Investigation of Some Medicinal Plants from Tunisia for Antimicrobial Activities

Abstract

Twenty-three selected plants belonging to 12 families were collected from different localities of Tunisia and dried and extracted with the solvents petroleum ether or hexane, ethyl acetate or acetone, and methanol to yield 72 extracts. The extracts were tested for their antimicrobial activity against 14 Gram-positive and Gram-negative bacteria using agar diffusion and microdilution methods. The antimicrobial profiles varied considerably. Fifty-six (77.78%) extracts showed some degree of activity against one or more of the bacterial strains. Antibacterial activity was demonstrated especially against Gram-positive bacteria. The greatest activity was exhibited by all Cistus monspeliensis. L. (Cistaceae) leaves and flowers extracts, Inula viscosa. (L.) Ait. (Asteraceae) and Juniperus phoenicea.L. (Cupresaceae), by the acetone and methanol extracts of Arbutus unedo. L. (Ericaceae) and Polygonum aviculare. L. Var. Bellardi (All.) Duby (Polygonaceae), by the hexane and acetone extracts of Hypericum crispum. L. (Hypericaceae), by the methanol extract of Satureia nervosa. Desf. (Labiateae), and by the ethyl acetate extract of Atriplex parvifolia. Lowe Var. ifiniensis (caball) Maire (Chenopodiaceae). The observation that each extract showed a distinctive permutation of target organisms suggested that different bioactive phytochemicals were present in each species.

Keywords:

• Antimicrobial activity
• medicinal plants
• Tunisia

Introduction

The ever increasing resistance of human pathogens to current anti-infective agents is a serious medical problem, leading to the need to develop novel antibiotic prototype molecules. The investigation of medicinal plants as a guide to biologically active extracts has been well established. Scientists observing these practices have facilitated the discovery of a host of economically important drugs (Cox & Balick, 1994). The screening of plant extracts and plant products for antimicrobial activity has shown that higher plants represent a potential source of new anti-infective agents (Amani et al., 1998; Salvat et al., 2001; Ordonez et al., 2003; Arias et al., 2004). Higher plants produce hundreds to thousands of diverse chemical compounds with different biological activities (Hamburger & Hostettmann, 1991; Pichersky & Gang, 2000). Of the estimated 250,000 higher plants in the world (Wilson, 1988), only 5–15% have been studied for a potential therapeutic value (Balandrin et al., 1985; Kinghorn, 1992). A large number remain to be investigated.

Tunisia has a great variety of plants used in folk medicine, and only a few of these have been studied for their antimicrobial activities. Most of the inhabitants of Tunisia rely on medicinal plant preparations for the treatment of many diseases, including those of an infectious nature. The preparations may be obtained from healers or they may be processed directly from raw materials according to traditional methods (Le Floc'h, 1983; Boukef, 1986).

The main objective of this study was to search for Tunisian medicinal plants with antimicrobial activity that could served as good candidates for the development of new antimicrobial agents. In this article, we reported on antibacterial activity of 23 plants used in Tunisian traditional medicine for the treatment of several diseases.

Materials and Methods

Plant material

The plants were collected from different localities of Tunisia between January and December 2004 and authenticated according to the flora of Tunisia (Pottier-Alapetite, 19791981) by the botanist Dr. Fethia Harzallah-Skhiri (Institut Supérieur Agronomique de Chott-Meriem, Sousse, Tunisia) (Table 1). Voucher specimens were deposited in our laboratory for future reference.

Table 1. List of plants screened for antibacterial activity.

Scientific name	Family	Part used^a.	Popular uses (Le Floc'h, 1983; Boukef, 1986)
Atriplex inflata. Muell.	Chenopodiaceae	L	Unknown
Atriplex parvifolia. Lowe var. ifiniensis (Caball) Maire	Chenopodiaceae	AP	Unknown
Arbutus unedo. L.	Ericaceae	AP	Astringent, diarrhea, diabete, hypotensor,
Artemisia campestris. L.	Asteraceae	AP	Burns, antivenin, diarrhea, snake bites, scorpion stings, urinary infections, fever, cough, stomach ulcer, Eczema, gastroentiritis, dysentery, pain killing, tonic, rheumatism
Chrysanthemum paludosum. Poiret ssp. glabrum (Maire) Q. and S.	Asteraceae	S + L	Unknown
Chrysanthemum Myconis. L.	Asteraceae	L	Unknown
Chrysanthemum grandiflorum. (L.) Batt.	Asteraceae	L	Unknown
Cistus monspeliensis. L.	Cistaceae.	F, L	Wounds, dog bites, depurative
Clematis cirrhosa. L.	Ranunculaceae	AP	Unknown
Clematis flammula. L.	Ranunculaceae	AP	Rheumatism, eczema
Erica multiflora. L.	Ericaceae	AP	Urinary antiseptic, tranquillizing, prostate cancer, astringent
Helianthemum lippii. (L.) Pers	Cistaceae	AP	Unknown
Hypericum crispum. L.	Hypericaceae	AP	Antiseptic, wounds healing, astringent
Inula viscosa. (L.) Ait	Asteraceae	AP	Unknown
Juniperus phoenicea. L.	cupressaceae	AP	Skin diseases, pharyngitis, rheumatism, diabete, diarrhea
Ononis natrix. L.	leguminosae	AP	Dental caries
Plantago coronopus. L. ssp. eu-coronopus Pilger var. vulgaris G.G.	Plantaginaceae	AP	Wounds, emolient
Polygonum aviculare. L. var. Bellardi (All.) Duby	Polygonaceae	AP	Gastric pains, asthma, diarrhea, astringent
Reichardia tingitana. (L.) Roth ssp.	Asteraceae	AP	Unknown
discolor. (Pom.) Batt.
Salicornia fruticosa. L.	Chenopodiaceae	AP	Eczema, skin diseases
Satureia nervosa. Desf.	Labiateae	AP	Unknown
Thymelaea hirsuta. (L.) Endl.	Thymeleaceae	AP	Unknown
Thymus capitatus. (L.) Hoffm. and Link.	Labiateae	AP	Antiseptic, wounds healing, analgesic, depurative, aromatic food

^a.Plant part used: AP, aerial parts; S, stems; F, flowers; L, leaves.

Plant extracts

Air-dried powdered plants were extracted by maceration (3 × 48 h) with solvents of increasing polarity: petroleum ether or hexane, ethyl acetate or acetone, and methanol, yielding crude extracts further evaporated to dryness at 45°C under reduced pressure. Yields of extracts in terms of dry starting materials are listed in Table 2.

Table 2. Antibacterial activity of plant extracts in agar diffusion assay.

			Growth inhibition zone diameter (mm)^b.
Plant species	Extract	Extract yield (%)^a.	P.a.1.	P.a.2.	S.a..	E.c..	E.f..	Sh.f..	S.e..	S.s..	Et.cl..	K.p..	A.h..	Se.m..	S.a.1.	E.c.1.
Atriplex inflata.	PE	0.6	–	–	7	–	8	–	7	7	–	–	–	7	–	8
	EA	3.4	–	–	7	–	8	–	7	7	–	–	–	7	–	8
	M	9	–	–	7	–	8	–	7	7	–	–	–	8	–	–
Atriplex parvifolia. var. ifiniensis	PE	0.6	–	–	9	–	8	–	8	8	–	7	–	7	–	–
	EA	1	12	8	26	20	12	18	25	29	17	20	19	16	20	20
	M	9	–	–	7	–	8	–	7	8	–	–	–	7	–	–
Arbutus unedo.	H	6.2	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	A	17	14	–	24	–	13	–	20	18	–	–	–	–	–	–
	M	28.4	–	–	19	–	14	–	16	13	–	–	–	–	–	–
Artemisia campestris.	H	5.1	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	A	11.2	–	–	13	–	–	–	10	10	–	–	–	–	–	–
	M	17.1	–	–	Ü	–	–	–	–	–	–	–	–	–	–	–
Chrysanthemum paludosum. ssp. glabrum	PE	17.3	–	–	10	–	9	–	8	8	7	–	–	9	8	–
	EA	3	–	–	7	–	9	–	7	9	7	–	–	8	7	–
	M	6.26	–	–	–	–	8	–	–	8	7	–	–	10	–	–
Chrysanthemum myconis.	PE	2	8	–	10	–	9	–	8	8	–	–	–	9	8	–
	EA	2.93	8	–	9	10	9	–	8	9	7	–	–	8	8	–
	M	13.4	9	–	–	–	9	–	–	9	7	–	–	8	–	–
Chrysanthemum.	PE	1.73	7	–	8	–	8	–	8	9	7	–	–	9	8	–
grandiflorum.	EA	2.66	8	–	11	9	10	–	9	9	10	–	–	10	8	–
	M	5.2	7	–	9	–	9	–	9	11	9	–	–	8	–	–
Cistus monspeliensis.	H	26.64	–	–	16	–	–	–	12	12	–	–	–	–	–	–
(leaves)	A	22.55	12	–	26	–	11	–	20	18	–	–	–	–	–	–
	M	21.63	18	–	20	–	15	–	22	18	–	–	–	–	–	–
Cistus monspeliensis. (flowers)	H	25.8	–	–	12	–	–	–	12	10	–	–	–	–	–	–
	A	18.53	12	–	23	–	10	–	20	20	–	–	–	–	–	–
	M	11.72	–	–	20	–	18	–	32	24	–	–	–	–	–	–
Clematis cirrhosa.	H	6.1	–	–	15	–	–	–	14	13	–	–	–	–	–	–
	A	10.3	–	–	12	–	–	–	10	10	–	–	–	–	–	–
	M	15.4	–	–	–	–	–	–	–	–	–	–	–	–	–	–
Clematis flammula.	H	7.02	–	–	15	–	–	–	15	14	–	–	–	–	–	–
	A	13.05	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	M	19.2	–	–	–	–	–	–	–	–	–	–	–	–	–	–
Erica multiflora.	H	6.86	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	A	23.43	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	M	38.9	–	–	12	–	9	–	8	10	–	–	–	–	–	–
Helianthemum lippi.	H	2.36	–	–	–	–	–	–	10	–	–	–	–	–	–	–
	A	3.94	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	M	4	–	–	–	–	–	–	–	–	–	–	–	–	–	–
Hypericum crispum.	H	17.13	–	–	26	–	14	–	15	10	–	–	–	–	–	–
	A	27.36	–	–	20	–	15	–	15	14	–	–	–	–	–	–
	M	36.26	–	–	12	–	13	–	11	12	–	–	–	–	–	–
Inula viscosa.	H	7.14	–	–	16	10	–	–	16	20	–	–	–	–	–	–
	A	11.75	–	–	16	–	–	–	12	12	–	–	–	–	–	–
	M	13.76	–	–	16	–	–	–	10	10	–	–	–	–	–	–
Juniperus phoenicea.	H	9.54	–	–	13	–	16	–	12	8	–	–	–	–	–	–
	A	19.4	–	–	19	–	11	–	14	13	8	–	–	–	–	–
	M	24.75	–	–	20	–	15	–	25	20	–	–	–	–	–	–
Ononis natrix.	H	5.9	–	–	15	–	–	–	12	12	–	–	–	–	–	–
	A	16.4	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	M	21.3	–	–	–	–	–	–	–	–	–	–	–	–	–	–
Plantago coronopus. ssp. eu-coronopus var. vulgaris	PE	1.06	–	–	11	–	–	10	7	–	–	–	–	–	10	–
	EA	13.02	–	–	10	–	–	9	–	–	–	–	–	–	9	–
	M	15.3	–	–	10	–	–	8	7	–	8	–	–	9	8	–
Polygonum aviculare. var. Bellardi	H	1.1	–	–	10	–	9	–	14	8	–	–	–	–	–	–
	A	5.43	–	–	16	–	15	–	12	11	–	–	–	–	–	–
	M	16.44	–	–	16	–	–	–	16	15	–	–	–	–	–	–
Reichardia tingitana.	PE	8.6	–	–	9	–	–	10	7	–	–	–	–	–	10	–
	EA	10.5	–	–	7	–	–	–	–	–	–	–	–	7	7	–
	M	19.08	–	–	9	–	–	–	–	–	–	–	–	7	7	–
Salicornia fruticosa.	PE	5.9	–	–	10	–	–	11	7	–	–	–	–	7	–	–
	EA	9.1	–	–	–	–	–	9	–	–	–	–	–	7	–	–
	M	11.8	–	–	–	–	–	8	–	–	–	–	–	7	–	–
Satureia nervosa.	H	0.98	–	–	–	–	–	–	8	–	–	–	–	–	–	–
	A	5.28	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	M	14.94	–	–	13	–	–	–	28	22	–	–	–	–	–	–
Thymelaea hirsuta.	PE	4	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	EA	3.9	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	M	11	–	13	15	14	–	–	13	11	–	–	–	–	–	–
Thymus capitatus.	PE	4	–	–	–	–	–	–	–	–	–	–	–	–	–	–
	EA	3	–	12	9	11	12	–	–	–	–	–	–	–	–	–
	M	6	–	9	10	9	–	–	–	–	–	–	–	–	–	–
Erythromycin (15 µg/disc)			–	–	24	–	17	–	n.t.	23	–	–	20	–	24	–
Oxacillin (5 µg/disc)			–	–	30	–	9	–	n.t.	29	–	–	–	–	32	–
Tetracyclin (30 UI)			10	13	24	26	22	26	n.t.	36	23	24	23	19	35	18

P.a.1.: Pseudomonas aeruginosa. ATCC 27950; P.a.2.: Pseudomonas aeruginosa. ATCC 27853; S.a..: Staphylococcus aureus. ATCC 25923; E.c..: Escherichia coli. ATCC 25922; E.f..: Enterococcus faecalis. ATCC 29212; Sh.f..: Shigella flexneri. ATCC 12022; S.e..: Staphylococcus epidermidis;. S.s..:Staphylococcus saprophiticus;. Et.cl..: Enterobacter cloaceae;. K.p..: Klebsiella pneumoniae;. A.h..: Aeromonas hydrophila;. Se.m..: Serratia marcescens;. S.a.1.: Staphylococcus aureus1;. E.c.1.: Escherichia coli. 1; –: no activity; n.t.: not tested. PE: petroleum ether; H: hexane; EA: ethyl acetate: A: acetone and M: methanol.

^a.Percentage extract yield (w/w) was estimated as (dry extract weight/dry starting material weight) × 100.

^b.Inhibition zones including the diameter of the paper disc (6 mm).

Microorganisms

A collection of 14 test organisms, including both Gram-positive and Gram-negative bacterial strains, was used. The groups included six organisms of American Type Culture Collection (ATCC): Pseudomonas aeruginosa. ATCC 27950, Pseudomonas aeruginosa. ATCC 27853, Staphylococcus aureus. ATCC 25923, Escherichia coli. ATCC 25922, Enterococcus faecalis. ATCC 29212, and Shigella flexneri. ATCC 12022. Eight clinical organisms were obtained from the Laboratory of Microbiology, Faculty of Pharmacy, Monastir, Tunisia: Staphylococcus epidermidis., Staphylococcus saprophiticus., Staphylococcus aureus., Enterobacter cloaceae., Klebsiella pneumoniae., Aeromonas hydrophila., Serratia marcescens., and Escherichia coli.. All tested organisms were stored at + 4°C on Muller-Hinton agar (MH), subcultured every two weeks, and checked for purity.

Antimicrobial activity

Disc diffusion assay

The dried plant extracts were dissolved in dimethylsulphoxide (DMSO; Merck) and then in sterile water, to reach a final concentration of 20 mg/ml, and sterilized by filtration by 0.45 µm Millipore filters. Antimicrobial tests were then carried out by the disc diffusion method (Murray et al., 1995). The media used were Mueller-Hinton agar (Biorad). The discs (6 mm in diameter) were impregnated with 10 µl of the extracts (200 µg/disc) at a concentration of 20 mg/ml and placed on the inoculated agar (for the preparation of the inocula, colonies of bacteria were suspended in Mueller-Hinton broth (Biorad); the suspensions contained 10⁸ CFU/ml of bacteria). Negative controls were prepared using the DMSO solvent employed to dissolve the plant extracts. Oxacillin (5 µg/disc; Gibco), tetracyclin (30 UI/disc; Gibco), and erythromycin (15 µg/disc; Gibco) served as positive reference standards to determine the sensitivity of bacterial strains tested. The inoculated plates were incubated at 37°C for 24 h. Antimicrobial activity was evaluated by measuring the zone of inhibition against the tested organisms. Each assay in this experiment was repeated twice.

Microdilution assay

The minimal inhibition concentration (MIC) values were also studied for the microorganisms that were determined as sensitive to the extracts in disc diffusion assay. The inocula were prepared in broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity. All the extracts dissolved in 10% dimethylsulphoxide (DMSO) were first diluted to highest concentration (2.5 mg/ml) to be tested, and then serial two-fold dilution were made in a concentration range from 2.5 to 0.0195 mg/ml in 10 ml sterile test tubes containing sterile water. MIC values of the extracts against bacterial strains were determined based on a microwell dilution method (Zgoda & Porter, 2001) and described with some modifications as follows.

The 96-well plates were prepared by dispensing into each well 95 µl of Mueller-Hinton broth and 5 µl of the inocula. 100 µl from extracts initially prepared at the concentration of 2.5 mg/ml was added into the first wells. Then, 100 µl from their serial dilutions was transferred into six consecutive wells. The last well, containing 195 µl of MH broth without compound and 5 µl of the inoculum on each strip, was used as negative control. The final volume in each well was 200 µl. Tetracyclin at the concentration range of 400–0.39 µg/ml was prepared in sterile water and used as standard drug for positive control, and the DMSO was maintained as negative control. The plates were incubated at 37°C for 24 h, and the MIC was determined from the lowest concentration of the compounds to inhibit the growth of microorganisms. Inhibition of proliferation was assessed by optical density measurements (625 nm). The extracts tested in this study were screened two times against each organism.

Results

A total of 72 extracts from 23 Tunisian plant species belonging to 12 families were investigated for their antibacterial activity against 14 common Gram-positive and Gram-negative bacteria using agar diffusion and microdilution methods. Table 1 shows the botanical names, families, plant parts used, and traditional uses of those plants in Tunisia. Antimicrobial activities of the investigated extracts are shown in Table 2 and Table 3.

Table 3. Minimum inhibitory concentrations (mg/ml) of plant extracts with greatest activity.

		MIC (mg/ml)
Plant material	Extract	P.a.1.	S.a..	E.c..	E.f..	Sh.f..	S.e..	S.s..	Et.cl..	K.p..	A.h..	Se.m..	S.a.1.	E.c.1.
Atriplex parvifolia.	EA	1.25	0.156	0.312	1.25	0.63	0.312	0.156	0.625	0.312	0.625	0.625	0.312	0.312
Arbutus unedo.	A	1.25	0.312	–	1.25	–	0.312	0.312	–	–	–	–	–	–
	M	–	0.625	–	1.25	–	0.625	1.25	–	–	–	–	–	–
Cistus.	H	–	0.625	–	–	–	1.25	1.25	–	–	–	–	–	–
monspeliensis.	A	1.25	0.156	–	1.25	–	0.312	0.312	–	–	–	–	–	–
(leaves)	M	0.625	0.312	–	1.25	–	0.312	0.625	–	–	–	–	–	–
	A	1.25	0.312	–	1.25	–	0.312	0.312	–	–	–	–	–	–
Cistus monspeliensis. (flowers)	M	–	0.312	–	0.625	–	0.078	0.312	–	–	–	–	–	–
Hypericum crispum.	H	–	0.156	–	1.25	–	1.25	1.25	–	–	–	–	–	–
	A	–	0.312	–	1.25	–	1.25	1.25	–	–	–	–	–	–
Inula viscosa.	H	–	0.625	1.25	–	–	0.625	0.312	–	–	–	–	–	–
	A	–	0.625	–	–	–	1.25	1.25	–	–	–	–	–	–
	M	–	0.625	–	–	–	1.25	1.25	–	–	–	–	–	–
Juniperus phoenica.	H	–	1.25	–	0.625	–	1.25	2.5	–	–	–	–	–	–
	A	–	0.625	–	1.25	–	1.25	1.25	–	–	–	–	–	–
	M	–	0.312	–	0.625	–	0.156	0.625	–	–	–	–	–	–
Polygonum.	A	–	0.625	–	1.25	–	1.25	1.25	–	–	–	–	–	–
aviculare.	M	–	0.625	–	–	–	0.625	1.25	–	–	–	–	–	–
Satureia nervosa.	M	–	1.25	–	–	–	0.156	0.312	–	–	–	–	–	–
Tetracyclin		0.1	0.025	0,01	0.025	0.01	0.001	0.001	0.025	0.025	0.025	0.05	0.001	0.05

P.a.1.: Pseudomonas aeruginosa. ATCC 27950; S.a..: Staphylococcus aureus. ATCC 25923; E.c..: Escherichia coli. ATCC 25922; E.f..: Enterococcus faecalis. ATCC 29212; Sh.f..: Shigella flexneri. ATCC 12022; S.e..: Staphylococcus epidermidis;. S.s..: Staphylococcus saprophiticus;. Et.cl..: Enterobacter cloaceae;. K.p..: Klebsiella pneumoniae;. A.h..: Aeromonas hydrophila;. Se.m..: Serratia marcescens;. S.a.1.: Staphylococcus aureus.1;.E.c.1.: Escherichia coli.1; –: no activity; EA: ethyl acetate; A: acetone; H: hexane and M: methanol.

Fifty-six (77.78%) of the plant extracts showed some degree of activity against one or more of the tested organisms (Table 2). The plants that exhibited significant antimicrobial activity in agar diffusion assay (defined as a perfectly clear zone with a diameter greater than 15 mm) were: Atriplex parvifolia. Lowe Var. ifiniensis (Caball) Maire (Chenopodiaceae), Arbutus unedo. L. (Ericaceae), Cistus monspeliensis. L. (Cistaceae), Hypericum crispum. L. (Hypericaceae), Inula viscosa. (L.) Ait. (Asteraceae), Juniperus phoenicea. L. (Cupressaceae), Polygonum aviculare. L. Var. Bellardi (All.) Duby (Polygonaceae), and satureia nervosa. Desf. (Labiateae) (Table 3). The most susceptible microorganisms were Staphylococcus epidermidis., Staphylococcus saprophiticus., and Staphylococcus aureus. ATCC 25923, which were strongly inhibited by 17 (23.61%) of the extracts (Table 3).On the other hand, none of the extracts inhibited Pseudomonas aeruginosa. ATCC 27853. The greatest zone of inhibition (32 mm) was displayed by C. monspeliensis. flower methanol extracts against S. epidermidi.s. The ethyl acetate extract of A. parvifolia. inhibited growth of all tested microorganisms with a low activity (diameter of inhibition zone was less than 15 mm) against P. aeruginosa. ATCC 27950, P. aeruginosa. ATCC 27853, and Enterococcus faecalis. ATCC 29212.

The 19 extracts of eight plants that exhibited significant antimicrobial activity according to the agar diffusion method (diameter of inhibition zone greater than 15 mm) were tested at various concentrations, ranging from 2.5 to 0.0195 mg/ml, and the MIC values were reported in Table 3. The ethyl acetate extract of A. parvifolia. possessed antibacterial activity against all bacterial strains, with MIC values ranging from 0.156 to 1.25 mg/ml. The strongest activity (MIC = 0.078 mg/ml) was shown by C. monspeliensis. flowers methanol extracts against S. epidermidis.. Among the bacteria tested, Escherichia coli. ATCC 25922, Shigella flexneri. ATCC 12022, Enterobacter cloaceae., Klebsiella pneumoniae., Aeromonas hydrophila., Serratia marcescens., S. aureus. (clinical strain), and E. coli. (clinical strain) proved to be the most difficult to inhibit with all extracts except the ethyl acetate extract of A. parvifolia..

Discussion

In the present investigation, all extracts were able to inhibit the growth of one or more of the tested strains. In classifying the antibacterial activity as Gram positive or Gram negative, it would generally be expected that a much greater number would be active against Gram-positive than Gram-negative bacteria (McCutcheon et al., 1992). In our study, the highest activity was shown against the three Gram-positive bacteria: Staphylococcus epidermidis., S. saprophiticus., and S. aureus. ATCC 25923, which was the most susceptible bacterium of all the tested strains. These results were very interesting since these bacteria can be commonly involved in skin infections with the exception of S. aureus. (Jones et al., 2003; Rennie et al., 2003).

The ethyl acetate extract of Atriplex parvifolia. contained active compound(s) against all the strains tested, with MIC ranging from 0.156 to 1.25 mg/ml. Increasing polarity of the extracting solvent increases the yield of the extract (Table 2). As a result, methanol, which was the most polar of all solvents used for fractionation, afforded the maximum yield. However, the methanol extract was not active against the test strains, and only the relatively polar fraction (ethyl acetate) was active against the bacteria. This result supported the fact that the active compounds are concentrated more in this fraction.

Only the polar fractions of Arbutus unedo. showed antibacterial activity, especially against Staphylococcus epidermidis., S. saprophiticus., and S. aureus. ATCC 25923. In folk medicine, A. unedo. fruits are reported to possess astringent and antiseptic properties (Baytop, 1984).

Cistus monspeliensis. leaf and flower extracts showed an interesting antibacterial activity, particularly against Gram-positive bacteria. The acetone extract of Cistus monspeliensis. leaves inhibited the growth of S. aureus. ATCC 25923, with MIC = 0.156 mg/ml. The methanol extract of C. monspeliensis. flowers inhibited the growth of S. epidermidis., with MIC = 0.078 mg/ml. The whole plant of C. monspeliensis. (“oumillia”) was used in different localities of Tunisia for the treatment of skin infections. The decoction of the plant was applied directly to the skin or taken orally in small quantities as a depurative (Boukef, 1986). Other popular uses found in the literature for C. monspeliensis. were for the treatment of asthma (Polunin, 1969). All Cistus. species were frequently used in many traditional medicines for their antimicrobial activity (Chinou et al., 1994; Demetzos et al., 1999). Previous pharmacological investigations showed that Cistus. leaf extracts have antimicrobial properties (Chinou et al., 1994; Bouamama et al., 1999) against many bacteria and fungi responsible for human infections (Koch, 1981; Russell, 1991), which proved some scientific basis for the utilization of this plant in folk medicine (Peyron & Alessandri, 1986).

Hexane and acetone extracs of Hypericum crispum. showed promising activity against S. aureus. ATCC 25923, with MIC, respectively, of 0.156 and 0.312 mg/ml. The activity of H. crispum. extracts provided preliminary scientific validation for the popular use of this plant in Tunisia. Among the traditional uses of the oil and tannin of this plant are as an antiseptic and as an astringent and for wounds healing (Le Floc'h, 1983).

Inula viscosa. extracts showed an interesting antibacterial activity against the three species of Staphylococcus.,with MIC ranging from 0.312 to 1.25 mg/ml. Le Floc'h (1983) and Boukef (1986) have not reported its possible uses in popular medicine in Tunisia. However, in other countries, this plant has been used for the treatment of respiratory and skin diseases and for wound healing (Dafni et al., 1984; Oran & Al-Eisawi, 1998; Ali-Shtayeh et al., 2000; Lentini, 2000). Phytochemical investigations of this plant resulted in the identification of flavonoids, sesquiterpenoids, and essential oils (Chiappini et al., 1982; Grande et al., 1985; Benayache et al., 1991; Sanz et al., 1991; Abu Zarga et al., 19982003).

Juniperus phoenicea. obtained data were in accordance with previous reports (Angioni et al., 2003) and with traditional uses of the plant. The decoction can also be taken for the treatment of some skin diseases, pharyngitis, diarrhea, and other conditions (Le Floc'h, 1983).

The polar fractions (acetone and methanol extracts) of Polygonum aviculare. exhibited activity against S. aureus. ATCC 25923, with MIC = 0.625 mg/ml. Only the methanol extract of Satureia nervosa. showed activity against S. epidermidis. (MIC = 0.156 mg/ml) and S. saprophiticus. (MIC = 0.312 mg/ml). This result supported the fact that the active compounds were concentrated more in polar fractions of P. aviculare. and S. nervosa..

The extracts of three tested species of Chrysanthemum. showed lower activity against all strains tested, with MIC > 1.25 mg/ml. However, in a previous study, other species belonging to the same genus were reported to have antibacterial (Khallouki et al., 2000; Urzua & Mendoza, 2003; Zhu Shunying et al., 2005) and antifungal activities (Alvarez Castellanos et al. 2001).

The extracts of the other plants, Atriplex inflata. Muell. (Chenopodiaceae), Clematis cirrhosa. L. (Ranunculaceae), Clematis flammula. L. (Ranunculaceae), Helianthemum lippi. (L.) Pers. (Cistaceae), Ononis natrix. L. (Leguminosae), Reichardia tingitana. (L.) Roth ssp. discolor. (Pom.) Batt. (Asteraceae), and Thymelea hirsuta. (L.) Endl. (Thymeleaceae) showed low activity at tested concentrations against all microorganisms. However, it was interesting to note that many other species, such as Artemisia campestris. L. (Asteraceae), Erica multiflora. L. (Ericaceae), Plantago coronopus. L. (Plantaginaceae), Salicornia. fruticosa. L. (Chenopodiaceae), and Thymus capitatus. (L.) Hoffm. and Link. (Labiateae), which have traditionally been used to treat wounds, burns, bites, and skin diseases, were found to be relatively inactive at tested concentrations and against bacterial strains used in our study.

At the same time, our results of antimicrobial assays also justified and partially supported the popular usage of the tested Tunisian plants. The screening of some medicinal plant crude extracts have shown that some of those were potentially rich sources of antibacterial agents. Work is currently being undertaken to isolate the active compound(s) by bioassay-guided fractionation from the species that showed high inhibitory activity during screening.

Acknowledgments

The authors are grateful to Professor Rachid Chemli, Mr. Mohamed Ben Salah (Laboratoire de Pharmacognosie, Faculté de Pharmacie, Monastir, Tunisia), and Misses Dalila Haouas and Lilia Trabelsi (Laboratoire de Biologie Végétale et Botanique, Institut Supérieur Agronomique de Chott-Meriem, Sousse, Tunisia) for their help with plant collection and extraction facilities.