Arockia Badhsheeba M and Vadivel V - paper 2

Views:
 
Category: Entertainment
     
 

Presentation Description

No description available.

Comments

Presentation Transcript

slide 1:

1146 Journal of Pharmacognosy and Phytochemistry 2018 76: 1146-1151 E-ISSN: 2278-4136 P-ISSN: 2349-8234 JPP 2018 76: 1146-1151 Received: 01-09-2018 Accepted: 05-10-2018 R Arockia Badhsheeba Research Scholar PG and Research Department of Botany VO Chidambaram College Tuticorin Tamil Nadu India V Vadivel PG and Research Department of Botany VO Chidambaram College Tuticorin Tamil Nadu India Correspondence V Vadivel PG and Research Department of Botany VO Chidambaram College Tuticorin Tamil Nadu India Evaluation of in vitro antioxidant activity of Acrostichum aureum Linn. Rachis R Arockia Badhsheeba and V Vadivel Abstract Acrostichum aureum Linn is a medicinal fern collected from Puthalam Kanyakumari District Tamil Nadu India. This plant is used by the local people for curing pharyngitis chest pain and diabetics. Hence in the present study petroleum ether benzene ethyl acetate methanol and ethanol extracts of the rachis of A. aureum have been tested for antioxidant activity by using 1 1-diphenyl-2-picryl-hydrazyl DPPH radical scavenging activity hydroxyl radical scavenging activity superoxide radical scavenging activity antioxidant activity by radial cation ABTS •+ and reducing power assays. The antioxidant potential is dose dependent in all the assays carried out. It is concluded that the A. aureum can be used as a medicine against free radical associated oxidative damage. Keywords: medicinal plant Acrostichum aureum antioxidant free radicals DPPH ABTS Introduction Antioxidants may be defined as compounds that inhibit or delay the oxidation of other molecules by inhibiting the initiation or propagation of oxidizing chain reactions 1 . Antioxidants can also protect the human body from free radicals and reactive oxygen species ROS effects. Antioxidants retard the progress of many chronic diseases as well as lipid peroxidation 2 . Also antioxidants have been widely used as food additives to provide protection against oxidative degradation of foods 3 . At the present time the most commonly used antioxidants are butylatedhydroxyanisole BHA butylatedhydroxytoluene BHT propyl gallate PG and tert butyl hydroquinone TBHQ. The safety of these antioxidants has recently been questioned due to toxicity 4 . Therefore there is a growing interest on natural and safer antioxidants 5 . Development of safer natural antioxidants from extracts of oilseeds spices and other plant materials that can replace synthetic antioxidants has been of interest 6 . Natural antioxidants are known to exhibit a wide range of biological effects including antibacterial antiviral antiinflammatory ant allergic antithrombotic and vasodilatory activities. Thus there is a need of antioxidants of natural origin because they can protect the human body from the diseases caused by free radicals 7 . Acrostichum aureum Linn Family- Pteridaceae common name: Swamp Fern Mangrove Fern occurs Worldwide in mangrove swamps salt marshes canal margins and low hammocks. It is widely distributed throughout South Florida 8 Brazil South and West Mexico Guyanas Central America Colombia Venezuela Ecuador Paraguay Barbados Trinidad South China Taiwan Japan North Australia India Sri Lanka and Bangladesh 9 . It is an evergreen shrub can be grown as annual which is locally used as choice of medicinal plant in the treatment of major and minor complaints. Several studies have reported the traditional use of A. aure um’s rhizome for curing wounds non-healing ulcers boils syphilitic ulcers sore throat chest pains elephantiasis purgative febrifuge cloudy urine in women and rheumatism in Malaysia 10 Bangladesh 11 India 12 and Yap islands and Micronesia 13 . In the present study the mode of antioxidant action in petroleum ether benzene ethyl acetate methanol and ethanol extracts of A. aureum was investigated. In vitro methods of assessment were used to determine the scavenging activity of the extract on 1 1-diphenyl-2-picryl hydrazyl DPPH hydroxyl radical superoxide scavenging 2 2-azinobis 3- ethylbenzothiazoline 6-sulfonate ABTS and reducing power. Materials and Methods Plant material Rachis of Acrostichum aureum Linn were collected from Puthalam Kanyakumari District Tamil Nadu and was authenticated at Botanical Survey of India Southern circle Coimbatore Tamil Nadu. The gathered rachis were cut into small pieces and shade dried at room

slide 2:

1147 Journal of Pharmacognosy and Phytochemistry temperature. The shade dried rachis were ground into coarse powder using mechanic grinder and stored in air tight container for further use. Extract preparation The coarsely powdered rachis 20g was extracted separately with petroleum ether benzene ethyl acetate methanol and ethanol in a Soxhlet apparatus for 24h. The extracts obtained were filtered through Whatman No. 41 filter paper. Then the filtrates were concentrated under vacuum using rotary evaporator Heidolph Schwabach Germany. The concentrated extracts were stored at 4ºC for further investigation of potential in vitro free radical scavenging activity. Determination of DPPH radical scavenging activity DPPH free radical scavenging activity of the extracts were measured in vitro using 1 1-diphenyl-2-picryl-hydrazyl DPPH assay described by Blois 14 . 50mg of the extract and ascorbic acid standard were taken and dissolve in methanol and final volume make up to 50ml which was used as a stock solution with the concentration 1000µg/ml. Then different concentrations like 50 100 200 400 and 800μg/ml were prepared by diluting with the methanol from stock solution. 3ml of different concentration 50 100 200 400 and 800μg/ml of test solution and standard was taken in different test tubes. To this add 1ml DPPH working solution 0.1mM DPPH in methanol and the mixtures were shaken vigorously and allowed to stand at room temperature for 30min. The absorbance was measured against methanol as blank at 517nm using a UV-VIS spectrophotometer Genesys 10S UV: Thermo electron corporation. The percentage inhibition was calculated by comparing the absorbance values of the test sample with those of the controls not treated with extract. The capability of scavenging the DPPH radical was calculated by using the following formula. DPPH scavenging effect inhibition A 0 −A 1 A 0 x 100 Where A 0 refers to the absorbance of the DPPH control and A 1 refers to the absorbance of DPPH in the presence of extract/ascorbic acid. The inhibitory concentration IC50 value was calculated. IC50 values denotes the concentration of extract/standard required to scavenge 50 of free radicals Determination of hydroxyl radical scavenging activity The effect of extracts on hydroxyl radical scavenging activity was assayed by using the deoxyribose method 15 with some modification. Stock solutions of EDTA 1mM FeCl 3 10mM ascorbic acid 1mM H 2 O 2 10mM and deoxyribose 10mM were prepared in deionized distilled water. 1ml of different concentration 50 100 200 400 and 800μg/ml prepared with deionized distilled water of test solution and control ascorbic acid was taken in different test tubes. To this 0.1ml EDTA 0.01ml of FeCl 3 0.1ml H 2 O 2 0.36ml of deoxyribose and 0.33ml of phosphate buffer 50mM pH 7.4 were added and the mixture was then incubated at 37ºC for 1h. 1ml of the incubated mixture was taken out and was mixed with 1ml of 10 trichloroacetic acid and 1ml of 0.5 thiobarbituric acid TBA in 0.025M NaOH containing 0.025 butylated hydroxyl anisole BHA to develop the pink chromogen. The absorbance of the test solution and standard were measured at 532nm. Deionized distilled water was used as blank. The percentage inhibition was calculated by comparing the absorbance values of the test sample with those of the controls not treated with extract. The hydroxyl radical scavenging activity of the extract was reported as percentage inhibition of deoxyribose. The degradation is calculated by using the following equation. Hydroxyl radical scavenging activity A 0 −A 1 A 0 x 100 Where A 0 is the absorbance of the control and A 1 is the absorbance test samples and reference. Determination of superoxide radical scavenging activity The superoxide anion scavenging activity was measured as described by Srinivasan et al 16 . 1ml of different concentration 50 100 200 400 and 800μg/ml prepared with methanol of test solution and control ascorbic acid was taken in different test tubes. To this 0.5ml of 16mM Tris-HCl buffer pH 8 0.5ml of 0.3mM nitroblue tetrazolium NBT 0.5ml of 0.936mM of Nicotinamide Adinie Dinucleotide Reduced NADH and 0.5ml of 0.12mM of phenazine methosulphate PMS were added and incubated at dark for 5 min. The absorbance was measured at 560nm. Distilled water used as blank. The percentage inhibition was calculated by comparing the absorbance values of the test sample with those of the controls not treated with extract. The inhibition percentage was calculated as superoxide radical scavenging activity as follows. Superoxide radical scavenging activity A 0 −A 1 A 0 x 100 Where A 0 is the absorbance of the control and A 1 is the absorbance test samples and reference. Determination of ABTS scavenging activity 2 2-Azinobis 3-Ethylbenzothiazoline 6-Sulfonate ABTS radical scavenging activity of A. aureum extracts was measured by Huang et al 17 method with some modifications. Unlike DPPH assay the assay that involves scavenging of ABTS radicals required generation of the radicals. The ABTS radical cation ABTS •+ was generated by mixing ABTS stock solution 7mM with potassium persulfate 2.45mM. The reaction mixture left in the dark for 12h at room temperature and the resulting dark coloured solution was diluted using ethanol to an absorbance of 0.70 ± 0.02 at 734nm. 0.lml of different concentrations 50 100 200 400 and 800µg/ml prepared with methanol of extracts and trolox reference standard was mixed with 3.9ml of radical solution in clean and labeled test tubes. The tubes were incubated in dark for 6min at room temperature followed by measuring the absorbance of the reaction mixture in spectrophotometer at 734nm. Methanol replacing the extract / trolox served as control i.e. 0.1ml methanol + 3.9ml ABTS radical solution. The ABTS radical scavenging activity of the extracts was calculated using the following formula and the results were expressed as trolox equivalent antioxidant capacity TEAC values. ABTS radical scavenging activity A 0 −A 1 A 0 x 100 Where A 0 is the absorbance of the ABTS solution without extract/trolox and A 1 is the absorbance the ABTS solution in the presence of extract/trolox. The IC 50 value was calculated.

slide 3:

1148 Journal of Pharmacognosy and Phytochemistry IC 50 denotes the concentration of extract required to scavenge 50 of the radicals. Determination of reducing power The reducing power of the extract was determined by the method of Kumar and Hemalatha 18 . 1ml of each extract of different concentration 50 100 200 400 and 800µg/ml prepared with methanol and standard ascorbic acid were mixed with 5ml of sodium phosphate buffer 0.2M pH 6.6 and 5ml of 1 potassium ferricyanide and the mixture was incubated at 50ºC for 20min. After incubation 5ml of 10 trichloroacetic acid TCA was added to the mixture and centrifuged at 980g for 10min in a refrigerator centrifuge. About 5ml of supernatant of solution was taken and diluted with 5ml of distilled water and shaken with 1ml of freshly prepared 0.1 ferric chloride and the absorbance was measured at 700nm in UV-VIS spectrophotometer. A blank was prepared without adding extract. This result indicates that increase in absorbance of reaction mixture indicates increase in reducing power. Statistical analysis Grap Pad PRISM software version 4.03 was used for calculating IC 50 values for DPPH radical scavenging activity hydroxyl radical scavenging activity superoxide radical scavenging activity and antioxidant activity by radical cation ABTS •+ . The results were expressed as mean and all the experiments were done in triplicate. Result and Discussion DPPH radical scavenging activity Free radicals are molecules usually of oxygen which have lost an electron and are continuously generated during human body metabolism. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule which is widely used to investigate to investigate radical scavenging activity. In DPPH radical scavenging assay antioxidants react with DPPH deep violet colour and convert it to yellow coloured α α-diphenyl-β- picryl hydrazine. The degree of discoloration indicate the radical-scavenging potential of the antioxidant 14 . The effect of different solvent extracts of rachis of A. aureum and standard ascorbic acid on DPPH radical scavenging activity were shown in figure 1. Fig 1: DPPH radical scavenging activity of different extracts of Acrostic hum aureum rachis The DPPH radical scavenging activity of all the samples was highly depend on concentration namely antioxidant activity increased with increase in extract concentration. Among the tested solvents benzene extract of rachis of A. aureum exhibited high DPPH radical scavenging activity. Results shows that 800μg/ml concentration of benzene extract from the rachis of A. aureum exhibited the highest DPPH 118.56 scavenging activity compared to other extracts. The IC 50 value of ascorbic acid Table 1 was 32.84µg/ml whereas methanol extract was found to contain 36.54µg/ml IC 50 value. Table 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 Hydroxyl radical scavenging activity Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the extracts for hydroxyl radicals generated from Fe 2+ /EDTA/H 2 O 2 system Fenton reaction. The hydroxyl radicals attack deoxyribose which eventually results in the formation of thiobarbituric acid reacting substance TBARS 19 . Among the reactive oxygen species the hydroxyl radicals are the most reactive and predominant radicals generated endogenous during aerobic metabolism. A single hydroxyl radical results in the formation of many molecules of lipid hydroperoxides in the cell membrane which may severely disrupts its function and leads to cell death 20 . The petroleum ether benzene ethyl acetate methanol and ethanol extracts of rachis of A. aureum were found to possess concentration dependent scavenging activity on hydroxyl radicals and the results were given in figure 2.

slide 4:

1149 Journal of Pharmacognosy and Phytochemistry Fig 2: Hydroxyl radical scavenging activity of different extracts of Acrostichum aureum rachis Among all the tested extracts ethyl acetate and methanol extracts 800µg/ml showed high levels of hydroxyl radical scavenging activity 126.92 and 126.32 respectively.The IC 50 value of ascorbic acid was 31.48 whereas IC 50 value of methanol extract was found to be 32.16. ABTS radical cation scavenging activity The effect of A. aureum rachis extracts and standard ascorbic acid on ABTS radical cation were compared and shown in figure 3. At 800μg/ml concentration of methanol ethanol ethyl acetate extracts of A. aureum rachis possessed 119.22 108.16 106.32 scavenging activity on ABTS. All the concentration of A. aureum rachis extract showed lower activity than the standard ascorbic acid 121.36. The IC 50 value of ascorbic acid was 33.06 whereas methanol extract was found to be 30.11. This scavenging activity of ABTS radical by the plant extracts were found to be appreciable this implies that the plant extract useful for treating radical related pathological damage especially at higher concentration 21 . Fig 3: ABTS radical cation scavenging activity of different extracts of Acrostichum aureum rachis Superoxide radical scavenging activity Superoxide anion plays an important role in the formation of more reactive species such as hydrogen peroxide hydroxyl radical and singlet oxygen which induce oxidative damage in lipids proteins and DNA 22 . Therefore estimating the scavenging activity of plant extracts on superoxide radical is one of the most important ways of clarifying the mechanism of antioxidant activity. In the present investigation different solvent extracts of rachis of A. aureum were found to possess concentration dependent scavenging activity on superoxide and the results were given in figure 4. Among all the solvent extracts ethanol extracts 800µg/ml of the plant material exhibited the highest superoxide radical scavenging activity whereas the standard ascorbic acid had 109.54 of superoxide radical scavenging activity. The mean IC50 value of ascorbic acid was 30.15 whereas ethanol extract was found to be 34.84. Fig 4: Superoxide radical scavenging activity of different extracts of Acrostichum aureum rachis.

slide 5:

1150 Journal of Pharmacognosy and Phytochemistry Reducing power Reducing power reflects the electron donating capacity of its bioactive compounds which serves as a significant indicator of its antioxidant activity. Reduced Fe 3+ / ferricyanide complex to the ferrous form which indicated existence of reductants in the sample solution. The reductants have been exhibiting antioxidative potential by breaking the free radical chain and donating a hydrogen atom 23 . The reducing power of the extracts of A. aureum rachis was shown in figure 5. The reducing ability of the extract increased with increasing concentration our results were in accordance to studies done by Vijaya et al 24 . In the present study 800μg/ml concentration of methanol extract showed the highest reducing power than the ascorbic acid. Fig 5: Reducing power ability of different extracts of Acrostichum aureum rachis Conclusion In the present study it can be concluded that different solvent extracts of rachis of A. aureum have wide range of antioxidant and/or free radicals scavenging activity. The antioxidant activity of plant may vary depending upon the geographical area seasonal variation and also the method and solvent used for extraction. Literature survey reveals that flavonoids 25 and phenolic compounds 26 are responsible for antioxidant activity. However we do not know what components in the plant extracts show these activity. More detailed studies on chemical composition of the plant extracts as well as in vivo assays are essential to characterize them as biological antioxidant. It should be kept in mind that antioxidant activity measured by in vitro methods may not reflect in vivo effects of antioxidant 27 . Many other factors such as absorption/metabolism are also important. References 1. Velioglu YS Mazza G Gao L Oomah BD. Antioxidant activity and total phenolics in selected fruits vegetables and grain products. Journal of Agricultural and Food Chemistry. 1998 46:4113-4117. 2. Pryor WA. The antioxidant nutrient and disease prevention –what do we know and what do we need to find out American Journal of Clinical Nutrition. 1991 53: 391-393. 3. Shahidi F. Antioxidants in food and food antioxidants. Nahrung. 2000 44:158-163. 4. Sun B Fukuhara M. Effects of co-administration of butylated hydroxytoluene butylated hydroxyanisole and flavonoids on the activation of mutagens and drug metabolizing enzymes in mice. Toxicology.1997 122:61- 72. 5. Moure A Cruz JM Franco D Dominguez JM Sineiro J Dominguez H et al. Natural antioxidants from residual sources. Food Chemistry. 2001 72:145-171. 6. van Ruth SM Shaker ES Morrissey PA. Influence of methanolic extracts of soybean seeds and soybean oil on lipid oxidation in linseed oil. Food Chemistry 2001 84:177-184. 7. Mishra j Srivastava RK Shukla SV Raghav CS. Antioxidants in aromatic and medicinal plants. Science Tech Entrepreneur. 2007 1-16. 8. Adams D Tomlinson P. Acrostichum in Florida. American Fern Journal. 1979 2:42-46. 9. Medina E Cuevas E Popp M Lugo A. Soil salinity sun exposure and growth of Acrostichum aureum the mangrove fern. Botanical Gazette. 1990 1:41-49. 10. Hossan MS Hanif A Agarwala B Sarwar MS Karim M Rahman MT et al. Traditional use of medicinal plants in Bangladesh to treat urinary tract infections and sexually transmitted diseases. Ethnobotany Research and Publication. 2010 8:61-74. 11. Pattanaik C Reddy CS Dhal NK Das R. Utilisation of mangrove forests in Bhitarkanika wildlife sanctuary. Indian Journal Tropical Knowledge. 2008 7:598-603. 12. Benjamin A Manickam VS. Medicinal pteridophytes from the Western Ghats. Indian Journal of Traditional Knowledge. 2007 6:611-618. 13. Defilipps RA Maina SL Pray LA. The Palauan and Yap medicinal plant studies of Masayoshi Okabe 1941-1943. Atoll Research Bulletin. 1988 317:1-25. 14. Bilos MS. Antioxidant determinations by the use of a stable free radical. Nature. 1958 26:1199-1200. 15. Halliwell B Gutteridge JMC Aruoma OI. The deoxyribose method: a simple test to be assay for determination of rate constants for reaction of hydroxyl radicals. Analytical Biochemistry. 1987 165:215–219. 16. Srinivasan R Chandrasekar MJN Nanjan MJ Suresh B. Antioxidant activity of Caesalpinia digyna root. Journal of Ethnopharmacology. 2007 113:284–291. 17. Huang MH Huang SS Wang BS Wu CH Sheu MJ Hou WC et al. Antioxidant and anti-inflammatory properties of Cardiospermum halicacabum and its reference compounds ex vivo and in vivo. Journal of Ethnopharmacology. 2011 133:743-750. 18. Kumar RS Hemalatha S. In vitro antioxidant activity of alcoholic leaf extract and sub-fractions of Alangium lamarckii Thwaites. Journal of Chemical and Pharmaceutical Research. 2011 3:259–267. 19. Abirami B Gayathri P Uma D. In vivo antioxidant potential of Pterocarpus marsupium bark. International Journal of Chemistry and Pharmaceutical Sciences. 2012 3:17-24. 20. Battu GR Kumar BM. In-vitro antioxidant activity of leaf extract of Aerva lanata Linn. International Journal of Pharmacetical Science. 2012 2:74-78.

slide 6:

1151 Journal of Pharmacognosy and Phytochemistry 21. Karthika K Paulsamy S Jamuna S Evaluation of in vitro antioxidant potential of methanolic leaf and stem extracts of Solena amplexicaulis Arm Gandhi. Journal of Chemical and Pharmaceutical Research. 2012 4:3254- 3258. 22. Pietta PG. Flavonoids as antioxidants. Journal of Natural Products. 2000 63:1035-1042. 23. Gordon MH. The mechanism of antioxidant action in vitro. In: BJF Hudson Ed. Food antioxidants. London: Elsevier Applied Science. 1990 1-18. 24. Vijaya G Doss A Parthipan B Mohan VR. Assessment of in-vitro antioxidant activity of various bark extracts of Crateva manga Lour DC. Capparaceae. Journal of Pharmacognosy and Phytochemistry. 2018 7:1596-1599. 25. Lamson DW Bringnal MS. Antioxidents and cancer therapy II. Quick reference guide. Alternative Medicine Review. 2000 5:152-163. 26. Stratil P Klejdus B Kuban V. determination of total content of phenolic compounds and their antioxidant activity in vegetables evaluation of spectrophotometric methods. Journal of Agriculture and Food Chemistry. 2006 54:607-616. 27. Sini KR Sinha BN Karpagavalli M. Determining the antioxidant activity of certain medicinal plants of Attapady Palakkad India using DPPH assay. Current Botany. 2011 1:13-16.

authorStream Live Help