Antifungal activity of five species of Polygala

Crude extracts and fractions of five species of Polygala – P. campestris, P. cyparissias, P. paniculata, P. pulchella and P. sabulosa – were investigated for their in vitro antifungal activity against opportunistic Candida species, Cryptococcus gattii and Sporothrix schenckii with bioautographic and microdilution assays. In the bioautographic assays, the major extracts were active against the fungi tested. In the minimal concentration inhibitory (MIC) assay, the hexane extract of P. paniculata and EtOAc fraction of P. sabulosa showed the best antifungal activity, with MIC values of 60 and 30 μg/mL, respectively, against C. tropicalis, C. gattii and S. schenckii. The compounds isolated from P. sabulosa prenyloxycoumarin and 1,2,3,4,5,6-hexanehexol displayed antifungal activity against S. schenckii (with MICs of 125 μg/mL and 250 μg/mL, respectively) and C. gattii (both with MICs of 250 μg/mL). Rutin and aurapten isolated from P. paniculata showed antifungal activity against C. gattii with MIC values of 60 and 250 μg/mL, respectively. In the antifungal screening, few of the isolated compounds showed good antifungal inhibition. The compound α-spinasterol showed broad activity against the species tested, while rutin had the best activity with the lowest MIC values for the microorganisms tested. These two compounds may be chemically modified by the introduction of a substitute group that would alter several physico-chemical properties of the molecule, such as hydrophobicity, electronic density and steric strain.


INTRODUCTION
In recent years, fungal infections have emerged as a major cause of disease and mortality, largely because of the growing population of immunocompromised patients. In patients affected by AIDS, opportunistic yeasts of the genus Candida, such as C. albicans, have accounted for approximately 60% of clinical isolates. The appearance of other species of this genus as agents of candidosis may reflect the selection of species that are less susceptible to the action of antifungal agents (39).
Approximately 90% of AIDS patients present with oropharyngeal candidosis at least once in the course of the disease, and two-thirds of these patients display the same characteristic at the onset of the symptoms (4).
lead to the development of drug-resistant strains (11).
Therefore, there is a distinct need for the discovery of new, safer and more-effective antifungal agents. Natural products from higher plants may provide a new source of antimicrobial agents with possibly novel mechanisms of action (27). In recent years, antimicrobial properties of medicinal plants have been increasingly reported from different parts of the world (17,18,24).
Various pharmacological activities have been reported for some species of Polygala, such as anti-inflammatory activity in P. japonica and P. cyparissias (10,19), anxiolytic-like effects in P. sabulosa (8,9) trypanocidal activity in P. sabulosa and P.
cyparissias (34,35), antinociceptive effects in P. sabulosa (40) and P. cyparissias (34,35), antibacterial activities in P. myrtifolia (20) and so on. Although the biological activities of various Polygala species have been investigated, their antifungal properties have not. Few studies have reported the antifungal activities of species belonging to the genus Polygala, such as P. myrtifolia (26), P. gazensis (3) and P.
In this paper, we report an evaluation of the antifungal activities of extracts, fractions and isolated compounds from different species of Polygala, all of them found in Santa

Plant material
The species P. cyparissias (856 g) and P. paniculata paniculata was first extracted three times at room temperature with hexane (the "hexane extract"), then with 96% ethanol (EtOH extract), to avoid the formation of coumarinic artifacts such as those previously described by Pizzolatti et al. (33). P. pulchella and P. campestris were successively extracted three times each with dichloromethane (CH 2 Cl 2 extract), ethyl acetate (EtOAc extract) and ethanol (EtOH extract). P. cyparissias and P. sabulosa were extracted exhaustively at room temperature with 96% ethanol (EtOH extract). The EtOH extract obtained from P. sabulosa was then partitioned into nhexane, dichloromethane (CH 2 Cl 2 ) and ethyl acetate (EtOAc) soluble fractions. Each extract or fraction was filtered, and the solvent was removed under reduced pressure to yield the respective extract.

Isolation of compounds
During evaporation of the ethanolic extract obtained from P. sabulosa (456.1 g), formation of a solid compound was observed. This compound was separated and washed with appropriate solvent (CH 2 Cl 2 and acetone). The compound was analyzed by IV, 1 H and 13 C NMR and identified as 1,2,3,4,5,6hexanehexol, previously described as occurring in this species by Pizzolatti et al. (31). The n-hexane fraction of P. sabulosa was subjected to column chromatography on silica gel eluted with n-hexane/EtOAc gradient. The fractions obtained were then monitored by TLC (Thin-layer chromatography, viewed by spraying with sulfuric vanillin reagent followed by heating at 110 °C) and similar fractions (fr) were combined. The combined fr. 14-26 were recrystallized from acetone to give the sterol α-spinasterol (91 mg).
The CH 2 Cl 2 soluble fraction was further subjected to silica gel column chromatography eluted with increasing amounts of ethyl acetate in n-hexane to give 95 fr. of 75 mL each. The

Culture media and inoculum
Saboraud dextrose agar was used for the bioautographic tests.  (29). To obtain Y cells of S. schenckii, the fungal culture was cultured on brain heart infusion agar containing 1% glucose (BHID) at 37°C and maintained by continuous weekly passages.
The cells were harvested 4 to 6 days after the last passage and packed into a small volume of sterile saline using a sterile cotton swab. The cells were then suspended in saline and thoroughly vortexed. If large aggregates existed, they were allowed to settle for several minutes, and the supernatant was collected. The suspension was then diluted in RPMI medium to obtain a final inoculum size of approximately a 1x10 5 -5x10 5

RESULTS AND DISCUSSION
Physical and spectroscopic data of the compounds isolated from P. sabulosa and P. paniculata are presented bellow.    gattii and 250 µg/mL against C. glabrata and C. parapsilosis.
The hexane and CH 2 Cl 2 fractions from P. sabulosa had a wide spectrum of action, and the amounts required to inhibit the fungal growth varied between 250 and 1000 µg/mL.
However, most of the fungal growth was inhibited at 500 and 1000 µg/mL. Only C. glabrata and S. schenckii were more susceptible at low concentration to the CH 2 Cl 2 fraction of P.
sabulosa, with an MIC of 250 µg/mL. The EtOH extract of P.
myrtifolia species, the presence of saponins was related to activity against C. albicans (26). Table 3 shows the antifungal activities (MIC values in µg/mL) of compounds isolated from two Polygala species, P. paniculata and P. sabulosa, along with the antifungal activities of the extracts from which they were isolated. The other three plant species did not show good activity; therefore, compounds were not isolated from these species. Scopoletin, DST 1, 2, 3 and STY 4, 5,  albicans (29). In the present work, this compound was active against C. albicans with an MIC of 500 µg/mL. Rutin was already shown to be active against Phytophthora megasperma, Cylindrocarpon destructans (2) and C. albicans (MIC of 1000 µg/mL) (14). In the present study, rutin not was active against C.
albicans, but it showed activity against C. krusei and C. gattii.
Regarding the lack of activity of the coumarins scopoletin and phebalosin, it has been reported that the pharmacological and biochemical properties of simple coumarins may depend on the substitution pattern of the benzene moiety. Sardari et al. (41) demonstrated that a free 6-OH is essential and that alkylated derivatives of 7-hydroxycoumarin may show both antifungal and antibacterial properties. Although the coumarins prenyloxycoumarin, phebalosin and aurapten do not possess a free 6-OH, they were not totally devoid of antifungal activity.
Nevertheless, the observed lack of activity of scopoletin (MICs > 1000 µg/mL) is in conflict with the results of Sardari et al. (41) because this coumarin has a free 6-OH. There are also many studies that describe the antifungal activity of this coumarin against phytopathogens. There is a correlation between scopoletin accumulation and resistance to microbial pathogens in some plants showing important antifungal activity, scopoletin being considered a phytoalexin (42,1,43).
As is clear from Table 3, most of the isolated compounds were devoid of antifungal activity up to 1000 µg/mL, and only αspinasterol, 1,2,3,4,5,6-hexanehexol, prenyloxycoumarin, rutin and aurapten (Figure 1) showed moderate activity toward some fungal species, albeit lower than that displayed by the original extracts or fractions. This interesting and unexpected result could be due to a synergistic action between the compounds of the extracts in the case of P. sabulosa and P. paniculata or could be ascribed to minor components of the extracts.
Of all the compounds screened, two of them showed antifungal inhibition, whereas eleven did not have significant activity. Rutin and aurapten isolated from P. paniculata showed antifungal activity against C. gattii with MIC values of 60 and 250 µg/mL, respectively. The compound -spinasterol showed a wide activity against the species tested and rutin, with the lowest MIC for the microorganisms tested, had the best activity. Chemical modification may improve the activity of these two compounds.