MATERIALS AND METHODS

Seaweeds collection: Seven [Codium tomentosum (Chlorophyta), Corallina mediterranea, Hypnea musciformis and Laurencia papillosa (Rhodophyta) and Padina pavonica, Sargassum vulgare and Dictyota dichotoma (Phaeophyta)] seaweeds species were collected along the Syrian Coast at 4 km North-Latakia, Syria (38°29'766"E Longitude and 34°37'734"N Latitude) and prepared for extraction as reported by Saleh et al.⁵.

Seaweed extracts preparation: The seven seaweeds were extracted using aqueous and six solvents (methanol, ethanol, chloroform, acetone, ethyl acetate and hexane). All solvents were purchased from Sigma-Aldrich-Germany. Extraction process had been done as described by Saleh and Al-Mariri⁴.

Fungal strains: Aspergillus niger and Candida albicans fungal strains were provided by the Microbiology and Immunology Division, Department of Molecular Biology and Biotechnology of Atomic Energy Commission of Syria (AECS) in Damascus, Syria. Fungal inoculations were carried out by incubation on Potato Dextrose Agar (PDA) at 28±3°C for 2 days as described by Saleh and Al-Mariri⁴.

Antifungal activity test
Disc-diffusion test: Seaweeds antifungal activity has been carried out based on disc-diffusion method described by Bauer et al.¹⁷ and Saleh and Al-Mariri⁴. Amphotericin B (40 mg mL^–1) (Sigma-Aldrich, St. Louis, USA) was used as standard for antifungal activity. For each extract, duplicate trials were conducted against each strain.

Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC): The Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) were estimated using the microplate method as reported by Bauer et al.¹⁷ and Saleh and Al-Mariri⁴. Amphotericin B (40 mg mL^–1) (Sigma-Aldrich, St. Louis, USA) was used as standard for antifungal activity.

The current study was carried out between April and August, 2016.

Statistical analysis: Statistical analysis was carried out using Statview 4.5 statistical package¹⁸ at the 5% significance level (p = 0.05). Data were subjected to Analysis of Variance (Two-way ANOVA) for the determination of differences in means between different tested solvents against selected isolates for each algae species. Differences between means were examined for significance by Fisher’s Least Significant Difference (PLSD) test. Data were expressed as Mean±Standard Deviation (SD).

RESULTS

Antifungal activity of seven seaweeds had been evaluated against two fungal strains based on ZI (Table 1), MIC (Table 2) and MFC (Table 3) values.

Zone of Inhibition (ZI): This parameter ranged from 3 mm with hexane L. papilosa extracts against A. niger strain to 17 mm with acetonic P. pavonica extracts against C. albicans strain (Table 1).

Minimum Inhibitory Concentration (MIC): The MIC value varied according to the examined seaweed and solvent (Table 2). In this regards, it ranged from 0.11 mg mL^–1 with methanol S. vulgare against A. niger and hexane against C. albicans, to 53.3 mg mL^–1 with ethyl acetate H. musciformis against C. albicans (Table 2).

Minimum Fungicidal Concentration (MFC): This value ranged from 1.67 mg mL^–1 with methanol S. vulgare against the both fungal strains to 20 mg mL^–1 with H. musciformis (all solvents except methanol one) and also with L. papillosa (chloroform and acetone against A. niger and also with ethyl acetate and hexane against the both fungal strains) (Table 3).

Table 1:	Seaweed extracts antifungal activity using disc-diffusion method expressed as Zone of Inhibition
Same capital letter (column) and lowercase letter (row) are not significantly different at p = 0.05 probability by Fisher’s PLSD test. LSD 0.05 solvent 0.863, solvent and isolate 0.581

Table 2:	Minimum inhibitory concentration (MIC) of the seven seaweed extracts against the examined microorganisms
Same capital letter (column) and lowercase letter (row) are not significantly different at P= 0.05 probability by Fisher’s PLSD test, LSD 0.05 solvent 6.742, solvent and isolate 6.742, ND: No activity

Table 3:	Minimum fungicidal concentration (MFC) of the seven seaweed extracts against the examined microorganisms
Same capital letter (column) and lowercase letter (row) are not significantly different at p = 0.05 probability by Fisher’s PLSD test, LSD 0.05 solvent 2.912, solvent and isolate 0.543. ND: No activity

DISCUSSION

In the current investigation, ZI, MIC and MFC values served to screen the seaweeds fungicidal effects as reported in many investigations. Data presented herein showed that aqueous extracts have not any activity against the two tested fungi regardless examined seaweeds species. Whereas, the other six organic extracts adversely affected fungal strains. It was noticed that D. dichotoma extracts were not active against the both fungal strains regardless tested solvent.

Susceptibility of the two tested fungal strains to the algal extracts expressed as Zone of Inhibition (ZI) was recorded as following: little susceptibility when ZI between 7-10 mm, middle susceptibility between 10.1-15 mm and high susceptibility between 15.1-31 mm. From data presented in Table 1, variance analysis showed that seaweed, solvent and interaction seaweed with solvent effect’s on ZI (Table 1) values was significantly different (p<0.001). This parameter varied according to the examined seaweed species and solvent. In this regards, little effect (ZI between 7-10 mm) has been observed with hexane C. tomentosum, hexane and ethyl acetate L. papilosa, ethyl acetate and acetone S. vulgare extracts against the two fungal strains (Table 1). Whereas, the remaining extracts exhibited in majority activity ranged between middle to high effectiveness.

Wefky and Ghobrial¹² reported antifungal activity of 5 seaweeds against A. flavus and A. niger fungal strains using methanol, ethanol and acetone solvents. The previous investigation showed that acetonic L. pinnatifida and S. hystrix extracts displayed the highest antifungal effect with ZI of 16 and 26 mm, respectively. Whereas, Kim et al.¹⁵ reported antifungal activity of Eisenia bicyclis edible brown seaweed extract against 8 Candida strains. The previous investigation showed that ZI ranged between 20-24 mm with methanol extracts.

Khallil et al.¹⁶ reported antifungal activity of 5 brown seaweeds (S. vulgare, Cystoseira barbata, Dictyopteris membranacea, D. dichotoma and Colpomenia sinuosa) against 8 fungal strains using acetone, ethanol, chloroform, cyclohexane and ethyl acetate solvents. The previous investigation showed that cyclohexanic S. vulgare extracts exhibited the highest inhibitory activity against F. oxysporum. Otherwise, ZI against A. niger was recorded to be 7 and 12 mm with S. vulgare ethanol and cyclohexane extracts. Whereas, no activity recorded with chloroform, acetone and ethyl acetate extracts against the same strain.

As for MIC (Table 2) and MFC (Table 3), variance analysis showed that seaweed, solvent and interaction seaweed with solvent effect’s on MIC (Table 2) and MFC (Table 3) values were significantly different (p<0.001).

Overall, methanolic S. vulgare extract was the strongest by showing the lowest MIC/MFC value of 0.11/1.67 and 0.13/1.67 mg mL^–1 against A. niger and C. albicans fungal strains, respectively. Followed by C. mediterranea, in this respect, the lowest MIC/MFC value was recorded to be 1.7/ 3.3 mg mL^–1 against the both tested fungal strains with methanolic extract.

Previously, Tariq¹¹ reported antifungal activity of 4 red seaweeds against Aspergillus sp. and C. albicans. The previous investigation showed that A. flavus, A. fumigatus or C. albicans were resistant to all the previous seaweeds extracts. Moreover, Ballesteros et al.¹⁹ reported biological activity of 71 Mediterranean macrophytes. The previous investigation showed that Codium spp. and Corallina elongata displayed a strong antifungal effect with a good antifungal effect recorded with Corallina granifera.

Whereas, Zheng et al.⁶ reported antifungal effect of 23 species of marine seaweeds (Chlorophyta, Phaeophyta and Rhodophyta). The previous investigation showed that Rhodophyta were the most potent extracts. Morales et al.⁷ reported antifungal effect of 6 seaweeds using ethyl acetate partition of methanol extracts. The previous investigation showed that Trichophyton mentagrophytes fungus growth was inhibited by L. obtusa, S. filipendula and S. hystrix extracts at 6.25-3.13 mg mL^–1. Moreover, Wefky and Ghobrial¹² reported antifungal activity of 5 seaweeds against A. flavus and A. niger fungal strains. The previous investigation showed that acetonic S. hystrix extract displayed the highest antifungal effect.

Nor Afifah et al.⁸ reported antifungal activity of Halimeda discoidea green seaweed extract against 6 fungal strains. The previous investigation showed that all the 6 tested fungal strains were resistant to the examined extracts. Whereas, Rodloff et al.²⁰ reported that not all tested Candida species similarly responded to antifungal.

Kim et al.¹⁵ reported antifungal activity of ethyl acetate edible brown seaweed Eisenia bicyclis extract against Candida species. The previous investigation showed that MIC/MFC value ranged between 4-32/16-64 mg mL^–1 against C. albicans. Overall, ethyl acetate-soluble extract was the most potent with MIC/MFC value ranged between 4-8/16 mg mL^–1 against the tested strain.

Khaled et al.¹⁴ reported methanolic P. pavonica and S. vulgare antifungal effect against Candida species strains. The previous investigation showed that S. vulgare extract has no activity against tested fungal strains. Whereas, ethyl acetate P. pavonica fraction extract had antifungal effect against C. krusei and more noticeable against C. glabrata strain. Indeed, Guedes et al.¹³ reported antifungal activity of different seaweeds species against Candida species. The previous investigation showed that MIC ranged between 0.008-0.016 mg mL^–1. Moreover, dichloromethane, methanol and ethanol extracts showed the strongest inhibition growth. Moreover, Chowdhury et al.⁹ reported antifungal effect of 10 freshwater and marine algae against C. albicans strain. The previous investigation showed that Chlorella spout the examined algae had the highest antifungal activity. Recently, Raj et al.³ reported hexane, chloroform, ethyl acetate, acetone and methanol extracts of Ulva sp. green algae against 10 fungal isolates. The previous investigation showed that ethyl acetate U. lactuca extracts were the most potent by showing the lowest MIC of 0.250 mg mL^–1 against C. parapsilosis.

More recently, Saleh and Al-Mariri⁴ reported the inhibitory effect of U. lactuca (Chlorophyta), Dilophus spiralis (Phaeophyta) and Jania rubens (Rhodophyta) marine seaweeds against 2 fungal (C. albicans and A. niger) strains using aqueous and six organic extracts. The previous investigation showed that the lowest MIC value was recorded to be 0.106 mg mL^–1 with U. lactuca methanolic extract against the both fungal strains and with acetone and hexane against C. albicans. Moreover, the lowest MFC (0.266 mg mL^–1) was observed with D. spiralis Chloroform against both fungal strains. Moreover, Saleh et al.⁵ reported antibacterial effect of C. tomentosum, C. mediterranea, H. musciformis and S. vulgare extracts against 10 Gram-negative bacterial isolates using aqueous and six organic solvents. The previous investigation showed that the methanolic S. vulgare extract had the strongest antibacterial effect by showing the lowest MIC/MBC values of 0.08/1.00 mg mL^–1 against E. coli O:157 isolate. Whereas, Peres et al.²¹ reported antifungal activity of ten seaweeds against Aspergillus flavus fungal strains. The previous study revealed that A. flavus growth did not significantly inhibited by the seaweed extracts.

As for lichens extracts, as natural resources have been proved their effectiveness as antifungal agent. In this respect, Rukayadi et al.²² reported antifungal activity of methanolic Usnea sp. lichen extract against Malassezia furfur fungi. More recently, Plaza et al.²³ reported also antifungal effect of Cladonia aff. rappii A. Evans (Cladoniaceae) lichen against 4 genus of Candida and 1 genus of Cryptococcus using aqueous, ethanol and dichloromethane extract. The previous investigation showed that the lowest MIC value was recorded to be 2.2 and 5.9 mg mL^–1 against C. glabrata with ethanol and dichloromethane extracts, respectively. Whereas, the lowest MFC value was recorded to be recorded 7.4 mg mL^–1 against C. neoformans followed by 8.9 mg mL^–1 against C. glabrata with dichloromethane extracts.

The strongest antifungal activity recorded in S. vulgare could be attributed to the abundance of phenolic components making them more active against examined strains compared to the other seaweed phyla in particularly, Chlorophyta. Indeed, this phenomenon could be attributed to the presence of other bioactive components that were not measured in the present investigation. Phenolic compounds could be used as antimicrobial agents in wild mushrooms²⁴ and Ficus sycomorus²⁵ against some micro-organisms resistant to antibiotics.

CONCLUSION

Antifungal effectiveness of seven seaweeds [1 (Chlorophyta), 3 (Rhodophyta) and 3 (Phaeophyta)] crude extracts has been mainly investigated against two fungal (A. niger and C. albicans) strains using aqueous, methanol, ethanol, chloroform, acetone, ethyl acetate and hexane solvents. Seaweeds antifungal activity has been screened based upon MIC/MFC value. All over, methanolic S. vulgare extract was the most effective by showing the lowest MIC/MFC value of 0.11/1.67 mg mL^–1 and 0.13/1.67 mg mL^–1 against A. niger and C. albicans fungal strains, respectively. Thereby, methanolic S. vulgare extracts fractions needs performance research for future.

SIGNIFICANCE STATEMENT

The current study highlighted the importance of some seaweed extracts using different solvents, as antifungal agent. Methanolic S. vulgare crude extracts as antifungal agent should be given more attention. Moreover, their fractions with an in-depth study regarding their fractions potency separately need more investigation. Seaweeds seem to be promising natural resources with low cost for antifungal treatment in pharmacology and medicine applications.

ACKNOWLEDGMENTS

We thank Dr. I. Othman (Director general of AECS) and Dr. N. Mir Ali (Head of Molecular Biology and Biotechnology Department in AECS) for their support and also the Molecular Biology and Biotechnology Group for technical assistance.