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ORIGINAL ARTICLE
Year : 2021  |  Volume : 9  |  Issue : 1  |  Page : 25-31

In vitro antioxidant activity of ethanol extract of soybeans (glycine max [l.] merill) seeds


Department of Applied Biochemistry, Faculty of Biosciences Nnamdi Azikiwe University, Awka, Anambra, Nigeria

Date of Submission16-May-2020
Date of Decision13-Jul-2020
Date of Acceptance27-Sep-2020
Date of Web Publication20-May-2021

Correspondence Address:
Dr. Chinenye Enoch Oguazu
Department of Applied Biochemistry, Faculty of Biosciences Nnamdi Azikiwe University, Awka, Anambra
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_16_20

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  Abstract 


Introduction: Soybean is widely grown for its edible bean. It is a legume that grows in the tropical, subtropical, and temperate climates of Nigeria. It has been shown to contain a number of antioxidants that are used in preventing and treating chronic diseases. Aims: The purpose of this study is to evaluate the in vitro antioxidant activity of ethanol extract of soybean seed using the following assays: DPPH (2,2 diphenyl-2-picryhydrazylhydrate) scavenging activity assay, hydrogen peroxide (H2O2) scavenging activity assay, inhibition of lipid peroxidation activity assay, reducing power capacity assay, and antioxidant enzyme assay, which include superoxide dismutase (SOD) and catalase activity assay. Materials and Methods: In the present study, the antioxidant activities of ethanol extract of soybeans seed were determined spectrophotometrically using methods that include 2, 2-diphenyl-picryl hydrazyl radical (DPPH) scavenging activity assay, hydrogen peroxide scavenging activity assay, inhibition of lipid peroxidation assay, reducing power activity assay, peroxidation assay, and catalase and SOD activity assays. Results: The result of the DPPH scavenging activity revealed that the soybean extract has an EC50 value of 1053.542 μg/ml, hydrogen peroxide scavenging activity with an EC50 of 420.1852 μg/ml, and the inhibition of lipid peroxidation of soybeans extract had an IC50 of 1168.771 μg/ml. The reducing power activity of the soybeans extract had an OD0.5 of 484U/mg, catalase activity of 0.12985 U/mg, and SOD activity of 0.004125 U/mg. The EC50/IC50/OD0.5 obtained for the standard butylated hydroxyanisol (BHA) was lower than those of the soybeans extract. Conclusions: The use of soybean as a source of natural antioxidants should be promoted since soybean component can inhibit lipid peroxidation and protect the human body from the oxidative damages by free radicals. Hence, the dietary intake of soybean can be linked to prevention and management of certain diseases.

Keywords: Antioxidant, free radicals, glycine max (L.) Merill, oxidative stress


How to cite this article:
Enemor V H, Oguazu CE, Okpalagu C O, Okafor S C. In vitro antioxidant activity of ethanol extract of soybeans (glycine max [l.] merill) seeds. Niger J Exp Clin Biosci 2021;9:25-31

How to cite this URL:
Enemor V H, Oguazu CE, Okpalagu C O, Okafor S C. In vitro antioxidant activity of ethanol extract of soybeans (glycine max [l.] merill) seeds. Niger J Exp Clin Biosci [serial online] 2021 [cited 2021 Jun 14];9:25-31. Available from: https://www.njecbonline.org/text.asp?2021/9/1/25/316523




  Introduction Top


Antioxidant compounds play an important role in human health. A diet rich in foods containing antioxidant molecules can reduce the burden of human diseases.[1] The potential health hazards caused by the use of synthetic antioxidants in food products have led to the scrutiny for natural antioxidants.[2] Soybean [Figure 1] has been shown to possess antioxidant ability.[2] Soybeans and their products are nutritionally rich foodstuff and they contain various amounts of phytochemicals that show functional, antioxidants, and radical scavenging properties.[3],[4] Soybean grain contains proteins with sulfhydryl groups which play an important role as antioxidants. Besides their role in trapping free radicals and prevention of stress,[5] sulfhydryl groups could reduce trypsin inhibitors.[6] The oxidative activity in soybean results in processing into many products such as soymilk,[7] textured vegetable protein (soy flour), nonfermented and fermented soy foods,[8] tofu, soy sauce,[9] and miso.[10]
Figure 1: Soybean seeds (source: phone camera)

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Many researchers are interested in the potential role of soy foods in preventing and treating chronic diseases.[11] Dietary intake of soybean has been linked to prevention of osteoporosis, cardiovascular disease, and cancer, including breast, colon, and prostate cancers.[12] Rivas et al.[7] found that soy beverage decreased blood cholesterol levels and LDL cholesterol levels. A higher level of soy phenolic compounds and tocopherols has been reported, which may perform as antioxidants to protect against oxidative stress from free radicals.[13],[14] Protection against free radicals can be enhanced by the intake of dietary antioxidant nutrients as soybeans.[15] Consumption of soybean has been linked to many health-promoting activities, especially in reducing the risk of various cancers[16] and coronary heart disease.[17] Many studies confirmed that unique components of soybean play an important role in protecting against oxidative stress that causes the development of those chronic diseases.[14] Soy isoflavones, genistein, glycitin, and daidzein and their derivatives have the potential to directly scavenge oxidants such as superoxide and nitric oxide and other free radicals.[10],[3] Soybean also contains various phenolic compounds and a significant amount of tocopherols, which have been reported to possess antioxidant function as well.[18] The purpose of this study is to evaluate the in vitro antioxidant activity of ethanol extract of soybean seed.


  Materials and Methods Top


Sample collection processing

Soybean seeds were purchased from first market in Ifite Awka, Anambra state, Nigeria. The dried sample was ground with a Corona manual grinder into a fine powder and was stored in an airtight container till further use.

Sample extraction (Harbone, (1973)

Twenty grams (20 g) of the ground sample was soaked in 100 ml of 70% ethanol and was placed in a shaker (HY-4A multipurpose Oscillator) for 1 h. It was then allowed to stand for 24 h at room temperature. The mixture was filtered through Whatman No. 4 filter paper and the filtrate was evaporated at 78°C using a water bath (Techmel and Techmel, 420. USA). The dried residue was weighed and reconstituted in 70% ethanol at a concentration of 10 mg/ml and stored at 4oC in a refrigerator till further use.

DPPH scavenging activity

The stable 2,2-diphenyl-1-picryl hydrazyl radical (DPPH) was used for the determination of free radical scavenging activity (RSA) of the ethanol extract of the sample. This was assayed using the method of Ebrahimzadem et al.[19] Exactly 0.3 ml of six (6) different concentrations of the extracts (0, 200, 400, 600, 800, and 1000 μg/ml) was mixed with 2.7 ml of ethanol solution of DPPH (100 μM) in test tubes. The mixture was shaken and kept in dark for 60 min. The absorbance was taken at a wavelength of 517 nm using a spectrophotometer. Butylated hydroxyanisol (BHA) was used as standard. The percentage scavenging activity was calculated using the following formula:

%RSA = ([ADPPH − As]/ADPPH) ×100

As is the absorbance of the test solution with the sample and ADPPH is the absorbance of DPPH solution. The EC50 (concentration of sample at 50% RSA) was calculated from the graph of %RSA against the sample concentration.

H2O2 scavenging activity

This was determined by the method of Barros et al.[20] The extracts (0, 200, 400, 600, 800, and 1000 μg/ml) were dissolved in 3.4 mL of 0.1 M phosphate buffer (pH 7.4) and mixed with 600 μL (0.6 ml) of H2O2 (40 mM) prepared in 0.1M phosphate buffer (pH 7.4). The absorbance value of the reaction mixture was recorded at 230 nm using BHA as standard. Percentage of H2O2 scavenging was calculated from the formula:

% Scavenged [H2O2] = [(As − Ac)/As] × 100

Inhibition of lipid peroxidation

This was determined by the method of Barros et al.[20] Determination of the extent of inhibition of lipid peroxidation was carried out using homogenate of a goat brain. The brain of a goat (b. w. 70 kg) was purchased from Kwata animal slaughter house at Awka, Anambra State. The brain was dissected and homogenized with pestle and mortar in an ice-cold Tris-HCl buffer (pH 7.4, 20 mM) to produce 50% w/v brain homogenate and was centrifuged at 3000 g for 10 min. An aliquot (0.1 ml) of the supernatant was incubated with 0.2 ml of the sample extract at various concentrations (0, 200, 400, 600, 800, and 1000 μg/ml) in the presence of 0.1 ml of 10 uM Ferrous sulphate and 0.1 ml of 0.1 nM ascorbic acid at 37°C for 1 h. The reaction was stopped by the addition of 0.5 ml of 28% TCA followed by the addition of 0.38 ml of 2% thiobarbituric acid (TBA). The mixture was then heated at 80°C for 20 min. After centrifugation to remove the precipitated protein, the color intensity of the malondialdehyde-TBA complex in the supernatant was measured by its absorbance at 532 nm. The inhibition ratio (%) was calculated using the following formula:

Inhibition ratio (%) = ([A − B]/A) ×100%

Where A and B were the absorbance of the control and the compound solution, respectively. The extract concentration providing 50% lipid peroxidation inhibition (IC 50) was calculated from the graph of antioxidant activity percentage against the extract concentrations. BHA was used as the standard.

Reducing power capacity

The reducing power was determined according to the method of Barros et al.[20] This method is based on the principle of increase in the absorbance of the reaction mixture.

Exactly 2.5 ml of various concentration of ethanol extract of the sample (0–1000 μg/ml) was mixed with 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min. Then, 2.5 ml of 10% trichloroacetic acid was added and the mixture was centrifuged at 1000 rpm for 8 min. The upper layer (5 ml) was mixed with 5 ml of deionized water followed by the addition of 1 ml of 0.1% ferric chloride. The absorbance was measured at 700 nM. The graph of absorbance at 700 nM against the extract concentrations was plotted. BHA was used as a standard antioxidant.

%Scavenged (H2O2) = ([As − Ac]/As) × 100

Antioxidant enzyme

Sample preparation

One gram of the ground sample was extracted in ice-cold 0.1 M phosphate buffer (pH 7.4) and was centrifuged at 1500 rpm for 10 min. The supernatant was used for the enzyme activity assay.

Superoxide dismutase

Superoxide dismutase (SOD) activity was determined by the method of Aksenes and Njaa.[21] This is based on its ability to inhibit the auto-oxidation of epinephrine determined by the increase in absorbance at 480 nm. Determination of the extent of SOD activity was carried out using homogenate of a goat liver. The liver of a goat (b. w. 85 kg) was purchased from Kwata animal slaughter house at Awka, Anambra State. The liver was dissected and homogenized with pestle and mortar in an ice-cold Tris-HCl buffer (pH 7.4, 20 mM) to produce 20% w/v liver homogenate and was centrifuged at 3000 g for 10 min. The reaction mixture (3 ml) contained 2.95 ml 0.05 M sodium carbonate buffer pH 10.2 in 0.02 ml of liver homogenate and 0.03 ml of 0.3 mM adrenaline in 0.005 N HCL was used to initiate the reaction. The reference cuvette contained 2.95 ml buffer, 0.03 ml of substrate (epinephrine), and 0.02 ml of water. Enzyme activity was calculated by measuring the change in absorbance at 480 nm for 3 min.

Catalase activity determination

The catalase activity was determined according to the method of Beers and Sizer as described by Aksenes and Njaa (1985)[21] by measuring the decrease in absorbance at 240 nm due to the decomposition of H2O2 in a UV spectrophotometer. The reaction mixture (3 ml) contained 0.1 ml of sample extract in phosphate buffer (50 mM, pH 7.0) and 2.9 ml of 30 mM H2O2 in phosphate buffer pH 7.0. An extinction coefficient for H2O2 at 240 nm of 40.0 M-1 cm-1 was used for the calculation. The specific activity of catalase was expressed as moles of H2O2 reduced per minute per mg.

Statistical analysis

The results were presented as mean ± standard deviation and analysis was carried out using the Student's t-test at 95% confidence level considered at P ≤ 0.05.


  Results Top


This result showed that the % radical scavenging activity of soybean increases with increase in concentration. The DPPH scavenging activity of BHA (the standard) is significantly higher than those of soybeans at P ≤ 0.05.

This result showed relatively similar scavenging activity between BHA and soybean, but in all instances, the standard hydrogen peroxide scavenging activity appeared to be higher than that of soybeans particularly at 600 μg/ml where significant high activity was observed.

This result shows that the BHA and soybean show little differences in percentage inhibition ratio (%IR) at 600 and 800 μg/mg. At 400 μg/mg, the soybeans showed a significant high %IR than the standard at P ≤ 0.05.

This result shows a higher reducing power activity of the soybean with increasing concentration, but at all the concentration levels, the standard had higher significant reducing power activity than the soybeans seeds at P ≤ 0.05.

This showed that soybeans have relatively the same level of catalase activity and SOD activity with the standard. Statistically, no significant difference was observed at P ≤ 0.05.

For DPPH radical scavenging activity, hydrogen peroxide scavenging activity and inhibition of lipid peroxidation activity of the ethanol extract of soybean were compared with that of a standard antioxidant, butylated hydroxyanisole (BHA). The result showed that the EC50 (Effective concentration) of DPPH scavenging activity and hydrogen peroxide scavenging activity of soybean was significantly higher than that of BHA at P ≤ 0.05 [Figure 2]. Also, the IC50 (inhibition concentration) of lipid peroxidation activity of soybean is significantly greater than that of BHA. The result also revealed that the range of EC50 between soybean and BHA is higher in DPPH radical scavenging activity than in hydrogen peroxide scavenging activity. The range of IC50 between BHA and soybean in inhibition of lipid peroxidation activity is very small compared to the EC50 of DPPH and hydrogen peroxide scavenging activity.
Figure 2: Bar chart showing the EC50/IC50

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Reducing power activity

[Figure 3] shows that the OD0.5 for reducing power activity of soybean is significantly higher than that of BHA at P ≤ 0.05.
Figure 3: Bar chart showing the OD0.5 for reducing power activity of soybean seed compared with butylated hydroxyanisol

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  Discussions of Results Top


The in vitro antioxidant activity of ethanolic extract of soybean [Figure 1] was analyzed and the results of the experiment obtained showed that the DPPH scavenging activity [Figure 4], hydrogen peroxide scavenging activity [Figure 5], inhibition of lipid peroxidation [Figure 6], and reducing power activity [Figure 7] of soybean increase with increasing concentration of the soybean extract. The results also showed that the soybean extract had a catalase activity and SOD activity [Figure 8]. Soybean (Glycine max (L.) Merr.) is one of the most important plant proteins sources consumed by humans and animals. It is attracting a growing interest as a source of high-protein forage around the world.[22],[23] Studies that have examined the antioxidant activities of soybean seed found that soybean varieties differ in their antioxidant properties.[24],[25],[26] The total antioxidant capacity and free radical scavenging activity have been determined in immature soybean seeds harvested at three reproductive stages.[27] There was a reduction in total antioxidant capacity and free radical scavenging activity in late-harvested seeds. At the same reproductive stages, DPPH scavenging activity decreased from 59% to 44%.[27] Kumar et. al.[28] assessed DPPH radical scavenging activity in soybean with varying seed coat color and found that soybean with green and yellow seed coat had lower free radical scavenging activity than black soybean.
Figure 4: DPPH scavenging activity

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Figure 5: Hydrogen peroxide scavenging activity

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Figure 6: Inhibition Of Lipid Peroxidation

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Figure 7: Reducing power activity

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Figure 8: Superoxide dismutase and catalase activity

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Malenˇci´c et. al.[29] evaluated the antioxidant activity of the seeds of 20 soybean hybrids and found a positive linear correlation between antioxidant activity and contents of total tannins, proanthocyanidins, and total phenolic contents. Higher levels of all polyphenol classes in the extracts of soybeans hybrids were observed in those with the highest antioxidant activity.[29] The IC50 values [Figure 2] for the extracts investigated varied in a range between 0.09 and 1.29 mg/mL,[30] indicating the presence of biologically active biomolecules with pronounced antioxidant activity.[31]

Whent et al.[32] studied the antioxidant properties and chemical composition of eight soybean cultivars grown in different locations and showed that an antioxidant property may respond to individual environmental factors differently. Malenčić et al.[33] showed that antioxidant activity increased proportionally to the polyphenol content: a linear relationship between IC50 values and contents of total polyphenols and anthocyanins was reported. Malenčić et al.[33] also showed no correlation between DPPH values and flavonoid and tannin contents. Seeds of black and brown color expressed the most significant antioxidant activities and showed combined characteristics of favorable features, having both strong antioxidant capacities and higher contents of anthocyanins and isoflavones. Šibul et al.[34] found for DPPH radical assay EC50 values under 0.12 mg/mL in all investigated cultivars. They reported that antioxidant activity increased proportionally to the phenolic content. The soybean seeds showed the stronger antioxidant potential at the beginning of maturity than at full maturity.[35] Malenčić et al.[33] showed that antioxidant activity increased proportionally to levels of most of the polyphenol classes: a linear relationship between IC50 values and contents of total polyphenols and anthocyanins was reported. In parallel, there was no positive correlation between IC50 values and tannin content. Regression analysis for the isoflavone content and DPPH scavenging activity showed low correlation.[33]

Prvulović et al.[36] showed that the antioxidant activity of extracts from soybean seeds as measured by DPPH assay ranged from 2.12 mg TE/g to 10.98 mg TE/g in different solvents. The best antioxidant activity measured with DPPH assay is obtained with 70% ethanol followed by 70% methanol. Among various samples, acetone extract possessed the highest radical scavenging activity (31.17 mgTE/g). Methanol extract showed the lowest radical scavenging activity (14.70 mgTE/g). Acetone extracts demonstrated higher scavenging activities then other extracts. FRAP test showed that soybean seeds have a significant reduction potential.[36] According to Prvulovic et al.,[36] showed that total antioxidant activity, total reduction capacity, inhibition of Nitric Oxide radical and superoxide anion (O2¯) radical scavenging activity of soybean seeds were affected by the solvent used for the extraction. The lowest bioactivity was measured in methanol extracts, while the total antioxidant activity of acetone extracts was found to be the highest.[36] The methanol extracts of all soybean cultivars expressed the highest scavenging activity for nitric oxide and superoxide radicals, while acetone extracts possessed the lowest scavenging activity for both radicals.[36] There was a statistically significant correlation between TP content and TT content (r = 0.806), as well as between TP content and antioxidant capacity measured (DPPH: r = 0.578; ABTS: r = 0.828; total antioxidant activity: r = 0.702; total reduction capacity: r = 0.713).[37]

Although some these values are comparable with our data but the differences observed was due to the different method used, difference soybeans species and different solvent used for the extraction.


  Conclusions Top


This result showed that soybean can be used as a good source of antioxidant and can act as a better source of natural antioxidants. Data on all the investigated antioxidant activity of soybean varieties extracts could be valuable to the pharmaceutical and food industries for the production of supplements.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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