Introduction

Recently, health beneficial effects of marine cynobacterium are attracting much attention as biological response modifier because of the wide range of Physiological activities.

National cancer institute (NCI) scientists have screened 18,000 extracts of marine organism for activity against tumors, viruses and fungi and for immune system stimulation properties. In 1996, the NCI began studying thousands of types of blue-green algae for effects against the AIDS virus and 100 types of cancer. In 1989, the NCI announced that chemicals from blue-green algae were found remarkably active against AIDS virus (Gustafson et al., 1989). These are naturally accuring sulfolipid portions of the Glycolipids. Sulfolipids can prevent viruses from either attaching or penetrating into cells, thus preventing viral infection. In 1996, NCI announced another extract from the blue-green algae nostac, cyanovirin-N could be a broad-spectrum virucidal agent against HIV. This unique antiviral protein was selected for further high priority preclinical development (Boyd and Gustafson, 1996).

It is now becoming evident that marine organisms are emerging as a significant chemical resource with novel compounds in clinical trials deriving from these microorganisms.

Natural products isolated from marine organisms have been found to be a very rich source of bioactive molecules. Reported biological effects of these compounds include anti-tumour, anti-inflammatory and antiviral as well as immunomodulatory and analgesic properties ( Sundaraj Rajmanikandan et al., 2011). Since the late 1980s, more than 5000 natural products have been discovered from marine organisms and now exceeds 18,000 with hundreds of new compounds being discovered every year (Gokulkrishnan et al., 2011).

Marine natural products offer challenging targets to synthetic chemists due to their complicated structure that exhibits remarkable biological activities.

In the present study, we focused our attention to evaluate the anti-oxidative activity of Oscillatoria annae, since oxidative stress plays an important role in pathogenesis of several diseases.

Oscillatoria is a genus of filamentous cynobacterium which is named for the oscillation in its movement. It is commonly found in watering - troughs waters, and is mainly blue-green or brown-green. Oscillatoria is an organism that reproduces by fragmentation. Oscillatoria forms long filaments of cells which can break into fragments called hormogonia. The hormogonia can grow into a new, longer filament. Break in the filament usually occur when dead cells are present. Oscillatoria uses photosynthesis to survive and reproduce. Each filament of Oscillatoria consists of trichome which is made up of rows of cells. The tip of the trichome oscillates like a pendulum (Rajagoundan Rajavel et al., 2009).

Human body cells utilize oxygen to breakdown biomolecules for the generation of energy. Free radicals are produced in this process in the form of byproducts. These free radicals are highly active because they possess unpaired electrons. Normally free radicals are beneficial for oxidative energy production and killing the invaded bacteria into the cells. Almost all cells possess antioxidant defense and repair system to protect them against oxidative damages but unfortunately not sufficient entirely all the time (Indira and Kausalya, 2010).

The oxygen centered free radicals are called reactive oxygen species (ROS) which includes superoxide anion radical (O^- ₂), hydroxyl radical (OH^-), nitric oxide (NO^-) and hydrogen peroxide (H₂O₂) and other molecules. In healthy individuals, the production of free radicals is balanced with the anti-oxidative defense system. When this equilibrium gets unbalanced, it results the generation of free radicals beyond the limit and ultimately depletion of antioxidant levels. Exogenous free radical when coupled with endogenous free radicals result the elevation of ROS which consequences oxidation of cellular lipids, nucleic acids, proteins, carbohydrates and other biomolecules and leads to a number of diseases like cancer, atherosclerosis, diabetes mellitus, coronary heart diseases and various other degenerative diseases (Saikat Dewanjee et al., 2008).

Antioxidants play important role against oxidative damage of ROS. Antioxidants are important in the prevention of human diseases by functioning either as free radical scavengers, complexers of Pro-oxidant metals, reducing agents or quenchers of singlet oxygen formation.

Materials and Methods

Materials

Preparation of Extract

Invitro Antioxidant Activity

DPPH free radical scavenging activity

The scavenging of DPPH free radicals was used for measuring the antioxidant activity of the extracts according to the method of Blois, 1958. Briefly, stock solutions of the extracts were prepared by dissolving 0.1g of dry extract in 50ml of 50% methanolic solution. The stock solution was diluted with 50% methanolic solution to obtain sample solutions at the concentrations of 1,10,50,100,250,500,1000µg/ml (Wanlop weecharangsan et al., 2006). The sample solutions were thoroughly mixed with freshly prepared 0.01% DPPH methanolic solution at the ratio of 1:1 and kept for 30 min in the dark at room temperature. The amount of the reaction mixture was determined by UV-visible spectrophotometer (U-2000,Hitachi,Japan) at 517nm. The percentage of free radical scavenging activity was calculated as in the equation below

Figure 1

The antioxidant activity of the extract was expressed as IC₅₀, defined as the concentration of the extract required to inhibit DPPH radicals by 50% using the exponential curve.

Nitric oxide scavenging activity

This Method is based on the principle that Sodium nitro prusside in aqueous solution at physiological pH spontaneously generates nitric oxide which further interacts with oxygen to produce nitrite ion that can be estimated using Griess reagent.

Scavenger of nitric oxide competes with oxygen leading to reduced production of nitrite ion. For experimental, sodium nitroprusside (10mM) in phosphate buffer saline (PBS, p^H 7.4) was mixed with different concentration of extract (1,10,50,100,250,500,1000µg/ml) in respective solvent and incubated at 25⁰C for 150 min. The same reaction mixture without extract but with equivalent amount of ethanol serves as control. After incubation period, 1.5ml of the incubated solution were removed and diluted with 1.5ml of Griess reagent (1% Sulphanilamide, 2% Phosphoric acid and 0.1% Naphthyl ethelene diamine dihydrochloride). The absorbance of the chromophore formed during diazotization of the nitrite with sulphanilamide and subsequent coupling with naphthyl ethelenediamine was measured at 546nm (Tenpe et al., 2008).Analysis of all sample were run in triplicate. The nitric oxide scavenging activity is calculated as

Figure 2

Hydroxyl Radical Scavenging Activity

Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and test compound for hydroxyl radicals generated by Fenton reaction (Fe³⁺/ascorbate/EDTA/H₂O₂System).

The hydroxyl radical attacks deoxyribose, which results in thiobarbituric acid reacting substance (TBARS) formation. The formed TBARS were measured (Tatjana Kadifkova Panovska et al., 2005).

The extracts were added to the reaction mixture containing 2.8mmol deoxyribose, 100µmol Fecl₃, 104µmol EDTA, 100µmol ascorbic acid, 1mmol H₂O₂ and 230mmol phosphate buffer (pH7.4), making a final volume of 100ml. The mixture was incubated at 37⁰C for 1 hour. One milliliter of thiobarbituric acid TBA (1%) and 1.0ml Thiochloro acetic acid (TCA 2.8%) were added to the test tube and incubated at 100⁰C for 20minutes. After cooling, absorbance was measured at 532nm against blank containing deoxyribose and buffer. Reactions were carried out in triplicate.

Invivo Antioxidant activity

Albino wistar rats weighing (150-200gms) were used for the study. The animals were procured from Agricultural University, manuthy. Trissur and were housed under standard environmental conditions. They were fed with standard diet and water adlibitum. Experiments on rats were carried out after approval from Institutional Animals Ethics Committee.

The animals were divided into five groups of six animals each. Group I served as normal control group received vechile 0.5% w/v carboxy methyl cellulose (CMC) 1ml/kg orally for seven days. Group II served as CCl₄ induced oxidative stress group received vechile 0.5% w/v CMC 1ml/kg orally for seven days. Group III and Group IV animals received the ethanol extract of Oscillatoria annae 200mg/kg/day orally and 400mg/kg/day orally respectively for seven days. On seventh day 30 minutes after treatment the animals in group II, III, IV and V received intraperitoneal injection of equal mixture of CCl₄ and olive oil 5ml/kg as single dose. On eighth day, all the animals were sacrificed by spinal dislocation. Immediately after sacrifice, the liver of all animals were dissected out and washed with ice-cold saline and homogenized in 0.1M Tris-Hcl buffer (pH 7.4) (Nano et al., 2008). The homogenate were centrifuged and the supernatent was used for the assay of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GP_x) Glutathione (GSH) and Glutathione Reductase (GR) (Roli Budhwar and Sushil Kumar et al., 2005).

Statistical Analysis

Results and Discussion

In the present study, the invitro antioxidant activity of ethanol extract of Oscillatoria annae was determined by DPPH scavenging, nitric oxide scavenging and hydroxyl radical scavenging activity.

DPPH Free Radical Scavenging Activity

The DPPH radical is considered to be a model of lipophilic radical. A chain reaction in lipophilic radical was initiated by lipid auto oxidation. Several concentrations of extract ranging from 1µg/ml to 1000 µg/ml were tested for DPPH scavenging activity. A steady increase in percentage scavenging activity was observed with ethanol extract of Oscillatoria annae which is shown in figure 1. The DPPH scavenging IC₅₀ value was found to be 80µg/ml (Table 1). The effect of extract on DPPH radical scavenging is thought to be due to their hydrogen donating ability.

Figure 3

Table 1: Free radical scavenging activity of ethanol extract of by DPPH radical inhibition.

Values are mean ± SD of Triplicate determinations

Figure 4

Figure 1.

Nitric Oxide Scavenging Activity

Invitro inhibition of nitric oxide radical is a measure of antioxidant activity. Ethanol extract of Oscillatoria annae showed a dose dependent inhibition which is shown in figure 2. Nitric oxide scavenging IC₅₀ value was found to be 220µg/ml (Table 2). The results show that the ethanol extract of Oscillatoria annae decreased the amount of nitrite generated from the decomposition of sodium nitroprusside invitro. This may be due to the antioxidant property in the extract which competes with oxygen to react with nitric oxide thereby inhibiting the generation of peroxynitrite.

Figure 5

Table 2: Nitric oxide scavenging activity of Ethanol extract of

Values are mean ± SD of Triplicate determinations

Figure 6

Figure 2.

Hydroxyl Radical Scavenging Activity

Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and test compound for hydroxyl radical generated for Fenton reaction. The result of hydroxyl radical scavenging activity of ethanol extract of Oscillatoria annae is shown in Table 3. A dose dependent hydroxyl radical scavenging activity was noted with ethanol extract of Oscillatoria annae (Figure 3). The IC₅₀ value was found to be 107.5 µg/ml.

Figure 7

Table 3: Hydroxyl radical scavenging activity of .

Values are mean + SD of Triplicate determinations

Figure 8

Figure 3.

Invivo Antioxidant Activity

The invivo antioxidant activity of ethanol extract of Oscillatoria annae was studied on carbon tetrachloride induced oxidative stress in rats. The effect of carbon tetrachloride treatment on the marker enzymes in pre-treated and untreated rats are shown in Table 4. The results show, the rats treated with CCl₄ decreased the levels of superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GP_x), glutathione (GSH) and glutathione Reductase (GR) enzymes significantly (P < 0.05). Pre-treatment of ethanol extract of Oscillatoria annae 200 mg/kg and 400mg/kg showed an antioxidant defense which prevented the fall in the levels of antioxidant enzymes. A significant difference in enzyme level was noted between extract treated and untreated animals. The protective effect of ethanol extract of Oscillatoria annae was compared with standard drug silymarin (25mg/kg). The protective effect of extract was comparable with silymarin. The extract at a dose range of 400mg/kg showed a remarkable defence against oxidative stress. From the results it is concluded that ethanol extract of Oscillatoria annae has a capacity to prevent the liver damage by keeping the levels of various antioxidant enzymes in defensive range.

Figure 9

Table 4: Invivo antioxidant activity of Ethanol extract of against CCl induced oxidative stress.

Values are mean ± SEM. n = 6 in each group

Values are significantly different from hepatotoxic control (CCl₄) by one-way ANOVA followed by Dunnetts test ^*P<0.05, ^**P<0.01, ^***P<0.001.

Acknowledgment

The authors are thankful to the Chairman Thiru. V. Shanmugam B.Com., Nandha Educational Trust, Erode and Mr. S. Nandhakumar Pradeep, Secretary, Nandha Educational Trust, Erode, Tamil Nadu, INDIA for providing necessary facilities to carry out the research work.