In Vitro. Antibacterial Activity of Four Leuzea. Species

Abstract

The crude ethanol extracts of the aerial parts and roots of Leuzea carthamoides. DC., Leuzea centauroides. (L.) Holub, Leuzea longifolia. Hoffmgg. & Link, and Leuzea rhapontica. subsp. bicknellii. (Briq.) Holub have been investigated for their antibacterial activity. Growth inhibition using a broth tube dilution test was determined against Bacillus cereus. Frankland and Frankland, Bacillus subtilis. (Ehrenberg) Cohn, Bacteroides fragilis. (Veillon and Zuber) Castellani and Chalmers, Enterococcus faecalis. (Andrewes and Horder) Scheifer and Kilpper-Bälz, Escherichia coli. (Migula) Castellani and Chalmers, Pseudomonas aeruginosa. (Schroeter) Migula, Staphylococcus aureus. (Rosenbach), Staphylococcus epidermidis. (Winslow and Winslow) Evans, Streptococcus pneumoniae. (Klein) Chester, and Streptococcus pyogenes. (Rosenbach). The results showed that extracts from aerial parts of all species tested exhibited significant antibacterial activity, especially against Bacillus cereus., Staphylococcus epidermidis. and Bacteroides fragilis.. In comparison with other Leuzea. species, L. carthamoides. was the most effective against the bacteria used in this study.

Keywords:

• Antibacterial activity
• Leuzea
• Rhaponticum

Introduction

The genus Leuzea. DC. (syn. Rhaponticum. Ludw.) belongs to the family Compositae and comprises about 30 species of perennial herbs distributed mostly in the Old World from Morocco and Portugal to Eastern Asia (Holub, 1973). The plants of the genus have been traditionally used in folk medicine as a tonic (Gammerman & Grom, 1976) or digestive remedies (Vazquez, 1997). Leuzea carthamoides. DC. [Bas.: Rhaponticum carthamoides. (Willd.) Iljin] is the most studied species of the genus because of its marked biological activities, for example immunomodulatory (Lamer-Zarawska et al., 1996) and antimicrobial (Kokoska et al., 2002) effects. The principal constituents of the whole plant are ecdysteroids (Girault et al., 1988) and flavonoids (Varga et al., 1990). Polyacetylenes have also been detected in different parts of the plant (Szendrei et al., 1984).

Despite isolation of polyacetylenes from L. centauroides. (L.) Holub (Christensen & Lam, 1989) and sesquiterpene lactones from L. longifolia. Hoffmgg. & Link (Santos et al., 1988) the phytochemistry and pharmacology of both of these species are poorly known. According to our best knowledge, the phytochemical and pharmacological studies on L. rhapontica. subsp. bicknellii. (Briq.) Holub are completely lacking.

In this study, we aimed to determine the in vitro. antibacterial activity of crude ethanol extracts from aerial parts and roots of four Leuzea. species against Gram-positive and Gram-negative bacteria.

Materials and Methods

Plant materials

The fresh aerial parts and roots of L. carthamoides., L. centauroides., L. longifolia., and L. rhapontica. subsp. bicknellii. were collected from plants grown on experimental fields of the Czech University of Agriculture Prague (CUA Prague) in September 2001. The voucher specimens authenticated by Dr. Kokoska have been deposited in the Institute of Tropics and Subtropics Herbarium, CUA Prague. The specimen numbers are given in Table 1.

Table 1.. Minimum inhibitory concentrations of crude extracts from four Leuzea. species (mg/ml).

	Plant species and specimen number/part tested/yield (g)
	LCA (Kok 001)		LCE (Kok 002)		LL (Kok 004)		LR (Kok 003)		Positive and negative controls
	AP	R	AP	R	AP	R	AP	R	CIP	DMSO
Microorganisms	10.02	3.31	5.28	7.93	10.19	10.38	10.57	13.65	(µg/ml)	(1%)
Gram-positive
Bacillus cereus.	6.3	12.5	12.5	12.5	6.3	50	6.3	25	1	N.A.
Bacillus subtilis.	25	12.5	100	25	25	25	25	100	2	N.A.
Enterococcus faecalis.	50	25	N.A.	50	25	N.A.	25	N.A.	1	N.A.
Staphylococcus aureus.	12.5	12.5	12.5	50	12.5	50	12.5	50	0.5	N.A.
Staphylococcus epidermidis.	6.3	6.3	12.5	50	6.3	50	12.5	12.5	1	N.A.
Streptococcus pneumoniae.	12.5	12.5	25	12.5	12.5	N.A.	12.5	N.A.	1	N.A.
Streptococcus pyogenes.	50	50	25	25	25	100	12.5	100	1	N.A.
Gram-negative
Bacteroides fragilis.	6.3	12.5	12.5	12.5	6.3	50	12.5	50	2	N.A.
Escherichia coli.	50	50	100	100	100	100	N.A.	N.A.	0.015	N.A.
Pseudomonas aeruginosa.	50	N.A.	100	100	100	N.A.	N.A.	N.A.	0.25	N.A.

LCA, Leuzea carthamoides.; LCE, Leuzea centauroides.; LL, Leuzea longifolia.; LR, Leuzea rhapontica. subsp. bicknellii.; AP, aerial part; R, root; CIP, ciprofloxacin; DMSO, dimethylsulfoxide; N.A.: not active (> 100 for extracts and > 1 for DMSO).

Preparation of extracts

Each sample of the dried plant material was ground to a fine powder. Forty-five grams of the powder were extracted at room temperature with 80% ethanol (1350 ml) for 5 days, subsequently filtered, and the extract evaporated to dryness in vacuo. at 40°C. The residue was resolved in 1% (v/v) solution of dimethylsulfoxide (DMSO) in Tris buffer saline (TBS) of pH 7.6 (Sigma-Aldrich, St. Louis, MO, USA) to create a concentration of 200 mg/ml stock solution. All samples were sterilized by filtration through a 0.23-µm membrane filter and stored at + 4°C until tested. The yields of dried residues are shown in Table 1.

Microorganisms and media

The following strains of Gram-positive bacteria were used: Bacillus cereus. ATCC 11778, Bacillus subtilis. ATCC 6633, Enterococcus faecalis. ATCC 29212, Staphylococcus aureus. ATCC 25923, Staphylococcus epidermidis. ATCC 12228, Streptococcus pneumoniae. ATCC 6305, and Streptococcus pyogenes. ATCC 19615. The Gram-negative strains used in this study were Bacteroides fragilis. ATCC 25285, Escherichia coli. ATCC 25922, and Pseudomonas aeruginosa. ATCC 27853.

Bacteroides fragilis. was grown in Wilkins-Chalgren anaerobe broth under anaerobic conditions using Anaerobic Jar HP11 (Oxoid, Basingstoke, UK). Streptococci were grown in brain-heart infusion broth, and other bacteria were tested in Mueller-Hinton broth. All microbial strains and cultivation media were purchased from Oxoid. The susceptibility of all bacterial strains to ciprofloxacin (Sigma-Aldrich) was checked as a positive control (Table 1).

Antibacterial test

In vitro. antibacterial activity was determined by the broth macrodilution method (Jorgensen et al., 1999). Two-fold dilutions (six) of each extract tested were carried out, starting from a concentration of 100 mg/ml. The tubes were inoculated with a bacterial suspension at a density of 10⁷ cfu/ml, incubated at 37°C for 24 h, and then observed for the minimum inhibitory concentration (MIC). The growth of microorganisms was observed as turbidity determined by the UV-Vis spectrometer Helios ε (Spectronic Unicam, Cambridge, UK) at 600 nm. The MIC was determined as the lowest dilution that completely prevented microbial growth. The solution of DMSO (1% v/v) in TBS was simultaneously assayed as the negative control. All samples were tested in triplicate.

Results

Table 1 gives a summary of the investigated Leuzea. species and the results of antibacterial screening. It was observed that the extracts from the aerial parts of all species showed significant antibacterial activity, especially against Bacillus cereus., Staphylococcus epidermidis., and Bacteroides fragilis. (with MICs values ranging from 6.3 to 12.5 mg/ml). The extracts from aerial parts of L. carthamoides. and L. longifolia. were the most active. The moderate or weak inhibition activity was observed for extracts from aerial parts and roots against Bacillus subtilis., Enterococcus faecalis., Staphylococcus aureus., Streptococcus pneumoniae., and Streptococcus pyogenes.. No significant activity was seen against Escherichia coli. and Pseudomonas aeruginosa.. In general, the extracts from aerial parts were found to be more effective than the root extracts (except L. centauroides.).

In comparison with other Leuzea. species tested against bacteria used in this study, L. carthamoides. was the most effective, whereas L. rhapontica. subsp. bicknellii. was the last active.

Discussion

20-Hydroxyecdysone (20E) is currently considered to play an important role in the action of L. carthamoides. (Syrov & Kurmukov, 1976). Within a wide range of biological activities, its antimicrobial property has also been observed (Volodin et al., 1999). However, Ahmad et al. (1996) reported inhibition activity of 20E at rather high concentrations (between 100 and 400 µg/ml).

Previously, antibacterial or antifungal activities of flavonoids (Afolayan & Meyer, 1997), sesquiterpene lactones (Panagouleas et al., 2003), triterpenoid saponins (Taylor & Towers, 1998), and polyacetylenes (Avato et al., 1997) isolated from some species of the family Compositae have been described. The thiophene polyine (polyacetylene) isolated from ethanol extracts of L. carthamoides. underground parts demonstrated significant antifungal activity (Chobot et al., 2003).

As it was stated in a report by Petkov et al. (1984), ethanol extract from roots of L. carthamoides. applied intraperitoneally and subcutaneously in doses up to 4 g/kg did not produce mortality in male albino mice either 24 h or 7 days after its application. Ecdysteroids, the principal constituents of the whole plant, have a very low toxicity in mammals: in the mouse, the LD₅₀ of 20E is 6.4 g/kg (for intraperitoneal injection) and it is >9 g/kg when given orally (Matsuda et al., 1970; Ogawa et al., 1974). Low embryotoxicity of 20E has also been reported (Kosar et al., 1997).

The data summarized above indicate that the four Leuzea. species under study showed antibacterial activity. The combination of antibacterial action and low toxicity of some extracts may indicate their potential as antibacterial herbal remedies. In light of previous phytochemical reports, we also suppose that one or more groups of previously identified compounds may participate in antibacterial action of the tested plants. However, further phytochemical studies are required to determine the types of compounds responsible for the antibacterial effects of these species.

Acknowledgments

This work was supported by grant GA CR 525/02/D107 and by a project of the Ministry of Education, Youth and Sports (FRVS 1523).