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Invitro evaluation of antioxidant potential of Acrostichum aureum Linn. rachis Arockia Badhsheeba M 1 Vadivel V 2 Research scholarRegister Number:9246 1 Assisstant professor 2 1 Research Department of Botany V.O.Chidambaram CollegeAffiliated to Manonmaniam Sundaranar University Abhishekapatti Tirunelveli Tamil Nadu India Abstract: Acrostichum aureum is a medicinal fern found in estuarine region.This taxon is used by the local people for curing various ailments such as pharyngitis chest pain and diabetics.In the present study antioxidant activity of petroleum ether benzene ethyl acetate methanol and ethanol extracts of the A. aureum rachis have been tested using various antioxidant model systems viz 22-diphenyl-1-picryl-hydrazylDPPH hydroxyl superoxide ABTS and resolving power. The methanol extract of A. aureum rachis was found to possess higher activity intheDPPH assay the hydroxyl assay and the superoxide assay and the ethanol extract of A. aureum rachis is found to possess higher ABTS radical cation scavenging activities.The antioxidant potential was dose dependent in all assays carried out. The results suggest that phytocomponents in the rachis provide considerable antioxidant activity. It is concluded that the A. aureum can be used as a medicine against free radical-associated oxidative damage. Keywords:Acrostichum aureum rachis phytocomponents antioxidant activity. INTRODUCTION Antioxidants are compounds produced in our body and found in foods. They help defend our cells from damage caused by potentially harmful molecules known as free radicals. When free radicals accumulate they may cause a state known as oxidative stress. This may damage our DNA and other important structures in your cells.Sadly chronic oxidative stress can increase our risk of chronic diseases such as heart disease type 2 diabetes and cancer. Epidemiological studies have brought into being that the intake of antioxidants such as vitaminC ascorbic acid reduces the risk of coronary heart disease and cancer. The use of synthetic antioxidants such as butylated hydroxytoluene butylated hydroxyanisole tert-butylhydroquinone and propylgallate has been negatively perceived by consumers due to safety and health effects Juntachote et al. 2005 1 . Hence there is an increasing interest in the search of natural antioxidants from plant sources. It is well known that many botanicals possess natural antioxidants with high antioxidant activitysultana et al.2007 2 and investigations on these were initiated based on their uses in traditional folkloric medicinesDaffaodil et al .2014 3 . Fortunately eating a diet rich in antioxidants can help increase our blood antioxidant levels to fight oxidative stress and reduce the risk of these diseases. Plant phenols are secondary metabolites with powerful antioxidant capacities. However primarily synthesized for the plants’ International Journal of Scientific Research and Review Volume 7 Issue 11 2018 ISSN NO: 2279-543X Page No: 268

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own resistance against oxidative stress and these compounds retain the ability to act as antioxidants and thus largely contribute to the pharmaceutical and dietary properties of plant-derived food. Consequently characterization of phenols as antioxidants is essential for both plant biology and human nutrient. Several studies revealed that phenols mainly the type of flavonoids from some medicinal plants are safe and bioactive have antioxidant properties and exert anticarcinogenicantimutagenicantitumoralantibacterialantiviral and anti-inflammatory effectsozgova et al.2003 4 . Furthermore in current years substantial attention has been directed toward credentials of plants with antioxidant ability that may be used for human expenditure due to their acting as reducing agents hydrogen donors singlet oxygen quenchers and chelating metalsTung et al. 2009 5 . Acrostichum aureum is a medicinal pteridophyte is used as an antihelmintic styptic in traditional systems of medicine.In the present study the in vitro antioxidant activity of raches of A.aureum is investigated by 22-diphenyl-1-picryl-hydrazylDPPH hydroxyl radical ABTS radical cation and SOD assays and by measuring reducing power ability. These assays proved the antioxidant ability of the selected plant extracts in comparison with the reference antioxidant ascorbic acid. MATERIALS AND METHODS The rachis of A. aureum was collected from Puthalam Kanyakumari District Tamil Nadu. The plant was identified with help of local flora and authenticated in Botanical Survey of India Southern circle Coimbatore Tamil Nadu. Preparation of extract for antioxidant activity The rachis of A. aureum was dried in shade and then coarsely powdered separately in a willy mill. The coarse powder 100 g was extracted successively with petroleum ether benzene ethyl acetate methanol and ethanol each 250 ml in a Soxhlet apparatus for 24 hours. The extracts were filtered through Whatman No41 filter paper. The extracts were concentrated in a rotary evaporator. The concentrated extracts were used for in vitro antioxidant activity assays. DPPH radical scavenging activity The DPPH is a stable free radical and is widely used to assess the radical scavenging activity of antioxidant component. This method is based on the reduction of DPPH in methanol solution in the presence of a hydrogen donating antioxidant due to the formation of the non-radical form DPPH-HShen et al.2010 6 . The free radical scavenging activity of all the extracts was evaluated by DPPH according to the previously reported methodShen et al.2010 6 . Briefly an 0.1 mM solution of DPPH in methanol was prepared and 1 ml of this solution was added to 3 ml of the solution of all extracts in methanol at different concentrations 50100200400and800 μg/ml.The mixtures were shaken vigorously and allowed to stand at room temperature for 30 minutes. Then the absorbance was measured at 517 nm using a UV–VIS spectrophotometer Genesys 10S UVThermo Electron International Journal of Scientific Research and Review Volume 7 Issue 11 2018 ISSN NO: 2279-543X Page No: 269

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Corporation. Ascorbic acid was used as the reference. Lower absorbance values of reaction mixture indicate higher free radical scavenging activity. The capability of scavenging the DPPH radical was calculated by using the following formula. DPPH scavenging effect inhibition A0  A1/A0 100 where A0 is the absorbance of the control reaction and A1 is the absorbance in presence of all of the extract samples and reference. All the tests were performed in triplicates and the results were averaged. Hydroxyl radical scavenging activity The scavenging capacity for hydroxyl radical was measured according to the modified method of Halliwell1987 7 . Stock solutions of EDTA 1 mM FeCl3 10 mM ascorbic acid 1 mM H2O2 10 mM and deoxyribose 10 mM were prepared in distilled deionized water. The assay was performed by adding 0.1 ml of EDTA 0.01 ml of FeCl3 0.1 ml H2O2 0.36 ml of deoxyribose 1.0 ml of the extract of different concentrations 50100200400and 800 μg/mldissolved in distilled water0.33 ml of phosphate buffer 50 mM pH 7.9 and 0.1 ml of ascorbic acid in sequence. The mixture was then incubated at 37 C for 1 hour. The 1.0 ml portion of the incubated mixture was mixed with 1.0 ml of 10TCA and 1.0 ml of 0.5 TBA in 0.025 M NaOH containing 0.025 BHA to develop the pink chromogen measured at 532 nm. The percentage of inhibition was calculated by comparing the results of the test with those of the control using the above formula. Superoxide radical scavenging activity The superoxide anion scavenging activity was measured as described by Srinivasan et al2007 8 . The superoxide anion radicals were generated in 3.0 ml of Tris–HCL buffer 16 mM pH 8.0 containing 0.5 ml of NBT 0.3 mM 0.5 ml of NADH 0.936 mM solution 1.0 ml of extract of different concentrations 50100200400and800 μg/ml and 0.5 ml of Tris–HCl buffer 16 mM pH8.0. The reaction was started by adding 0.5 ml of PMS solution 0.12 mM to the mixture andincubated at 25 C for 5 minutes and the absorbance was measured at 560 nm against a blank sample ascorbic acid. The percentage inhibition was calculated by comparing the results of the test with those of the control using the above formula . Antioxidant activity by radical cation ABTS+ ABTS assay was based on the slightly modified method of Haung et al 2011 9 . ABTS radical cation ABTS+ was produced by reacting 7 mM ABTS solution with 2.45 mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 12–16 hours before use. The ABTS+ solution was diluted with ethanol to an absorbance of 0.70 + 0.02 at 734 nm. After the addition of 100 μl of sample to 3.9 ml of diluted ABTS+ solution absorbance was measured at 734 nm by Genesys 10S UV–VIS Thermo Scientific exactly after 6 minutes. Results were expressed as trolox equivalent antioxidant capacity TEAC. The percentage of International Journal of Scientific Research and Review Volume 7 Issue 11 2018 ISSN NO: 2279-543X Page No: 270

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inhibition was calculated by comparing the results of the test with those of the control using the above formula. Reducing power The reducing power of the extract was determined by the method of Kumar and Hemalatha2011 10 . A total of 1.0 ml of solution containing 50100200400and 800 μg/ml of extract was mixed with sodium phosphate buffer 5.0 ml 0.2 M pH6.6 and potassium ferricyanide 5.0 ml 1.0.The mixture was incubated at 50 C for 20 minutes. Then 5 ml of 10 trichloroacetic acid was added and centrifuged at 980  g 10 minutes at 5 C in a refrigerator centrifuge. The upper layer of the solution 5.0 ml was diluted with 5.0 ml of distilled water and ferric chloride and absorbance read at 700 nm. The experiment was performed thrice and results were averaged. Statistical analysis Antioxidant activities such as DPPH radical scavenging activity hydroxyl radical scavenging activity superoxide radical activity ABTS radical cation scavenging activity and reducing powers were estimated in triplicate determinations. Data were analyzed using the statistical analysis system SPSS SPSS software for windows release 17.5 SPSS Inc. Chicago IL USA. Estimates of mean and standard error for aforesaid parameters were calculated. RESULTS AND DISCUSSION DPPH radical scavenging activity Several methods are available to assess antioxidant activity of compounds. DPPH free radical scavenging assay is an easy rapid and sensitive method for the antioxidant screening of plant extracts. In presence of an antioxidant DPPH radical obtain one more electron and the absorbance decreasesSudhanshu et al.2012 11 . The effect of petroleum ether benzene ethyl acetate methanol and ethanol extracts of Acrostichum aureum rachis and standard ascorbic acid on DPPH radical scavenging activity were compared and shown in Figure 1.In this present study the scavenging effect increased with the concentration both in standard and extracts. Among the solvent tested methanol extract of A.aureum rachis exhibited highest DPPH radical scavenging activity. At 800 μg/ml concentration methanolic extract of A.aureum rachis shows 126.31 of DPPH scavenging activity.The IC 50 value of ascorbic acid was 32.84µg/ml whereas methanol extract was found to be 36.54µg/ml. Hydroxyl radical scavenging activity The hydroxyl radical scavenging activity is measured as the percentage of inhibition of hydroxyl radicals generated in the Fenton’s reaction mixture by studying the competition between deoxyribose from Fe 3+ /EDTA/H2O2 systems.The hydroxyl radicals attack deoxyribose which eventually results in TBARS formationAbirami et al.2012 12 . The effect of petroleum ether benzene ethyl acetate and methanol and ethanol extracts of and standard ascorbic acid on hydroxyl radical scavenging activity were compared and shown in Fig 2.The results indicate the increase effect with the concentration of standard and samples. At 800 μg/ml of concentration ethyl acetate methanol ethanol and benzene extract of A.aureum rachis showed 126.92126.31118.31119.67 scavenging activity on hydroxyl radical International Journal of Scientific Research and Review Volume 7 Issue 11 2018 ISSN NO: 2279-543X Page No: 271

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respectively. All the concentration of A.aureum rachis extracts showed higher activity except petrolrum ether. Among the tested extracts of A.aureum rachis ethyl acetate extract showed the strongest hydroxyl radical scavenging effect 126.92 at 800µg/ml While standard ascorbic acid showed 113.86 at 800µg/ml radical scavenging activity.The IC 50 value of ascorbic acid was 31.48 where as methanolextract was found to be 32.16. ABTS radical cation scavenging activity Free radical scavenging activity of plant samples was determined by ABTS radical cation decolorization assaypellagrini et al .1999 13 The effect of A.aureum rachis extracts and standard ascorbic acid on ABTS radical cation were compared and shown in Figure 3. At 800 μg/ml concentration of methanol ethanolethyl acetate extracts of A.aureum rachis possessed 119.22108.16106.32 scavenging activity on ABTS. All the concentration of A.aureum rachis extract showed lower activity than the standard ascorbic acid 121.36.The IC 50value of ascorbic acid was 33.06 where as methanol extract was found to be 30.11. This scavenging activity of ABTS radical by the plant extracts were found to be appreciablethis implies that the plant extract useful for treating radical related pathological damage especially at higher concentrationkarthika et al.2012 14 . Superoxide radical scavenging activity Superoxide radical plays an vital role in plant tissues extract and it is involved in the formation of other cell damaging free radicals. The A.aureum rachis extract were subjected to be superoxide scavenging assay and the results were shown in Figure 4.The result indicate that ethanol and methanol extract of A.aureum rachis 800 μg/ml exhibited the maximum superoxide radical scavenging activity of 128.13112.67 respectively which is higher than the standard ascorbic acid whose scavenging affect is 109.54. The IC 50 value of ascorbic acid was 30.15whereas ethanol extract was found to be 34.84. Reducing power The reducing power of A.aureum rachis extracts was compared with the standard ascorbic acid .The reducing power increases with the increasing concentration .The reducing power of the methanol ethanol ethyl acetate petroleum ether and benzene extracts of A .aureum rachis extracts was shown in Figure 5. At 800 μg/ml concentration of methanol and ethanol extracts of showed higher reducing power than the ascorbic acid. This reducing capacity of compounds could serve as an indicator of potential antioxidant properties and increase in absorbance could indicate an increase in reducing power .Among the extracts methanol extract exhibited higher reducing power activity as compared with ascorbic acid. CONCLUSION In this study it can be concluded that all the extracts of A .aureum rachis is capable of scavenging wide range of free radicals .The in vitro antioxidant activity of the petroleum ether benzene ethyl acetate and methanol and ethanol extracts of rachis of A.aureum was investigated in the present study by DPPH hydroxyl superoxide and ABTS radical cation scavenging activities. These methods had proven the effectiveness of the extracts in comparison to that of the reference standard antioxidants ascorbic acid. These results revealed that the selected taxon is more potential as an antioxidant. Further in vivo assessment is also needed to confirm antioxidant nature of A .aureum rachis . International Journal of Scientific Research and Review Volume 7 Issue 11 2018 ISSN NO: 2279-543X Page No: 272

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Fig1: DPPH radical scavenging activity of different extracts of Acrostichum aureum rachis Fig2: Hydroxyl radical scavenging activity of different extracts of Acrostichum aureum rachis Fig3: ABTS radical cation scavenging activity of different extracts of Acrostichum aureum rachis 0 50 10 0 15 0 DPPH Radical Scavanging activity Concentration µg/mL 50 10 0 20 0 40 0 80 0 0 50 10 0 15 0 Hydroxyl scavanging activity Concentration µg/mL 50 10 0 20 0 40 0 80 0 0 10 0 20 0 ABTS radical cation scavanging activity Concentration µg/mL 50 10 0 20 0 40 0 80 0 International Journal of Scientific Research and Review Volume 7 Issue 11 2018 ISSN NO: 2279-543X Page No: 273

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Fig 4: Superoxide radical scavenging activity of different extracts of Acrostichum aureum rachis Fig5: Reducing power ability of different extracts of Acrostichum aureum rachis 3.6.1 IC50 values of different solvent extracts of rachis of A.aureum Solvent DPPH Hydroxyl radicals ABTS Superoxide Petroleum ether 31.56 25.16 26.12 26.18 Benzene 34.13 30.18 22.46 24.16 Ethyl acetate 30.36 31.48 27.16 28.16 Methanol 36.54 32.16 30.11 30.96 Ethanol 32.16 30.84 28.36 34.84 Ascorbic acid 32.84 29.93 33.06 30.15 0 50 10 0 15 0 superoxide scavanging activity Concentration µg/mL 50 10 0 20 0 40 0 80 0 0 0.2 0.4 0.6 Reducing powerOD Concentration µg/mL 50 10 0 20 0 40 0 80 0 International Journal of Scientific Research and Review Volume 7 Issue 11 2018 ISSN NO: 2279-543X Page No: 274

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14 Karthika K Paulsamy S Jamuna S Evaluation of in vitro antioxidant potential of methanolic leaf and stem extracts of Solena amplexicaulis Arm Gandhi. 2012 J chem Pharmaceu Res. 4: 3254-3258 International Journal of Scientific Research and Review Volume 7 Issue 11 2018 ISSN NO: 2279-543X Page No: 276

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