Anti-Sporothrix spp. activity of medicinal plants

1Departamento de Veterinária Preventiva, Faculdade de Veterinária, Universidade Federal de Pelotas, UFPEL, Pelotas/ RS, Brasil,2Centro de Controle de Zoonoses, CCZ, Prefeitura Municipal de Pelotas, Pelotas-RS, Brasil,3Departamento de Clínicas Veterinária, Faculdade de Veterinária, Universidade Federal de Pelotas, UFPEL, Pelotas/RS, Brasil,4Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, UFRGS, Porto Alegre, RS, Brasil.

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.

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 CONVENTIO-NAL 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).
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.
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.
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).
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 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 .
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.
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.
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.
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).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 -1 and 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  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.

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

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 (cont.)

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 (cont.)

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 *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).

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

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 Suleiman et al., 2009 * The extrats types of considered plants were those with minimum values of inhibitory concentration at 24 hours.

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 (cont.) of leaves from Combretum imberbe, C. nelsonii, C. albopunctatum and Terminalia sericea (Combretaceae)