Egypt. J. Exp. Biol. (Bot.), 8(2): 199 – 204 (2012) © The Egyptian Society of Experimental Biology

RESEARCH ARTICLE

Mohammed A. Deyab Lotfy Z. Habbak* Fatma M. Ward**

ANTITUMOR ACTIVITY OF WATER EXTRACT AND SOME FATTY ACIDS OF ORNATA (TURNER) J. AGARDH

ABSTRACT: Boiled water extract of the brown macroalga for 72 h and water extract at INTRODUCTION: room temperature for 6 days reduced the The brown seaweed, Turbinaria ornata viability of tumor cells to 68.9% and 81.8%, belongs to Kingdom: Chromlveolata, Division: respectively. 1017.5 µg oleic acid and Heterokontophyta, Class: Phaeophyceae, 1406.01 µg palmitic acid were extracted by Order: , Family: Saragassaceae (Guiry organic solvents from 100 grams air dried M. D. and Guiry G.M., 2011). Turbinaria has powder of T. ornata. Different concentrations green chlorophyll but this is masked by other of these acids showed antitumor activities pigments, in particular fucoxanthin, an orange after 5 minutes against Ehrlich ascites carotenoid pigment that gives their distinctive carcinoma cells. Hundred µg/ml oleic acid or brown colour (Bell and Hemsley, 2000). The palmitic acid reduced tumor cells viability to Turbinaria species is hardy stiff, erect plant. approximately 0-1%. All tumor cells were non Blades are conical, hard, and thick with double viable after treating by different row of stiff spines around the irregularly concentrations of oleic acid after 40 min and triangular margin of the blade when viewing palmitic acid at 30 min. Also, the present from above (Cooksley, 2007). Turbinaria ornata study indicates that antitumor activity of is a very common brown alga found intertidally palmitic acid was higher than that of oleic throughout the Red sea, Pacific and Indian acid. The effect of water extract of T. ornata, Ocean. The morphological characteristics of as well as oleic acid and palmitic acid upon this alga enable it to survive extreme tumor cells in vitro were shown to be environmental conditions (Russell and Balazs, concentration and time dependent. Although 2000). Density of T. ornata varied among sites the studies showed medical importance of T. and seasons. The highest density was found on ornate in tumor treatment, more scientific the semi-exposed shore during October with 21 2 research is required on the level of molecular fronds/m (Stewart, 2004). biology to determine genes responsible for Approximately 16,000 marine natural these natural products in the alga and how to products have been isolated from marine increase the effect of these active substances organisms. Hence, humans use secondary in tumor treatment. metabolites of algae as medicines, flavorings, recreational drugs (Bhadury and Wright, 2004; KEY WORDS: Smit, 2004; Selim, 2012) and antitumor activities (Lee and Sung, 2003). The seaweeds Antitumor fatty acids, natural products, also contain high amounts of polyphenols such seaweeds, Turbinaria ornata as catechin, epicatechin, epigallocatechin

gallate, and gallic acid (Yoshie et al., 2002). Mohammed Ali Deyab Fatty acids are carboxylic acids with a Botany Department, Faculty of Science at new long unbranched aliphatic tail (chain), which are Damietta, Egypt either saturated or unsaturated. Oleic acid is a E-mail: [email protected] monounsaturated omega-9 fatty acid that has the formula CH3(CH2)7CH=CH (CH2)7COOH (Schneiter et al., 2000). Palmitic acid is a Lotfy Z. Habbak* saturated fatty acid that has formula Fatma M. Ward** CH3(CH2)14COOH (Beare-Rogers, et al., 2001). * Zoology Department, Faculty of Science, Damietta Human Cancer treatments do not have University, Egypt. potent medicine as the currently available drugs are causing side effects in some instances. In **Botany Department, Faculty of Science, Damietta this context, the natural products derived from University, Egypt. medicinal plants have gained significance in the cancer treatment (Gurib-Fakim, 2006). ARTICLE CODE: 24.02.12

ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org 200 Egypt. J. Exp. Biol. (Bot.), 8(2): 199 – 204 (2012)

Concerning , polysaccharides and terpenoids are considered as promising bioactive molecules in the search for anticancer drugs (Zubia et al., 2009). Marine algae are also rich sources of unsaturated fatty acids and these fatty acids were also reported to block growth and systemic spread of human breast cancer via mechanisms independent of the host immune system, perhaps by peroxidation of intracellular lipids (Devery et al., 2001). Fucosterol, 24ȟ-hydroperoxy-24- vinylcholesterol , 29-hydroperoxystigmasta- 5,24(28)-dien-3ȕ-ol, 24-ethylcholesta-4,24 (28)- dien-3-one, 24ȟ-hydroperoxy-24-ethylcholesta- 4,28(29)-dien-3-one, 24-ethylcholesta-4,24(28)- dien-3,6-dione, 24ȟ-hydroperoxy-24- ethylcholesta-4,28(29)-dien-3,6-dione, 6ȕ- hydroxy-24-ethylcholesta-4,24(28)-dien-3-one, and 24ȟ-hydroperoxy-6ȕ-hydroxy-24-ethylcholesta -4, 28(29)-dien-3-one were isolated from the marine brown alga Turbinaria. The structures of these compounds were established by spectral analysis. Isolated for the first time Fig. 2. Photograph of brown seaweed; Turbinaria from a natural source, the oxygenated ornata. fucosterols exhibit cytotoxicity against various Extraction of oleic acid and palmitic acid cancer cell lines (Sheu et al., 1999). from T. ornata: In the present study, we investigated the First, Lipids content in known weight of antitumor activities of boiled and cold water predried powder of T. ornata was determined extracts of T. ornate, as well as oleic acid and according to Bligh and Dyer (1959). The lipids palmitic acid extracted from this seaweed. were separated into major classes according to Romano et al. (1976). Separation of oleic MATERIAL AND METHODS: acid (monounsaturated fatty acid) and palmitic acid (saturated fatty acid) was carried out Preparation of water extracts of Turbinaria ornate: according to Fulco (1970).Concentrations and Turbinaria ornata was collected along purity of oleic acid and palmitic acid were the semi- exposed shores of Hurghada, Red determined by Reversed Phase High sea, Egypt (Fig. 1) during October 2010. T. Performance Liquid Chromatography (RP- ornata was identified according to Guiry and HPLC). The chromatography model, Agilent Guiry (2011). Twenty grams of dry T. ornata technologies 1200 series was used. The (Fig. 2) powder were soaked in 100 ml bi- column was Eclipse XDB C 18 (4.6×150mm). distilled water for different times (1, 2, 3, 4, 5, Gradient solvent was 100% MeOH with flow and 6 days). Another 20 g of T. ornata powder rate of 0.5 ml/min. The detector was UV at were boiled in 100 ml bi-distilled water for wave length 254 nm. different times (6, 12, 24, 36, 48, and 72 Source of tumor cells: hours).Soaked and boiled T. ornata extracts was used to detect antitumor activity. Ehrlich ascites carcinoma cells (EAC) were injected intrapertoneally in Swiss albino mice (its weight was 22 - 25 grams; its age was 70-80 days). The parent cell line was kindly supplied by the National Cancer Institute, Cairo University, Egypt. The tumor cell line was maintained in mice through serial intraperitoneal transplantation of 1x106 viable tumor cells in 0.2 ml of saline. The tumor is characterized by moderately rapid growth, which kills mice in 16 to 18 days due to accumulation of ascetic fluid and seldom shows distal metastasis or spontaneous regression. EAC cells were collected 7 days after intraperitoneal implantation. The harvested cells were diluted with saline to obtain a concentration of 5 x 106 viable EAC cells per ml (Zahran, 2009).

Fig. 1. Map showing sampling site (Hurghada, Egypt).

ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org Deyab et al., Antitumor Activity of Water Extract and Some Fatty Acids of Turbinaria Ornata (Turner) J. Agardh 201

Assay of antitumor activities of water extract of T. ornata and some of its fatty acids: Antitumor activities were assayed for water in which T. ornata was soaked as well as boiled for different times. Oleic acid and palmitic acid extracted from T. ornata were dissolved and diluted in ethanol to concentrations of 1, 5, 10, 20, 40, 50, and 100 µg/ml to be used for antitumor assay. In Trypan Blue Exclusion test (Mo et al., 2011), 100µl of the test solution (Different concentrations of oleic acid, palimtic acid and water extract of T. ornata) was added to 100 µl of tumor cells suspension for different times (5- 40 min) at 37oC and then mixed with 200 µl 0.4% trypan blue solution and incubated for 30 min at 37oC. The integrity of the cells was determined by counting the number of unstained (viable) and stained (nonviable) cells separately in the Fig. 4. HPLC-RP spectrum of oleic acid extracted haemocytometer (Fig. 3). from Turbinaria ornate. The percentage of viable cells was calculated as follows: total number of viable cells Viable cells (%) = u 100 total number of cells

Fig. 5. HPLC-RP spectrum of palmitic acid extracted from Turbinaria ornata. Antitumor activity of water extract of Fig. 3. Viable ( ) and non viable ( ) tumor cells using light microscope Turbinaria ornata: Antitumor activity of water extracts of T. Statistical analysis: ornata were assayed after 15 minutes. Water Data were statistically analyzed for in which Turbinaria ornata was soaked for 1-4 variance and the least significant difference days had no antitumor activity, while the (LSD) using one-way analysis of variance water extract of T. ornata reduced viability of (ANOVA). A software system SPSS version 13 tumor cells to 88.8% and 81.8% after 5 and 6 was used for these calculations. days respectively. Water in which Turbinaria ornata was boiled for times less than 48 hours RESULTS AND DISCUSSION: had no antitumor activity, while this water extract reduced viability of tumor cells to Oleic acid and palmitic acid were 74.8% and 68.9% after 48 h and 72 h, extracted from T. ornata. Hundred grams of respectively (Table 1). These results showed dry T. ornata powder contains 1017.50 µg that the water extract of T. ornata caused oleic acid. Its purity was 51.15% (Fig. 4); highly significant (LSD at 0.01 level) decrease while hundred grams of dry T. ornata powder (F= 1403.35) in tumor cells viability after 15 contains 1406.01 µg palmitic acid. Its purity minutes. was 36.9% (Fig. 5).

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Boiled water extract of Turbinaria ornata The anti-cancerous activity of seaweed showed higher antitumor activity than water extracts has been attributed to the cytotoxic extract at room temperature. This was due to the nature of palmitic acid found in these crude polysaccharides are extracted from brown seaweeds (Harada et al., 2002). The present seaweeds by boiling. The antitumor activities of results (Table 2) showed that the antitumor aqueous extracts of T. ornata may be due to its activity of oleic acid was lower than that of content of polyphenolic compounds that have palmitic acid; this was in agreement with Ito antitumor activities (Boonchumi et al., 2011). It et al. (1982).This was related to the was in agreement with (Kuda and Ikemori, 2009) cytotoxicity effect of palmitic acid was higher that many hydrophilic polyphenolic compounds than that of oleic acid (Martins et al., 2006). were reported in aqueous extract of Turbinaria, After 5, 10, 15, and 30 minutes, the for example peptides, fucoidan and Maillard viability of tumor cells decreased significantly reaction products. Moreover, crude by increasing concentrations of oleic acid in polysaccharides were extracted by boiling the o the studied range of concentrations from 1 dried brown seaweed samples in water at 100 C µg/ml to 100 µg/ml (Table 2). After 5 minutes, (Kantachumpoc and Chiraport , 2010). The 1 µg/ml oleic acid reduced the viability of sulfated polysaccharides, viz, fucans, tumor cells to 63% and it continuously carrageenans and ulvans are compounds which reduced by increasing concentrations till 0% posses several biological activities like as by 100 µg/ml oleic acid. After 10 minutes, 1 antitumor (Pomin, 2010). µg/ml oleic acid reduced the viability of tumor Table 1. Tumor cells viability after affecting by water extract cells to 49.3%; while 50 µg/ml oleic acid and of T. ornata after 15 minutes. higher concentrations reduced it to 0%. After Water extract Water Water Boiled water Boiled water 15 minutes, 1 µg/ml oleic acid reduced the extract of T. extract of extract of T. extract of T. viability of tumor cells to 21%; while 40 µg/ml ornata for 5 T. ornata ornata for ornata for oleic acid and higher concentrations reduced days for 6 days 48h 72h Tumor cells it to 0%. After 30 minutes, 1 µg/ml oleic acid Number of viable cells /ml (×105) 191 184 172 160 reduced the viability of tumor cells to 8%; Number of non viable followed by 2% after treatment with 5 µg/ml 5 24 41 58 72 cells /ml (×10 ) and 10 µg/ml oleic acid; while 20 µg/ml oleic Total viability % 88.8% 81.8% 74.8% 68.9% acid and higher concentrations reduced it to 0% (Table 2). Statistical analysis (F) = 1403.35; P= 0.000 Table 2. Percentages of tumor cells viability after treatment The mean difference is significant at the 0.01 level. with different concentrations of oleic acid and palmitic Antitumor activity of oleic acid and palmitic acid extracted from T. ornata at different times. acid extracted from Turbinaria ornata: Time (min.) Statistical 5 10 15 30 40 In the present study, the reduction in analysis tumor cells viability was directly proportional to Percentages of viability of tumor Concentrations F P the concentration of fatty acids and time of (µg/mL) cells exposure. Wang et al. (2011) concluded that 1 63.0 49.3 21.0 8.0 0.0 13410.0 0.00 palmitic acid and oleic acid-induced cytotoxicity 5 42.0 27.0 9.0 2.0 0.0 4842.2 0.00 in a concentration and time-dependent manner. Consequently, in the present study, high 10 35.0 20.8 6.0 2.0 0.0 1954.9 0.00 concentrations (100 µg/ml) of oleic acid and 20 27.0 14.0 7.0 0.0 0.0 1877.0 0.00 palmitic acid extracted from T. ornata have the 40 19.0 3.0 0.0 0.0 0.0 673.8 0.00 ability to reduce tumor cells viability to 0% after Oleic acid 5 minutes. Moreover, the complete reduction of 50 8.2 0.0 0.0 0.0 0.0 434.1 0.00 tumor cells viability (0%) (Fig. 6) was obtained 100 0.0 0.0 0.0 0.0 0.0 0.5 0.00 after treatment with low concentrations (1 1 41.6 22.5 6.3 0.0 0.0 6140.8 0.00 µg/ml) after 40 minutes for oleic acid and 30 5 29.4 22.5 6.3 0.0 0.0 19394.3 0.00 minutes for palmitic acid (Table 2). 10 20.7 6.1 0.0 0.0 0.0 3390.5 0.00 20 16.0 4.6 0.0 0.0 0.0 987.6 0.00 40 12.0 3.0 0.0 0.0 0.0 501.22 0.00 Palmitic acid 50 7.3 0.0 0.0 0.0 0.0 86.1 0.00 100 0.0 0.0 0.0 0.0 0.0 0.25 0.00 The mean difference is significant at the 0.01 level. After 5, 10, and 15 minutes, the viability of tumor cells decreased significantly by increasing concentrations of palmitic acid in the studied range of concentrations from 1 µg/ml to 100 µg/ml (Table 2). After 5 minutes, 1 µg/ml palmitic acid reduced the viability of Fig. 6. The complete reduction of viability of tumor cells tumor cells to 41.6% and it continuously reduced by increasing concentrations till 0%

ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://www.egyseb.org Deyab et al., Antitumor Activity of Water Extract and Some Fatty Acids of Turbinaria Ornata (Turner) J. Agardh 203 by 100 µg/ml palmitic acid. After 10 minutes, /or ATP/ADP antiporter. This uncoupling 1 µg/ml and 5 µg/ml palmitic acid reduced the effect of fatty acids prevents the increase of viability of tumor cells to 22.5%; while 50 the trans-membrane electrochemical potential µg/ml palmitic acid and higher concentrations and the subsequent increase in mitochondrial reduced it to 0%. After 15 minutes, 1 µg/ml reactive oxygen species (ROS) production and 5 µg/ml palmitic acid reduced the viability (Duval et al., 2002).During times of of tumor cells to 6.3%; while 10 µg/ml palmitic environmental stress, ROS levels can acid and higher concentrations reduced it to increase dramatically. This may result in 0%. After 30 and 40 minutes, different significant damage to cell structures concentrations of palmitic acid reduced the (Devasagayam et al., 2004). Inhibition of tumor cells viability to 0% (Table 2). respiration of tumor cells has been correlated The mechanisms of fatty acids to induce with the ability of fatty acids to cause tumor cells death involved changes in decreased cell viability and cell count. The mitochondrial transmembrane potential and effect of fatty acids to inhibit respiration of intracellular neutral lipid accumulation tumor cells was related to their ability to (Martins et al., 2006).In isolated mitochondria stimulate oxygen uptake as the causative of tumor cells, fatty acids lower the production factor in uncoupling of phosphorylation in the mitochondria (Katchman et al., 1963). of H2O2 and induce mitochondrial uncoupling, possibly mediated by mitochondria UCPs and

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ϡ΍έϭϷ΍ΪοΎΗΎϧέϭ΃ΎϳέΎϨϴΑήΘϟ΍ϦϣΔμϠΨΘδϤϟ΍ΔϴϨϫΪϟ΍νΎϤΣϷ΍ξόΑρΎθϧ 3ΩέϭϯϮΒϨϟ΍ΪϤΤϣΔϤρΎϓˬ 2 ϖΒΣΎϳήϛίϰϔτϟˬ 1ΏΎϳΩϰϠϋΪϤΤϣ ήμϣˬΓΪϳΪΠϟ΍ρΎϴϣΪΑϡϮϠόϟ΍ΔϴϠϛˬϥ΍ϮϴΤϟ΍Ϣδϗ (2)ήμϣˬΓΪϳΪΠϟ΍ρΎϴϣΪΑϡϮϠόϟ΍ΔϴϠϛ ˬΕΎΒϨϟ΍Ϣδϗ (3ˬ1)

ξϤΣϭϚϴϟϭϷ΍ξϤΣϰϟ·ΔϓΎοϻΎΑ ΎΗΎϧέϭ΃ΎϳέΎϨϴΑήΘϠϟϰ΋ΎϤϟ΍ 72 ΓΪϤϟ ΎΗΎϧέϭ΃ ΎϳέΎϨϴΑήΘϠϟ ϲϠϐϤϟ΍ ϲ΋ΎϤϟ΍ κϠΨΘδϤϟ΍ ϞϠϗ ϢϏέϭ .ΖϗϮϟ΍ϭΰϴϛήΘϟ΍ΓΩΎϳΰΑΩ΍Ωΰϳϡ΍έϭϷ΍ΎϳϼΧϰϠϋϚϴΘϤϟΎΒϟ΍ 6ΓΪϤϟΔϓήϐϟ΍Γέ΍ήΣΔΟέΩϲϓϲ΋ΎϤϟ΍κϠΨΘδϤϟ΍ϭΔϋΎγ ϡ΍ΪΨΘγ·ϰϓΓήϴΒϛΔϴΒρΓΩΎϔΘγ·ΖΘΒΛ΃Δγ΍έΪϟ΍ϥ΃ ϰϠϋ %81,8ϭ %68,9ϰϟ·ϞμΘϟϡ΍έϭϷ΍ΎϳϼΧΔϳϮϴΣ˭ϡΎϳ΃ ϥ΃ϻ·ˬϡ΍έϭϷ΍ΝϼϋϰϓΎΗΎϧέϭ΃ΎϳέΎϨϴΑήϴΘϟ΍ΐϠΤρκϠΨΘδϣ ϭ ϚϴϟϭϷ΍ ξϤΣ ϡ΍ήΟϭήϜϴϣ 1017,5 ιϼΨΘγ· ϢΗ .ΐϴΗήΘϟ΍ ΎϴΟϮϟϮϴΒϟ΍ ϯϮΘδϣ ϰϠϋ ΏϮϠτϣ ϰϤϠόϟ΍ ΚΤΒϟ΍ Ϧϣ ΪϳΰϤϟ΍ ΔϳϮπϋΕΎΒϳάϣΔτγ΍ϮΑϚϴΘϤϟΎΒϟ΍ξϤΣϡ΍ήΟϭήϜϴϣ 1406 ,01 ΕΎΠΘϨϤϟ΍ ϩάϫ Ϧϋ ΔϟϮΌδϤϟ΍ ΕΎϨϴΠϟ΍ ΪϳΪΤΘϟ ΔϴΌϳΰΠϟ΍ ΕήϬυ΃ . ˯΍ϮϬϟ΍ϰϓΔϔϔΠϣϭΔϧϮΤτϣΎϳέΎϨϴΑήϴΗϡ΍ήΟ 100Ϧϣ Ω΍ϮϤϟ΍ΝΎΘϧ·ΔϔϋΎπϤϟΎϬΘϔϋΎπϣΔϴϔϴϛϭΐϠΤτϟ΍ϰϓΔϴόϴΒτϟ΍ Ϧϣ ΔμϠΨΘδϤϟ΍ νΎϤΣϷ΍ ϚϠΗ Ϧϣ ΔϔϠΘΨϤϟ΍ Ε΍ΰϴϛήΘϟ΍ .ϡ΍έϭϷ΍ΝϼϋϰϓΔϟΎόϔϟ΍ ΎϳϼΧ Ϊο ϖ΋ΎϗΩ 5 ΪόΑ ϡ΍έϭϷ΍ Ϊο ρΎθϧ ΎΗΎϧέϭ΃ ΎϳέΎϨϴΑήΘϟ΍ 100 ΰϴϛήΗ ωΎτΘγ· .Ϛϴϟήϳ· ˯ΎϘδΘγ· ϥΎρήγ ϥ΃ ϚϴΘϤϟΎΒϟ΍ ξϤΣ ϭ΃ ϚϴϟϭϷ΍ ξϤΣ Ϧϣ ϲϠϠϣ/ϡ΍ήΟϭήϜϴϣ

ϞϛΖΤΒλ΃ .%1-0ϰϟ·ΎΒϳήϘΗϞμΘϟϡ΍έϭϷ΍ΎϳϼΧΔϳϮϴΣϞϠϘϳ :ϥϮϤϜΤϤϟ΍ ξϤΣϦϣ ΔϔϠΘΨϣ Ε΍ΰϴϛήΘΑ ήϴΛ΄Θϟ΍ΪόΑΔϴΣήϴϏ ϡ΍έϭϷ΍ΎϳϼΧ Ύπϳ΃ .ΔϘϴϗΩ 30ΪόΑϚϴΘϤϟΎΒϟ΍ξϤΣϭΔϘϴϗΩ 40ΪόΑϚϴϟϭϷ΍ ΓήϫΎϘϟ΍ϡϮϠϋˬΕΎΒϨϟ΍Ϣδϗ ϪϧΎΒηϲϤϬϓΖϔϋ .Ω.΃ ϡ΍έϭϸϟΩΎπϤϟ΍ϚϴΘϤϟΎΒϟ΍ξϤΣήϴΛ΄Ηϥ΃ΔϴϟΎΤϟ΍Δγ΍έΪϟ΍ΖΘΒΛ΃ ΎτϨρϡϮϠϋˬΕΎΒϨϟ΍Ϣδϗ Φϴθϟ΍ΪϤΤϣϰϔτμϣ .Ω.΃ κϠΨΘδϤϟ΍ήϴΛ΄Λϥ΃ ΪΟϭ .ϚϴϟϭϷ΍ξϤΣρΎθϧϦϣϰϠϋ΃

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