Screening of Antibacterial Activity of South Brazilian Baccharis. Species

Abstract

South Brazilian Baccharis. species were studied for antibacterial activities against Gram-positive and Gram-negative bacteria using disk diffusion and broth dilution assays. The results showed that the n.-BuOH fraction (100 µg) from Baccharis usterii. Heering exhibited inhibitory activity against Staphylococcus aureus., Enterococcus faecalis., and Enterococcus faecium.. The n.-BuOH fraction of Baccharis spicata. (Lam.) Bailon (1000 µg) was effective against S. aureus., Escherichia coli., Enterococcus faecalis., and Enterococcus faecium.. The crude extract of Baccharis trimera. (Less) A. P. de Candolle (in doses of 1000 µ/disc) showed activity against S. aureus.. The minimum bactericidal concentration (MBC) obtained from n.-BuOH fraction of B. spicata. were 50 mg/ml against S. aureus., E. coli., E. faecalis., and E. faecium.. From the crude extract of B. trimera., a MBC of 25 mg/ml was obtained against S. aureus.. The n.-BuOH fraction of B. usterii. showed a MBC of 25 mg/ml against S. aureus. and 50 mg/ml against E. faecalis. and E. faecium., while the crude extract of this plant showed a MBC of 12.5 mg/ml against S. aureus..

Keywords:

• Antimicrobial activity
• Baccharis
• broth dilution assay
• disk diffusion method
• MBC
• MIC

Introduction

Multiple-drug-resistant organisms that are resistant to major classes of antibiotics have created an urgent need for new antibacterial agents (American Society of Microbiology, 1995). Natural products from plants provide a tremendous variety of lead structures, which are used as templates for the development of new products by the pharmaceutical industry. Although microbial products have been the mainstay of industrial natural products discovery (Borris, 1996), in recent years, many researchers have turned to discover the scientific basis of effects related to traditional folk medicine, and some natural products have been approved as new antibacterial drugs (Cragg et al., 1997).

Several species of Baccharis. (Asteraceae) are used in the folk medicine in the form of infusions for curing wounds or locals infections, and as digestives, suggesting possible antibacterial activity (Palacios et al., 1983; Zardini, 1984; Gené et al., 1996). These include B. anomala., B. crispa., B. glutinosa., B. grisebacchii., B. microdonta., B. notosergila., and B. pedunculata. (Palacios et al., 1983; Anesini & Perez, 1993; Rahalison et al., 1995; Verástegui et al., 1996; Feresin et al., 2001; Coelho de Souza et al., 2004), and reports indicate that the antibacterial activity described from Baccharis. genus are attributed to flavonoids like gencawanin and apigenin (Palacios et al., 1983; Xu & Lee, 2001). In southern Brazil, the main species of Baccharis. used in folk medicine are B. trimera. and B. articulata. for digestives properties (Verástegui et al., 1996; Oliveira et al., 20032004). Considering that there are few chemical and pharmacological reports on these plants, the current study was carried out to screen some native South Brazilian Baccharis. species in order to detect new sources of antibacterial agents.

Materials and Methods

Plant material

The aerial parts of Baccharis articulata. (Lam.) Persoon and Baccharis trimera. (Less) A. P. de Candolle were collected in Camargo, State of Rio Grande do Sul, Brazil, in April 2003. The voucher specimens were identified by Profa. Msc. Branca Severo from Universidade de Passo Fundo and are deposited in the herbarium of Instituto de Ciências Biológicas of this university. Baccharis spicata. (Lam.) Bailon and Baccharis usterii. Heering were collected in Porto Alegre, State of Rio Grande do Sul, Brazil, in February 2002. The voucher specimens were identified by Marcos Sobral from Universidade Federal do Rio Grande do Sul and are deposited in the herbarium of the Botanical Department of this university.

Preparation of plant extracts

Aerial parts (50 g) of B. articulata., B. spicata., B. trimera., and B. usterii. were crushed and extracted under reflux (90°C) with 500 ml of ethanol for 30 min. After cooling, each extract was filtered separately, the volume was adjusted to 500 ml with water, and the preparation was separated in two fractions of 250 ml. One fraction was evaporated under reduced pressure to dryness to obtain the crude extract. The ethanol content of the second fraction was removed under reduced pressure, its volume was adjusted to 250 ml with water, and this aqueous suspension was successively partitioned (3 × 50 ml) with ethyl acetate (yielding the ethyl acetate fraction) and n.-BuOH (yielding the n.-BuOH fraction). These fractions were evaporated to dryness and redissolved with sterile water in concentrations of 50, 10, 5, 2.5, and 1.25 mg/ml for biological tests.

Phytochemical characterization of plants extracts by thin-layer chromatography

The presence of different constituents in crude extracts from B. articulata., B. spicata., B. trimera., and B. usterii. was established by thin-layer chromatography (TLC) on silica gel plates (Merck 60 F₂₅₄ 20 × 20 cm) using as mobile phases CHCl₃─EtOH─HOAc (100:40:6 v/v) and n.-butanol:acetic acid:water (BAW) (5:1:3 v/v). Detection was performed, respectively, with chlorosulfonic acid–glacial acetic acid reagent spraying and heating for terpenoids, and for phenolic compounds, fluorescence at 365 nm after spraying with 1% diphenylboryloxyethylamine in CH₃OH (Wagner, 1983).

Antibacterial assays

The disk diffusion method was used as a screening test for antibacterial activity. Filter-paper disks (6 mm in diameter) impregnated with extract solutions were placed on Müeller-Hinton agar plates (Merck®, Darmstadt, Germany), which were inoculated with test organisms according to the standard protocol described by the National Committee of Clinical Laboratory Standards (2004). The filter-paper disks were impregnated with 20 µl of the extract solutions in order to obtain final concentrations of 1000, 200, 100, 50, and 25 µg of extract in the disks. The microorganisms from the American Type Culture Collection (ATCC) used for the biological evaluation were Staphylococcus aureus. (ATCC 25923), Escherichia coli. (ATCC 25922), Pseudomonas aeruginosa. (ATCC 27853), Enterococcus faecalis. (ATCC 14506), and Enterococcus faecium. (ATCC 10541) (Rios et al., 1988).

The plates were incubated at 35°C (±1°C), and after 18 h the diameters of the inhibition zones were measured. Filter-paper disks containing sterile water without any test compound were negative control and no inhibition was observed. Standard antibiotic disks were selected according to the sensitivity of the bacteria tested. Thus ampicillin (10 µg), chloramphenicol (30 µg), and ceftazidin (30 µg) were used (Rios et al., 1988).

The minimum inhibitory concentration (MIC) of the extracts was determined by the macrobroth dilution assay. The bacterial suspensions were diluted from the original suspensions, which had a turbidity of 0.5 MacFarland standard (1.5 × 10⁸ CFU/ml). The tubes were incubated at 35°C (±1°C) for 18 h. For the evaluation of the active extract, four dilutions at 100, 50, 25, and 12.5 mg/ml were prepared. MIC values were determined as the lowest concentration of the extract that completely inhibited growth of the test organisms (Rios et al., 1988; Mazzanti et al., 2000).

The tube that showed negative visible growth after 18 h of incubation was replated on BHI (brain-heart infusion) plates. The viability of bacteria was assessed from colonies after 18 h incubation at 35°C (±1°C). The lowest concentration of the extracts giving negative growth of bacteria was recorded as the minimum bactericidal concentration (MBC) (Wongkham et al., 2001).

Results and Discussion

The development of new drugs with antimicrobial properties has been the mainstay in recent years. Natural products from plants and microorganisms traditionally have provided many lead compounds in the search for new drugs and medicines (Borris, 1996).

Investigation of the antimicrobial activity of medicinal plants has encountered some problems because of the diverse criteria and techniques employed and the lipophilic properties of some samples. Diffusion and dilution methods have been employed to study the antimicrobial activity of medicinal plants (Rios et al., 1988).

Considering the large use of the genus Baccharis. in folk medicine as an anti-inflammatory and antibacterial, as well as a lack of reports describing the screening for antibacterial activity of native South Brazilian Baccharis. species, and the potential therapeutic importance of the title plant, we decided to verify the antibacterial activity of B. articulata., B. spicata., B. trimera., and B. usterii..

The extracts of plants were prepared in order to obtain polar compounds considering ethnopharmacological uses of the plants in rural areas for antibacterial activity. The inhibition zone diameters of 16 extracts were measured and are shown in Table 1. We verified that four extracts showed antibacterial activity, displaying varying degrees and specificity of inhibition against different bacterial species.

Table 1.. Results of screening of Baccharis. extracts/fractions using the disk diffusion method.

			Inhibition zone (mm)
			B. trimera. (µg)					B. spicata. (µg)					B. usterii. (µg)
			Doses (µg)					Doses (µg)					Doses (µg)
Extracts	Bacterial	Positive controls (mm)	1000	200	100	50	25	1000	200	100	50	25	1000	200	100	50	25
Crude extract	S. aureus.^a.	28	12	−	−	−	−	−	−	−	−	−	12	−	−	−	−
	E. coli.^b.	26	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	E. faecium.^a.	27	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	E. faecalis.^a.	30	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	P. aeruginosa.^c.	28	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
EtOAc fraction	S. aureus.^a.	28	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	E. coli.^b.	28	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	E. faecium.^a.	28	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	E. faecalis.^a.	28	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	P. aeruginosa.^c.	27	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
n.-BuOH fraction	S. aureus.^a.	27	−	−	−	−	−	15	−	−	−	−	21	11	8	−	−
	E. coli.^b.	26	−	−	−	−	−	8	−	−	−	−	−	−	−	−	−
	E. faecium.^a.	27	−	−	−	−	−	24	−	−	−	−	16	11	9	−	−
	E. faecalis.^a.	29	−	−	−	−	−	10	−	−	−	−	29	15	12	−	−
	P. aeruginosa.^c.	25	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
Aqueous residue	S. aureus.^a.	28	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	E. coli.^b.	25	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	E. faecium.^a.	28	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	E. faecalis.^a.	29	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−
	P. aeruginosa.^c.	26	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−

−, No activity.

^a.Ampicillin (10 µg).

^b.Chloramphenicol (30 µg).

^c.Ceftazidin (30 µg).

The n.-BuOH fraction of B. usterii. exhibited good activity against Gram-positive bacteria (S. aureus., E. faecium., and E. faecalis.) at a concentration of 1000 µg, with a similar inhibition zone for E. faecalis. when compared to a positive control (ampicillin). The same fraction also had antibacterial activity in concentrations of 100 and 200 µg, although with inhibition zones lower than positive control.

B. spicata. showed activity against Gram-positive (S. aureus., E. faecium., and E. faecalis.) and Gram-negative (E. coli.) bacteria at a concentration of 1000 µg. However, herein, the higher activity was verified against E. faecium., with an inhibition zone similar to the positive control.

The weakest activity was found with the crude extract of B. trimera. and B. usterii. against S. aureus., which showed an inhibition zone lower than the positive control at a concentration of 1000 µg.

The difference in the antibacterial spectra suggests that the active components present among fractions are different. TLC analyses of n.-BuOH fractions of B. spicata. and B. usterii. showed a predominance of polar compounds, mainly flavonoids and terpenoids. These components could contribute to the inhibitory effect displayed by these fractions (data not shown). The extracts from B. articulata. were not active against microorganisms tested. Unfortunately, none of the extracts tested showed activity against P. aeruginosa., which have in clinical cases a high degree of resistant forms against different antibacterial drugs.

The extracts and fractions that showed antibacterial activity by the disk diffusion method were further tested by the macro broth dilution to determine the MICs. Considering that in this method turbidity is taken as an indication of bacterial density and that color of the active extracts could influence the results obtained, we decided to replate on BHI plates the tubes that showed negative visible growth after 18 h of incubation, in order to obtain the MBC of the extracts. The MBCs obtained for the active extracts were 50, 25, and 12.5 mg/ml, and that obtained for standard chloramphenicol was 4 mg/ml (Table 2). These findings suggest that results obtained herein with crude extracts and fractions have potential antimicrobial activity, suggesting that this activity should be higher when the compounds are isolated.

Table 2.. Results of minimum bactericidal concentration (MBC) of active extracts from Baccharis. species.

		MBC (mg/ml)
Plants	Extracts	S.. aureus.	E.. coli.	E.. faecium.	E.. faecalis.	P.. aeruginosa.
B. spicata.	n.-BuOH fraction	50	50	50	50	−
B. trimera.	Crude extract	25	−	−	−	−
B. usterii.	Crude extract	12.5	−	−	−	−
	n.-BuOH fraction	25	−	50	50	−
Chloramphenicol		4	4	4	4	4

−, Activity higher than 100 mg/ml.

The antibacterial activity presented herein with tested Baccharis. species must not be considered high in relation to drugs used as references but are similar to the results observed with Baccharis glutinosa. (Persoon) extract against Clostridium perfringens., Listeria monocytogenes., Proteus vulgaris., and other microorganisms (Verástegui et al., 1996). In Baccharis. species, the antibacterial activity was attributed to the presence of flavonoids such as gencawanin and apigenin and to diterpenes (Palacios et al., 1983; Anesini & Perez, 1993; Rahalison et al., 1995; Verástegui et al., 1996; Cowan, 1999; Feresin et al., 2001). The antibacterial activity of some chalcones, isoflavones, and biflavones (Xu & Lee, 2001) was also reported. Although the literature confirms that flavonoids gencawanin and apigenin are present in extracts of B. articulata. (Gianello & Giordano, 1984), and that these compounds are potential antimicrobial agents, our results showed no activity for this extract with the bacteria tested.

This study presents the screening of antibacterial compounds from South Brazilian Baccharis. species used in South American traditional medicine. Bioguided fractionation must be carried out using the active fractions detected herein in order to identify the active compounds.

Acknowledgments

We are grateful to Marcos Sobral (Programa de Pós-Graduação em Ciências Farmacêuticas, UFRGS), Prof. Dr. Sérgio Bordignon (ULBRA), and Profa. Msc. Branca Severo (ICB-UPF) for locating, collecting, and identifying the plant material. This work was supported by FAPERGS (02/0057.0-PROADE2-RS/BRAZIL) and a CNPq (Brazil) fellowship to G.G.