Antioxidant and Antiprolifereative Capacity of ... - ispacs

International Journal of Cellular & Molecular Biotechnology 2014 (2014) 1-9

Available online at www.ispacs.com/ijcmb Volume 2014, Year 2014 Article ID ijcmb-00013, 9 Pages doi:10.5899/2014/ijcmb-00013 Research Article

Antioxidant and Antiprolifereative Capacity of Dichloromethane Extract of Holoturia leucospilota sea cucumber Mozhgan Soltani1, Javad Baharara2* (1) Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran (2) Research Center for Animal Development Applied Biology&Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran

Copyright 2014 © Mozhgan Soltani and Javad Baharara. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract The antioxidant and anti-proliferative effects of dichloromethane extract from methanol extract of H.leucospilota were investigated in this study. Scavenging effects on the DPPH and power reducing were evaluated. The dichloromethane fraction of H.leucospilota exhibited antioxidant activities to scavenge DPPH, The reducing capacity enhanced with increasing concentration of the extract and the activity was comparable with the Butylated hydroxyanisole (BHA) as a standard. Furthermore, the dichloromethane fraction of H.leucospilota exhibits activity against MCF-7 and A549 cancer cell lines. These results demonstrate potential antioxidant activities and anti-proliferative effect of H.leucospilota, as a natural antioxidant and antitumor agent. Keywords: Sea cucumber, Antioxidant, Antiproliferation.

1 Introduction Cancer is one of the major causes of death worldwide. It is estimated between the years 2005 to 2015 around 180 million people will die due to cancer. Studies reveal that free radicals are involved in the initiation and promotion of cancer [1]. Free radicals are normally produced during metabolic activities in the body, but excessive production of these free radicals can cause oxidative stress and Finally leads to cause of many diseases such as cancer [2]. The function of oxidative stress has been shown in different types of cancers like lung and breast cancers [3]. Antioxidants are compounds that inhibit free radicals, and prevented from oxidative stress. Therefore it is possible to prevent the occurrence of some diseases, including cancer [2]. Endogenous antioxidant are not enough to inhibit the production of free radicals in the body. Therefore, for excessive free radical scavenging, a dietary antioxidant is required [4]. Most of the

* Corresponding Author. Email address: [email protected]

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foods contain antioxidants compounds are derived from naturally sources, especially from plants and marine organisms. Marine organisms are valuable sources of natural products with biologically active components. Sea cucumbers are one of the marine animals of the phylum Echinoderm and the class Holothuroidea, with a leathery skin and gelatinous body. There are used as a traditional treatment in Chinese and Malaysian for the treatment of some diseases such as hypertension, eczema and cancer [5]. Many studies have shown that the sea cucumber contains antioxidants and anti-cancer compounds, however, lack information about these activities of the Iranian species of sea cucumbers are available. Therefore, this study was aim to assay the anti-proliferative and antioxidant activities of dichloromethan extract from H.leucospilota sea cucumber as part of an investigation hopes to find novel potential anticancer compounds in Iranian sea cucumbers[6]. 2 Material and Methods 2.1. Chemicals All of the chemicals and solvents were obtained from reputable suppliers such as Sigma-Aldrich Chemical Company (St. Louis, MO, USA), and E. Merck (Darmstadt, Germany). 2.2. Extraction and fractionation Fresh sea cucumber (H.leucospilota) were collected from the Coast of Bandar Abbas, Iran and transferred to Mashhad and was maintained at -80 ° C until testing. Air-dried body walls (200 g) of H.leucospilota were powder and extracted with absolute methanol (0.10 g/ml) by shaking for 72 h at room temperature. Then the extracts were filtered with a Whatman no.1 filter paper and evaporated in vacuo. The crude residue partitioned between water and dichloromethane. The dichloromethan fraction was evaporated in vacuo and used for later analysis [7]. 2.3. Evaluation of Antioxidant Activity of Dichloromethan Extract 2.3.1. DPPH Scavenging Assay The scavenging capability of the dichloromethan extract against of DPPH free radicals was measured by the method of [8]. REAGENTS 1. DPPH – 2, 2-diphenyl-2-picryl hydrazyl hydrate (0.1mM in methanol) 2. Methanol PROCEDURE The dichloromethan extract (500μl) were added to 500μl of methanolic solution of DPPH. Absorbance at 517 nm was determined after 30 min at room temperature. The radical scavenging activity was calculated as follows: % of cell viability = (OD of treated cells / OD of control cells) × 100 2.3.2. Power reducing Assay In this way, antioxidant compound, react with potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+), which then potassium ferrocyanide reacts with ferric chloride to form ferric ferrous complex. REAGENT phosphatebuffer (0.2 M, pH 6.6) , 2% potassium ferricyanide [K3Fe (CN) 6] , FeCl3 (0.1%) , trichloroacetic acid 10% PROCEDURE Different concentrations of the dichloromethan extract in 1.0 ml of distilled water were mixed with phosphate buffer (2.5 ml) and potassium ferricyanide (2.5 ml) and incubated for 20 min at 50C. After International Scientific Publications and Consulting Services


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incubation, trichloroacetic acid (2.5 ml) were added to the mixture and then centrifuged at 3000 rpm for 10 min. The upper layer was mixed with equal volume of distilled water and ferric chloride solution (0.1 %). The absorbance was measured at 700 nm. BHA was used as a standard. Phosphate buffer (pH 6.6) was used as blank solution [9]. 2.4. Evaluation of Antioxidant Activity of Dichloromethan Extract 2.4.1. Cell Cultures Two human cancer cell lines; MCF7 (human breast carcinoma) and A549 (human non-small lung carcinoma); were obtained from National cell bank of Iran (Pasteur institute, Iran) and cultured in RPMI 1,640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin streptomycin. The cells were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37ºC. 2.4.2. MTT Assay When the cells reached ~90% confluency were detached with 0.05% trypsin/EDTA and seeded in 96-well microtitre plate (200 μl/well). After reaching confluent, the cells were exposed to different concentration of the dichloromethan extract (μg/mL) for 24, 48, 72 hours. At the end of incubation, 20 μl MTT solution (5 mg/ml in PBS) was added to each well and Cells were maintained in 37°C with 95% air, 5%CO2 and for 4h. After incubation, the medium was discarded and 100 μl DMSO was added in each well. Eventually, the absorbance was measured at 517 nm using a Microplate reader. The percentage of cell viability was determined using the formula: Viability %=(optical density of sample/optical density of control) ×100 Furthermore, the cytotoxicity of the extract was measured using the formula: CI%= [1-(optical density of sample/optical density of control)] ×100 The concentrations needed for inhibition of 50% of cell viability (IC50) were calculated [10]. 3 Results Different concentration of the dichloromethan extract of H.leucospilota were tested for their antioxidant activity in different systems. It was observed that free radicals were scavenged by the extract in a dose dependent manner in all of the models. the result represented in figure 1-2 [11]. 3.1. DPPH Free Radical Scavenging Activity The anti-oxidative potential of the dichloromethan extract was evaluated using the DPPH free radical scavenging activity assay. As shown in Figure 1, dichloromethan extract showed dose dependent activity and also the DPPH scavenging effect was 51.9 % at a concentration of 1 mg/ml. Therefore, depending on concentration, the dichloromethan extract exhibited a moderate DPPH-scavenging activity as compared to the BHA as a standard compound [7].

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100 90 80 70 60 50 40 30 20 10 0

Extract Standard Log. (Extract) Linear (Standard) 0

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Concentration ϻg/ml Figure 1: Comparative effect of dichloromethan extract (sample) and BHA on DPPH assay. The values are significantly different (P < 0.05) when compared to the control.

3.2. Reducing Ability The reducing power activity of dichloromethan extract and BHA as a standard compound increases absorbance with increasing dose dependent manner.

Absorbance 700 nm

2.5 2 1.5

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Concentrationϻg/ml Figure 2: Comparative effect of dichloromethan extract (sample) and BHA on reducing power. The values are significantly different (P < 0.05) when compared to the control.

3.3. Cytotoxic activity The time and dose dependent effects of dichloromethan extract on proliferation of MCF-7 and A549 cell lines investigated by MTT procedure, has been shown in Figure 3, 4 via plots of viability and cytotoxicity index percentages versus concentrations of dichloromethan extract. Treated group compared with the control group showed that a dose and time dependent decrease in viability. so that the highest decrease in MCF7 and A549 cells viability were exhibited to 6.8%, 3% respectively, by 0.5mg/mL of the extract after 72h incubation [12]. As the results show (Fig 3, 4), with increasing the concentration of the extract, increased toxicity and reduced viability cells. Higher doses caused cell lysis as time dependent. MCF7 and A459 cell lines survival in the presence of dichloromethan extract was prominently suppressed in concentration between 30-60, 60-125 µg/ml (respectively) dose dependently in 24h, 48h, 72h relative to the untreated control groups.



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Figure 3: Effect of dichloromethan extract of H.leucospilota sea cucumber on Cell Proliferation of A549 Cell Line Presented as Percentage of, a) Cell Viability and b) Cytotoxicity Index, Versus Concentration of the Extract. The values are significantly different (P < 0.001). 100 90 80 70 60 50 40 30 20 10 0

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100 90 80 70 60 50 40 30 20 10 0

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Concentrationϻg/ml Figure 4: Effect of dichloromethan extract of H.leucospilota sea cucumber on Cell Proliferation of MCF7 Cell Line Presented as Percentage of, a) Cell Viability and b) Cytotoxicity Index, Versus Concentration of the Extract. The values are significantly different (P < 0.001).

4 Discussion & Conclusion The selection of methods and solvents for the extraction of active metabolites from marine organisms are important steps to assess the biological activity, because it allows the active molecules to be separated from the biological matrix of the marine organisms. Specifically, Alcohol-based extraction method are used for the preparation of total extracts from marine organisms [14]. In this study, using dichloromethan solvent, non-polar compounds such as lipids and non-polar metabolites were extracted and it extract was studied as an antioxidant and antitumor agent. There is a relationship between antioxidants and cancer. Free radicals with induce oxidative damage are involved in initiation and promotion of cancer. On the other hand, antioxidants protect the human body against free radical damage [15]. Results from previous studies have shown that the use of natural antioxidants is associated with reduced risk of cancer. The purpose of this study was to assay the anti-oxidative and anti-proliferative potentials of dichloromethan extract of H.leucospilota sea cucumber. DPPH and power reducing assay were used to evaluate antioxidant activities of dichloromethan extract [6]. The DPPH method is a fast method for measuring antioxidant activity. Some free radicals such as hydroxyl radical and superoxide anion are laboratory-generated free radicals that be influenced by side effects such as metal ion chelation and enzyme inhibition but unlike the radicals, DPPH is not affected by these side reactions [16]. The antioxidant activities of dichloromethan extract were evaluated using DPPH procedure based on the inhibition of the stable DPPH free radical, while for distinguish the reducing potentials of the extract was used of the Fe3+-Fe2+ reductive procedure. Because antioxidants can act with various mechanisms, different antioxidant systems are employed to illustrate the antioxidant activity of fractions. Evidently, the comprehension of the mechanism of antioxidant action is necessary to select appropriate test methods. Therefore, antioxidant activity should be evaluated using different methods that is proportional to the mechanisms of antioxidant action [18]. On the other hand, radical choice and selection of solvent extract could be involved in the capacity of the extract to react with different radicals. DPPH assay provides beneficial information on the reactivity of the fractions with stable free radicals [7]. In this study the antioxidant activity of the extract was compared with BHA as a standard. The results show that with increasing concentration, increases free radical scavenging [6]. Some studies have shown different biological and pharmacological activities of sea cucumber species such as anti-angiogenic, anti-nociceptive, anti-hypertension, antibacterial, antioxidant, antitumor, and wound healing promoters [5]. Results from previous studies have shown that some of sea



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cucumber species consist various potentially anti-proliferative substances. In a study conducted in 2005, the effect of hot water extracts from sea cucumber, Stichopus japonicus on cytotoxicity was evaluated and the results showed that human colon adenocarcinoma cells growth was inhibited in a dose-dependent manner and showed that the toxicity caused by increasing concentrations induce apoptosis and decrease in the growth of Caco-2 cells [19, 20]. In another study, glycoproteins isolated from sea cucumber was studied to investigate the anti-proliferative effect and the results showed that these compounds could significantly inhibit the growth of Sarcoma 180 cells implanted subcutaneously in mice. Many studies have shown that saponins isolated from sea cucumbers have anti-tumor effects [21, 22]. In addition, in a study of mucopolysaccharide extracted from sea cucumber (Stichopus japonicus) were studied and the results showed that Stichopus japonicus acid ingredients mucopolysaccharide to be a potential antitumor agent [23]. In this study, the inhibition effect of the dichloromethan extract from the H.leucospilota sea cucumber on the growth of two cancer cell lines, i.e. A549 and MCF7, was demonstrated by using MTT assay. The present study found that MCF7 cell line was more sensitive to the dichloromethan extracts than A549 cells [6] These results demonstrate potential antioxidant activities and anti-proliferative effect of H.leucospilota, as an natural antioxidant and antitumor agent. References [1] O. Y. Althunibat, B. H. Ridzwan, M. Taher, J. M. Daud, S. J. A. Ichwan, H. Qaralleh, Antioxidant and cytotoxic properties of two sea cucumbers, Holothuria Edulis lesson and Stichopus horrens selenka, Acta Biologica Hungarica, 64 (2013) 10-20. http://dx.doi.org/10.1556/ABiol.64.2013.1.2 [2] D. S. Patel, P. B. Shah, N. B. Managoli, Evaluation of in-vitro anti-oxidant and free radical scavenging activities of withania somnifera and Aloe vera, AJPTech, 2 (2012) 143-147. [3] S. Sen, R. Chakraborty, C. Sridhar, Y. S. R. Reddy, B. De, Free radicals, antioxidant, diseases and phytomediciines: current sattus and future prospect nitrogen species, Int J Pharm Sci Rev Res, 3 (2010) 91-100. [4] P. Li, L. Huo, W. Su, R. Lu, C. Deng, L. Liu, et al; Free radical-scavenging capacity, antioxidant activity and phenolic content of Pouzolzia zeylanica, J. Serb. Chem. Soc, 76 (2011) 709-717. http://dx.doi.org/10.2298/JSC100818063L [5] S. Bordbar, F. Anwar, N. Saari, High-value components and bioactives from sea cucumbers for functional foods--a review, Mar. Drugs, 9 (2011) 1761-805. http://dx.doi.org/10.3390/md9101761 [6] O. Y. Althunibat, R. B. Hashim, M. Taher, In vitro antioxidant and antiproliferative activities of Three malaysian sea cucumber species, Eur. J. Sci. Res, 37 (2009) 376-387. [7] L. J. Mampuru, P. K. Chokoe, M. C. Madiga, A. Theron, R. Anderson, M. P. Mokgotho, Antioxidant and anti-proliferative capacity of a dichloromethane extract of dicerocaryum senecioides leaves, Phytochemicals as Nutraceuticals Global Approaches to Their Role in Nutrition and health, book, (2011). [8] S. Rajamanikandan, T. Sindhu, D. Durgapriya, D. Sophia, P. Ragavendran, V. K. Gopalakrishnan, Radical scavenging and antioxidant activity of ethanolic extract of Mollugo nudicaulis by invitro assays, IJPER, 45 (2011) 310-316. International Scientific Publications and Consulting Services


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