08 mg mL-1	Masoko, Picard, Eloff, 2007
	C. apiculatum; C. microphyllum; C. mossambicense; C. paniculatum	Dichloromethane	Equine	0.02 to 0.04 mg mL-1	Masoko, Picard, Eloff, 2007
	C. bracteosum; C. hereroense; C. mossambicense	Hexane	Equine	0.02 to 0.04 mg mL-1	Masoko, Picard, Eloff, 2007
	C. celastroides spp. orientale; C. collinum spp. suluense; C. woodii	Acetone	Equine	0.08 mg mL-1	Masoko, Picard, Eloff, 2007
	C. caffrum	Methanol	Equine	0.32 mg mL-1	Masoko, Picard, Eloff, 2007
	C. celastroides spp. celastroides	Methanol	Equine	0.16 mg mL-1	Masoko, Picard, Eloff, 2007
	C. collinum spp. taborense; C. kraussii	Hexane, Dichloromethane	Equine	0.16 mg mL-1	Masoko, Picard, Eloff, 2007
	Combretum erythrophyllum	Hexane	Equine	0.32 mg mL-1	Masoko, Picard, Eloff, 2007
	Combretum imberbe	Dichloromethane	Equine	0.32 mg mL-1	Masoko, Picard, Eloff, 2007
	Combretum padoides	Acetone	Equine	0.32 mg mL-1	Masoko, Picard, Eloff, 2007
	Combretum vendae	Hexane	Unspecified	0.13 mg mL-1	Suleiman et al., 2009
	Terminalia brachystemma; T. mollis	Dichloromethane	Equine	0.02 mg mL-1	Masoko, Picard, Eloff, 2005
	Terminalia gazensis; T. prunioides; T. sambesiaca	Acetone	Equine	0.02 to 0.08 mg mL-1	Masoko, Picard, Eloff, 2005
	Terminalia prunioides	Hexane	Equine	0.04 mg mL-1	Masoko, Picard, Eloff, 2005
Combretaceae	Terminalia sericea	Methanol	Equine	0.02 mg mL-1	Masoko, Picard, Eloff, 2005
Kirkiaceae	Kirkia wilmsii	Acetone	Unspecified	0.26 mg mL-1	Suleiman et al., 2009
Meliaceae	Khaya anthotheca	Acetone	Unspecified	0.11 mg mL-1	Suleiman et al., 2009
Ochnaceae	Ochna natalitia	Hexane	Unspecified	0.08 mg mL-1	Suleiman et al., 2009

• * The extrats types of considered plants were those with minimum values of inhibitory concentration at 24 hours.

Suleiman et al. (2009) showed the anti-Sporothrix spp. activity of different extracts from leaves of native species trees of South Africa: Combretum vendae (Combretaceae), Commiphora harveyi (Burseraceae), Khaya anthotheca (Meliaceae), Kirkia wilmsii (Kirkiaceae), Loxostylis alata (Anacardiaceae), Ochna natalitia (Ochnaceae) and Protorhus longifolia (Anacardiaceae). In different extracts at 10% of L. alata, acetonic extract of L. alata exhibited better antifungal activity in MIC of 0.04 mg mL^-1 in 24 hours and 0.08 mg mL^-1 in 48 hours (Suleiman et al., 2009) and other fractions of the L. alata had synergistic activity against Sporothrix spp. in the MIC of 0.2 to 1.88 mg mL^-1 (Suleiman et al., 2012). These activities can be explained by the lupeol compound, which showed anti-Sporothrix spp. activity when individually tested at the MIC values of 57 μg mL^-1 (Suleiman et al., 2013). Lupeol isolated from leaves and stem barks of Curtisia dentata B. (Cornaceae) showed higher activity (12 μg mL^-1) when compared with betulinic acid, ursolic acid and 2-α-hydroxy ursolic acid isolated, that had also activity between 16 to 32 μg mL^-1 (Shai et al., 2008).

In another study, the ethanolic extract of the rhizomes of Agapanthus africanus L. (Liliaceae) had activity against Sporothrix spp. with MIC value of 250 μg mL^-1, and the compound saponin was isolated from this extract and was responsible for antifungal activity in MIC of 15.6 μg mL^-1 (Singh et al., 2008).

Bioautographic Assay

Many researchers carry out bioautographic assay to detect new or unidentified compounds in plants of interest, in order to pursue and deepen the studies in cases of confirmed antimicrobial action (Betina, 1973). This test is based on assessing the inhibition zone afforded by the extract of interest, through the addition of this extract in silica gel plates and further immersion in a fungal suspension and addition of p-iodonitrotetrazolium violet (Rahalison et al., 1993). Among eight plants popularly used in traditional Brazilian medicine, Johann et al. (2007) measured the MIC values by broth microdilution technique only of six plants of different extracts, because those were effective against a several number of pathogens tested by bioautographic assay and the ethanolic extract of leaves from S. terebinthifolius (Anacardiaceae) showed better activity against S. schenckii at 15 μg mL^-1.

In plants from Polygala genus, five species presented activity in the bioautographic assay and were submitted to broth microdilution test, demonstrating that ethanolic extract of P. paniculata (1000 μg mL^-1) and the fractions of dichloromethane and ethyl acetate of P. sabulosa (250 μg mL^-1and 30 μg mL^-1, respectively) showed good antifungal activity against S. schenckii (Johann et al., 2011).

Agar Cup Method

Brazilian propolis produced by bees that collected pollen from Baccharis dracunculifolia (Asteraceae) and araucaria (Araucaria spp. - Araucariaceae) showed antifungal activity in the 30% ethanolic extract at the concentration of 16 to 2.0 mg mL^-1 by the agar cup method, in which the antifungal activity is measured by inhibition zone diameter. The inhibitory action of both plants occurred when tested individually and jointly against S. schenckii, whose inhibition diameter ranged between 16 to 24 mm and the lowest concentration tested (2 mg mL^-1) of B. dracunculifolia was able to inhibit the fungal growth up to 19 mm diameter (Salomão et al., 2008).

The small annual herb Cuminim cyminum L. (Apiaceae), popularly known as cumin, is the second most popular spice in the world, after black pepper, and showed activity against S. schenckii in the zone of inhibition of 18 mm to essential oil, 12 mm to methanol extract and 0.3 mm to hydroalcoholic extract, but aqueous extract of this plant did not show anti-Sporothrix spp. activity when the products where tested at concentration of 0.5 mg mL^-1 (Chaudhary, Husain, Ali, 2014).

IN VIVO STUDIES

In vivo studies with aqueous creams containing 20% of acetone extracts (2 g of extract in 10 g of cream) from the leaves of different species of Combretaceae were evaluated in experimental cutaneous sporotrichosis and showed wound-healing properties when administered topically three times per week in rats Wistar. The preparations of leaves from Combretum imberbe, C. nelsonii, C. albopunctatum and Terminalia sericea (Combretaceae) have produced reversal of lesions in 15 days, confirming in vivo antifungal activity. Irritating and damaging effects from the same plants on skin wounds were not observed, indicating the absence of adverse effects on healthy skin of the tested animals (Masoko et al., 2010a; Masoko, Picard, Eloff, 2010b).

The promising use of medicinal plants in sporotrichosis encourages scientific researchers to find a new antifungal product. However, there are few studies proving the anti-Sporothrix spp. activity of medicinal plants. More studies about safe concentrations of their use must be performed because these plants can present chemical elements with capacity to cause toxic effects (Veiga Jr., Pinto, 2005; Leal et al., 2013). More studies are needed to determine medicinal plants´ safe use with little or no adverse effects in the patient.

CONCLUSION

In vitro studies suggest plants as sources of new promising molecules in the control and treatment of sporotrichosis, especially species belonging to the families Combretaceae, Asteraceae and Lamiaceae. Due to various protocols and methodologies used in the studies, as well as to the different tested concentrations, we emphasize the difficulty in comparing the fungistatic and fungicidal results of plant extracts and it is not possible to attribute the plants with better activity. Furthermore, the lack of in vivo studies of plants was noticed, which reveals the need for deeper studies focussed on the action mechanism of the plants, as well as their toxicity, side effects and possibility of drug interactions in order to understand the safe use of medicinal plants for the treatment of sporotrichosis.

ACKNOWLEDGEMENTS

The authors acknowledge José A. Curbelo Knutson (B.A. International Affairs, The George Washington University Elliott School of International Affairs, Washington, District of Columbia, U.S.A.) for the English translation and revision of the text. Additionally, the authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), to Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), and to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for student and research scholarships.

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Publication Dates

• Publication in this collection
  Apr-Jun 2016

History

• Received
  22 June 2015
• Accepted
  02 May 2016