INTRODUCTION:

Barnyard millet (Echinochola frumentacea) belongs to the family of poaceae and species of Echinochola. It is widely distributed in India, Japan, Pakistan and Nepal ¹. It has good sources of protein and nutrient content. The major advantage of barnyard millet is easily digestible in human body. The physiological uses were to reduce the cholesterol level, blood sugar level and to control the diabetes ². Oxidation is a process of chemical reaction which produces free radicals. Free radicals are harmful to all living organism which will damage the normal cells and brain function. It cause several diseases such as chronic disorder, cardio heart diseases, Alzheimer’s diseases, cataracts, neurodegenerative disorder and inflammation³.

Antioxidant compounds act as defense mechanisms in all living organisms to prevent the oxidation reaction. It plays a major role in food industry to preserve the colour, flavour and vitamins during storage. Synthetic antioxidant, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are commonly used in food to retard lipid peroxidation during storage and increases the shelf life of food ⁴.

Antioxidant plays an important role in physiological process. The common function of antioxidant is to decreases the DNA damage and lipid peroxidation. It maintain the immune system and inhibit the malignant transformation of cells⁵. Antioxidant components like polyphenols, phenolic acid and flavonoids have the ability to scavenge free radicals such as peroxide and hydroperoxide and inhibit the oxidative mechanisms that leads to degenerative diseases. This millet contains phytochemical component such as dietary fibers, soluble and insoluble fractions⁶. In this study we investigated the phytochemical, total phenolic content and antioxidant capacities of different forms of barnyard millet.

METHODS AND MATERIALS:

Chemicals:

Gallic acid, sodium bicarbonate, Folin–Ciocalteu reagent, Dragendorff’s reagent, ferric chloride, lead acetate, NaOH, Fehling’s solution, Millon's reagent reagent, chloroform, Conc. H₂SO₄, methanol.

Sample collection and preparation:

The millet sample was collected from cultivable land at Vellore district, Tamil Nadu during the period of June-July in the year 2015. The sample was prepared into three different forms as given below:

Raw millet:

The sample was washed and dried in atmospheric condition for one day and homogenized.

Soaked millet:

The sample was soaked in sufficient amount of distilled water for 3hrs, then it was dried under room temperature for one day and later the sample was grinded into powder form.

Sprouted millet:

The sample was soaked in distilled water for 12 hours, later it was tied in muslin cloth and allowed it to germinate for 12 hours, then the sample was dried under room temperature for one day and homogenized.

Preparation of extract:

The dried samples of barnyard millet were mixed with 25 ml of distilled water and 25 ml of methanol at different concentration and kept in orbital shaker for 24 hours at room temperature. After incubation the mixture was centrifuged at 10,000 rpm for 10 minutes. Supernatants were filtered from the residue through filter paper (Whatman No. 1). The extract was stored at 4 ºC for further use ⁷.

Phytochemical analysis:

The aqueous and methanol extract was used for phytochemical screening to analyse the presence of various secondary metabolites such as alkaloids, phenols, flavonoids, steroids, terpenoids, saponins, tannins, proteins, carbohydrates and the detailed procedure is given below⁸.

Test for Phenols:

To 1 ml of extract few drops of neutral 5% ferric chloride solution was added. A dark green colour indicates the presence of phenolic compounds.

Test for alkaloid:

The extract was treated with 2ml of with Dragendorff’s reagent. Formation of turbid orange colour indicates the presence of alkaloid.

Test for flavonoids:

To 1 ml extract a few drops of 20% NaOH solution was added. A change to yellow colour was observed; on addition of acid it changed to colorless solution indicates the presence of flavonoids.

Test for tannins:

To the extract alcoholic FeCl₃ reagent was added. A bluish black colour, which disappears on addition of dilute H₂SO₄, was followed by the formation of yellowish brown precipitate indicated the presence of tannins.

Test for saponins:

The extract was mixed with 5ml of distilled water in a test tube and it was shaken vigorously. The formation of stable foam was taken as an indication for the presence of saponins.

Test for carbohydrate:

Fehling test:

The aqueous extract was treated with Fehling A solution. Formation of green colour at the bottom of test tube indicates the presence of carbohydrate. The Fehling B solution was mixed in the extract. As brown colour indicates the presence of carbohydrate.

Test for protein:

The extract was treated with few drop of millon’s reagent. Formation of white precipitate indicate the presence of protein (it turns red while heating).

Estimation of total phenolic content:

Phenolic content was estimated by using Folin–Ciocalteu phenol reagent. About 100µl of extract was mixed with 2.5ml of Folin–Ciocalteu phenol reagent in the test tube and allowed to react for 5 min. Then 2.5 ml of Na₂CO₃ solution was added to the mixture and allowed to stand for 1 hour at room temperature. After incubation the absorbance of mixture was measured at 725nm using UV-spectrometer. The same procedure was done with aliquots of 5, 10 to 50 μg/mL gallic acid solutions used as standard for calibration curve. Total phenolic value was obtained from the regression equation: y=0.209x +0.2695 with R²=0.9684 and expressed as mg/g gallic acid equivalent ⁹.

Estimation of DPPH free radical scavenging activity:

The free radical scavenging activity of the extracts were measured in terms of hydrogen donating or radical scavenging ability using the stable free radical DPPH. The aqueous and methanol extract was used for determination of free radical scavenging activity. Different concentration of barnyard millet extract was mixed with 2ml of DPPH (0.1 mM).The reaction mixture was shaken well and incubated for 10 mins at room temperature. The absorbance was measured at 517 nm against a blank. The DPPH scavenging activity was calculated using the following equation¹⁰. DPPH scavenging activity (%) = ((A₀ - A₁)) X 100, Where A₀ is the absorbance of the control and A₁ is the absorbance of the sample.

Statistical Analysis:

All results were reported as Mean± SD. The statistical significance was determined by analysis of variance (ANOVA). The P value was found to be <0.05, which is considered to be significant.

RESULTS:

Phytochemical screening of barnyard millet:

The aqueous and methanol extracts of all samples were subjected to phytochemical analysis to check the presence of phenols, alkaloids, flavonoids, steroids, saponins, terpenoids, tannins, proteins, carbohydrates and the results were presented in Table 2. These chemical constituents were responsible for different medicinal properties of the extracts.

Determination of Total phenolic content:

The total polyphenol content of all extracts at different concentrations were determined by Folin-Ciocalteu method. The highest concentration of phenol was observed in methanol extract of soaked millet. The standard curve obtained was shown in figure 1.

Figure 1: The Standard curve for total phenolic content of gallic acid at different concentration (µg) on X-axis and Absorbance on Y-axis.

DPPH radical scavenging activity:

The stable DPPH was used to identify the antioxidant property of the millet. The absorbance was measured at 517nm in UV spectrometer. The DPPH free radical scavenging activitiy of all extracts were determined at different concentration. The greatest scavenging ability of 87.90% was observed in methanol extract of soaked millet at higher concentration. Ascorbic acid was taken as standard showing 96.28% antioxidant activity. Percentage of DPPH radical scavenging activity of all extracts and ascorbic acid is shown in Figure 2.

Table 1: Phytochemicals present in aqueous and methanol extract of different forms of Barnyard millet

Concentration	Total phenolic content (mg of GAE/g of extract)
	Raw millet		Sprout millet		Soaked millet
	Aqueous	Methanol	Aqueous	Methanol	Aqueous	Methanol
1%	10.74±0.06	11.26±0.15	15.23±0.04	16.3±0.04	20.16±0.12	24.7±0.10
3%	10.85±0.15	11.42±0.12	14.09±0.07	16.9±0.15	20.45±0.16	29.9±0.06
5%	11.44±0.22	12.26±0.08	14.19±0.12	17.4±0.04	23.32±0.08	36.5±0.10
7%	11.62±0.16	12.87±0.18	14.28±0.15	17.8±0.12	29.86±0.09	43.7±0.15
9%	11.28±0.08	13.93±0.18	14.50±0.08	17.9±0.16	36.46±0.01	57.5±0.00

Table 2: Total phenolic contents of aqueous and methanol extracts

Phytochemicals	Sprouted millet		Soaked millet		Raw millet
Phytochemicals	Aqueous	Methanol	Aqueous	Methanol	Aqueous	Methanol
Alkaloid	-	+	-	-	-	+
Phenol	-	+	+	+	-	+
Saponins	+	+	+	+	-	-
Tannins	-	-	-	-	-	-
Flavanoid	-	+	-	+	-	-
Terpenoid	-	-	-	-	-	-
Carbohydrate	-	-	-	+	-	+
Protein	-	+	+	+	-	+

Figure 2: DPPH free radical scavenging activity of aqueous and methanol extracts of barnyard millet (Concentration on X axis, Inhibition percentage on Y axis).

DISCUSSION:

All kind of millets contains bioactive compounds which have more beneficial and nutritional value. Barnyard millet is one of the nutritious minor millet because it contain good source of macronutrient, micronutrient, protein content and nutraceutical components. Dietary fiber (13%) is one of the main components of barnyard millet. It is mainly taken as a diet in the case of diabetes mellitus, obesity and hyperlipidemia etc¹¹. This millet contains 11.2g/100g protein, 10.1g/100g crude fiber, 4.4g/100g mineral and 15.2mg/100g iron¹². In this study antioxidant activity of the millet was analyzed using aqueous and methanol extract. The sample was prepared in three different forms (Raw millet, Sprouted millet , Soaked millet) and analyzed for phytochemical, total phenolic content and DPPH scavenging activity using aqueous and methanol extract. The barnyard millet showed phytochemical component like alkaloid, tannins, saponins, phenols, carbohydrate, protein, flavonoid and terpenoid in both aqueous and methanol extract . The total phenolic content of the millet extract was determined by Folin-Ciocalteu method. It is also called as gallic acid equivalence method (GAE). Folin Ciocalteu is a mixture of phosphomolybdate and phosphotungstat.¹³

Polyphenolic compounds are one of the most effective antioxidant constituents. It is important to quantify polyphenolic content and their contribution to antioxidant activity. It transfers electron in alkaline medium from phenolic compounds to phosphomolybdic acid complexes. Phenolic compounds are a class of antioxidant agents which act as free radical terminators and their bioactivities may be related to their abilities to chelate metals, inhibit lipoxygenase and scavenge free radicals¹⁴.It is based on the mechanism of oxidation-reduction reaction. Folin's method measures -OH groups in a sample based on the fact that light absorption increases as OH group in a sample increases. So, this method do not measure the exact amount of polyphenol because polyphenol are not the only compound, has OH group, other OH group containing compounds such as protein, carbohydrate, vitamin C, and saponin are also present in the same medium¹⁵.

The soaked millet contain high phenolic content than sprout millet and raw millet. DPPH free radical scavenging assay is the most widely used method to detect the antioxidant activity of natural compounds. DPPH radical is reduced to colourless compounds by donating a hydrogen molecule to non-radical DPPH-H and it happens in the presence of antioxidant ¹⁶. Antioxidant activity is high in aqueous and methanol extract of soaked millet while comparing to raw millet and sprout millet. The extract with high scavenging activity had a highest and significant amount of total phenol concentration.

CONCLUSION:

The present study concludes that the barnyard millet extract exhibited antioxidant activity due to the presence of bioactive natural compounds. Different forms of millet (Raw millet, soaked millet and sprouted millet) were used for analysis of Phytochemical, total phenolic content and free radical scavenging activity. A high correlation between antioxidant activity and total phenolic content indicate that phenolic compounds are one of the main components in the antioxidant capacities of this millet. So consuming barnyard millet has many health benefits and it can act as a good antioxidant source.

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Received on 01.02.2016 Modified on 14.02.2016

Research J. Pharm. and Tech. 9(3): Mar., 2016; Page 262-266

DOI: 10.5958/0974-360X.2016.00048.2