Evaluation of Anti-oxidant and Bio Active Quantification of Vigna unguiculata Seed Extracts

Introduction

Vigna unguiculata, also known as ‘Cowpea’ is one of the ancient crops known. It is an edible legume belonging to the family Fabaceae and has high protein content. ¹ It emerged in Africa and is now cultivated in Brazil, Nigeria, India, Central America, China.^2,3 In India, it is commonly grown in Uttar Pradesh, Assam, Tamil Nadu (Madras), Karnataka (Mysore), Andhra Pradesh, and Mumbai.^4,5

Antioxidant activity

Oxidative stress is one of the primary cause for many of the chronic diseases. Free radicals and reactive oxygen species are linked to the aetiology of asthma, diabetes, Parkinson’s, Alzheimer’s disease, atherosclerosis and cancer.⁶ An antioxidant is a molecule that hinders or impedes oxidative damage to a designated molecule.⁷ The ability of an anti-oxidant to capture free radical is its primary character. Polyphenols, phenolic acid, and flavonoids scavenge free radicals such as peroxide/ hydroperoxides and lipid peroxide, restricting oxidative pathways that cause degeneration.⁸ The study aims to Evaluate Anti-oxidant and Bio Active Quantification of Vigna unguiculata Seed Extracts

Materials and Methods

The Vigna unguiculata seeds were purchased from local markets of Bengaluru and was certified by Dr. V Rama Rao, Research Officer, Department of Botany, Central Ayurveda Research Institute, Bengaluru with certificate no. Authentication/SMPU/CARI/BNG/2021-22/781.

Extraction

The extraction was done by maceration method. The Vigna unguiculata seeds were shade dried and pulverized. They were then macerated with distilled water, 50% ethanol, and 70% acetone for 18 hours. The macerate was filtered using Whatmann paper grade No.1 and concentrated by evaporation in a water bath. All the three extracts were preserved in airtight containers and kept at 4-5⁰ C for further use.

Preliminary Phytochemical Screening of Extracts

The phytochemical screening was conducted for all the extracts of V. unguiculata seeds for the detection of phytoconstituents present as per standard methods.^9,10

Chemicals and Reagents

Gallic acid, Quercetin, Sodium hydroxide, Methanol, Sodium nitrite, Ascorbic acid, Sodium nitroprusside, Aluminum chloride.

Folin-Ciocalteau reagent, Griess reagent, DPPH (1,1-Diphenyl-2-picrylhydrazy) reagent.

Antioxidant Assay

The anti-oxidant activity of each of the extracts was determined by the methods, such as DPPH free radical and Nitric oxide scavenging assay.

DPPH Radical Scavenging Assay^11,12

Principle

The scavenging reaction between DPPH and an antioxidant (H-A) can be given as:

The delocalization of a spare electron across molecule distinguishes DPPH as stable free radical which contributes to its deep violet color. As a result, this free radical does not dimerize like the others. When antioxidants bind to DPPH, it is converted to DPPHH, or 1-1 diphenyl -2-picryl hydrazine, and the violet colour is lost. The level of discoloration reflects the extract’s antioxidant activity based on hydrogen donating capability.

Procedure

1 mL of DPPH (0.1 mM) prepared in methanol was combined with 3 mL of extracts of different concentration (5 – 30 μg/mL) and incubated for 30 min at room temp. Then absorbance was recorded at 517 nm. The ascorbic acid was utilised as standard. The IC₅₀ value, i.e., the amount of extract necessary to suppress 50% DPPH radical was calculated. Lower IC₅₀ of reaction mixture indicates better activity.

The DPPH scavenging effect was computed using the equation mentioned below:

% inhibition = [(A_o – A_i ) / A_o ] × 100

A_o = absorbance of control and

A_i = absorbance of extract or vitamin C

Nitric-oxide Scavenging Assay¹³

Principle

The Nitric-oxide scavenging activity was calculated by employing Griess IlIosvoy reaction. At pH 7.2, Sodium nitroprusside degrades in water, releasing NO. This NO reacts with O₂ in aerobic condition to form nitrates and nitrites. Griess reagent then determines this. Scavengers of nitric oxide compete with oxygen resulting in lower nitrite production.

Procedure

5 mM Sodium nitroprusside in phospho-buffered saline was combined with various concentrations (25 – 150 μg/ mL) of extracts prepared in methanol and incubated at 30⁰ C for 2 hours to which 0.5 mL Griess reagent was mixed followed by absorbance reading at 550 nm. Reduced nitrite formation by extracts and the ascorbic acid were calculated and the IC₅₀ was determined. 

% Inhibition was computed using the below mentioned equation:

% inhibition = [(A_o – A_i )/A_o )] × 100

A_o = absorbance of control, and

A_i = absorbance of extract or vitamin C

Bio Active Quantification of Extracts

Total Phenolic Content^{14, 15, 16}

Principle

The Folin-Ciocalteau method was used for the quantification of phenols. Phosphotungstic or Phosphomolybdic acid complex is present in FC reagent. This method is based on transfer of electron in an alkaline media from phenolic compound to generate blue chromophore comprised of a phospho molybdenum complex, with maximum absorbance determined by concentration of phenolics. A decrease in the FC reagent is detected in the range of 690–710 nm. Gallic acid was utilised as standard and the results were reported as gallic acid equivalents (mg/GAE).

Procedure

0.5 mL of test solution (1 mg/mL with methanol) was taken in 25 mL volumetric flasks. 1 mL of methanol served as Blank. 1.5 mL of FC reagent was added and after 5 min, 4 mL 20% w/v Na₂CO₃ was mixed. The volume was made with distilled water and incubated in dark for 45 min and the absorbance was computed at 765 nm. The total phenolic content (TPC) was estimated using a calibration curve of gallic acid with concentrations ranging between 20 to120 μg and results were expressed as gallic acid equivalent (mg/g of GAE). The quantity of phenols in extracts was computed by-

T = (V * C) / M

Where,

T = TPC in mg/g of seed extract

C = Gallic acid concentration

V = volume of extract

M = weight of extract

Total Flavonoid Content^14,15

Principle

The Aluminium chloride technique was used to determine the flavonoid concentration of the extracts. The theory behind this method is that AlCl₃ forms an acid-stable combination with the C-4 keto group or the C-3 / C-5 hydroxyl groups of flavonoids, resulting in a pink coloured complex.

Procedure

1 mL of test solution (1 mg/mL) was added to 4 mL of distilled water along with 0.3 mL of 5% w/v NaNO₂ solution, followed by 5 min incubation and then 0.3 mL of 10% AlCl₃ was combined. This was then incubated for six minutes. The volume was then increased to 10 mL with distilled water after 2 mL of 0.4% w/v NaOH was added. After 15 min, the absorbance was recorded at 510 nm. The total flavonoid content (TFC) was calculated from quercetin’s calibration curve with concentrations ranging between 10-60 μg, and the result was expressed in quercetin equivalent (mg/g of QE).

The flavonoid concentration in extracts was computed by-

Where,

T = TFC in mg quercetin/g dry weight of seed extract

C = quercetin concentration

V = volume of extract

M = weight of extract

Results

Extracts

The percentage yield of extracts is given in Table 1. The maximum percentage was obtained when seeds were extracted in 50% ethanol.

Preliminary Phytochemical Screening

The preliminary phytochemical screening discovered the existence of alkaloid, tannin, flavonoid, carbohydrate, protein & glycoside contents in the extracts of Vigna unguiculata seeds and the results are shown in Table 2.

Anti-oxidant activity

DPPH radical scavenging assay

The DPPH assay showed that the extracts of V. unguiculata seeds possessed radical scavenging activity (Figure 3). The IC₅₀ values are shown in Table 2. 

Nitric oxide radical scavenging activity

The NO scavenging assay showed that extracts of V. unguiculata seeds possesses radical scavenging ability (Figure 4). The IC₅₀ values are shown in Table 3.

Total Phenolic Content

The phenolic content was specified in mg gallic acid / gram of dry extract and was obtained from standard curve of gallic acid (Figure 5). The findings are presented in Table 1.

Total Flavonoid Content

The flavonoid content was specified in mg of quercetin/ gram of dry extract and was obtained from standard curve of quercetin (Figure 6). The findings are presented in Table 1. 

Discussion

In the present study, seeds of Vigna unguiculata were macerated with aqueous, 50% ethanol, and 70% acetone as solvents and the yield was found to be 5.86%, 11.27% and 9.10%, respectively. All the extracts were screened for phytochemicals. It was evident from these tests that the extract consisted of alkaloids, flavonoids, tannins, phenols, etc.

The extracts were also subjected to in vitro determination of anti-oxidant activity by adopting DPPH and Nitric oxide scavenging assays. The IC₅₀ value in the DPPH method was found to be 265.34, 112.72 & 28.94 mcg/ mL, and that of NO free radical scavenging activity was found to be 398.73, 354.75 & 124.46 mcg/mL for aqueous, 50% ethanolic and 70% acetone extracts, respectively. As a part of the bioactive quantification, the total phenolic and flavonoid contents were estimated as gallic acid and Quercetin equivalents, respectively. The total phenolic content of aqueous, 50% ethanolic and 70% acetone extracts were found to be 0.49 ± 0.0068, 0.55 ± 0.0103, and 2.02 ± 0.0137 mg GAE/gm of extract and the flavonoid content was found to be 14.38 ± 0.7592, 19.33± 0.3796, and 69.91 ± 0.5603 mg QE/ gm of extract, respectively. It was clear from the reports that the quantity of phenols and flavonoids is directly proportional to the anti-oxidant property of the plant.^17,18

As per the study, it was found that 70% acetone extract exhibited better flavonoid content and showed promising anti-oxidant properties in both the DPPH and NO scavenging assays, in comparison to other extracts.

Conclusion

The estimation of the anti-oxidant properties and bio-active quantification of seed extracts of Vigna unguiculata was conducted. It was revealed that different extracts of Vigna unguiculata exhibited polyphenols, and flavonoids. The acetone extract was found to contain better quantities of flavonoids and phenols and hence it can be concluded that the anti-oxidant capability is proportional to flavonoid and phenolic contents. Thus, acetone extract could be explored for its various pharmacological properties in view of its antioxidant activity and could be a worthy candidature for its usage as a natural antioxidant for the benefit of humans.

Conflict of interests

None

Acknowledgements

The authors extend their thanks to Principal, Government College of Pharmacy, Bengaluru for providing facility to carry out this research work.