Potent Antibacterial Activity of Eugenia umbelliflora.

Abstract

The current work describes the antibacterial activity of the crude extract (CE) and some fractions (dichloromethane and ethyl acetate) of fruits and leaves from Eugenia umbelliflora. Berg. (Myrtaceae) against eight pathogenic bacteria. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined using the agar dilution method. The results showed that almost all the material tested exhibited excellent activity, comparable to the activity of some typical antibiotics, particularly against the Gram-positive bacteria. In summary, the results obtained with this plant extract may be considered very promising, indicating the potential for obtaining new antiinfectious agents against Gram-positive microorganisms.

Keywords:

• Antibacterial activity
• Eugenia umbelliflora
• medicinal plant

Introduction

The Myrtaceae is a large family of plants consisting of trees and shrubs found in the tropics and subtropics and comprises about 150 genera and 3600 species (Cronquist, 1981). The genus Eugenia. possesses approximately 600 species, of which about 400 grow in Brazil (Cronquist, 1981; Henriques et al., 1993). Experimental studies have revealed that several of these plants exhibit pharmacological action, including anticancer (Gu et al., 2001), antidiarrheic (Almeida et al., 1995), antimutagenic (Miyazawa & Hisama, 2003), gastroprotective (Ramirez & Roa, 2003) and hypoglycemic properties (Costa et al., 2000; Grover et al., 2002; Ravi et al., 2004). Moreover, it has been reported that many species of this genus are potential antimicrobial sources (Bruneton, 1995; Almeida et al., 2000; Shafi et al., 2002; Souza et al., 2002).

Compounds such as flavonoids, triterpenes, tannins, and especially essential oils have already been isolated from the genus Eugenia. (Scalbert, 1991; Henriques et al., 1993; Slowing et al., 1994; Lee et al., 1997; Djipa et al., 2000; Hernandez et al., 2000; Lis-Balchin et al., 2000; Lunardi et al., 2001). However, few reports have been found on the species Eugenia umbelliflora. that relate to chemical (Kuskoski et al., 2003) or biological aspects. E. umbelliflora. is a shrub or a small tree that measures 3–10 m, being popularly known as ‘baguaçu.’, ‘guapê.’, or ‘guamirim.’ in Brazil (Legrand & Klein, 1969).

In this study, we investigated the antibacterial activity of extracts and fractions obtained from leaves and fruits of E. umbelliflora. Berg. using the agar dilution method.

Materials and Methods

Plant material

Leaves and fruits of E. umbelliflora. Berg. were collected in May 2002 in Itapema (Meia Praia), State of Santa Catarina, Brazil, and identified by Dr. Ademir Reis (Department of Botany, UFSC, Florianópolis, SC, Brazil). A voucher specimen was deposited in the Barbosa Rodrigues Herbarium (BRH, Itajaí, SC, Brazil) under number VC Filho 50.

Phytochemical analysis

Air-dried material from different parts of the plant [leaves (1.1 kg) and fruits (250 g)] were powdered and macerated separately with methanol at room temperature for 7 days. After filtration, the solvent was removed by rotary evaporation under reduced pressure yielding the respective crude extracts (CE) (103.28 g and 15.63 g). They were then suspended in water and successively partitioned with dichloromethane (DCM) and ethyl acetate (EtOAc), giving the respective fractions [22.8 g (DCM); 13.3 g (EtOAc)] to the leaves, and [0.881 g (DCM); 2.062 g (AE)] to fruits from E. umbelliflora..

Evaluation of biological activity

Microorganisms

To determine the antimicrobial activity, the following microorganisms were used: Bacillus cereus. (ATCC 14579), Enterobacter cloacae. (ATCC 35030), Escherichia coli. (ATCC 11775), Pseudomonas aeruginosa. (ATCC 27853), Salmonella typhimurium. (ATCC 14028), Staphylococcus aureus. (ATCC 6538P), Staphylococcus saprophyticus. (ATCC 35552), and Streptococcus agalactiae. (ATCC 13813). They were purchased from the tropical culture collection of “André Tosello Technology and Research Tropical Foundation”, Campinas, SP, Brazil.

Quantitative antimicrobial evaluation

The minimal inhibitory concentration (MIC) of the extract and fractions of E. umbelliflora. was determined against Gram-positive and Gram-negative bacteria, using the twofold serial agar dilution assay in concentrations ranging from 1 to 1000 µg/ml. The extracted components of the plant were added to sterile Mueller-Hinton agar medium (Merck, 5437) as solution in dimethyl sulfoxide (DMSO)/water (1:1) (Pretto et al., 2004). The antibacterial agent vancomycin (Sigma, V2002) was included in the assay as a positive control. Drug-free solution was also used as a blank control.

The MIC values were taken as the lowest concentration of extract and fractions that inhibited the growth of the organism after 18 h of incubation at 37°C, and the minimum bactericidal concentration (MBC) was determined by subculture of the tube with inhibition in appropriate agar plate. When microorganisms did not grow, the component was considered a bactericide.

Results and Discussion

We initially prepared the crude extracts (CE) and two fractions from two different parts of the plant, fruits and leaves of E. umbelliflora., in order to determine where the possible active compounds are located. Table 1 shows the results of antibacterial activity obtained by the in vitro. agar dilution method. In extracts that displayed MIC values below 10 µg/ml, the antibacterial activity was considered excellent; from 10 to 100 µg/ml, the antibacterial activity was considered good; from 100 to 500 µg/ml, it was considered moderate; from 500 to 1000 µg/ml, the antibacterial activity was considered weak; over 1000 µg/ml, the extracts were considered inactive.

Table 1.. Antimicrobial activity of extracts and fractions from Eugenia umbelliflora. against Gram-positive bacteria.

	Antibacterial activity (µg/ml)
	B. cereus.		S. aureus.		S. saprophyticus.		S. agalactiae.
Material tested	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
CE Fruits	10	20	6	9	10	30	3	10
DCM Fr. Fruits	7	10	8	10	20	20	2	10
EtOAc Fr. Fruits	20	20	10	15	30	50	4	20
CE Leaves	30	50	50	80	50	100	20	50
DCM Fr. Leaves	50	50	20	40	80	100	10	90
EtOAc Fr. Leaves	300	300	100	100	200	300	400	800
Vancomycin	0.7	—	2	—	2	—	0.8	—

Minimal inhibitory concentration (MIC); minimum bactericidal concentration (MBC); crude extract of fruits (CE Fruits); dichloromethane fraction of fruits (DCM Fr. Fruits); ethyl acetate fraction of fruits (EtOAc Fr. Fruits); crude extract of leaves (CE Leaves); dichloromethane fraction of leaves (DCM Fr. Leaves); ethyl acetate fraction of leaves (EtOAc Fr. Leaves); Bacillus cereus. (B. cereus.); Staphylococcus aureus. (S. aureus.); Staphylococcus saprophyticus. (S. saprophyticus.); Streptococcus agalactiae. (S. agalactiae.).

Both crude extracts (leaves and fruits) and respective fractions (DCM and EtOAc) exhibited antimicrobial activity. However, the extract and fractions of fruits exhibited higher activity than the leaves of E. umbelliflora.. The starting material (CE) of fruits and leaves from E. umbelliflora. inhibited the growth of B. cereus., S. aureus., S. saprophyticus., and S. agalactiae. strains, with MIC values between 3 to 50 µg/ml. Similar activity was also verified for the DCM and EtOAc fractions. However, the EtOAc fraction from the leaves was less active against the microorganisms tested.

As expected, the profile of minimal bacterial concentrations (MBC), was in general, similar to the MIC values (Table 1).

Antimicrobial activity in species of the Mirtaceae family has been reported for essential oils, flavonoids (Slowing et al., 1994; Hernandez et al., 2000; Lis-Balchin et al., 2000) and tannins (Scalbert, 1991; Djipa et al., 2000). No activity was observed against Gram-negative bacteria (Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa., and Salmonella typhimurium.), with exception of the extract of fruits, which inhibited the growth of E. cloacae. strains with activity considered weak (MIC = 900 µg/ml). This can be explained because the outer membrane of Gram-negative bacteria is known to present a barrier to the penetration of numerous antibiotic molecules, and the periplasmic space contains enzymes that are able to break down foreign molecules introduced from outside (Poole, 1994; Schaechter et al., 1999; Duffy & Power, 2001), and efflux pumps that reduce the cellular level of antibiotics (Nikaido, 1989; Köhler et al., 1999; Van Bembeke et al., 2003).

It is important to emphasize that plant compounds are routinely classified as “antimicrobial” based on susceptibility tests that produce MICs ranging from 100 to 1000 µg/ml (Tegos et al., 2002). However, the E. umbelliflora. showed superior activity against Gram-positive bacteria, comparable to the activity of some typical antibiotics, indicating the possibility of the presence of strong active compounds, and the results obtained with this plant may be considered clinically promising, and it may be a valuable source for obtaining new anti-infectious agents, particularly against specific Gram-positive bacteria. Studies are currently in progress to isolate and identify the active principles responsible for the antibacterial properties of E. umbelliflora..

Acknowledgments

The authors are grateful to CNPq, FAPESC, and ProPPEC/UNIVALI for financial support and to Prof. Oscar Benigno Iza (BRH and UNIVALI) for the collection of plant material.