ANTIOXIDANT ACTIVITY OF ETHYL ACETATE EXTRACT OF GENUS AMARANTHUS LINN.

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)

B=SX.Y/ SX2

A= ¯y-b¯x

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.

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.

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

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

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.

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    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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