ANTIOXIDANT ACTIVITY OF ETHYL ACETATE EXTRACT OF GENUS AMARANTHUS LINN.HTML Full Text
ANTIOXIDANT ACTIVITY OF ETHYL ACETATE EXTRACT OF GENUS AMARANTHUS LINN.
Pinkie Cherian* and D. Sheela
Department of Botany, St. Teresa’s College, Ernakulam - 682011, Kerala, India.
ABSTRACT: Plants possess good antioxidant capacity to get rid of free radicals that cause many lifestyle diseases especially cancer. Assessment of Antioxidant activity of Amaranthus Linn. leaves were evaluated by using in- vitro model. In the present study evaluation of ethyl acetate of extract of Amaranthus Linn. leaves with the help of two In-vitro antioxidant models were carried out for Nitric-oxide scavenging Activity and DPPH method. IC50 value was calculated and compared with standard Ascorbic acid. Ethyl acetate extract was found to be extremely effective in scavenging nitric-oxide (IC50 584.4 μg/ml to 1072 μg/ml). In inhibition of DPPH Radical Scavenging activity (IC50 260.6 μg/ml to 957 μg/ml). Ethyl acetate extract showed different levels of antioxidant activities in tested models.
Antioxidant activity, DPPH, Nitric-oxide, Amaranthus
INTRODUCTION: India, rich with wide variety of under- utilised plants can be explored in the modern medicinal system for curing ailments related to oxidative stress. Plants acting as a dietary supplement can convert the free radical components in the body to an healthy molecule that can function effectively in the human physiological activities 21. Many medicinal plants act as these supplement and can function as a target molecule to convert the Reactive oxygen species (ROS) by scavenging the molecule and get rid of diseases associated with it. All aerobic organisms can easily react with most biological molecules including proteins, lipids, lipoproteins and DNA by producing Reactive oxygen species (ROS).
Reactive oxygen species (ROS), which consist of free radicals such as superoxide anion (O2-) and hydroxyl (HO·) radicals and non-free radical species such as H2O2 and singled oxygen (O2), are different forms of activated oxygen. Antioxidants regulate various oxidative reactions naturally occurring in tissues. Antioxidants can terminate or retard the oxidation process by scavenging free radicals, chelating free catalytic metals and also by acting as electron donors. Free radicals formation can result in many different diseases including nerve disorders, cancerous cell formation, inflammations of internal organs and other patho-physiological disorders.
Recently, there has been a considerable interest in finding the natural antioxidants from plant materials to replace synthetic ones. Plant phytochemicals not only counteract free radical induced oxidative stress but also overcome the side effects of synthetic antioxidants 25. Amaranth grows rapidly and has a high tolerance to arid conditions and poor soils where traditional cereals cannot be grown.
Amaranth has been touted as a miracle grain, a super grain, and the grain of the future 18. Grain Amaranth has got neutraceutical property that the flour obtained were used to prepare bakery products and can act as a good health supplement 3. Amaranth, a legacy of the Atecs, Mayas, and Incas, continues to be an under-exploited plant with a promising economic value due to the variety of uses it can have and the benefits it can provide to producers, processors, and consumers 11.
The Amaranth plant is also attractive since it adapts itself to a large number of environments, grows with vigor, produces large amounts of biomass, and resists drought, heat, and pests 26, 23.
The pharmacological properties of Amaranth products are considered of vital importance 22. Increasing the Antioxidant activity, Total Phenolic and flavonoid contents were observed in germination conditions during the seedling formation, as the plant mature the content varies 10. For reducing tissue swelling the leaves are well thought-out to be constructive, and they have a cleansing effect too. The plant has also been used curatively for diarrhea, dysentery, excessive menstrual flow, ulcers and intestinal hemorrhaging.
The present study is an attempt to evaluate the antioxidant activity of Amaranthus spinosus Linn., Amaranthus caudatus Linn., Amaranthus tricolor Linn., Amaranthus dubius Mart., and Amaranthus viridis Linn.
MATERIALS AND METHODS:
Materials: The plant specimen proposed for the study was collected from places around Ernakulam city Kerala, India. The plants were authenticated and voucher specimen were placed in KFRI Peechi Thrissur, Kerala India.
Extraction: The plant material was washed, shade dried coarsely powdered. Then the drug was dried and was extracted with Ethyl acetate and extract was filtered. Then the filtrate was concentrated over water bath and dried in a vaccum desicator.
Phytochemical Investigation of Extract: Different chemical constituents present in Ethyl acetate extract were subjected to the tests by Kokate and Trease & Evans 13, 7.
In-vitro Antioxidant Method:
DPPH Radical Scavenging Activity: 4, 11 In different vials, 1ml of extract solution and standard were taken. To these solutions, 5 ml of methanolic solution of DPPH was added, shaken well and the mixture was incubated at 37 0C for 20 min. The absorbance was measured against methanol as a blank at 517 nm. The absorbance of DPPH taken as a control was measured. Percentage anti-radical activity can be calculated by using following formula:
% Anti-radical activity = Control Absorbance - Sample Absorbance/ Control Absorbance× 100
IC50 value was calculated using formula:
IC50= a + b (50)
Nitric oxide Scavenging Activity: Nitric oxide (NO) was generated from sodium nitroprusside (SNP) and was measured by the Griess reagent. SNP in aqueous solution at physiological pH spontaneously generates NO which interacts with oxygen to produce nitrite ions that can be estimated by the use of Griess Reagent. Scavengers of NO compete with oxygen leading to reduced production of NO 5. SNP (10 mM) in phosphate buffer saline (PBS) was mixed with different concentration of extracts (100-1000μg/ml) and incubated at 25 °C for 180 minutes.
The samples from the above were reacted with Griess reagent (1% sulphanilamide, 0.1% naphthyl-ethylene-diamine dichloride and 3% phosphoric acid). The absorbance of the chromophores formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with naphthyl-ethylene-diamine-dichloride was read at 546 nm. Inhibition of nitrite formation by the plant extracts and the standard antioxidant ascorbic acid were calculated relative to the control.
Nitric Oxide scavenged (%) = A control − A test A control X 100
Where, A control = Absorbance of control reaction and A test = Absorbance in the presence of the samples of extracts/Ascorbic acid.
IC50 Value of the extracts: The antioxidant activity of the extract was expressed as IC50. The IC50 value was defined as the concentration (in) μg/ml of extracts that inhibits the formation of DPPH radicals by 50%. IC50 was calculated for all the extracts by plotting the percentage of DPPH radicals/NO scavenged versus the concentration of extract. Lower the IC50 value, higher the radical scavenging effect.
Statistical Analysis: All the experiments were evaluated statistically with SPSS version 20.0, the results were represented in mean±SEM (standard error of mean). One way analysis of varience (ANOVA) followed by DMRT (Duncan's Multiple Range test) to find out any significant difference in the antioxidant potential among genus Amaranthus Linn.
RESULTS AND DISCUSSION: In the phyto- chemical and phamacological studies, Amaranthus Linn. posses many secondary metabolites which has got numerous function both for plants and animals. In humans, these metabolites have beneficial effects including antioxidant, anti-inflammatory effects, modulation of detoxification enzymes, stimulation of the immune system, modulation of steroid metabolism and antibacterial and antiviral effects 12. A. spinosus showed the presence of different phytochemicals that has got antimicrobial and antioxidant activity and work support that the plant posses antioxidant activity 2. For the determination of antioxidant potential various in vitro assay systems was used like DPPH radical scavenging assay and Nitric oxide scavenging assay since, evaluation of antioxidant properties of plants cannot be carried out accurately by single universal method.
So, therefore it is important to carry out more than one type of antioxidant capacity measurement to cover the various mechanism of antioxidant action 8. Thus the present work undertake in evaluating the antioxidant activity of Amaranthus Linn. species using two in vitro antioxidant models.
In phytochemical screening of ethyl acetate extract showed positive test for Flavonoids, Tannins, Saponins, Phenolic compounds. Phenol, flavonoid metabolites are correlated with the antioxidant activity and these metabolite have direct role in freeradical scavenging activity 25. These secondary metabolite has got immense role in acting against variety of biological activity concerned with human metabolism.
Inhibition of DPPH radical: Free radical molecule DPPH accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants. The potential decrease in the concentration of DPPH radical due to the scavenging ability of Amaranthus Linn. and Ascorbic acid (reference standard) showed significant free radical scavenging activity A.viridis 95.1%, A. spinosus 98.2%, A. dubius 91.4%, A.caudatus 64.7% and A. tricolor 53.8% of inhibition, respectively, at 100 μg/ml. The IC 50 (the inhibitory concentration at which there is 50% reduction of free radical) of Amaranthus Linn. was in the range 260.6 to 957 μg/ml in Table 1. The significant decrease in the concentration of the DPPH radical is due to the scavenging ability of Amaranthus Linn. Another report showed that the peptides derived from Amaranth species got antifungal and antioxidant property 6. Table 3 showed the mean and standard error of five selected plants showing significant difference among antioxidant capacity with p value is less than 0.05.
Nitric oxide scavenging activity: The scavenging of nitric oxide by Amaranthus Linn. and Ascorbic acid was concentration dependent. There was a moderate inhibition of nitric oxide formation, with the maximum inhibition being of A. viridis 79.1% and A. caudatus 78% at 100 μg/ml and Ascorbic acid showing 83.5% respectively. The IC50 of Amaranthus Linn. was found to be in the range of 584.4 μg/ml to 871.1 μg/ml respectively as shown in Table 2.
Similar work done to evaluate antioxidant activity of hydroalcoholic extract of A. spinosus L. showed IC50 value of 525µg/ml 17, 16. Amaranthus viridis Linn., A. lividus Linn. and A. paniculatus Linn. also showed antioxidant activity 27, 19, 15. A. viridis showed good antioxidant potential and showed the presence of phenol in phytochemical profiling of methanol extract 1, 20.
Table 4 showed the significant difference among antioxidant capacity since p value is less than 0.05.
Nitric oxide was generated from sodium nitroprusside and measured by the Greiss reduction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrate ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide compete with the oxygen, leading to reduced production of nitric oxide 9. Significant scavenging activity was observed for A. dubius and A. caudatus, though all the other species of Amaranthus Linn. also exhibited the activity 24. Major compounds like squalene and tocopherols content in the leaf extract of Amaranthus Linn. plants exhibited antioxidant property from few reported works 14. Thus work has to be carried out in the detection of these compounds responsible for similar property. Thus the Amaranthus Linn. has potent antioxidant and free radical scavenging effects in different in vitro antioxidant models.
TABLE 1: COMPARISON OF DPPH ASSAY ON GENUS AMARANTHUS LINN.
|Name of Plants||IC50 µg/ml|
AV- Amaranthus viridis Linn. AS- Amaranthus spinosus Linn. AD - Amaranthus dubius, Mart.
AC- Amaranthus caudatus, Linn. AT- Amaranthus tricolor Linn.
TABLE 2: COMPARISON OF NITRIC OXIDE SCAVENGING ASSAY ON GENUS AMARANTHUS LINN.
|Nitric Oxide Scavenging Activity|
|Name of Plants||IC50µg/ml|
AV- Amaranthus viridis Linn. AS - Amaranthus spinosus Linn. AD - Amaranthus dubius, Mart.
AC- Amaranthus caudatus, Linn. AT- Amaranthus tricolor Linn.
TABLE 3: DPPH SCAVENGING ACTIVITY OF FIVE AMARANTHUS SPECIES
|Amaranthus spinosus Linn.||Amaranthus dubius Mart.||Amaranthus caudatus Linn.||Amaranthus tricolor Linn.||Ascorbic
|100||25.408 e ±0.413||32.812 e ±0.20||36.278 e ±0.179||22.626 e ±0.150||15.666 e ±0.152||28 e ±0.707|
|200||40.632 d ±0.418||47.996 d ±0.336||43.604 d ±0.168||27.088 d ±0.327||21.812 d ±0.31||36.8 d ±1.01|
|500||64.308 c ±0.434||60.688 c ±0.173||74.208 c ±0.412||42.462 c ±0.217||29.224 c ±0.345||57.4 c ±0.4|
|700||84.256 b ±0.337||83.068 b ±0.306||81.436 b ±0.126||53 b ±0.291||38.168 b ±0.134||70.6 b ±0.5|
|1000||94.854 a ±0.331||98.036 a ±0.209||91.284 a ±0.157||62.666 a ±1.991||53.642 a ±0.130||87.2 a ±0.37|
Each value is expressed as mean±Std. Error done in triplicates. Data analysed by SPSS version 20.0 by Duncan's Multiple Range test(α=0.05).Mean values followed by different Superscript in the columns are significantly different
TABLE 4: NO SCAVENGING ACTIVITY OF FIVE AMARANTHUS SPECIES
|Amaranthus viridis Linn.||Amaranthus spinosus Linn.||Amaranthus dubius Mart.||Amaranthus caudatus Linn.||Amaranthus tricolor Linn.||Ascorbic Acid|
|100||25.408 e ±0.413||32.812 e ±0.204||36.278 e ±0.179||22.626 e ±0.150||15.666 e ±0.152||28 e ±0.707|
|200||40.632 d ±0.418||47.996 d ±0.336||43.604 d ±0.168||27.088 d ±0.321||21.812 d ±0.311||36.8 d ±1.01|
|500||64.308 c ±0.434||60.688 c ±0.137||74.208 c ±0.412||42.462 c ±0.217||29.224 c ±0.345||57.4 c ±0.40|
|700||84.256 b ±0.337||83.068 b ±0.306||81.436 b ±0.126||53 b ±0.291||38.168 b ±0.134||70.6 b ±0.509|
|1000||94.854 a ±0.331||98.036 a ±0.209||91.284 a ±0.157||62.666 a ±1.991||53.642 a ±0.130||87.2 a ±0.374|
Each value is expressed as mean±Std. Error done in triplicates. Data analysed by SPSS version 20.0 by Duncan's Multiple Range test(α=0.05). Mean values followed by different Superscript in the columns are significantly different
By comparing the R2 value between DPPH assay and Nitric oxide scavenging activity, the result showed that R2 value of Nitric oxide scavenging activity was 0.952 and while the R2 of DPPH assay were 0.870.Thus from the present study conducted the R2 value Nitric oxide scavenging activity were more positively correlated than the DPPH assay in the selected Amaranthus species.
CONCLUSION: In brief summarizing of the above results, it is well clear that all the five Amaranthus species posses antioxidant capacity and can act as antioxidant plant substitutes from the present work. This under-utilised plant can throw light to ethno-pharmacological importance further confirming the pharmacological basis in the use of the plants in traditional medicine for the treatment of diseases associated with oxidative stress. Further studies are still needed on the isolation and identification of bioactive components responsible and to clarify the in vivo potential of this plants in the management of human diseases resulting from oxidative stress.
ACKNOWLEDGEMENT: We would like to show the gratitude to all the authors supported and advised us in improving the work. We also kindly send heartfelt thanks to MANF 2015-16 for providing financial support in undergoing work.
CONFLICT OF INTEREST: There is no conflict regarding the result availed in the data provided.
- Ahmed SA, Hanif S and Iftkhar T: Phytochemical profiling with antioxidant and antimicrobial screening of Amaranthus viridis Linn. leaf and seed extracts. Open Journal of Medical Microbiology. 2013; 3(3):164.
- Amabye TG: Evaluation of Physiochemical, Phytochemical, Antioxidant and Antimicrobial Screening Parameters of Amaranthus spinosus Nat Prod Chem Res. 2015; 4:199. doi:10.4172/2329-6836.1000199
- Anshula Bhat, Gouri Satpathy and Rajinder K. Gupta: Evaluation of Nutraceutical properties of Amaranthus hypochondriacus grains and formulation of value added cookies. Journal of Pharmacognosy and Phytochemistry 2015; 3(5): 51-54.
- Badami S, Omprakash, Dongre SH and Suresh B: In vitro antioxidant properties of Solanum pseudocapsicum leaf extracts. Indian J Pharmacology 2005; 37 (4): 251-52.
- Balakrishnan N, Panda AB, Raj NR, Shrivastava A and Prathani R: The evaluation of nitric oxide scavenging activity of Acalypha indica root. Asi J Res Chem 2009; 2:148-50.
- Delgado MC, Galleano M, Añón MC and Tironi V: Amaranth peptides from simulated gastrointestinal digestion: antioxidant activity against reactive species. Plant foods for human nutrition. 2015; 70(1): 27-34.
- Evans WC: Trease and Evans Pharmacognosy, Singapore: Hartcourt Brace and Company Asia Pvt. Ltd. 1997: 565.
- Gan RY, Xu XR, Song FL, Kuang L and Li HB: Antioxidant activity and total phenol content of medicinal plants associated with the prevention and treatment of cardiovascular and cerebrovascular diseases. J Med Plants Res 2010; 4: 2438-44.
- Govindarajan R, Rastogi S, Vijayakumar M, Shirwaikar A, Rawat AK and Mehrotra S: Studies on antioxidant activities of Desmodium gangeticum. Biol Pharm Bull 2003; 26: 142-47.
- Janitzio X. K. Perales-Sánchez, Cuauhtémoc Reyes-Moreno, Mario A. Gómez-Favela: Increasing the Antioxidant Activity, Total Phenolic and Flavonoid Contents by Optimizing the Germination Conditions of Amaranth Seeds. Plant Foods for Human Nutrition, 2014; 69(3):196.
- Joaquin A, Sofia S, Mercedes PB, Pablo JL, Teris AV, Manuel Nogueras and Aldolfo Sanchez: Preliminary assay on radical scavenging activity of olive wood extracts. Fitoterapia 2002; 76: 348-351.
- Johanna WL: Spicing up a vegetarian diet: Chemopreventive effects of phytochemicals. Am J Clin Nutr 2003; 78: 579S-583.
- Kokate CK, Purohit AP and Gokhale SB: Text book of Pharmacognosy. Nirali Prakashan, Pune. 24th edition, 2004: 107-111.
- Kraujalis P and Venskutonis PR: Supercritical carbon dioxide extraction of squalene and tocopherols from amaranth and assessment of extracts antioxidant activity. The Journal of Supercritical Fluids. 2013; 80: 78-85.
- Kumar A, Lakshman K, Jayaveera KN, Vamshikrishna N, Manjunath M and Suresh MV: Estimation of rutin and quercetin in Amaranthus viridis by HPLC. Asian Journal of Experimental Science 2009; 23: 51-54.
- Kumar BS, Lakshman K, Jayaveera KN, Shekar DS, Kumar AA and Manoj B: Antioxidant and antipyretic properties of methanolic extract of Amaranthus spinosus leaves. Asian Pacific Journal of Tropical Medicine. 2010; 3(9): 702 - 6.
- Manik Baral, Subrata Biswas, Subrata Chakraborty, Ashoke Kumar Ghosh, Jaime A. Teixeira da Silva, Subhamay Panda and Pranabesh Chakraborty: In vitro Antioxidant Activity of the Whole Plant of Amaranthus spinosusInternational Journal of Biomedical and Pharmaceutical Sciences 2010; 5(1):75-78.
- Monica WM, Nicholas, Gikonyo K, Glaston Kenji M and Alfred M: Properties of a Complementary Food based on Amaranth Grain (Amaranthus cruentus) Grown in Kenya. Journal of Agriculture and Food Technology 2011; 1: 153-178.
- Ozsoy N, Yilmaz T, Kurt O, Can A, Yanardag R: In vitro Antioxidant activity of the Amaranthus lividus Food chemistry 2009; 116: 867-872.
- Pająk P, Socha R, Gałkowska D, Rożnowski J and Fortuna T: Phenolic profile and antioxidant activity in selected seeds and sprouts. Food chemistry 2014; 143: 300-6.
- Pedersen B, Kalinowski LS and Eggum BO: The nutritive value of amaranth grain (Amaranthus caudalus) Protein and minerals of raw and processed grain. Plant Foods Human Nutrition 1987; 36: 309-324.
- Rosa Nilda CJ, Raul Dias S and Alessandra M: Effects of defatted amaranth (Amaranthus caudatus ) snacks on lipid metabolism of patients with moderate hypercholesterolemia. Ciência e Tecnologia de Alimentos 2010; 30: 1007-1010.
- Slabbert MM and Krüger GH: Antioxidant enzyme activity, proline accumulation, leaf area and cell membrane stability in water stressed Amaranthus leaves. South African Journal of Botany 2014; 95:123-8.
- Sravanthi S and Gangadhar Rao: Antioxidant - Phytochemical Studies in Amaranthus varieties. Int. J. Pharm. Sci. Rev. Res. 2015; 32(1): 188-193.
- Subedi L, Timalsena S, Duwadi P, Thapa R, Paudel A and Parajuli K: Antioxidant activity and phenol and flavonoid contents of eight medicinal plants from Western Nepal. J Tradit Chin Med 2014; 34: 584-90.
- Teutonico RA and Knorr D: Amaranth: composition, properties, and applications of a rediscovered food crop. Food Tech 1985; 39: 49-61.
- Yadav RK: Modulation of radiation induced biochemical changes in testis of swiss albino mice by Amaranthus paniculatus Asian Journal of Experimental Science. 2009; 18:63-74.
How to cite this article:
Cherian P and Sheela D: Antioxidant activity of ethyl acetate extract of genus Amaranthus Linn. Int J Pharm Sci Res 2017; 8(12): 5199-04. doi: 10.13040/IJPSR.0975-8232.8(12).5199-04.
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