Antifungal Activity of Extracts and Prenylated Coumarins Isolated from Baccharis darwinii Hook & Arn. (Asteraceae)

The petroleum ether extract of Baccharis darwinii showed activity against Cryptococcus neoformans and dermatophytes. Bioactivity-guided fractionation of Baccharis darwinii has resulted in the isolation of three coumarins: 5’-hydroxy aurapten (anisocoumarin H, 1), aurapten (7-geranyloxycoumarin, 2) and 5’-oxoaurapten (diversinin, 3). The structures of these compounds were characterized by spectroscopic methods. These compounds were evaluated for their antimicrobial activity against a panel of each, bacteria and fungi. Compound 3 showed the best activities against Microsporum gypseum, Trichophyton rubrum and Trichophyton mentagrophytes with MICs = 15.6 µg/mL, followed by compound 1 whose MICs against the same fungi were 62.5 µg/mL. In addition they showed fungicidal rather than fungistatic activity. Both compounds showed moderate activity (MICs = 125 µg/mL) against Cryptococcus neoformans. This is the first report of the presence of compound 1 in B. darwinii.


Introduction
Currently, medicinal plants are widely used as home remedies or as alternative treatments by both rural and urban inhabitants in developing countries [1], which can be explained in part by the high cost of industrialized medicine. In turn, many extracts from plants have shown biological activities of interest, which justifies the search of potentially active compounds in them.
A matter of concern in public health in the last decades is the increasing resistance of bacteria and fungus to antimicrobial drugs and the growing number of immunocompromised patients undergoing fatal fungal infections, since the available antifungals are scarce, sometimes ineffective and not always safe.
The genus Baccharis L. possesses the highest number of species (between 400 and 500) within the Asteraceae family [2]. It was found only in American countries from the south of United States to the austral end of Argentina and Chile, occupying a great variety of environments. In the Argentine Republic, it is represented by 96 species [2], and among them, Baccharis darwinii, commonly known as "chilca", can be found in the far south of South America that is, in the arid Patagonia region of Argentina .
Studies on the biological activities of species of this genus have shown antimicrobial, antioxidant, anti-inflammatory and antifeedant activities [6][7][8][9][10][11][12][13][14][15][16][17]. Regarding the chemical constituents found in the genus, flavonoids and terpenoids are the most frequently reported [17][18][19][20]. Regarding B. darwinii, the presence of β-farnesene, aurapten, 5'-oxoaurapten, and 6',7'-epoxy-and 6',7'-dihydroxyaurapten were reported by Zdero et al. in a sample collected in the Argentinean Neuquén province [21]. Nevertheless, to the best of our knowledge there are no reports on the biological activities of Baccharis darwinii. We report here the antimicrobial activity of crude extracts of this plant collected in Chubut province, a southernmost province of Argentina, and the bioassay-guided fractionation of its most active extract which led to the isolation of the main compounds responsible for the activity.

Results and Discussion
In the first step of our work, we evaluated the antimicrobial (antibacterial and antifungal) activity of the petroleum ether (PE), dichloromethane (DCM) and methanol (MeOH) extracts from aerial parts of Baccharis darwinii. Regarding antibacterial activity of the different extracts, MeOH did not show any activity (MIC > 1,000 µg/mL), PE showed only marginal activity (MIC ≥ 1 ,000 µg/mL) and DCM extract showed a very low activity against only two bacteria (MIC = 750 µg/mL) and marginal or null activity against the rest of bacteria of the panel.
In contrast, the three extracts showed better antifungal activities, mainly against dermatophytes and, in addition, they were shown to possess not only fungistatic but also fungicidal capacity (Table 1). Nevertheless, some differences could be observed among the activity of the three extracts (Table 1): the PE and MeOH extracts were the most active extracts mainly against C. neoformans. Between them, PE extract showed four-fold higher potency than MeOH extract against the same fungi. Among the different extracts, PE is of great interest since it showed a selective and good activity against C. neoformans and dermatophytes. On the basis of these results, PE was submitted to fractionation guided by antifungal activity as described in the Experimental section.
Table1. Antifungal and antibacterial activity to Baccharis darwinii extracts (MIC/MFC, µg/mL). The nine sub-fractions obtained from PE extract (I-IX) were evaluated for antifungal properties against the same panel used for the crude extracts. Results (Table 2) showed that the best activity was displayed by sub-fraction VI against dermatophytes (MIC and MFC = 62.5 µg/mL). From this subfraction for successive fractionations, three known coumarins: 5'-hydroxyaurapten (anisocoumarin H, 1), aurapten (7-geranyloxycoumarin, 2) and 5'-oxoaurapten (diversinin, 3) were isolated and their antifungal activities were evaluated. Of these, 1 has not been reported previously in B. darwinii while 2 and 3 have been reported previously in this plant [21]. The compounds were identified by comparison of their NMR data with literature vales (see Experimental). The corresponding structures are shown in Figure 1.

Microorganisms
It is interesting to note that two of these coumarins, 1 and 3, displayed antifungal activity against C. neoformans and dermatophytes ( Table 2) and therefore both compounds would be the responsible for the antifungal activity observed in the PE extract of B. darwinii. Compound 3 possess a high fungistatic activity against the three dermatophytes tested, with MICs = 15.6 µg/mL and a fungicide capacity. This result is very interesting since T. rubrum and T. mentagrophytes are responsible for approximately 80-93% of chronic and recurrent dermatophytes infections in human beings. They are the ethiological agent of tinea unguium (producer of invasive nail infections), tinea manuum (palmar and interdigital areas of the hand infections) and tinea pedis (athlete's foot), the last one being the most prevalent fungal infection in developed countries, and the first one accounting for 50% and 90% of all fingernail and toenail infections respectively [22].  In turn, although the activity displayed by 1 and 3 against C. neoformans (MIC = 125 µg/mL) is moderate, it is worthy to take into account that C. neoformans remains an important life-threatening complication for immunocompromised hosts, particularly for patients who have undergone transplantation of solid organs and, therefore, new compounds acting against this fungus are highly welcome [23]. The antimicrobial activity is considered very interesting in the case of MICs < 100 µg mL -1 for extracts and 10 µg mL -1 for isolated compounds [24]. Another interesting contribution of this work is that, since the three coumarins have been previously isolated from other species, such as anisocoumarin H (1) in Clausena anisata [25] and Notopterygium forbesii [26]; auraptene (2) in Ferulago capillaris [27], Paliurus ramosissimus [28]; diversinin (3) in Ferula diversivittata [29], this work encourages the beginning of antifungal studies of these species in order to evaluate their antifungal potentiality.

General
IR spectra were recorded in a FTIR Shimadzu Prestige-21 instrument; 1D and 2D NMR spectra were recorded using a Bruker Avance 300 in deuterated chloroform solutions, using standard Bruker software. GC-MS were run in a Perkin Elmer Autosystem XL Gas Chromatograph coupled to a Turbo Mass Spectrometer. Column: SE-30, 25 m × 0.22 mm ID, Scientific Glass Engineering (Ringwood, Victoria, Australia).

Extraction
The dried powdered aerial parts (450 g) were extracted successively with petroleum ether (PE), dichloromethane (DCM) and methanol (MeOH) at room temperature (3 × 1,000 mL, for 96 h each time). After evaporation to dryness under reduced pressure, PE, DCM and MeOH extracts were obtained in yields (w/w in terms of dry starting material) of 3.46%, 2.26% and 14,14%, respectively.

Isolated compounds
7-(5-Hydroxy-3,7-dimethylocta-2,6-dienyloxy)-chromen-2-one (5'-hydroxy aurapten, 1). The NMR data were in agreement with those reported by Ngadjui et al. [25]. (aurapten, 2). The NMR data were in agreement with those reported by Zdero et al. [21]. (5'-oxoaurapten, 3). The NMR data were in agreement with those reported by Zdero et al. [21].  [30] and adjusted to 1-5 × 10 3 cells/spores with colony forming units (CFU) /mL 3.6.2. Antibacterial susceptibility testing Cultures less than 30 h old were touched with a loop and transferred to sterile Mueller-Hinton broth. The broth was incubated at 37 ºC until the growth reaches turbidity equal to, or greater than that of 0.5 Mc Farland standards. The culture was adjusted with sterile physiological solution to give final organism density of 5 × 10 5 CFU/mL [31,32]. The antibacterial activity was evaluated with the agar dilution method using Mueller Hinton agar medium for Gram (+) and Gram (-) bacteria. Stock solutions of the compounds/extracts in DMSO were diluted to give serial two-fold dilutions that were added to each medium resulting in concentrations ranging from 10 to 1,000 µg/mL. The final concentration of DMSO in the assay did not exceed 1%. The antimicrobial agent cefotaxime (Argentia Pharmaceutica) was included in the assays as positive control. The plates were incubated for 24 h at 37 ºC. Minimum Inhibitory Concentration (MIC) was defined as the lowest compounds/extracts concentration showing no visible bacterial growth after incubation time. Tests were carried out in triplicate. Extracts and compounds with MICs ≤ 250 µg/mL were considered of interest.

Antifungal susceptibility testing
Minimum Inhibitory Concentration (MIC) of each extract or compound was determined by using broth microdilution techniques according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards, NCCLS) for yeasts (M27-A2) and for filamentous fungi (M 38 A), [30]. MIC values were determined in RPMI-1640 (Sigma, St Louis, MO, USA) buffered to pH 7.0 with MOPS. Microtiter trays were incubated at 35 ºC for yeasts and hialohyphomycetes and at 28-30 ºC for dermatophyte strains in a moist, dark chamber. MICs were visually recorded at 48 h for yeasts, and at a time according to the control fungus growth, for the rest of fungi. Extracts with MICs ≤ 1 ,000 µg/mL and sub-extracts and compounds with MICs ≤ 250 µg/mL were considered active.
For the assay, stock solutions of pure compounds were two-fold diluted with RPMI from 1,000 or 250 µg/mL to 0.98 (final volume = 100 µL) and a final DMSO concentration ≤ 1%. A volume of 100 µL of inoculum suspension was added to each well with the exception of the sterility control where sterile water was added to the well instead. Ketoconazole (Sigma Chem. Co.), terbinafine (Novartis, Argentina) and amphotericin B (Sigma Chem. Co.), were used as positive controls. Endpoints were defined as the lowest concentration of drug resulting in total inhibition (MIC 100 ) of visual growth compared to the growth in the control wells containing no antifungal.
The Minimum Fungicidal Concentration (MFC) of each extract or compound was determined as follows: After determining the MIC, an aliquot of 5 µL sample was withdrawn from each clear well of the microtiter tray and plated onto a 150-mm RPMI-1640 agar plate buffered with MOPS (Remel, Lenexa, Kans.). Inoculated plates were incubated at 30 ºC, and MFCs were recorded after 48 h. The MFC was defined as the lowest concentration of each compound that resulted in total inhibition of visible growth in these plates.

Conclusions
Phytochemical study of B. darwinii extracts, bioguided by the antifungal assay, led to the isolation of the known prenylated coumarins 5'-hydroxyaurapten (1), aurapten (2) and 5'-oxoaurapten (3). This is the first report of the presence of compound 1 in B. darwinii. Among the three isolated compounds compound 3 showed the best activities against M. gypseum, T. rubrum and T. mentagrophytes with MICs = 15.6 µg/mL, followed by compound 1 whose MICs against the same fungi were 62.5 µg/mL. In addition their activity was shown to be fungicidal rather than fungistatic. Both compounds showed moderate activity (MICs = 125 µg/mL) against C. neoformans.