Antimicrobial Activity of Three Baccharis Species Used in the Traditional Medicine of Northern Chile

The antimicrobial activities of aqueous ethanol and chloroform extracts of three Baccharis species currently used in Northern Chile folk medicine for the treatment of several infectious and inflammatory disorders were tested against Gram-positive and negative bacteria and fungal spp. using the agar-disc diffusion assay. The results indicated that the activity was more pronounced against Gram-positive than against Gram-negative bacteria and yeast. No significant differences on the antibacterial activity were observed in the aqueous ethanol versus chloroform extracts. None of the plant extracts evaluated exhibited any activity against ten fungi tested.


Introduction
In Northern Chile, as well as in other developing countries, medicinal plants still represent the main therapeutic tool in traditional medicine [1]. The genus Baccharis, one of the most important genera of Asteraceae, is widespread throughout South America and is represented in Chile by 48 species [2]. Several of these are used in traditional medicine. The species selected for the present study -Baccharis microphylla, Baccharis petiolata and Baccharis santelicis -are popularly known as "chilca" and they are commonly used to protect stomach and liver, restore blood circulation, reduce inflammatory processes and cure ulcers, burns and skin wounds [3][4][5][6][7][8]. The Baccharis species under study grow at 3,200-3,800 meters above sea level in the Puna de Atacama, which is characterized by areas of arid climate, low atmospheric pressure, high solar radiation and extremely broad seasonal and diurnal temperature ranges. No chemical studies on B. microphylla have been carried out. B. petiolata [9] and B. santelicis [10] were previously reported to contain labdane and clerodane diterpenoids and flavonoids (flavones and flavanones); other terpenoids (C-15 and C-30) and mixed biogenesis metabolites have also been isolated from these species [10]. The main objective of this study was to identify Northern Chilean medicinal plants with antimicrobial activity which might serve as good candidates for the development of new antimicrobial agents and/or standardized phytomedicines.

Results and Discussion
The results of the antibacterial and antifungal screening of aqueous ethanol and chloroform extracts from the three studied Baccharis species against seven bacterial species and ten fungal strains are summarized in Table 1 (inhibition zones in the agar disc diffusion assay at 25 mg extract /disc) and Table 2 (MIC values). The following tested microorganisms are omitted from Tables 1 and 2 because no extract inhibited their growth: E. coli, P. aeruginosa, C. albicans, C. tropicalis, C. neoformans, S. cerevisae, A. fumigatus, A. flavus, A. niger, M. gypseum, T. tubrum and T. mentagrophytes. The extracts exhibited a broad spectrum of activity. Gram-positive bacteria are more sensitive to the extracts than Gram-negative ones [11]. None of the plant extracts evaluated inhibited the growth of the Gram-negative E. coli and P. aeruginosa, nor did they exhibit any activity against the ten fungi tested, including C. albicans. The most susceptible bacterium was B. subtilis. If extracts displayed an MIC less than 100 μg/mL, the antimicrobial activity was considered as good; from 100 μg/mL to 500 μg/mL the antimicrobial activity was moderate; from 500 μg/mL to 1000 μg/mL the antimicrobial activity was weak; over 1000 μg/mL the extract was considered inactive. Aqueous ethanol and chloroform extracts of B. microphylla, B. petiolata and B. santelicis presented a good activity against Bacillus subtilis, with MIC values of 7.8, 15.6, 62.5, 31.2, 15.6 and 31.2 μg/mL, respectively. The aqueous ethanol extract of B. santelicis showed good activity against S. aureus (31.2 μg/mL), but was inactive against E. faecalis (> 1000 μg/mL), while the chloroform extract of this plant that displayed good activity against E. faecalis (15.6 μg/mL) and was inactive against S. aureus (> 1000 μg/mL).

Conclusions
Although this study investigated the in vitro antimicrobial activity, the results showed that the extracts from Baccharis microphylla, Baccharis petiolata and Baccharis santelicis possessed good antibacterial activity, confirming the great potential of these plants used in Northern Chilean ethnomedicine for the production of bioactive compounds and are useful for rationalizing the use of these medicinal plants in primary health care. In vivo data may be helpful in determining the real potential usefulness of these plants for the treatment of infectious diseases.

Plant materials
Aerial parts of Baccharis microphylla, Baccharis petiolata and Baccharis santelicis were collected near Socaire, in Northern Chile (23º 36´40" S; 67º 50´33" W) at 3,500 m above sea level. The plant's classifications were confirmed by Professor Clodomiro Marticorena, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Chile. A sample of each plant was deposited at the Herbario de la Universidad de Concepción.

Preparation of extracts and dry residues
The aqueous ethanol extract were prepared by adding EtOH-H 2 O (1:1, 50 mL) to ir-dried and coarsely powdered plant (5.0 g), and heating the mixture at reflux condenser for 10 min with continuous magnetic stirring. Afterwards, the extract was covered with a watch glass, left standng for 30 min to cool, centrifuged and filtered through sterilized cellulose nitrate. The dry residue from the extracts was obtained by vacuum-evaporation followed by lyophilization at 5 mm Hg pressure and -50ºC in a Freezone lyophilizer (Labconco). For chloroform extraction, samples (5.0 g) were ground to a fine power in a mill and mixed with chloroform (50 mL). The obtained extracts were filtered and the solvent evaporated in a rotary evaporator at 50ºC to afford the corresponding dry extracts. Extracts were tested at a concentration of 100 mg/mL in water-DMSO (9:1).

Microorganisms tested
The

Antibacterial testing
Antimicrobial activities of the aqueous ethanol and chloroform extracts were determined using the agar-disc diffusion method [12]. The media used were Mueller-Hinton agar for the bacteria and Sabouraud dextrose agar for the fungi. After 24 h, the culture of selected bacteria/yeast was mixed with physiological saline solution and the turbidity was corrected by adding sterile physiological saline to a MacFarland turbidity standard of 0.5 (10 8 CFU/mL). Afterwards, a top layer of Mueller-Hinton agar inoculated with 0.2% microbial suspension was poured over the Petri dishes. Sterile filter discs 6 mm in diameter were impregnated with 250 μL of solution of extract (100 mg/ mL, equivalent to 25 mg of extract) per disc and then placed onto the inoculated plates. The plates were then incubated at 37 ± 0.1ºC for 24 h. The test was carried out in triplicate. Antimicrobial activity was measured as the diameter (mm) of clear zone of growth inhibition. Ampicillin (10 μg/disc), streptomycin (25μg/disc) and chloramfenicol (30 μg/disc) were used as positive control antibiotics in all plates and their inhibition zones against E. coli were 17, 9 and 18 mm, respectively. Nystatin (100 μg/disc) and Amphoterecin B(200 μg/disc) were used as positive control for the fungi in all plates and their inhibition zones against C. albicans were 26 and 17 mm, respectively. DMSO, ethanol and methanol were included in every experiment as negative controls. The Minimum Inhibitory Concentration (MIC) values of extract from Baccharis were determined in triplicate by using the microdilution broth method in 96-well microplates [13]. The extracts were dissolved in DMSO (1 mg/mL), and then diluted in tryptone soya broth to achieve concentrations ranging from 1000 to 7.8 μg/mL. The final concentration of DMSO was 10% (v/v). The inoculum was adjusted to each microorganism to yield a cell concentration of 10 8 CFU/mL. Standard antibiotics were used as positive controls and their concentrations ranged from 5.9 to 0.01 μg/mL. The microplates were incubated at 37ºC for 24 h (bacteria) or at 30ºC for 48 h (yeast), and aqueous 2,3,5-triphenyltetrazolium chloride solution (0.5%, 20 μL) was added to indicate the viability of bacteria and yeast. MIC values were determined as the lowest concentration of the extract capable of inhibiting visible microorganism growth.