Int J Pharm Pharm Sci, Vol 7, Issue 3, 47-51Original Article

SCREENING OF ANTIBACTERIAL AND ANTIFONGICAL ACTIVITY IN MARINE MACROALGAE AND MAGNOLIOPHYTEA FROM THE COAST OF TUNISIA

RIHAB BEN ABDALLAH KOLSI*, DONYEZ FRIKHA**, IMED JRIBI**, ASMA HAMZA***, LOTFI FEKI*,
KARIMA BELGHITH*

*Laboratoire des Biotechnologies Végétales Appliquées à l’Amélioration des Cultures, **Unité Biodiversité et écosystèmes aquatiques/ Faculté des Science de Sfax, BP 1171, Sfax 3000, Tunisia, ***Institut National des Sciences et Technologies de la Mer, Sfax 3000, Tunisia.
Email: rihab_b86@hotmail.com

Received: 04 Aug 2014 Revised and Accepted: 08 Sep 2014

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ABSTRACT

Objective: The present study was conducted to evaluate the antimicrobial and antifongical activity of Hexane, ethyl acetate and methanol extracts of thirteen marine spices from Tunisian coastline (Chebba and Sfax): five pheophytea, five cholorophytea and three Magnoliophytea.

Methods: These spices were tested against eight human pathogenic bacteria: Gram- (Escherichia coli, DH5 (alpha), listeria monocytogéne, Salmonella enterica, Agrobacterium tumefaciens, Pseudomonas aerigunosa), Gram+ (Staphylococus aureus, Micrococcus luteus), and two human pathogenic yeast (Candida tropicalis, saccharomyces cerevisiae) and a fungi (Aspergillus niger), using the agar disk diffusion assay method.

Results: The results of thirteen marine spices extracts collected from the coast of Tunisia have shown significant antimicrobial activity and the maximum inhibitory activities were observed in the brown algae, it was more active on bacteria than the fungi.

Conclusion: The results of the present study revealed that the seaweeds and magnoliophytea from Tunisia appear to have immense potential as a source of antibacterial and antifongical compounds, they could be used in treating diseases caused by these organisms test.

Keywords: Antibacterial activity, Antifungal activity, Disc diffusion method.

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INTRODUCTION

The marine environment is an exceptional reservoir of bioactive natural compounds, which exhibit structural/ chemical features not found in terrestrial natural products [1].

These compounds are synthesized by different metabolic pathways those observed in terrestrial environments. Among marine organisms, algae, marine magnoliophytea that synthesize a wide variety of secondary metabolites chemically active, which are used for defense against other organizations predators or colonizers. These active metabolites produced by several species of and macro marine micro algae, show antibacterial and anti-inflammatory properties, which are effective in the therapeutic field [2-9]. Several studies indicated that there has bioactivity in marine organisms origins [10-16]. The synthesis of substances Side by marine algae is influenced by seasonal variation and geographical distribution [17, -19]. With the emergence of resistant bacteria to several forms of certain antibiotics, of research new active molecules has become a necessity.

The purpose of this work was to evaluate the antibacterial and antifungal activity of ten Mediterranean marine macroalgae and three Magnoliophytea collected from the coast of Chebba and Sfax (Tunisia), with solvents of increasing polarity extraction (Hexane, Ethyl Acetate, Methanol) against eight human pathogenic bacteria and three human pathogenic yeast in order to discover new antibacterial or / and antifungal metabolites.

MATERIALS AND METHODS

Sample collection

Fresh ten seaweeds: five pheophytea (Dictyota dichotoma, Cystoseira crinita, Cystoseira barbata, Dictyopteris membranaceae and Sargussum vulgare), five cholorophytea (Flabellia petiollata, Anadypméne stellata, codium fragile, Halimeda tuna and Ulva rigida) and three Magnoliophytea: Cymodocea nodosa, Posidonea oceanica and Halophila stipulacea, were collected at Tunisian coastline (Chebba and Sfax), during Mars 2013 by hand picking using Scuba diving or snorkeling (1 – 4 m depth) and preserved on ice until further processing. These species were identified at the National Institute of Oceanology of S fax (INSTM). After collection, the samples were rinsed with fresh seawater and distilled water to remove associated debris and epiphytes. Then seaweeds were dried for 24 h under an artificial light in 24 °C and finally in a heater. The dry seaweeds were crushed in an electric mill until a fine powder was obtained.

Preparation of extracts

For extraction of bioactive in shade dried seaweeds, 600 g of finely powdered algal material were packed in small bags (5x 10 cm) of Whatman filter paper and all bags were sealed and soaked three times in an organic solvent bath for steeping during 24h. The extraction was carried out, separately, with different organic solvents in the order to increase polarity: Hexane, ethyl acetate and methanol. The organic extracts were concentrated to solvent free by evaporation in a rotary vacuum evaporator rotavable at 45 °C. The extracts obtained for each species were stored at 4 °C for testing different biological activities.

Microorganisms

The strains used were Eight bacteria: Gram-positive Escherichia coli (ATCC 8739), Escherichia coli DH5 (alpha), listeria monocytogéne (BUG 496), Salmonella enteria (ATCC 43972), Agrobacterium tumefaciens, Pseudomonas aerigunosa (ATCC 49189), Gram-negative: Staphylococus aureus (ATCC6538), Micrococcus luteus (LB 14110); Antifungal activity was determined against the filamentous fungi Aspergilus niger, unicellular yeast saccharomyces cerevisiae and Candida tropicalis R2 CIP203 (CTR2), which is a strain resistant to both reference polyene antifungal major: amphotericin B and nystatin. were obtained from the Microbiology Departement, Faculty of science, S fax.

Disk diffusion method

The antibacterial activity of extracts was performed using the agar-disk diffusion method [20]. The suspensions of organisms were initially adjusted with sterile distilled water to a density equivalent to the 0.5 McFarland standards. 0.20 ml of a 24 h-broth culture (106 cfu/ml) of the bacteria species were spread on the surface of gelled sterile Mueller-Hinton Agar plates (pH 7, 4 ± 0, 2 à 25 °C) at 37 °C for 24h prior to use. Several colonies of similar morphology of the respective bacteria were transferred into API suspension medium. The different extracts were prepared and then absorbed onto the sterile disks (20 and 30 μl) and the same volume of solvent was used as the negative control. The antibacterial activity was evaluated by measuring the diameter of the inhibition zones surrounding the disks. The experiment was performed in triplicate.

Anti fongical activity

For screening the antifungal activity of algae extracts, the agar-disk diffusion method was used as previously described [21]. Three strains (saccharomycete, Candida tropicalis and Aspergillus niger). All strains were first grown on Sabouraud chloramphenicol agar plate at 30°C for 18-24h. Several colonies were transferred into API suspension medium and adjusted to 2 McFarland turbidity standards with a densimat. The inocula of the respective yeast was streaked into Sabouraud chloramphenicol agar plates at 30°C using a sterile swab and then dried. A sterile filter disk, diameter 5 mm (Whatman paper # 3) was placed in the plate. An amount of 10 µl of the extract were dropped on each paper disc (10 mg/ disk). The treated Petri dishes were incubated at 30 °C for 18- 24h. The antifungal activity was evaluated by measuring the diameter of the inhibition zones around the disks. The same volume of solvent was used as the negative control. Each experiment was carried out in triplicate and the mean diameter of the inhibition zones was recorded.

RESULTS

Anti bacterien test

Currently, nosocomial infections are a major problem in public health. The bacteria is considered among the bacteria most responsible for these infections. the deadliest bacteria we can cite Staphylococcus aureus (gram+) who shares with Escherichia coli (gram-) the forefront of germs responsible for nosocomial infections. To this worrying situation it is essential to find new antibiotics active from natural resources. The results of antibacterial tests of the species studied are given in table 1, present inhibition diameters obtained on bacteria pathogens references. The antibacterial activity was classified from less active (+: Diameter of inhibition 10 mm) to moderately active (++:10 mm Diameter of inhibition 15 mm), to highly active (+++Diameter of inhibition 15 mm) and no active (-: resistant).

Table 1: Antimicrobial activity of macroalgae extracts

Algea	Extractant	1	2	3	4	5	6	7	8
Gram		-	-	-	-	+	+	-	-
Phéophytea
I	Hexane	-	+	-	+	-	+	-	-
	Ethyl Acetate	-	+	+	+	+	++	-	-
	Methanol	-	+	+	+	++	++	-	-
II	Hexane	-	-	-	-	+	-	-	-
	Ethyl Acetate	-	-	+	-	++	-	-	-
	Methanol	-	-	-	-	+++	+	-	-
III	Hexane	-	-	-	-	-	-	-	-
	Ethyl Acetate	-	-	-	+	-	-	+	-
	Methanol	-	-	-	-	+	+++	-	-
IV	Hexane	-	-	-	-	-	+	-	--
	Ethyl Acetate	-	+	-	-	+	-	-	-
	Methanol	+	-	-	-	+++	-	-	-
V	Hexane	-	-	-	-	-	-	+	+
	Ethyl Acetate	-	+	-	-	-	+	-	-
	Methanol	++	-	-	-	++	+++	-	+
Chlorophytea
VI	Hexane	-	-	-	-	-	+	-	-
	Ethyl Acetate	+	+	+	-	+	+	-	-
	Methanol	-	-	-	-	++	-	-	-
VII	Hexane	-	-	+	-	-	-	-	-
	Ethyl Acetate	+	-	-	+	++	-	-	-
	Methanol	+	-	-	-	++	+	-	-
VIII	Hexane	-	-	-	-	-	-	-	-
	Ethyl Acetate	-	-	-	-	-	-	-	-
	Methanol	-	-	-	-	-	-	-	-
IX	Hexane	-	-	-	-	-	-	++	-
	Ethyl Acetate	+	-	-	-	+	-	-	-
	Methanol	++	-	+	-	++	+	-	-
X	Hexane	-	-	+	-	-	-	-	-
	Ethyl Acetate	+	+	-	-	-	-	-	-
	Methanol	-	-	-	-	+++	-	-	+
Magnoliophytea
XI	Hexane	-	-	-	-	-	-	-	-
	Ethyl Acetate	-	+	-	-	-	+	-	-
	Methanol	+	+	+	-	+	+	-	-
XII	Hexane	-	-	-	-	-	-	-	-
	Ethyl Acetate	-	-		-	-	+	-	-
	Methanol	-	+	+	-	+	++	-	-
XIII	Hexane	-	-	-	-	-	-	-	-
	Ethyl Acetate	-	-	+	+	-	-	-	-
	Methanol	-	-	-	-	++	++	-	-

I -Dictyota dichotoma (Dd) from Chebba II- Cystoseira crinita (Cc) from Chebba III- Cystoseira barbata (Cb) from Sfax IV- Dyctéopteris menbranacea (Dm) from Sfax V- Sargussum vulgare (S v) from Sfax VI- Flabelia petiolata (Fp) from Chebba VII -Anadyoméne stelleta (As) from Chebba VIII- Halimeda tuna (Ht) from Sfax IX- Ulva rigida (Ur) from Sfax X-Codium fragile (Cf) from Sfax XI-Posidonea oceanica (Po) from Chebba XII-Cymodocea nodosa (Cn) from Sfax XII-Halophila stipulacea (Hs) from Sfax; 1. Escherichia coli, 2. Escherichia coli DH5(alpha), 3. listeria monocytogéne, 4. Salmonella enterica, 5. Staphylococus aureus, 6. Micrococcus luteus, 7. Agrobacterium tumefaciens, 8. Pseudomonas aerigunosa.

Table 2: Antifongical activity of macroalgae extracts

Algea	Extractant	Micro organisms
		1	2	3
Phéophytea
I	Hexane	-	-	-
	Ethyl Acetate	-	-	-
	Methanol	-	-	-
II	Hexane	-	-	-
	Ethyl Acetate	++	-	-
	Methanol	+	-	+
III	Hexane	-	-	-
	Ethyl Acetate	-	-	-
	Methanol	-	-	++
IV	Hexane	-	-	-
	Ethyl Acetate	-	+	-
	Methanol	+	-	-
V	Hexane	-	-	+
	Ethyl Acetate	-	-	-
	Methanol	-	-	+
Chlorophytea
VI	Hexane	-	-	-
	Ethyl Acetate	-	-	-
	Methanol	-	-	-
VII	Hexane	-	-	-
	Ethyl Acetate	-	-	-
	Methanol	-	-	-
VIII	Hexane	-	-	-
	Ethyl Acetate	-	-	-
	Methanol	-	-	-
IX	Hexane	-	-	-
	Ethyl Acetate	-	-	-
	Methanol	-	-	-
X	Hexane	-	-	-
	Ethyl Acetate	-	-	-
	Methanol	-	-	-
Magnoliophytea
XI	Hexane	-		-
	Ethyl Acetate	-	+	-
	Methanol	-	+	-
XII	Hexane	-	-	-
	Ethyl Acetate	-	-	-
	Methanol	+	-	++
XIII	Hexane	-	-	-
	Ethyl Acetate	-	-	-
	Methanol	-	-	-

I -Dictyota dichotoma(Dd) from Chebba II- Cystoseira crinita(Cc) from Chebba III- Cystoseira barbata(Cb) from Sfax IV- Dyctéopteris menbranacea (Dm) from Sfax V- Sargussum vulgare (S v) from Sfax VI- Flabelia petiolata (Fp) from Chebba VII -Anadyoméne stelleta (As) from Chebba VIII- Halimeda tuna (Ht) from Sfax IX- Ulva rigida (Ur) from Sfax X-Codium fragile (Cf) from Sfax XI-Posidonea oceanica (Po) from Chebba XII-Cymodocea nodosa (Cn) from Sfax XII-Halophila stipulacea (Hs) from Sfax; 1. Candida tropicalis, 2. saccharomyces cerevisiae, 3. Aspergillus niger.

Antifongical activity

Algal extracts are subjected to screening antifongical activity. The fungi studied are harmful to crops and even humain health. These results are shown in the following table 2

DISCUSSION

Antimicrobial activity

In this study, all algal extracts tested showed an important antibacterial activity. Our results showed that the methanol extracts caused better halo-zones then followed by the ethyl acetate than Hexane for all strains. The type of extraction solvent had a big influence on the antimicrobial properties of obtained extracts, suggesting that antimicrobial activity depends on both algal species and the efficiency of the extraction method. Some studies concerning the effectiveness of extraction methods highlight that methanol extraction yields higher antimicrobial activity than hexane and ethyl acetate, whereas others report that chloroform extraction is better than methanol and benzene [22]. A good number of authors [23] have documented the antimicrobial potency of organic extracts and of some compounds isolated from marine algae of the genus of Cystoseira: such as terpenes [24, 25]. From our results it seems that the antibacterial actions of the organic extracts were more pronounced with brown algae are the best actives. The best activities were detected against S. aureus and micrococus for most algal species or magnoliophytea (Dd, Cc, Dm, Sv, Cb, Cf, Fp, As, Ur, Cn, Po, Hs) species and against S. aureus for Cc species. these results are correlated with those of [26]. who reported that The seaweed extracts are responsible for its activity against Gram (+) bacteria, especially Bacillus subtilis and Staphylococcus aureus and There are some reports regarding the antimicrobial activity of seaweeds from the Aegean Sea, Turkey [27–29] The previous reports showed that the algal extracts were generally more effective against Gram (+) than Gram (–) bacteria, probably due to the more complex structure of the cell wall of Gram (–) bacteria. Antibacterial activity found by other authors in Mediterranean seaweeds was considerably larger; it ranged between 13% [30] and nearly 50% [31, 32] of the species tested.

The Gram-negative bacteria (E. coli, Salmonella, and Listeria) reveal themselves more resistant to the majority of algal extracts obtained with a few exceptions. There are some reports regarding the antimicrobial activity of seaweeds, [33] reported showed that Of the 14 brown algae tested, only nine species showed a positive activity against at on a Gram+ bacterium and Of the 13 green algae tested, the presence of a positive activity on a Gram+ bacterium was observed in seven species. Diameter of inhibition higher than 15 mm was observed in the chloroformic extract of C. clathrata against S. aureus ssp. aureus, and in the acetonic extract of R. riparium against S. aureus ssp. aureus and that of E. compressa toward S. aureus, and for the green algae tested, no activity was detected against the Gram-negative bacteria.

The most sensitive to the action of algal extracts bacterial strains are strains gram (+) essentially Staphylococcus aureus and Micrococcus luteus which correlates with previous studies [34, 22].

The difference in results for antimicrobial activity in these marine algae shows the involvement envirnnementals factors in the metabolism of algae and significant way affects the presence of such bioactive compound responsible for these activities. Finally we conclude that macroalgae from Tunisian coast are potential sources of bioactive compounds and should be investigated from natural antibiotics. This study has shown that the production antibactérial subtance by macroalgae is a regular occurrence among those found on the coast of Tunisia. Biochemical analyses are currently undertaker to determine the structure and nature of these compounds.

Antifongical activity

After the results of the biological screening, we can note that low activity towards the fungi and human pathogenic yeast. Only 5 algal extracts that showed antifungal activity against 13 extracts for antibacterial activity (considering only the upper zones of inhibition to 10 mm). These results show that the extracts are more active on bacteria on champignon. Similar results were obtained by [35], they found that 61% of algae screening conducted inhibit bacteria gram positive against 15% for gram-negative and 19% are active in fungies. Similar findings have been reported by other groups [36]. For after this study we also found that the magnoliophytea have an important antifungal than the antimicrobial activity, these results are in accordance with [37], reported a high level of antifungal activity, coupled with a low level of antibacterial activity, exhibited by the magnoliophytea. For the antifungal activity, our results showed that the brown algae, a diameter of inhibition ranging between 10 and 15 mm was observed in the ethyl acetate extract of Cystoseira crinita against Candida tropicalis, and in the methanolic extract of Cystoseira barbata toward Aspergillus niger. For the green algae studied, no activity was detected against fungi. From the species tested, those belonging to Phaeophyceae were the most active in comparison with Chlorophyceae; the same result was reported by [38] and [39]. The seaweeds belonging to the genus of Cystoseira possess a wide variety of compounds with different biological activities. These results are of interest as we are dealing with an extract and not a pure product; therefore the antimicrobial activity may be due to different compounds and related to the presence of bioactive metabolites.

CONCLUSION

Marine organisms have several active chemicals such as antioxidant and antimicrobial Compounds. Marine organisms are currently undergoing detailed investigations with the objective of isolating biologically active molecules along with the search for new compounds.

This study reports the presence of antibacterial and antifongical compounds in the algae and magnoliophytea collected from the coast of Chebba and Sfax.

We hope that the present results will provide a starting point of investigations aimed at exploiting new natural pharmaceutical substances present in the extracts of the species collected from the coast of Tunisia.

CONFLICT OF INTERESTS

Declared None

REFERENCES

1. Carter BK. Biomedical potential of marine natural products. Biosci 1996;46:271-86.
2. Paul VJ, Puglisi MP. Chemical mediation of interactions among marine organisms. Nat Prod Rep 2004;21:189-209.
3. Kubanek J, Jensen PR, Keifer PA, Sullards MC, Collins DO, Fenical W. Seaweed resistance to microbial attack: a targeted chemical defense againstmarine fungi. Proc Natl Acad Sci USA 2003;100:6916-21.
4. De Nys R, Steinberg PD, Linking ES, Jensen PR, Fenical W. Chemical ecology of marine microbial defense. J Chem Ecol 2002;28:1971-85.
5. Farid Y, Chennaoui M, Assobhei O, Etahiri S. Evaluation de l’effet du lieu de recolte des algues marines des cotes atlantiques marocaines sur l’activite antibacterienne et anti inflammatoire. Microbiol Ind Sanit Environ 2012;6:54-66.
6. Engel S, Jensen PR, Fenical W. Chemical ecology of marine microbial defense. J Chem Ecol 2002;28:1971-85.
7. Hay ME. Marine chemical ecology: what’s known and what’s next? J Exp Mar Biol Ecol 1996;200:103-34.
8. Hay ME, Steinberg PD. The chemical ecology of plant herbivore interactions in marine versus terrestrial communities In: Rosenthal J, Berenbaum M (eds) Herbivores: their interactions with secondary plant metabolites, vol II. Evolutionary and ecological processes. Academic Press, New York; 1992. p. 317–413.
9. Bakus GJ, Targett NM, Schulte B. Chemical ecology of marine organisms: an overview. J Chem Eco 1986;12:951–87.
10. Sridhar KR, Vidyavathi N. Antimicrobial activity of seaweeds. Acta Hydrochim Hydrobiol 1991;19:455-96.
11. Iken KB, Baker BJ. Ainigmaptilones, sesquiterpenes from the Antarctic gorgonian coral Ainigmaptilon antarcticus. J Nat Prod 2003;66:888-90.
12. Peters L, Konig GM, Wright AD, Pukall R, Stackebrandt R, Eberl L, et al. Secondary metabolites of Flustra foliacea and their influence on bacteria. Appl Environ Microbiol 2003;69:3469-75.
13. Tziveleka LA, Vagias C, Roussis V. Natural products with anti-HIV activity from marine organisms. Curr Top Med Chem 2003;3:1512-35.
14. Yim JH, Kim SJ, Ahn SH, Lee CK, Rhie KT, Lee HK. Antiviral effects of sulfated exopolysaccharide from the marine microalga Gyrodinium impudicum strain KG03. Mar Biotechnol 2004;6:17-25.
15. El Gamal AA. Biological importance of marine algae. J Saudi 2010;18(1):1-25.
16. Valls L, Banaigs B, Piovetti L, Archavlis A, Artaud J. Linear diterpene with antimitotic activity from the brown alga Bifurcaria bifurcata. Phytochem 1993;34:1585-8.
17. Maréchal JP, Culioli G, Hellio C, Thomas-Guyon H, Callow ME, Clare AS, et al. Seasonal variation in antifouling activity of crude extracts of the brown alga Bifurcaria bifurcate (Cystoseiraceae) against cyprids of Balanus Amphitrite and the marine bacteria Cobetia marina and Pseudoalteromonas haloplanktis. J Exp Mar Biol Ecol 2004;313:47-62.
18. Younes F, Etahiri S, Assobhei O. Activité antimicrobienne des algues marines de la lagune d’Oualidia (Maroc): Criblage et optimisation de la période de la récolte. J Appl Biosci 2009;24:1543–52.
19. Perez C, Agnese AM, Cabrera JL. The essential oil of Senecio graveolens (Compositae): chemical composition and antimicrobial activity tests. J Ethnopharmacol 1999;66:91-6.
20. Nilsson L. New rapid bioassay of gentamicin based on luciferace assay of extracellular ATP in bacterial cultures. Antimicrob Agents Chemother 1978;14:812-6.
21. Turney I, Cadirci BH, Nal D, Sukatar A. Antimicrobial activities of the extracts of marine algae from the coast of Urla (Izmir, Turky). Turk J Biol 2006;30:171-5.
22. Caccamese S, Azzolina R. Screening for antimicrobial activities in marine algae from Eastern Sicily. Planta Med 1979;37:333-9.
23. Amico V, Oriente G, Piattelli M, Ruberto G, Tringali C. Novel acyclic diterpenes from the brown algea Cystoseira crinita. Phytochem 1981;30:171-5.
24. Amico V, Neri P, Piattelli M, Ruberto G. Geranylgeraniol-derived diterpenes from the brown alga Bifurcaria Bifurcata: comparison with two other Cystoseiraceae species. Phytochem 1987;26:2637-9.
25. Zeliha D, Ferda F, Yilmaz K, Ulku N, Karabay Y, Guven O, et al. Antimicrobial and antioxidant activity of brown algae from Aegean sea. J Serb Chem Soc 2009;74(6):619-28.
26. Tuney I, Cadirci BH, Unal D, Sukatar A. In antimicrobial activities of crude extracts of marıne algae from the coast of Izmir (Turkey). Fres Environ Bull 2007;16:428-34.
27. Ozdemir G, Horzum Z, Sukatar A, Karabay-Yavasoglu NU . Antimicrobial activities of volatile components and various extracts of Dictyopteris membranaceae and Cystoseira barbata from the Coast of Izmir, Turkey. Pharm. Biol 2006; 44: 183-8.
28. Taskin E, Ozturk M, Kurt O. Turkiye'nin yararlanilabilir denizel alg potansiyeli. XI. Ulusal Su Urunleri Sempozyumu, 4-6 Eylul, Antakya; 2001. p. 225-32.
29. Pesando D, Garam B. Screening of marine algae from French Mediterranean coast for antibacterial and antifungal activity. Botanica Marina 1984;27:381-6.
30. Barbagallo C, Cormaci M, Furnari G, Gismondo R, Majorana G. Attivita antibatterica di estratti di alghe marine delle coste orientali della Sicilia: I contribute. Pubbl 1st Bot Univ Catania; 1979. p. 1-5.
31. Caccamese S, Toscano RM, Furnari G, Cormaci M. Antimicrobial activities of red and brown algae from Southern Italy coast. Bot Mar 1985;28:505–7.
32. Khadija O, Nabila B, Samira E, Omar A. Screening of antibacterial and antifungal activities in green and brown algae from the coast of Sidi Bouzid (El Jadida, Morocco). Afr J Biotechnol 2012;104:16831-7.
33. Chiheb I, Riadi H, Matinez L, Dominger S, José F, Gomez Vidal A, et al. Screening of antibacterial activity of marine green and brown macroalgae from the coast of morocco. Afr J Biotechnol 2009;8:1258-62.
34. Ballantine DL, Gerwick WH, Velez SM, Alexander E, Guevara P. Antibiotic activity of lipid-soluble extracts from caribbean marine algae. Hydrobiologia 1987;151/152:463-9.
35. Zeng C, Zhang J. Chinese seaweeds in herbal medicine. Hydrobiol 1984;116/117:152-4.
36. Ballesteros E, Martin D, Uriz MJ. Biological activity of extracts from some Mediterranean macrophytes. Bot Mar 1992;35:481-5.
37. Caccamese S, Azzolina R, Funari G, Cormaci M, Grasso S. Antimicrobial and antiviral activities of extracts from mediterranean algae. Bot Mar 1980;23:285–8.
38. Pesando D, Caram B. Screening of marine algae from the french mediterranean coast for antibacterial and antifungal activity. Bot Mar 1984;27:381-6.