Antimicrobial Activities of Volatile Components and Various Extracts of Dictyopteris membranaceae. and Cystoseira barbata. from the Coast of Izmir, Turkey

Abstract

The methanol, dichloromethane, hexane, chloroform, and volatile components of Dictyopteris membranaceae. (Stackhouse) Batters (Dictyotaceae) and Cystoseira barbata. (Good et Woodw.) J. Agardh (Cystoseiraceae) were tested for their antimicrobial activities (four Gram-positive bacteria, four Gram-negative bacteria, and Candida albicans. ATCC 10239). Five compounds were identified in the volatile oil of D. membranaceae. accounting for >85% of the composition of the volatile oil. Twenty-eight compounds were identified in the volatile oil of C. barbata. accounting for >67% of the composition of the volatile oil. Major components were 6-butyl-1,4-cycloheptadiene (43.21%) for D. membranaceae. and docosane (7.61%) and tetratriacontane (7.47%) for C. barbata.. Many compounds in the volatile oil of C. barbata. were identified as hydrocarbon compounds. The volatile oils of these algae did not remarkably inhibit the growth of tested microorganisms. However, the hexane extracts showed more potent antimicrobial activity than methanol, dichloromethane, and chloroform extracts.

Keywords:

• Antimicrobial activity
• brown algae
• Cystoseira barbata
• Dictyopteris membranaceae
• extracts
• volatile compounds

Introduction

Seaweeds are considered to be an important nutrient source for diet and food additives because of their high content of essential and free amino acids (Heiba et al., 1993). The ability of seaweed to produce secondary metabolites of potential interest has been extensively documented (Faulkner, 1993). However, there are numerous reports of compounds derived from macro algae with a broad range of biological activities such as antibiotics (Scheuer, 1990). In spite of an extensive literature, studies on biocide production refer normally to a limited number of algal species with some exceptions (Ballantine et al., 1987). Furthermore, the compounds previously studied were extracted directly from field-collected samples of seaweeds, so there are no reported data concerning the ability of these organisms to produce different compounds under different conditions.

Terpenoids, polyphenols, and C₁₁ metabolites are broadly distributed among brown seaweeds. Most of brown algal secondary metabolites are defensive against a broad spectrum of larger herbivores (Schnitzler et al., 2001). Dictyopteris. Lamouroux genus belonging to the Dictyotaceae family and Cystoseira. C. Agardh genus belonging to the Cystoseiraceae family show distribution in the Atlantic ocean and the Mediterranean, Aegean, and Black seas. Brown algae of the genus Cystoseira. are known to contain enzyme inhibitors, cell division inhibitors, antibacterial and antitumor constituents (Faulkner, 1986; Amico et al., 1988).

Turkey has an extensive coastline along which marine algae are well represented. However, the distribution of such antimicrobial activity within algal thalli has not been studied. The current study investigated the antimicrobial activities within volatile components and various extracts from selected Dictyopteris membranacea. (Stackhouse) Batters and Cystoseira barbata. (Good et Woodw.) J. Agardh from Izmir Bay, Turkey.

Materials and Methods

Organisms

Field collections of seaweed were made from several reefs (depths of 1–2 m) along the Izmir coast during September and November 2004 and seaweed identified by Dr. Atakan Sukatar. Voucher specimens were deposited at the Hydrobiology Laboratory of Ege University, Faculty of Science, Department of Biology (Izmir, Turkey). Samples were frozen immediately after harvesting and stored at −30°C until they were freeze-dried.

Preparation of various extracts

Freeze-dried samples were pulverized. Pulverized samples (15 g for each) were extracted as reported by Khan et al. (1988) and Vlachos et al. (1996) in 150 ml methanol, dichloromethane, hexane, and chloroform for 24 h using a Soxhlet extraction apparatus (yield: 12%, 0.3%, 0.9%, 12% for D. membranacea. and 6%, 0.4%, 0.3%, 18% for C. barbata., respectively). The resulting extracts of D. membranacea. and C. barbata. from these different solvents were kept at 4°C until use.

Extraction of the volatile components

For the volatile compounds, the dried samples of each alga (50 g) were subjected to steam distillation for 4 h using a Clevenger-type apparatus. The distillate was diluted with ethyl acetate and the volume was reduced 100-fold prior to analysis.

Gas chromatography–mass spectrometry (GC-MS) analysis

The steam-distilled components were analyzed by gas chromatography (GC) and gas chromatography and mass spectrometry (GC-MS). A HP 6890 gas chromatograph equipped with a FID and a 5 m × 0.2 mm HP-1 capillary column (0.33 µm coating) was employed for the GC analysis. GC-MS analysis was performed on a HP 5973 mass selective detector coupled with a HP 6890 gas chromatograph, equipped with a HP-1 capillary column. The column temperature was programmed from an initial temperature of 70°C to a final temperature of 280°C at 10°C/min. The injector temperature was 150°C (1 µl injection size), whereas the detector temperature was 250°C. The carrier gas was helium (2 ml/min). Identification of the individual components was performed by comparison of mass spectra with literature data and by a comparison of their retention indices (RI) relative to a C8–C32 n.-alkenes mixture (Adams, 1995). A computerized search was carried out using the Wiley 275 L. GC-MS library and ARGEFAR GC/MS library created with authentic samples.

Test microorganisms

In vitro. antimicrobial studies were carried out against ten bacterial strains (Streptococcus faecalis. ATCC 8043, Bacillus subtilis. ATCC 6633, Staphylococcus aureus. ATCC 6538 p, Staphylococcus epidermidis. ATCC 12228, Streptococcus faecalis. ATCC 8043, Pseudomonas aeruginosa. ATCC 27853, Enterobacter cloacae. ATCC 13047, Escherichia coli. ATCC 29998, Salmonella typhimurium. CCM 583) and one yeast strain (Candida albicans. ATCC 10239), which were obtained from the Microbiology Department Culture Collection of Ege University, Faculty of Science.

Antimicrobial testing

The paper disk diffusion method was employed for determination of antimicrobial activities of volatile oils and extracts of samples (Collins & Lyne, 1989; Bradshaw, 1992). Briefly, sterile, 6-mm-diameter filter paper disks (Schleicher and Schül, no 2668, Dassel, Germany) were impregnated with 20–30 µl of three different concentrations (1, 2, 4 mg disk⁻¹) of the D. membranacea. and C. barbata. extracts.

The bacteria strains were inoculated on nutrient broth (Oxoid) and incubated for 24 h at 37 ± 0.1°C, while the yeast strain was inoculated on malt extract broth (Oxoid Ltd., Hampshire, UK) and incubated for 48 h at 28 ± 0.1°C. Adequate amounts of autoclaved Muller-Hinton agar (Oxoid) and malt extract agar were dispensed into sterile plates and allowed to solidify under aseptic conditions. The counts of bacteria strains and yeast strain were adjusted to yield approximately 1.0 × 10⁷ to 1.0 × 10⁸ ml⁻¹ and 1.0 × 10⁵ to 1.0 × 10⁶ ml⁻¹, respectively, using the Standard McFarland counting method. The test organisms (0.1 ml) were inoculated with a sterile swab on the surface of appropriate solid medium in plates.

The agar plates inoculated with the test organisms were incubated for 1 h before placing the extract impregnated paper disks on the plates. Following this, the sterile disks impregnated with the different extracts were placed on the agar plates. The bacterial plates were incubated at 37 ± 0.1°C for 24 h, and the yeast plates were incubated at 28 ± 0.1°C for 48 h. After incubation, all plates were observed for zones of growth inhibition, and the diameters of these zones were measured in millimeters. All tests were performed under sterile conditions in duplicate and repeated three times. Tobramycin disks (10 µg/disk) and nystatin disks (30 µg/disk) were used as positive controls.

Results and Discussion

The GC-MS method was used to determine the composition of D. membranacea. and C. barbata. volatile compounds. Different groups of compounds were identified, but many of the compounds in the volatile oil of C. barbata. were identified as hydrocarbon compounds. The profile of volatile oils obtained from the steam distillations of D. membranacea. culture was fairly simple. Five compounds were identified from the distillate, accounting for 85.83% of the total composition of the volatile oil. The components are listed in Table 1.

Table 1 Volatile components of D. membranacea. (GC-MS analysis).

Rt (min)	Compound	Area (%)
0.61	3-Allycyclobutane	20.16
11.55	Bicyclo-(2,2,1)-heptadiene	4.07
11.92	6-Butyl-1,4-cycloheptadiene	43.21
20.38	Thiophene-D3	15.72
21.49	(E.)-Farnesene	2.67
	Total	85.83

The major component was 6-butyl-1,4-cycloheptadiene (43.21%). Butenyl-1,4-cycloheptadiene and 6-butyl-1,4-cycloheptadiene have been reported to be common major volatile components in other Dictyopteris. species such as D. prolifera. (Okamura) Okamura, D. latiuscula. (Okamura) Okamura, and D. undulata. Holmes (Dictyotales, Dictyotaceae) (Yamamoto et al., 2001). Also, occurrence and composition of the cyclopentene was explored by Boland et al. (1983) in essential oils of D. prolifera. (Okamura) Okamura, D. undulata. Holmes, and D. divaricata. Okamura. As an interesting compound, structurally-related to the pheromones, cis.-3-butyl-4-vinylcyclopentene was identified by GC-MS analyses in essential oils of D. membranacea. (Stackhouse) Batters and D. prolifera. (Okamura) Okamura (Boland & Müller, 1987; Kajiwara et al., 19891997). The second most abundant component was 3-allycyclobutane (20.16%). Thiophene-D3 (15.72%), bicyclo-(2,2,1)-heptadiene (4.07%), and (E.)-farnesene (2.67%) were also found to be components of the volatile oil of D. membranacea.. Several simple C₁₁ hydrocarbons, such as neodictyoprolene, dictyoprolene, and dictyopterenes (Yamamoto et al., 2001; Song et al., 2005), and C₁₁ sulfur compounds, were derived from Dictyopteris. spp. (Moore, 19761977; Schnitzler et al., 2001). The genus Dictyopteris. seems unusual in that C₁₁ hydrocarbons occur not only in the gametes but are also produced in rather large concentrations in the thalli. Subsequent chemical investigation of the ethyl acetate–soluble fraction of the ethanol extract of D. divaricata. Okamura has led to the isolation of seven cadinane sesquiterpenes and a new sesquiterpene-substituted phenol named dictyvaric acid together with nine known compounds such as 3-farnesyl-p.-hydroxybenzoic acid (Song et al., 2004). However, in this study, we did not identify some compounds previously identified in the volatile oil of D. membranacea.. These observations indicate that the changing composition of the D. membranacea. extracts from the different regions investigated may be due to the seasonal and environmental variations in the production of volatile compounds by the algae. Further investigations showed the release of volatile compounds by macro algae from various climate regions and different seasons (Moreau et al., 1988; Itoh & Shinya, 1994; Laturnus, 1996). In the polar regions and coastal areas, where macro algae occurred down to considerable depth (>30 m) along thousands of kilometers of coastlines, higher concentrations of volatile compounds were detected as compared with the open oceans (Giese et al., 1999).

Twenty-eight compounds were identified in the volatile oil of C. barbata. accounting for >67% of the total composition of the volatile oil. Components are listed in Table 2. Many of the compounds in the volatile oil of C. barbata. were identified as hydrocarbon compounds. C. barbata. consists of docosane (7.61%) and tetratriacontane (7.47%) as major compounds. The other most abundant components were identified as eicosane (5.05%), tricosane (4.43%), hexadecane (4.16%), and heptadecane (1.35%). Heptadecane and hexadecane have been reported to be common major volatile components in many other alga (Tellez et al., 2001). However, chemical composition studies of Cystoseira. genus are very limited. Fucosterol, 22-trans.-dehydrocholesterol, brassicasterol, 24-methylene cholesterol, as well as cystosterol, a new C27 sterol, were identified in different Cystoseira. species (Francisco et al., 1977; Fadli et al., 1991). Also, 14 sterols have been identified by Kamenarska et al. (2002) in the chemical composition of the C. crinita. Bory (Fucales, Cystoseiraceae) from the eastern Mediterranean.

Table 2 Volatile components of C. barbata. (GC-MS analysis).

Rt (min)	Compound	Area (%)
6.27	Octane	0.65
6.51	Heptane	0.78
6.72	2,4-Dimethyl-1-heptene	1.15
10.13	Undecane	0.74
10.98	Tridecane	0.71
11.23	Tetradecane	0.23
14.65	Pentadecane	1.61
15.35	1-Octene	2.01
15.55	Hexadecane	4.16
15.76	Heptadecane	1.35
15.96	Octadecane	0.74
18.60	Nonadecane	0.94
18.89	Eicosane	5.05
19.06	Heneicosane	1.03
19.32	Phenol, 2,4 bis. (1,1-dimethyl ethyl)	2.74
19.70	Docosane	7.61
19.90	Tricosane	4.43
20.04	Tetracosane	2.11
20.11	Pentacosane	1.99
20.27	Hexacosane	2.32
20.37	Heptacosane	0.61
20.47	Octacosane	1.44
20.55	Nonacosane	0.78
22.89	Triacontane	3.18
23.05	Tritriacontane	0.96
23.75	Tetratriacontane	7.47
24.05	1-Heptadecanamin	6.31
24.26	Pentatriacontane	4.51
	Total	67.61

Rt = retention time.

The antibacterial activities are reported in Tables 3, 4, and 5. In this study, it was reported that the volatile oils of D. membranacea. and C. barbata. did not remarkably inhibit the growth of tested microorganisms. Whereas volatile oil of C. barbata. has weak antimicrobial activity against the test organisms, volatile oil of D. membranacea. does not have antimicrobial activity with the test organisms except P. aeroginosae.. However, the hexane extracts of both algae showed more potent antimicrobial activity than methanol, dichloromethane, and chloroform extracts. Generally, when compared with the standard, tobramycin, all extracts exhibited low antimicrobial activity.

Table 3 Antimicrobial activity of D. membranacea. and C. barbata. volatile components.

		Inhibition zone (mm)^a.
		0.015 µg/disk		Standard
Microorganism	G	D. membranacea.	C. barbata.	Tob	Nys
S. epidermidis.	+	–	–	7	nt
S. aureus.	+	–	7	16	nt
S. faecalis.	+	–	–	9	nt
B. subtilis.	+	–	–	24	nt
S. typhimurium.	–	–	7	10	nt
P. aeruginosa.	–	7	–	12	nt
E. cloacea.	–	–	–	13	nt
E. coli.	–	–	7	10	nt
C. albicans.		–	–	nt	18

Tob, tobramycin (10 µg/disk); Nys, nystatin (30 µg/disk); nt, not tested; G, Gram reaction; –, no activity.

^a.Zone of inhibition, including the diameter of the filter paper disk (6 mm); mean value of three independent experiments.

Table 4 Antimicrobial activity of D. membranacea. extracts.

				Diameter of zone of inhibition (mm)
	Methanol (mg/disk)			Dichloromethane (mg/disk)			Hexane (mg/disk)			Chloroform (mg/disk)
Microorganism	1 mg	2 mg	4 mg	1 mg	2 mg	4 mg	1 mg	2 mg	4 mg	1 mg	2 mg	4 mg
S. epidermidis.	—	—	—	—	—	—	—	—	8	—	—	—
S. aureus.	—	—	—	—	—	—	11	12	13	—	—	—
S. faecalis.	—	—	—	—	—	—	—	—	—	—	—	—
B. subtilis.	—	—	—	—	—	—	—	—	7	—	—	7
S. typhimurium.	—	—	—	—	—	—	—	7	8	—	—	7
P. aeruginosa.	—	—	—	—	—	—	—	—	—	7	7	8
E. cloacea.	—	—	—	—	—	—	7	—	—	—	—	8
E. coli.	—	—	—	—	—	—	—	7	—	—	—	8
C. albicans.	—	—	9	—	—	—	—	11	16	—	—	—

Table 5 Antimicrobial activity of C. barbata. extracts.

				Diameter of zone of inhibition (mm)
	Methanol (mg/disk)			Dichloromethane (mg/disk)			Hexane (mg/disk)			Chloroform (mg/disk)
Microorganism	1 mg	2 mg	4 mg	1 mg	2 mg	4 mg	1 mg	2 mg	4 mg	1 mg	2 mg	4 mg
S. epidermidis.	—	7	7	—	7	7	7	7	8	7	7	8
S. aureus.	—	—	—	—	—	—	10	12	16	—	7	—
S. faecalis.	—	—	—	—	—	—	—	—	—	—	—	—
B. subtilis.	—	—	—	—	—	—	—	—	—	—	—	7
S. typhimurium.	—	—	—	—	—	—	—	—	—	—	—	8
P. aeruginosa.	—	—	—	—	7	7	7	7	8	—	—	—
E. cloacea.	—	—	—	—	7	7	7	7	8	—	—	—
E. coli.	—	—	—	—	—	—	—	—	—	—	—	—
C. albicans.	—	—	—	—	—	—	—	—	—	—	—	—

Although methanol and hexane extracts of D. membranacea. showed activity on the yeast C. albicans., but less than that of the standard nystatin, none of the extracts from C. barbata. showed activity against C. albicans.. The antimicrobial effect of several types of extracts (methanol, ethanol, diethyl ether, hexane, chloroform, and water) of Cystoseira tamariscifolia. (Hudson) Papenfuss was carried out on yeasts and only ethanol extracts showed antimicrobial activity against yeasts (Zinedine et al., 2004). Also, the Et₂O fraction of C. tamariscifolia. showed more potent antibacterial activity than hexane and CH₂Cl₂ fractions (Abourriche et al., 1999).