Phenolic Compound, Antioxidant Activity and Nutritional Components of Five Legume Seed

Phenolic Compound, Antioxidant Activity and Nutritional Components of Five Seed. Abstract Legume is a major source of antioxidant and nutritional component that is very helpful for human health. In recent years, the functional properties of legume seed have received attention, particularly with respect to antioxidant, antitumor, anti-diabetic and anti-cholesterol effects. The objective of this study was to investigate the phenolic compound, flavonoid, antioxidant capacity and nutritional profiles of five legume seeds. The phenolic content determined according to the Folin Ciocalteu method, for five legume samples varied from 13.68 to 35.5 mg Tannic acid equivalent/g of extract. Flavonoid content was measured using 2% aluminum chloride varied from 1.21 to 4.81 mg quercetin equivalent/g of extract. Red bean contained maximum phenolic (35.5 mg/g extract) and flavonoid (4.81 mg/g extract) content that was statistically different from other legume seed (p<0.05). Antioxidant activities were comparatively assessed by ABTS (2, 2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) and DPPH (2,2-diphenyl-1- picrylhydrazyl) free radical decolorization assay method. Red bean, soybean and black bean showed good free radical scavenging capacity by ABTS and DPPH. Red bean contained highest amount of protein (262.73 ± 8.87 mg/g), free amino acid content (27.19 ± 0.40 mg/g) and reducing sugars (35.78 ± 0.76 mg/g) whereas black bean contained highest amount of total soluble sugar content (570.52 ± 5.44 mg/g). Obtained results suggest that red bean, soybean and black bean can be regarded as promising candidates for natural plant sources of antioxidants with high nutritional value.


Introduction
The term legumes refer to the plants whose fruit is enclosed in a pod. A legume is a plant in the family Leguminosae (or Fabaceae).
The Leguminosae contain more than 700 genera and 25,000 species, yet fewer than 20 of these are grown as major world crops. The nutritional value of legume seeds is frequently less than ideal, however, because their proteins contain lower concentrations of certain "essential" amino acids than do animal proteins. The fact that "free" amino acids frequently constitute more than 10% of the weight of legume seeds is often overlooked when considering their nutritional value, as free amino acids tend to be lost in traditional methods of cooking.
The objective of this study was to investigate the phenolic compound, antioxidant activity and nutritional compound in five legume seed.

Materials Method Plant Materials
Five legume seeds were selected for the study namely Soybean

Preparation of Extraction
Dried seed samples were ground with a laboratory grinder to make powder or flour and sieved with a 100-mesh sieve. One gram of sample was extracted by 10mL 70% ethanol for three hours shaking at 180 rpm, and then centrifuged 10 min on 3000 rpm. Extract sample filtered by syringe and preserved in refrigerator for chemical analysis.

Total Phenolic Compounds Content
Total phenolic content (TPC) of legume seed extract was determined by the reported method [18] with slight modification. The results are expressed as mg Tannic acid equivalents (TA eq) per g of extract of legume seeds. All determinations were carried out in triplicate.

Estimation of Flavonoid
The aluminum chloride colorimetric method was used for the determination of the total flavonoid content of the sample [19][20][21][22].
For total flavonoid determination, quercetin was used to make the standard calibration curve. Stock quercetin solution was prepared by dissolving 10 mg quercetin in 1.0 mL methanol, then the standard solutions of quercetin were prepared by serial dilutions using methanol (0-500 g/mL). An amount of 100μL diluted standard quercetin solutions and all sample extracts was separately mixed with 500μL of 2% aluminum chloride. After mixing, the solution was incubated for 60 min at room temperature. The absorbance of the reaction mixtures was measured against blank at 420 nm wavelength with spectrophotometer. The concentration of total flavonoid content in the test samples was calculated from the calibration plot ( = 0.0039 + 0.0079, 2 = 0.9915) and expressed as mg quercetin equivalent (QE)/g of extract legume seeds. All the determinations were carried out in triplicate.

Free Radical-Scavenging Ability by the use of ABTS Radical
Antioxidant activity was determined by the ABTS (2, 2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) free radical decolorization assay method developed by Re [23]. The ABTS positive (+) radical cation was progenerated by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and incubating for 12-16 h in the dark at room temperature until the reaction was complete and the absorbance was stable. The absorbance of the ABTS. + solution was equilibrated to 0.70 (± 0.02) by diluting with water at room temperature, then 100μl was mixed with 50 μl of the test sample and the absorbance was measured at 734 nm after 6 min. All experiments were repeated three times. The radical scavenging activities of legume sample were calculated by the following equation:

Determination of Protein Contents
The protein content was measured by Lowry Assay [25] with The results are expressed as mg BSA equivalents per g of extract or per 100 g of seeds. All determinations were carried out in triplicate.

Detection of Free Amino Acid Contents
Free amino acid content of legume seed was detected by the method described by Setsuro Matsushita [26] with minor modification. For free amino acid content determination, L-tyrosine was used to make the standard calibration curve. Stock L-tyrosine solution was prepared by dissolving 10 mg L-tyrosine powder in 400μL, 1N HCl then add 600 μL distilled water. Standard solutions of L-tyrosine were prepared by serial dilutions using distilled water The results are expressed as mg L-tyrosine equivalents per g of extract or per 100 g of seeds. All determinations were carried out in triplicate.

Determination of Total Sugar
Total sugar content was determined by the phenol-sulfuric acid method described by DuBois [27] with minor modification. For

Determination of Reducing Sugar
Reducing sugars were measured by the method of Somogyi & Nelson [28,29] with minor modification. For reducing sugar content determination, Glucose (Dextrose) was used to make the standard calibration curve. Stock glucose solution was prepared by dissolving 10 mg glucose in 1mL distilled water. Standard solutions of glucose were prepared by serial dilutions using distilled water

Statistical Analysis
All the experiments for determination of total phenolics, total flavonoids, and antioxidant properties using ABTS and DPPH and all nutritional value were conducted in triplicates. The values are expressed as the mean ± standard deviation (SD). The statistical analysis of the results was done by using -statistical software package version 2.2.1 [30]. Analysis of variance and significance of difference among means were tested by one-way ANOVA and least

Results and Discussion
The extraction yield of five legume seeds were shown in Table 1.
Maximum extraction yield was observed in soybean (11.15%) then in chickpea (9.44 %) that was statistically similar with black bean (8.98%) and minimum was in red bean (5.30%). Hakime Hulya et al. [31] reported that the extraction yield of Mungbam was 10.70%. anti-inflammatory, anti-allergic and anti-cancer activities [36]. The result of flavonoid content of legume seeds were also presented in

Consumption of foods containing antioxidant phytoconstituents
is beneficial to human health since they can protect the human body from detrimental free radicals and inhibit the progress of many chronic disease [38]. Previous studies by Schlesier et al. [39] showed that when analyzing the antioxidant activity, it is preferable to use at least two methods. In their experiments, the analysis of the antioxidant activity of legume seed was performed using two methods: ABTS and DPPH. The ABTS and DPPH radical is widely used to evaluate the free-radical scavenging capacity of antioxidants according to their hydrogen donating ability [40].
In addition to that, reactions involved in these methods are fully unaffected by side reactions [41]. These methods are distinguished by their mechanism of action and would be complementary to the study of the antioxidant potential of legume seed. The antioxidant proprieties of extracts were measured in terms of their efficient IC50 concentration corresponding to the sample concentration that reduced the initial DPPH absorbance of 50%. These IC 50 values for ABTS and DPPH are given in Table 3.
In this study, it was investigated that the five legume extracts had different antioxidant activity levels that the IC50 values for the test samples lie in the range between 375.64 to 1335.05 μg/ml for ABTS and 500.29 to 10041.30 μg/ml for DPPH (Table 3). Among the sample, red ben showed good free radical scavenging capacity by ABTS (IC50 375.64 μg/ml) and by DPPH (IC50 500.29 μg/ml).
Radical scavenging activity of soybean and black bean was more or less similar. Red bean, soybean and black bean showed a stronger scavenging activity than another legume. Red bean, soybean and black bean might be a potential material for antioxidants. Yildirim et al. [42] reported that some potent antioxidants applied in food might cause a serious of side effects. Therefore, it is important to find more natural antioxidative ingredients for food industries.

The deviation in results can be due to the difference in extraction
technique and assay method.
The different nutritional parameters were found in varying concentrations in all samples taken in present studies. Proteins represent the major storage compound which is a common trait of most legume seeds. Red bean seed is a rich source of protein content.
As compared to different legume seed samples, protein content was highest in red bean seeds (262.73 ± 8.87 mg/g) whereas lowest in chickpea seeds (71.75 ± 4.34 mg/g). Protein content of soybean was 153.25 mg/g which was statistically similar with black bean protein content (145.33mg/g) ( Table 4). Sucheta Sharma et al. [35] described that protein content of soybean is 41.4 ± 1.82 %. Amresh Kumar et al. [43] reported that protein content of faba bean is with the range of 20-32 %.
Amino acid is the main components in forming protein. Amino acids content of red bean is much higher than other legume seed.
The free amino acid content of red bean was 27.19 ± 0.40 mg/g which was significantly different from other legume seed. Soybean showed the lowest amount of free amino acid (4.09 ± 1.09 mg/g) (Table 4). Although free amino acids have little effect on the nutritional value of the seeds [44], but as seeds mature, these stored free amino acids are converted to storage proteins and/or non protein constituents. Amresh Kumar et al. [43] reported that free amino acid content of faba bean is with the range of 188-348 mg/ 100 g. Sucheta Sharma et al. [35] described that free amino acid content of soybean is 0.55 ± 0.17 %.   [45].
With respect to reducing sugars, red bean (35.78 ± .76 mg/g) and black bean (28.95 ± 0.07 mg/g) were found to contain highest and lowest amount respectively though reducing sugar content of all legume seeds were statistically similar except red bean (Table 4).
Amresh Kumar et al. [43] reported that reducing sugar content of faba bean with the range of 85-188 mg/100 g. Sucheta Sharma et al. [35] described that reducing sugar content of soybean is 0.27±0.04 %. Cowpea seed contain 9.11 ± 2.42 mg/g reducing sugar content described by Maina Antoine N et al. [45].
It is hypothesized that carbon from starch is used either for lipid synthesis or for protein synthesis especially in pulses seed where proteins are by far the major storage compounds. Amino acids needed for reserve protein synthesis. Although different soluble sugars were not determined separately in present study but major soluble sugars present in seeds are sucrose and raffinose family oligosaccharides (RFOs).

Conclusion
Among five legume seeds, red bean contains maximum phenolic and flavonoid component. On the other hand, red bean, soybean and black bean showed a stronger scavenging activity than another legume seed. Red bean, soybean and black bean might be a potential material for antioxidants. Red bean also contains maximum nutritional value such as protein, free amino acid and reducing sugar content in this experiment. On the basis of the results obtained in the present experiment, it is concluded that soybean, black bean and red bean have maximum capacity of antioxidant activity and nutritional value among five legume sample. Obtained results suggest that red bean, black bean and soybean can be used as functional ingredient with high antioxidant activity. More research is needed on red bean, black bean and soybean to adequately know their bioactive component and their functionality, which is needed in order to be able to produce food products with maximum health benefits.