IN VITRO ANTIOXIDANT ACTIVITY OF METHANOLIC EXTRACT OF CHLORELLA VULGARIS

Bhuvana P 1 , * Anuradha. V 1 , Syed Ali.M 2 , Suganya V 1 and Sangeetha P 1 . 1. Department of Biochemistry, Mohamed Sathak College of Arts & Science, Sholinganallur, Chennai, Tamil Nadu, India. 2. Department of Biotechnology, Mohamed Sathak College of Arts & Science, Sholinganallur, Chennai, Tamil Nadu, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


Sample preparation
1 gram dried powder of Chlorella vulgaris was mixed with 10 ml of methanol solvent and it is kept at room temperature for 24 hours. This mixture was filtered using Whatmann No.1 filter paper and stored at 4ºC for further analysis.

Quantification of Total Phenolics and Flavonoids
Total phenolic contents were assessed by following strategy: 100 μl of methanol extract of Chlorella vulgaris was blended with 2.0 ml of 2% Na 2 CO 3 and permitted to remain for 2 min at room temperature. After incubation, 100 μl of 50% FolinCiocalteau's phenol reagent was supplemented and was mixed thoroughly. It is then allowed to stand at room temperature for 30 min. Absorbance of all the samples were measured at 720 nm using spectrophotometer. Gallic acid was used as standard to determine total phenolic activity. (Taga, et al., 1984).
The total flavonoid content of tests was detected by the aluminum chloride colorimetric method (Chang, et al., 2002). 0.5 ml of methanol extract of Chlorella vulgaris were mixed with 250 µl of 5% sodium nitrite (NaNO 2 ) solution and 150 µl of 10% AlCl 3 solution and incubated for 5 mins. At that time, 0.5 ml of 1 mol/L sodium hydroxide (NaOH) solution was added, and was brought to 2.5 ml with double-distilled water. The mixture was allowed to stand for 15 min which was measured at 510 nm. The total flavonoid content was calculated from a calibration curve, and the result was expressed as mg. Rutin was used as standard (equivalent per g dry weight).

In vitro Antioxidant and Free Radical Scavenging Assays (Suganya, et al., 2017) Total antioxidant activity
The total antioxidant capacity of methanol extract of Chlorella vulgariswas evaluated by Prieto P et al., 1999. At first, 7.45 ml of sulphuric acid (0.6 mM solution), 0.9942 g of sodium sulphate (28 mM solution) and 1.2356 g of ammonium molybdate (4mM solution) was dissolved in distilled water and made up to 250 ml which was marked as TAC reagent. Then, to 300 µl of test sample 3 ml of TAC reagent was added. Reaction mixture was incubated at 95° C for 90 minutes. Absorbance was measured at 695 nm and ascorbic acid was used as standard. The concentration in the range of 100 to 500 µl were taken for both standard and test samples.

DPPH radical scavenging assay
The scavenging activity of methanol extract of Chlorella vulgaris for DPPH radical were identified by the method of (Yen and Chen, 1995). Concisely, 2.0 ml of test samples and 2.0 ml of 0.16 mM DPPH methanolic solution was mixed together. The mixture was vortexed for 1 min and then allowed to stand at room temperature for 30 min in the dark. The absorbance of all the sample solutions was measured at 517 nm. The varied concentration in the range of 100 to 500 µl were taken for both standard and methanol extract of Chlorella vulgaris. The scavenging effect (%) was calculated by using the formulae: Scavenging effect (%) = (Absorbance of control -Absorbance of test solution)/Absorbance of control] × 100 Hydrogen peroxide scavenging activity The hydrogen peroxide scavenging assay was done based on the procedure of Ruchet al., 1989. A solution of hydrogen peroxide (H 2 O 2 , 10 mM) was prepared in phosphate buffer (0.1 M, pH 7.4). 3.4 ml of phosphate buffer was mixed with 0.6 ml of H 2 O 2 solution (0.6 ml, 43 mM) and 1ml (0.25 mg) of methanol extract of Chlorella vulgaris was added to it. The absorbance value of the mixture was recorded at 230 nm after 10 minutes and incubation at room temperature. Blank solution contains sodium phosphate buffer without H 2 O 2 was used. Ascorbic acid was used as the standard. The percentage of H 2 O 2 scavenging of crude extract and standard compounds were calculated using the following equation: H 2 O 2 scavenging effect (%) = (Absorbance of control -Absorbance of test solution)/Absorbance of control) × 100 Nitric oxide scavenging activity Nitric oxide scavenging activity was performed based on the following procedure: 3ml of 10 mM of sodium nitroprusside was prepared in phosphate buffer saline (pH 7.4, 0.2 M) which was mixed with 1 ml of methanol extract of Chlorella vulgaris and incubated at 25°C for 180 mins. By mixing equal amounts of 1% sulphanilamide in 2.5% phosphoric acid and 0.1% naphthylethylenediaminedihydrochloride in 2.5% phosphoric acid the Griess reagent was prepared immediately before use. The sample was mixed with an equal volume of freshly prepared Griess reagent. The absorbance was measured at 546 nm. Ascorbic acid was used as the positive control. The 1467 percentage inhibition of the extract and standard was calculated and recorded (Fadzai Booraet al., 2014). The percentage nitrite radical scavenging activity of the ethanol extracts and Gallic acid were calculated using the following formula: Nitric oxide activity (%) = Absorbance of control-Absorbance of test / Absorbance of control × 100.

Ferric reducing antioxidant Power (FRAP):-
Reducing power of methanol extract of Chlorella vulgaris was carried out by the method prescribed by Oyaizu M, 1986. To 1.0 ml of methanol extract of Chlorella vulgaris, 2.5 ml of Phosphate buffer (0.2 M, pH 6.6) and 2.5 ml Potassium ferricyanide (1%) was mixed. Reaction mixture was incubated at 50°C for 20 min. After incubation, 2.5 ml of Trichloroacetic acid (10%) was added and centrifuged (650 g) for 10 min. From the upper layer, 2.5 ml solution was taken and mixed with 2.5 ml distilled water and 0.5 ml FeCl 3 (0.1%). Absorbance of all the sample solutions was measured at 700 nm. Increased absorbance is indicated increased reducing power.
Deoxyribose Radical Scavenging Activity:-Deoxyribose non-site specific hydroxyl radical scavenging activity of methanol extract of Chlorella vulgaris was estimated (Indu and Seenivasan, 2013). Briefly, 2.0 ml aliquots of test samples were mixed to the test tube containing reaction mixture of 2.0 ml FeSO 4 .7H 2 O (10mM), 0.2 ml EDTA (10mM) and 0.2 ml deoxyribose (10mM). The volume was made up to 1.8 ml with phosphate buffer (0.1M, pH-7.4) and to that 0.2 ml H 2 O 2 (10mM) was added. The mixture was incubated at 37°C under dark for 4 hours. After incubation, 1 ml of TCA (2.8%) and TBA (1%) were added to the mixture, and then kept under boiling water bath for 10 min. After the treatment the samples were absorbed at 532nm. If the mixture was turbid, the absorbance was measured after filtration. Ascorbic acid was used as standard. Scavenging activity (%) was calculated using the equation: Deoxyribose radical scavenging activity (%) = Absorbance of control-Absorbance of test / Absorbance of control × 100

ABTS [2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] Radical Cation Scavenging Assay:-
Free radical scavenging activity was determined by ABTS radical cationdecolorization assay (Re R et al., 1999). ABTS radical cation was creäted by mixing 20mM ABTS solution with 70mM potassium peroxodisulphate and kept to stand in dark at room temperature for 24 hours before use. To, 0.6 ml of methanol extract of Chlorella vulgaris(0.25 mg), 0.45 ml of ABTS reagent was added and absorbance of these solutions was measured at 734 nm after 10 min. ABTS radical cation scavenging assay [%] = Absorbance of control-Absorbance of test / Absorbance of control × 100 Superoxide radical scavenging activity:-Scavenging of superoxide radical was calculated using the method elaborated by (Winterbournet al., 1975). Assay tubes contained 0.2 ml of methanol extract of Chlorella vulgaris(corresponding to 20 mg extract) with 0.2 ml EDTA (12mM), 0.1 ml Nitro blue tetrazolium, 0.05 ml riboflavin (20µg) and 2.64 ml phosphate buffer (50 mM, 7.6 pH). The control tubes were set up with DMSO (Dimethyl sulfoxide) solution instead of the test solution. The initial optical densities of the solutions were recorded at 560 nm and the tubes were illuminated uniformly with the fluorescent lamp for 30 mins. A560 was measured again and the difference in O.D was taken as the quantum of superoxide production. The percentage of inhibition was calculated by comparing with O.D of the control tubes.
SOD activity:-Measurement of superoxide anion scavenging activity was performed based on the method (Nishimikiet al., 1972), with slight modifications. To, 1ml of NitroblueTetrazolium (NBT) solution containing 156μM NBT dissolved in 1.0 ml of phosphate buffer (100mM, pH 7.4) and 1ml of NADH solution containing 468 μM of NADH which is dissolved in 1ml of phosphate buffer (100 mM, pH 7.4) with 0.1 ml of test samples were added and the reaction was started by adding 100 μl of Phenazinemethosulphate (PMS) solution containing 60 μM of PMS 100 μl of phosphate buffer (100 mM, pH 7.4). The reaction mixture was incubated at 25°C for 5 min and the absorbance at 560nm was measured against the control samples. BHT was used as the reference compounds (200 to 1000 µg/ ml). The percentage of inhibition was calculated as mentioned below.
% of SOD = (Absorbance of control-Absorbance of test) / Absorbance of control × 100 1468 Estimation of lipid peroxidation using egg yolks:-Inhibitions of lipid peroxidation in the egg of hen were determined using a modified method thiobarbituric acidreactive species (TBARS) assay (Badmuset al., 2013). Egg homogenate (0.5 ml, 10% in distilled water, v/v) was mixed with 0.1 ml of methanol extract of Chlorella vulgaris in a test tube and the volume was made up to 1 ml, by adding distilled water. Lastly, 0.05 ml FeSO 4 (0.07 M) was added to the above mixture to induce lipid peroxidation and incubated for 30 min. Then, 1.5 ml of 20% acetic acid and 1.5 ml of 0.8% TBA (w/v) in 1.1% sodium dodecyl sulfate (SDS) and 0.05 ml 20% TCA was added, vortexed and then heated in a boiling water bath for 60 min. After cooling, 5.0 ml of butanol was added to each tube and centrifuged at 3000 rpm for 10 min. The absorbance of the organic upper layer was measured at 532 nm. % Inhibition= Absorbance of control-Absorbance of test / Absorbance of control × 100 β-carotene linoleic acid assay:β-Carotene linoleic acid assay was carried out based on (Zargaret al., 2011). Briefly, in 10 ml of chloroform, 2 mg β-carotene, 200 mg linoleic acid and 20 mg Tween 40 were dissolved which was taken in flask. Chloroform was evaporated using vacuum evaporator apparatus. At that time, 50 ml of distilled water saturated with oxygen by shaking for 30 mins. This mixture is used as stock solution. 200 µl of methanol extract of Chlorella vulgaris were mixed with 2.5 ml of stock solution in the test tube. After that, the samples were placed in an oven at 50°C for 3 hours. The absorbance was read at 470 nm. The percent of antioxidant activity was calculated from the following equation: % Inhibition= Absorbance of control-Absorbance of test / Absorbance of control × 100   Table 2 denotes the total antioxidant activity of Ascorbic acid standard and methanol extract of chlorella vulgaris. The total antioxidant activity of chlorella vulgaris at the concentration of 500 μg/ml (0.021 ± 0.009) indicates higher activity whereas, at the concentration of 100 μg/ml (0.06 ± 0.004) indicates lower activity and the concentration of 300 μg/ml and 400 μg/ml (0.013 ± 0.123 to 0.017 ± 0.012) indicates moderate activity when related with standard ascorbic acid (0.008 ± 0.009 to 0.012 ± 0.005). In present study, the DPPH activity of ascorbic acid and methanol extract were determined and the results (OD values and percentage) are presented in The hydrogen peroxide scavenging activity of test samples along with standard the concentration in the range of 100 -500μg/ml was shown in Table 4. The minimum activity was founded for methanol extract at the concentration 100 μg/ml (26.41%). Similarly, the standard ascorbic acid possesses 23.68%. At concentration, 500 methanol extract (87.28%) showed higher activity than standard (84.26%). The IC 50 values of both standard and test samples were calculated which was found to be 267.800 μg/ml and 250.829 μg/ml based on their percentage of inhibition.

Nitric oxide scavenging activity:-
The OD values, Percentage of inhibition and IC 50 values were noticed in Table 5. The control values were also recorded (1.934) which is used to calculate the percentage of samples. The nitric oxide scavenging activity was done for methanol extract of Chlorella vulgaris along with standard ascorbic acid. The highest inhibition was shown at the concentration 500 μg/ml with inhibition of 74.30 %. The lowest inhibition was found at the concentration 100 μg/ml (12.66%). At all concentration test samples possesses lower percentage of inhibition when compared with standard ascorbic acid which produced 14.53% to 78.75% of inhibition. 0.062 ± 0.158 0.055 ± 0.146 Standard in the range of 0.012 ± 0.322 to 0.062 ± 0.158 was recorded at concentration 100 -500 µg/ml which shows higher activity than methanol extract whose OD ranges from 0.006 ± 0.101 to 0.055 ± 0.146. The values were denoted in Table 6.

 Deoxyribose Radical Scavenging Activity
From the    Table 8 indicates the percentage of inhibition against concentration in the range of 100 -500 μg/ml for both Gallic acid standard and test samples. The methanol extract of chlorella vulgaris showed maximum of 81.25 % of inhibition at 500 μg/ml concentration with IC 50 values 236.682 µg/ml and they are slightly higher than that of the standard Gallic acid (78.66%) with IC 50 values of 260.203 µg/ml. Table 9 explains the superoxide radical scavenging activity of standard and test samples which shows 29.14 % at 100 μg/ml and 88.70% at 500 μg/ml with IC 50 value 230.799 μg/ml for standard and shows 30.72 % at 100 μg/ml and 89.90 % at 500 μg/ml which is slightly higher than the standard drug with IC 50 values of 214.507 μg/ml.

Estimation of lipid peroxidation using egg yolks
Percentage of inhibition by standard BHT was founded to be 78.01% which is slightly higher than the test sample 76.07%. Similarly, the IC 50 value of standard was 313.030 μg/ml which is lower than the IC 50 value of methanol extract (326.024 μg/ml)( Table 11).  15.37 % at 100 μg/ml and 80.65% at 500 μg/ml concentration which is compared with the methanol extract of Chlorella vulgaris which possess18.48% at 100 μg/ml and 87.74% at 500 μg/ml concentration respectively. Hence, percentage of inhibition by test sample is higher than the standard drug (Table 12).

Discussion:-
In present investigation, methanol extract of Chlorella vulgaris shows higher antioxidant activity in all the tests except Nitric oxide scavenging, Ferric reducing, LPO and β-Carotene when compared with the different standard drug. This was related with other findings whose results were similar to our present analysis.
In (Dantaset al., 2015). studies, the solvents with higher efficiency of extraction of compounds with antioxidant activity of the species Chlorella vulgaris were done. The solvents DMSO (dimethyl sulfoxide) and water showed the DPPH percentage of inhibition of 64.6% and 68.5%, respectively, higher than the standards Catechin (49.6%) and Gallic acid (28.7%), showing that they are potential inhibitors of cellular oxidation by free radicals.
Antioxidant activity of the methanol extracts of Chlorella vulgaris was determined in terms of IC50 value based on the percentage of free radical scavenging activity. In both the extracts, higher scavenging activity was observed at the concentration of 1000 μg/mL. IC50 values for the C. vulgaris determined by H 2 O 2 and Thiocyanate assay were 26.31μg/mL and 28.18μg/mL respectively. The presence of flavonoids and phenols in the methanol extract might been responsible for free radical scavenging activity individually or by synergistic action (Annamali and Nallamuthu, 2014).
Based on our present result, the highest phenolic content, hydrogen peroxide Radical Scavenging Activity was also obtained by Anantharamanet al., 2013. The highest phenolic content was observed in methanolic extract of C. marina (0.78 ± 0.032 mg/g gallic acid equivalent) and methanol extract of C. marina (23.08%) and D. salina(17.66%) and acetone extract of C. marina (20.54%) was found to be the most potent scavenger. Hydrogen peroxide Radical Scavenging Activity (%) was found to be maximum in methanol (61.33%) and hexane extract (55.3%) of C. marina, whereas minimum in acetone extract (15.54%) of N. clavata. The reducing power was found to be higher in methanolic extract of C. marina (0.73 ± 0.026 mg/g). The highest nitric oxide scavenging activity was also observed in methanol and hexane extract of C. marina (25.76% and 21.73%) respectively.

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According to Manivannanet al., 2012 methanol extract of Chlorella marina exhibited higher activity which as followed by diethyl ether and hexane extract. This may be due to the differencesin the polarity of the solvents used.The highest antioxidant activity of C. marina methanol extract is (1.03 ± 0.02 mg/g).
Antioxidant potential of microalgae in relation to their phenolic and carotenoid content was carried out by Goiriset al., 2012 and founded that industrially cultivated samples of Tetraselmissuecica, Botryococcusbraunii, Neochlorisoleoabundans, Isochrysis sp., Chlorella vulgaris and Phaeodactylumtricornutum possessed the highest antioxidant capacities and thus could be a potential new source of natural antioxidants. The results from various studies with different types of extracts clearly indicated that next to the well-studied carotenoids, phenolic compounds also contribute significantly to the antioxidant capacity of microalgae.
Similarly, Sivakumar andRajagopal, 2011 reported that the highest antioxidant activity was observed in methanol extract from eight green algal species. Uma et al., 2011 observed that the methanolic extracts displayed greater potential in all antioxidant assays when compared to ethanolic and acetone extract of green microalgae Desmococcusolivaceousand Chlorococcumhumicola and it also clearly explain that the methanolic extract found to be having higher phenolic content in D. olivaceousand flavonoid content was high in acetone extract of C. humicola. The hexane extract of N. clavatashowed the minimum DPPH radical scavenging activity at 9.1% respectively. Similarly, both methanolic and acetone extracts of D. olivaceousand C. humicolashowed a significant dose dependent reduction of DPPH radicals. The methanolic extracts of D. olivaceousexhibited 39% scavenging activity and the acetone extracts of C.humicolaexhibited 15% scavenging activity.
The present study was related with the finding of Lee et al., 2010, who found that the ethyl acetate of H. porphyrae(30.1%) and the 80% of methanol extract of O. unicellularis(49.3%) exhibited significantly higher nitric oxide radical scavenging effects than those of the commercial antioxidants. The implications are important as radical scavengers may protect cell tissues from free radicals, thereby preventing diseases such as cancer.
Wang et al., 2010 studied the species Chlorella vulgaris and they observed that in the DPPH assay using ultrasonic extraction with ethanol found the lowest percentage of inhibition (0.74%). This fact might be related to the variation in strains of microalgae and with the appropriate constituents. The result found using aqueous extract proves satisfactory because in addition to reducing processing costs, resulting in a product without the potentially toxic residues found in other solvents.
Similarly, Kudaet al., 2005 reported that the highest amount of reducing power was observed in the highly polar water extract of S. lomentariaand the minimum reducing power was observed in ethanol extract and crude fucoidan, these were dose dependent. Supportively, Herreroet al., 2005 explained that these polar compounds can be extracted to a higher extent, in this way increasing the yield of extract.

Conclusion:-
The antioxidant activities of various microalga species, their phenolic and carotenoid contents were evaluated extensively during the past two decades. In the present study, methanolic extract of Chlorella vulgaris showed potent antioxidant activity. Microalga biomass can therefore be considered as potential source of natural antioxidants, such as the carotenoids and the phenolic compounds.