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ISSN: 2155-9546
Journal of Aquaculture Research & Development
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Cytotoxicity, Antioxidants and Antimicrobial Activities of Lipids Extracted from Some Marine Algae

Abd El Baky HH1*, El-Baroty GS2, Ibrahim AE1 and El Baz FK1
1Plant Biochemistry Department, National Research Centre, Dokki, Cairo, Egypt
2Biochemistry Department, Faculty of Agriculture, Cairo University, Cairo, Egypt
Corresponding Author : Abd El Baky HH
Plant Biochemistry Department
National Research Centre, Dokki, Cairo, Egypt
Tel: +20 2 33371211
E-mail: [email protected]
Received: October 19, 2014; Accepted: October 31, 2014; Published: November 03, 2014
Citation: Abd El Baky HH, El-Baroty GS, Ibrahim AE, El Baz FK (2014) Cytotoxicity,Antioxidants and Antimicrobial Activities of Lipids Extracted from Some Marine Algae. J Aquac Res Development 5:284. doi:10.4172/2155-9546.1000284
Copyright: © 2014 Abd El Baky HH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

The lipids of five marine algae species, i.e., two from the Red sea, Rhodophyta (Laurencia popillose, Galaxoura cylindriea), and three from the Mediterranean sea (Chlorophyta, Ulva fasciata; and Phaeophyta, Dilophys fasciola and Taonia atomaria) were evaluated as anticancer, antiviral, antimicrobial and antioxidant activities. Total lipid content was varied significantly, and the values ranged from 0.66 to 2.20%. The highest lipid content was found in U. fasciata (2.2% dw). Among fatty acids of all algae species, palmitic was dominating fatty acid and C14:0, C17:0, C18:0, C18:1, C20:4 were presented in significant levels. The polyunsaturated fatty acids C18:2, C22:5 and C20:3 were identified in most algal species. The biological activities of algal crude lipids were assessed in vitro. The crude lipids at a concentration of 10 μg/ml inhibited HSV-1 virus growth (in vitro) and % inhibition ranged from 12.5 to 74.4%. While, total lipids at concentration of 20 μg/ml induced toxic effect in host cells. The algal lipids exhibited a potent inhibitory effect on both breast and liver human cancer cell lines with IC50 values ranged from 0.34 to 7.11 μg/ml. All algal lipids induced remarkable antimicrobial activity Aspergillus niger and Candida albicans. Marine algal lipids exhibited moderate scavenging activity toward DPPH. radical, and high activity was found in lipids of fractionD. fasciol.

Keywords
Marine macroalgae; Algal lipids; Anticancer; Antiviral; Antibacterial; HSV-1; MCF7 Cell; Hepg2 cell
Introduction
Algae represent valuable sources of a wide spectrum of complex lipids with different potential applications in food, cosmetic and pharmaceutical industries [1-3]. Lipids of marine macroalgae possess antibacterial, antiviral, antitumor, antiinflammatory, antiprolierative and antioxidant activity [4-6]. Algal lipids contained high concentrations of polyunsaturated fatty acids (PUFA) and vitamins as well as bioactive molecules, e.g. phenolic and terpens compounds. Marine algae are known to be a good source of healthy food. PUFA prevent atherosclerosis development, and they reduce a frequency of heart and blood vessel diseases [4]. Bhaskar [7] found that the lipids of brown algae.
Sargassum marginatum possessed an inhibition effects against human promelocytic leukemia. Lipid extracts of Ginsens marc showed potent inhibitory activity on human hepatoma (HepG2) and breast (MCF7) cancer cell proliferation in vitro [8]. The aim of current work was to study lipid potential of some Egyptain marine algae as antiviral, anticancer, antimicrobial and antioxidant agents.
Materials and Methods
Collection of algal samples
Samples of Laurencia popillose and Galaxoura cylindriea were collected from Red Sea (Faied and Ein Al-Sokhna in Suez both in April 2007). Ulva fasciata and Taonia atomaria were collected from the Mediterranean Sea from Abu-Qir near Alexandria in August 2007 and Dilophys fasciola from Marsa Matrouh in August 2005. All algal sample were washed several times with tap water and then left in air for dryness. Samples were grinded and stored in glass containers at room temperature for further experiments.
Identification of algae species
After preparation of herbarium specimens of the algae species,they were identified by Dr. Rauhaiya Abdul-Latif, Professor of Botany Department of Botany, Faculty of Science, El-Azhar University, to whom the authors are very indebted.
Extraction and determination of total lipids
Total lipids of marine algae (100 g) were extracted according to the method described by Roughan and Bratt [9].
Identification of algal lipid fatty acids
The algal lipids were subjected to direct transmethylation in 1.5% sulfuric acid: methanol at 95°C for 2 h [10]. Fatty acid methyl esters were analyzed by gas chromatography (Perkin Elmer Auto system XL) equipped with a flame ionization detector and fused silica capillary column (DB-5 (American) 60 m×0.32 mm, i.d.) with a film thickness of 0.25/25 μm. The column temperature was initially set up at 150°C and gradually increased at rate of 3oC/min up to 250°C. The injector and detector temperatures were 230 and 250°C respectively. The helium was used as a carrier gas at 1ml/min. The split ratio was 1/100. Fatty acids were identified by comparison between retention times of samples and with those of methyl fatty acid standard mixture ( Sigma, >99% purity by GLC).
Biological evaluation of algal lipids
Preparation of algal extract for biological bioassays: The algal lipids (100 mg) of lipids were dissolved in 10 ml of DMSO in water (90:1, v/v) and kept at 4°C until used, appropriate dilution of solution were used in each assay.
Antiviral screening of algal lipids: Algal lipids were evaluated as antiviral against herps simplex virus type 1 (HSV-1). The virus was obtained from Virology Laboratory, Water Pollution Research Department National Research center (NRC), Egypt. The virus was propagated in virocell cultures. Inhibition % of virus was calculated as plaque reduction as a result of being subjected to a given extracts [11].
Antitumor activity of algal lipids: Potential antitumor activity of algal lipids was evaluated using the method of Skehan [12]. Human HEPG2 (liver carcinoma cell lines) and MCF7 (breast carcinoma cell lines) cells were plated in 96 multiwell plate for 24 h before treatment with the algal lipids to allow attachment of cells to the wells of the plate. Lipids and antitumor reference drug (Novantron) were added at serial concentrations to cell monolayer. After incubation for 48 h at 37°C the cytotoxicity was determined spectrophotometrically by measureing the developed color at 570 nm by ELISA reader (Tecan Sunrise absorbance reader (No. 3008746), software Magllan V.4 was used).
Antimicrobial activity of algal lipids: The antimicrobial activities of algal lipids were determined by conventional agar diffusion assay [13] using one gram positive (Bacillus subtilis NRRL B- 94) one gram negative (Escherichia coli NRRL B- 3703) bacteria, fungi (Aspergillus niger NRRL 313), and yeast (Candida albicans NRRL 477). The microbial growth inhibition zone was measured after incubation at 30°C by the appearance of clear microbial free inhibition zone, beginning within 24 h for yeast, 24-48 h for bacteria and 72-96 h for fungus.
Antioxidant activity of macro algal lipids using DPPH free radical-scavenging assay
Quantitative measurement of radical scavenging properties of algal lipids was determined according to Ye [14] method.
Ethanolic DPPH• solution (0.1 mM) was prepared, to give the initial absorbance value of 0.993 at 517 nm. The different concentrations of lipid samples (in 0.1 ml) of each sample (with appropriate dilution if necessary) was added to 3.0 ml of ethanolic DPPH solution. After incubation for 30 min in the dark, the absorbance was measured at 517 nm. The percentage of DPPH which was scavenged was calculated using the following formula: Scavenging (%)=[1–(A sample–A blank/A control)]×100%.
Statistical analysis
Data were statistically analyzed through analysis of variance (Anova) and Duncans test at 0.01 probability level [15].
Results and Discussion
Lipid content of marine algae
Results illustrated in Table 1 showed that the total lipid content (TL) of five macroalgae species (Ulva fasciata, Dilophys fasciola, Taonia atomaria, Laurencia popillose and Galaxoura cylindriea)] were ranged from 0.09 to 2.2% (D.W). Total lipids showed significant differences amongst all algae species. The maximum lipid content was found in U. fasciata (2.2%) followed by D. fasciola (1.1%) then, L. popillose (0.81%) and T. atomaria (0.66%). Whereas the minimum value was found in G. cylindriea (0.090%). These results are in agreement with those obtained by [16], they found that the twelve species of seaweeds which includes from Chlorophyceaean, species from Phaeophyceae (Brown algae) and species from Rhodophyceae (Red algae) had lipid values ranged from 1.33 to 4.6%. In another study carried out by [17], they reported that the lipid contents of Rhodophyta species, Chlorophyta species and Phaeophyta species were ranged from 1.33 to 4.6% based on D.W. On the other hand [18] found that the macroalgal total lipid content was dependent on species specific and genetic origin. Also, accumulation of lipids in macroalgal species was dependent on climate and geography development [16].
Fatty acid composition of macroalgal lipids: Fatty acid composition of marine macroalgae under investigation is illustrated in Table 2. L. papillose had the highest plamitic acid (C16:0) content (59.35%) followed by G. cylindria (49.4%), T. atomaria (36.27%) and D. fasciola (32.58%). While, U. faciata had the minimum C16:0 content (19.2%).
It is very interesting that, all macroalgae species under investigation had an odd fatty acid margaric acid (C17:0). U. fasciata had the highest margaric acid content (16.03%) followed by L. papillose (8.1%). Moreover, all marine macroalgal had high content of long chain polyunsaturated fatty acids such as eicosatrienoic (C20:3) and docosapentaenoic (C22:5). However, all macroalgal species contained a low amounts of docosapentaenoic (C22:5) except T. atomaria (11.89%). Hence, the predominant fatty acid is dependent on the marine algae species. The highest content of eicosatrienoic (C20:3) was detected in U. fasciata (54.41%) and oleic acid (C18:1) was abundant in D. fasciola (37.49%) (Table 3).
These results are in agreement with those obtained by Shehnaz (2003), who found that the major fatty acid of U. fasciata was palmitic acid (16:0) (50%) followed by oleic acid (18:1) (12.5%), margaric acid (17:0) (9-10%) and stearic acid (18:0) (4%). Orhan [19] found that palmitic and oleic acids were the most abundant fatty acids in several of marine algae. Among all lipid extracts analyzed, H. scoparia contained capric acid, however myristic acid was found in C. fracta and V. sessilis, which present in freshwater origin. Fatty acid composition of H. incrassate lipids were found to be rich of C16:0, C18:1, C 20:1, C20:3, C24:0 and (Lignoceric) C24:1 (Nervonic) fatty acid [20].
Biological evaluation of marine macroalgal lipids
Antiviral activity for marine algal crude lipid extracts against HSV-1: Table 4 showed the antiviral potential of marine algal lipids against HSV-1. The maximum inhibition of HSV-1 was recorded for the lipid extract of T. atomaria (74.4%) followed by D. fasciola (72%) and G. cylindriea (72%) at a concentration of 10 μg/ml. The complete inhibition effect was observed in T. atomaria, D. fasciola and G. cylindriea lipids at the concentration of 20 μg/ml.
These results are similar to that obtained by Chirasuwan, they found that lipid extracts of S. platensis possessed antiviral activity against HSV-1 with an IC50 value of 25.1 μg/ml. On the other hand, the 1- docosanol (C22:0) was found to inhibit viral replication by interfering with the early intracellular events surrounding viral entry into target cells. It is possible that interaction between the highly lipophilic compound and components of target cell membranes renders such target cells less susceptible to viral fusion and/or entry.
If this mechanism proves to be correct, 1-docosanol (C22:0) may provide a broad spectrum activity against many different viruses, especially those with lipid-containing envelopes [21]. Pope [22] reported the C22:0, n-docosanol exhibited in vitro antiviral activity against several lipid enveloped viruses including herps simplex viruses 1 and 2 by a mechanism that interferes with normal viral entry into target cells.
Antitumor activity of marine algal lipids extract against human breast carcinoma (MCF-7) and hepato carcinoma cells (HepG2): Antitumor potential of macroalgal lipids was evaluated against breast carcinoma cell line (MCF-7) and hepato carcinoma cells (HepG2) at different concentrations of algal lipids (1, 2.5, 5 and 10 μg/ml) and tumor cells viability after 48 h incubation and the results are presented in Tables 5 and 6. The inhibition % of algal crude lipids on MCF-7 and HepG2 cells was ranged between 17.21- 82.34% and 24.0- 61.0%, respectively. The T. atomaria crude lipids showed the highest inhibition % at all concentrations ranged from 77.21 to 82.34%. However, T. atomaria lipid extract had the lower IC50 value against MCF-7 cell line (0.34 μg/ml) compared with that of the antitumor drug Novantron (1.40). While, G. cylindriea lipids caused high potential antitumor activity with IC50 of 4.09 μg/ml against HepG2 cell, compared to Novantron antitumor drug (IC50=4). The results of the present study demonstrated that the marine algal lipids of T. atomaria showed significantly higher antitumor activity against human breast carcinoma (MCF-7) and the human hepato carcinoma (HepG2) compared with that of Novantron antitumor drug. Similar results were obtained from lipids of brown algae Sargassum marginatum, which possessed an inhibition effect against human pro-melocytic leukemia [7]. Lipid extracts of Ginseng marc showed a potent inhibitory activity on human hepatoma (HepG2, IC50=41.7 μg/ml) and breast (MCF-7, IC50=54.4 μg/ml) cancer cell proliferation [8]. Some macroorganisms such as mushroom lipid extract from G. sinensis possessed antitumor activity against leukemic (U937) and hepato carcinoma cells (HepG2). All these symptoms induced throughout the apoptotic changes in cells, including decreased cell volume and chromatin condensation [23].
Antimicrobial activity of marine algal crude lipid extracts
The antimicrobial potential of macroalgal crude lipids were assayed against one fungi (Aspergillus niger NRRL313), one yeast (Candida albicans NRRL477), one gram positive bacteria (Bacillus subtilis NRRL B-94) and one gram negative bacteria (Escherichia coli NRRL B-3703) Table 6 by measuring the inhibition zones (IZs) at the concentration of 100 μg/well. Lipid extract of L. popillose had antifungal effect against A. niger with 12.0 mm IZs followed by G. cylindriea (10 mm IZs). Lipid extracts of L. popillose and T. atomaria induced antiyeast effect against C. albicans with IZs values of 16 and 15 mm, respectively. However, lipids extract of U. fasciata and D. fasciola had a moderate activity against both microbes. D. fasciola lipids had the maximum antibacterial activity against E. coli (13.0 mm IZs) followed by T. atomaria (11 mm IZs). All macroalgal lipids extract did not show any effect against Bacillus subtilis.
The results of the present experiment showed that the algal crude lipids of L. papillose and T. atomaria induced the highest antiyeast activity, which may be attributed to the presence of unsaturated fatty acids. The results of the present study are in agreement with the findings of Ballantine [24], they found that some of the algal lipid extracts caused antifungal activity against C. albicans, gram-negative P. aeruginosa and E. coli. Ramadan [25] found that the lipid extract of Spirulina platensis induced antimicrobial activity against A. niger and C. albicans at concentration of 48 μg/disck, with IZs 28.3 mm and 21.3 mm, respectively. In another study carried out by Patra [26], they found that the lipid extract of E. compressa exhibited antibacterial activity against B. subtilis and E. coli with 14 mm and 12 mm IZs, respectively.
Desbois and Smith [27] attributed the prime target of total lipids may be due to the constituents of fatty acids which may disrupting the electron transport chain and oxidative phosphorylation of microbe in addition to its interference with cellular energy production. Moreover, fatty acids action may be resulted from the inhibition of enzyme activity, impairment of nutrient uptake, generation of peroxidation and autooxidation degradation products, or direct ana lysis of bacterial cells.
Antioxidant activity of marine algal crude lipid extracts
The antioxidant effects were evaluated by scavenging of 2, 2 diphenyl- picryl-hydrazyl (DPPH.) radical as shown in (Table 7). Most of the macroalgal lipid extracts possessed the ability of scavenging of DPPH. radical with different degrees. The scavenging % was ranged from 12.17 to 90.88% at the concentration of 100 μg algal lipids. The highest antioxidant effect was obtained with D. fasciola (90.88%) total lipids. Whereas, the lipid extracts of U. fasciata (39.47%), L. papillose (35.93%), G. cylindriea (32.97%) and T. atomaria (19.17%) exhibited the weakest DPPH. radical scavenging activity. Shanab [28] found that S. dentifolium extracted with di-chloromethane induced the greatest free radical (DPPH) scavenging activity (86%) at concentration 100 μg/ ml. Lipids of L. papillosa caused the greatest anti-lipid peroxidation efficiency (87%) comparing with silymarin (96%) as a standard antioxidant agent. However, the highest antioxidant activity of crude lipids of several macroalgae species extracted with hexane may be due to the presence of many substances such as carotenoids, soluble lipids, phenolic compounds, steroids and terpenoids [29]. Ramadan [25] found that the Spirulina platensis lipids exhibited free radical scavenging activity (from 21 to 27%) against DPPH with various degrees.
In conclusion, crude lipids of algal species under investigation was biologically evaluated. The results indicated that the algae showed different biological properties as antitumor, antioxidant, antimicrobial and antivirus with various degrees. Most of the algal crude lipids possessed an inhibition effect against human hepato carcinoma (HepG2) and human breast carcinoma (MCF-7), specially T. atomaria which induced the greatest antitumor activity with IC50 0.34 and 1.68 μg/ml, respectively. Moreover, it is worth mentioned that the crude lipids of D. fasciola showed a significant higher scavenging radical activity against the DPPH (90.88). The antioxidant activity may be due to the presence of lipophilic antioxidants (total carotenoids and α-tocopherol, phenolic and terpenoid compounds) and PUFAs. The highest antimicrobial activity of algal crude lipids was found in T. atomaria and L. papillose against A. niger and C. albicans, which might be attributed to the presence of different polyunsaturated fatty acids, phenolic and flavonoids.
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