International Journal of Drug Development and Research

Keywords

Padina boergesenii, antioxidant, cytotoxicity

INTRODUCTION

Seaweeds are a group of photoautotrophic, multicellular algae occurring in marine environments. They can perform photosynthesis and the term includes some members of the red, brown and green algae. Seaweeds are commercially important renewable resource and they are used as food, medicine, fertilizer [1]. Seaweeds also possess a wide application in food and pharmaceutical industry [2]. It has become an untapped resource for the potential bioactive compounds as compared to terrestrial plants for food and health benefits. Among the diverse group of marine organisms, algae are considered as a most nutritious and possess wide range of bioactive compounds. Consumption of the marine algae is thought to ameliorate some inflammatory disorders; breast cancer and high cholesterol level. Seaweeds are considered as excellent sources of bioactive compounds such as carotenoids, dietary fibre, protein, vitamins [3] essential fatty acids and minerals [4]. Seaweed lipids has been drawn attention because of the presence of important bioactive molecules like conjugated fatty acids and pigments that have wide range of physiological effects in the treatment of tumours and other cancer related problems [5]. It possesses various pigments such as Fucoxanthin, Zeaxanthin, Carotene and Anthocyanin derivatives [6]. It considers as an important resource of long chain polyunsaturated fatty acid [PUFA] (n-3; n-6) which is fundamental for the formation of structural lipids and components of cell membrane [6]. Excessive intake of PUFA n6:n3 ratio will promote the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune disease [7]. Recent decades, Seaweeds have been recognized as valuable source of antibiotic production, could be an indicator for the synthesizing secondary metabolites [8]. Padina boergesenii -abrown alga, member of the class Phaeophyceae commonly found in coastal regions along continental shelf. Brown algae are said to be a rich source of various bioactive components like polyphenols, alkaloids, flavonoids, terpenoids etc. These secondary metabolites are helpful in studying various physiological effects i.e. either it is harmful or curative on human health [9]. The main objective of the present study is to evaluate the antioxidant and cytotoxic activity of brown algaP. boergesenii collected from Rameswaram, Gulf of Mannar, India.

MATERIALS AND METHODS

Sample collection:

Brown algaPadina boergesenii (Allender & Kraft)were collected from Mandapam region, Gulf of Mannar lying along the longitude from 78°08 ‘N to 79°30’E and along the latitude 8°35´ to 9°25’ during the low tide (temperature was 34- 36°C) in the month of September 2013. The samples were cleaned and washed with sterile water to remove the dust particles, shade dried and then powdered with the help of a mechanical grinder.

Preparation of the crude extract

Ten grams of the powered sample was extracted with 100 mL of four different solvents petroleum ether, dichloromethane, ethyl acetate and methanol. The mixture was kept in the shaker at 150 rpm for 48 hours. The extracts were centrifuged for 3 minutes at 3000 rpm. The supernatant was stored at 4°C which was used for further analysis [10].

SCREENING OF PHYTOCHEMICALS

Phytochemicals present in the extract of P. boergesenii were carried out by following the standard method [11].

Test for alkaloids (Meyer’s reagent)

3 mL of extract was stirred with 3 mL of 1% HCl on water bath. Mayer’s reagent was then added to the mixture. Turbidity of the resulting precipitate was taken as an evidence for the presence of alkaloids.

Test for Tannins using ferric chloride

2 mL of extract was stirred with 2 mL of distilled water, and few drops of FeCl3 were added. Formation of green precipitate indicates the presence of tannins.

Test for flavonoids

1 mL of lead acetate solution was added to 1 mL of extract. The Yellow precipitate formation was considered to be a positive test for flavonoids.

Test for Terpenoids

2 mL of extract was dissolved in 2 mL of chloroform and evaporated to dryness. 2 mL of conc. H2SO4 was then added and heated for about 2 minutes. A greyish colour development indicates the presence of terpenoids.

Test for Glycosides(Libermann’s test)

2 mLof extract was dissolved in 2 mL of chloroform, followed by addition of 2 mL of acetic acid. The solution was then cooled in ice and few drops of H2SO4 were added. A colour change from violet to blue to green indicates the presence of glycosides.

Test for Steroids

2 mL of extract is combined with 2 mL of chloroform and 2mL of conc. H2SO4. A red colour produced in lower chloroform layer was taken as the positive test for presence of steroids.

Total Phenolic content

Total Phenolic content present in the extract was determined using the Folin Ciocalteau reagent [12]. 0.5 mL of sample was added with 1mL of Folin Ciocalteau (1:10) reagent followed by 3 minutes incubation at room temperature. 3 mL of 1% sodium carbonate solution was then added to the mixture. The content was thoroughly mixed and kept for incubation in dark for 2 hours. The absorbance was measured at 760 nm spectrophotometrically. Gallic acid was used as standard. Results were expressed as mg/g Gallic acid equivalents.

Total Flavonoid content

Total flavonoid content was estimated using the method of Ebrahimadeh [13]. 0.5 mLof extract was mixed with 0.1 mL of 10% temperature for 30 minutes. The absorbance of aluminium chloride, 0.1 mL of 1M potassium acetate and 2.8mL of distilled water. The contents were then incubated at room temperature and then the mixture was measured at 450nm using UV Spectrophotometer. Quercetin was used as a standard. Results were expressed as mg/g Quercetin equivalents.

Total Antioxidant activity

The total antioxidant activity wasmeasured using Phosphomolybdenum method [14]. Different concentration of extract was prepared. 1mL of extracts was added to 3 mL of reagent(0.6M sulphuric acid, 28mM sodium phosphate, 4mM ammonium molybdate) a reagent without the extract was used as the control. The reaction mixtures were incubated at 950C for 90 minutes. The absorbance was measured at 635nmspectrophotometrically.

Reducing potential

Different concentration of the extracts in 1mL of distilled water was prepared which were with 2.5 mL of 0.2 M phosphate buffer and 2.5ml of 1 % potassium ferric cyanide.The mixture was incubated for 20 minutes at 500C. After incubation 2.5 mL of 10% TCA was added to the mixtures and centrifuged for 10 minutes at 3000 rpm. 2.5 mL of supernatant was mixed with 2.5mL of distilled water and 0.5mL FeCl3 (0.5mL,0.1%). The absorbance was measured at 700nm using spectrophotometer [15].

DPPH free radical scavenging test

The free radical scavenging activity of the extracts was studied using 1, 1-Diphenyl-2-picryl hydrazyl radical test [16].1mL of different concentration of the extract was added with 0.5mL of DPPH (0.16mM DPPH in methanol). Reagent without the extract was used as control.The mixture was Vortexed and then it was incubated at room temperature at 30 minutes. After incubation 2mL of distilled water was added to it. The absorbance was measured at 517nm spectrophotometrically.

Nitric oxide scavenging activity

Different concentration (250, 500, 750, and 1000) μg / mL of the extract were prepared. 100ml Griess reagent was taken and added to 300μl of sample. To this 2.6 mL of distilled water was added.Reagent without the extract was taken as control. The solution was incubated at room temperature for 30 minutes. Absorbance was measured at 548nm spectrophotometrically [17].

Cytotoxic activity against liver cancer Hep G2 cell line

Cytotoxic activity of the methanol extracts of P. boergesenii was screened against the liver cancer cell line Hep G2 by following the method of Zandiet al.,with little modifications [18]. 5x103 Hep G2 cells in 100μl DMEM medium with 10% FBS per well plated in a 96 well plate and incubated overnight at 370C in 5% CO2 incubator. 10mg/ml stock of the test samples was prepared in DMEM. Samples were serially diluted from the stock using DMEM. 100 μl of these diluted samples were added to cells in triplicate wells containing 100 μl of medium in accordance with the following layout as shown in the table 1.100 μl of DMEM was used as negative control and 5μg/ ml Doxorubicin was added as internal positive control for the assay. Wells without any cells are used as blank. Plate was gently shaken and incubated at 37oC at 5% CO2 for 48 hours. 20 μl of 5mg/ml MTT in PBS was added to each well and incubated at 37oC at 5% CO2 for 4 hours. Then the medium was aspirated out and 200μl of Dimethylsulfoxide (DMSO) to each well. The optical density of each well was measured using Microplate reader at 570 nm. Then, the inhibition of growth is a measure of cytotoxicity and the percentage inhibition is calculated as follows:

% Inhibition = 100 - [(Mean OD for test sample/ mean OD for the control) x 100]

RESULTS

P. boergeseniiextracts were screened for the qualitative phytochemical analysis. The results revealed the presence of alkaloids, phenolics, flavonoids, tannins, terpenoids, glycosides and sterol in the methanol extract as shown in the table 2.

The total phenolic content of the methanol extract P. boergeseniiwas determined by performing Folin-Ciocalteau reagent method spectrophotometrically. The phenolic content present in the extract was found to be 84.96±0.40 mg/g expressed as Gallic acid equivalent. The total flavonoid content of the extract P.boergeseniiwas estimated by aluminium chloride method spectrophotometrically. The flavonoid content present in the extract was found to be 85.42±0.97 mg/g expressed as Quercetin equivalent.The total antioxidant activity of P. boergeseniiwas determined using the Phosphomolybdenum method. It was found that the antioxidant activities increased with the increasing concentration. Higher antioxidant activity was exhibited at a concentration of 1000 μg/mL is (58.5 ± 0.48) % as shown in the figure 1.

The reductive capability of the methanol extracts of P.boergesenii the reductionof ferric to ferrous iontransformation. Reducing power assaywas an electron donor and it terminates the oxidation chainreaction by reducing the oxidized intermediates in to the stable form [19]. Increasing absorbance indicates the increasing reducing power. The reducing power increased with the increase in concentration.as shown in the figure 2.

The DPPH assay is a quick and cost effective method which has frequently been used for the estimation of the ant oxidative potential of different natural products. It shows that by using DPPH radical scavenging assay methanol extracts of brownalga P. boergesenii exhibited potent antioxidant activity in a dose-dependent manner. It also reveals that the methanol extract of P. boergesenii also contains a high amount of phenolic compounds. The higher scavenging activity of P. boergesenii may be attributed to hydroxyl groups in the phenolic compounds, which might provide the essential component [20]. It was found that the scavenging effect increased with the increase in concentration of the sample. The value obtained was 59.70 ± 0.21 % at 1000 μg/ml.

Nitric oxide plays a major role in promoting inflammatory response and the toxicity when they react with oxide radicals to form peroxynitrite which can damage the biomolecules. Nitric oxide generates when sodium nitroprusside reacts with oxygen to form nitrite. Seaweeds reduce the formation of nitrite by competing with oxygen to react with nitric oxide [21]. The scavenging activity of P. boergesenii was found to be 54.3 ± 0.34 % at a maximum concentration of 1000 μg/ml as shown in the figure 4. Marine algae have been used from ancient times in Chinese herbal medicine for the treatment of cancer [22].The activity againstcancer cell lines is one of the major activities of marine algae, and many algae have showed cytotoxic and antitumor activities [23]. The viability of Hep G2 liver cancer cell line treated with the methanolic extract of P. boergesenii wasdetermined by MTT assay. It has shown the strong cytotoxic activity as shown in the table 3 and the IC50 value was found to be 1.67 mg/ml.

DISCUSSIONS

Recent decades, seaweeds have been considered as a rich source of reactive oxygen species (ROS) inhibitors and can be used as additives in food and also provide a protection against oxidative damage in tissue induced by ROS[24]. The present study has shown that methanol extract of P. boergesenii possess antioxidant activity and potential to scavenge free radicals. Few evidences are also available that seaweeds have certain bioactive compounds with a high antioxidant and antiproliferative activity. Seaweeds contain some bioactive compound which is not found in the terrestrial plants [25].

Antioxidant compounds has an ability to scavenge free radicals leads to the reduces the level of risk associated with oxidative stress related disease [26]. Seaweeds have been attracted attention as a rich natural antioxidant source due to the toxic and mutagenic effects caused by the synthetic antioxidants [27].

This method used for the analysis of phenolic content, flavonoid content, antioxidant activity and scavenging assays such as DPPH, Nitric oxide and reducing potential refers as a convenient and accurate method for the detection of potential source of antioxidant compounds [28].The total phenolic content of P. boergeseniiwas found to be84.96±0.40mg gallic acid equivalents/g extract.The total flavonoid content of P. boergesenii was found to be 85.42±0.97 mg/g expressed as Quercetin equivalent. Phenolic compounds are having potential to acts as free radical scavengers. It’s commonly found in the edible marine algae in which the antioxidant property has been correlated to their phenolic content [29]. Previous studies were reported phenolic compounds play an important role in preventing the oxidative damage to tissue [30]. The relationship between the total phenols and antioxidant activity has been observed in many seaweed species [31-32]. Flavonoid acts as an antioxidant and free radical scavenger due to its unique chemical structure. The position of hydroxyl groups and other features in the chemical structure of flavonoids are very important for their antioxidant and free radical scavenging activities [33]. Hence, the potential scavenging activity of the P. boergeseniicould be linked to the phenolics and flavonoids present in it.

Total antioxidant activity of P. boergesenii was determined by the Phosphomolybdenum method. The results observed that P. boergeseniihave the maximum antioxidant activity of (58.5 ±0.48) %. Oxidative damage within the tissues is a prolonged process involving free radical chain initiation and propagation steps [34]. One of the mechanisms by which antioxidants bring about their action is by scavenging free radicals [35]. So, it becomes necessary to assess the scavenging ability of the brown alga P. boergeseniiextract. Reducing potential of methanol extracts of P. boergeseniiwas evaluated usingpotassium cyanoferrate. The reducing ability of a compound widely varies based on the presence of reductones, which have exhibit antioxidative potential by breaking the free radical chain by donating a hydrogen atom [36]. The reducing potential of the extracts is a significant indicator of antioxidant and scavenging activity. Similar reports have also been suggested by Kumaran and Karunakaran [37], Kumaret al., [38], Vijayabhaskar [3] in methanolic extracts of higher plants, brown seaweeds from India.

In this study, DPPH, Nitric oxide free radical assays were done for the evaluation of scavenging potential of methanol extract of P. boergesenii. 1, 1- Diphenyl-2-picrylhydrazyl (DPPH) is stable nitrogen centered free radical which can be effectively scavenged by antioxidants [39-40]. Hence, it has been widely used to test the ability of compounds as free radical scavengers or hydrogen donors [41].The present study has shown that the P. boergesenii extract has exhibited maximum DPPH scavenging activity [59.70 ± 0.21] % at 1000 μg/ml. It indicates the hydrogen donating ability of P.boergesenii.Since the effect of antioxidant on DPPH might be due to their hydrogen donating capability [42]. Nitric oxide radicals plays an vital role in inducing inflammatory response and their toxicity increases when they react with O2 radicals to form peroxynitrite, which leads to the damage in biomolecules such as proteins, nucleic acids, lipids. [21]. Nitric oxide is generated when sodium nitroprusside reacts with oxygen to form nitrite. The methanolic extract of P.boergeseniihas exhibited higher scavenging activity as [54.3 ± 0.34] % at 1000 μg/ml. These results suggest that P. boergesenii could be a capable and novel therapeutic agent for scavenging of NO– and the regulation of pathological conditions caused by excessive generation of NO– and its oxidation product peroxynitrite. It is believed that the antioxidant activity of phenolics is a result of their ability to act as reducing agents, free radical scavengers [43]. It may be possible that the antioxidant activity of P.boergesenii could be the result of their high concentration of phenolic compounds.

The cytotoxic activity of the P.boergesenii’s methanol extract against Hep G2 liver cancer cell line was studied and the IC50 was found to be 1.67 mg/ml. Brown algae represent a rich source of polysaccharides and glycosides and phenolics, the activity could be connected with these compounds. P. pavonica aqueous extract showed cytotoxic activity against breast cancer cell line in vitro [44].

CONCLUSION

In the present study, the extracts of P. boergesenii showed high antioxidant activity as well as cytotoxic activity to the best of our knowledge. Very few works have been carried out with this seaweed from the region of Gulf of Mannar, Rameswaram. Hence, P. boergesenii can be used as a potential therapeutic intervention due to its bioactive properties.

ACKNOWLEDGEMENT

We are thankful to the authority of VIT University for the facilities and their constant support.

Tables at a glance

Table 1 Table 2 Table 3

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4

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