Total phenolic, total flavonoid contents and antioxidant potential of Common Bean (Phaseolus vulgaris L.) in Vietnam

In this study, the antioxidant properties of total phenolics and flavonoids extracts from different parts (leaves, pods, and seeds) of common bean were evaluated. Specifically, the highest total phenolic content was recorded with methanol extracts of pods (95.41 ± 1.18 mg GAE/g), whereas methanolic extract of seeds contained the lowest content of phenols (6.87 ± 1.45 mg GAE/g). The highest total flavonoid content was found in methanol extracts of leaves (44.59 ± 2.15 mg RE/g). Meanwhile, the methanol extract of seeds and pods contained less flavonoid content (9.29 ± 1.65 mg RE/g and 3.64 ± 0.87 mg RE/g, respectively). The GC-MS analysis showed the presence of 29, 18 and 29 different plant compounds in methanol extracts of leaves, seeds and pods, respectively. The methanol extracts of leaves showed the highest antioxidant capacity with an inhibitory percentage of 48.74 ± 0.32% at a concentration of 100 μg/mL and the EC50 value of 137.4 μg/mL. The methanol extracts of seed had the lowest antioxidant capacity with an inhibitory percentage of 13.99 ± 1.22% at a concentration of 100 μg/mL and the EC50 value of 486.2 μg/mL. The results showed that the extract from leaves of common bean had the highest antioxidant activities as well as total contents of flavonoid in comparison with an extract from seeds and pods and the positive relationship between total flavonoids content and antioxidant activities in this plant.


Introduction
Nowadays, the development of chronic (e.g., renal failure, myocardial infarction, and heart failure) and neurogenerative (e.g., Parkinson's, multiple sclerosis, and Alzheimer's) diseases have determine the total phenolic and flavonoid contents as well as the antioxidant potential of common bean (Phaseolus vulgaris L.) in Vietnam.

Plant material and extract preparation
Leaves, pods, and seeds of common bean (GRIS2) (Figure 1) were collected at Genomic Research Institute and Seed (GRIS), Ton Duc Thang University, Ho Chi Minh City, Vietnam. Before extraction, they were cleaned to eliminate soil and damaged seeds, dried and ground into a fine powder. The sample extract was extracted using the method according to [20]. The leaves, pods, and seeds extract were individually prepared in methanol (plant: solvent ratio [1: 10], w/v), and extracted for shaken overnight at 28 ℃. The extract was then filtered through filter paper. The solvent was then removed by evaporation in a vacuum to obtain dry extracts. This process was repeated once. The extracts were stored at 4 ℃ in a refrigerator prior to use.

Total phenolic contents
The total phenolic content of each leaf, pods, and seeds extract was determined using the Folin-Ciocalteu reagent as described by the method of Singleton et al. [21] with slight modification. Approximately 0.5 mL of each extract was dissolved in methanol (100 µg/mL) was mixed with 2.5 mL of Folin-Ciocalteu reagent (0.2 N). This mixture was shaken well and was kept at room temperature for 5 min and then, 2 mL of sodium carbonate solution (75 g/L) was added. After 2 h of incubation in the dark, the absorbencies were measured at 760 nm against a water blank using a UV-Vis spectrophotometer. The same procedure was repeated by gallic acid solutions used as a standard for the calibration curve. The concentrations of the standard were set at 0, 0.02, 0.04, 0.06, 0.08 and 0.1 mg/mL. The determination was performed in triplicate and the results were expressed as terms of gallic acid equivalent (mg of GAE/g of extract).

Total flavonoid contents
The total flavonoid content of each leaf, pods, and seeds extract was determined using the Dowd method as described by Sawadogo et al. [22] with slight modification. In brief, 2 mL of 2% AlCl 3 in methanol was mixed with 2 mL of each extract (100 µg/mL), shacked well, and hold for 10 minutes. Absorption was read at 415 nm against a blank sample consisting of 2 mL of methanol and 2 mL of each extract without AlCl 3 using a UV-vis spectrophotometer. The same procedure was repeated by rutin solutions used as a standard for the calibration curve. The concentrations of the standard were set at 0, 0.02, 0.04, 0.06, 0.08 and 0.1 mg/mL. The determination was performed in triplicate and the results were expressed as terms of rutin equivalents (mg of RE/g of extract).

GC-MS analysis
Methanol extract from leaf, pod, and the seed of common bean was subjected to gas chromatography-mass spectrometry (GC-MS) analysis. The GC-MS was equipped with a DB-5MS column (30 m, 0.25 mm, and 0.25 µm) (Agilent Technologies, J & W Scientific Products, Folsom, CA, USA.). Helium gas was used as the carrier gas with a split ratio of 5:1. The temperature program was as follows: initial temperature of 50 ℃ without hold time and gradually increased to 300 ℃ at a rate of 10 ℃/min for 20 min of hold time. The injector and detector temperatures were set to 300 ℃ and 320 ℃, respectively. The mass spectra were scanned from 29 to 800 amu. The identification and characterization of chemical compounds in various crude extracts were based on the JEOL's GC-MS Mass Center System software, version 2.65a (JEOL Ltd., Tokyo, Japan).

Determination of free radical scavenging activity by DPPH method
The DPPH (2, 2-Diphenyl-1-picrylhydrazyl) assay is one of the most commonly employed methods because it is simple, efficient, and inexpensive. DPPH radical scavenging method was used to evaluate the antioxidant properties of each leaf, pods, and seeds bean extract. The standard procedure for the DPPH assay was performed based on Bakasso et al. [23] with minor modifications. The samples were added of 1.5 mL DPPH solution (80 µg /mL) to 0.75 mL various concentrations (6.25, 12.5, 25, 50, and 100 µg/mL) of each extract. The solution was mixed vigorously and left to stand at room temperature for 30 minutes in the dark after which its absorbance was measured spectrophotometrically at 517nm, the analysis was done in triplicate. A positive control (ascorbic acid) was prepared in the same way as samples, while the blank solution by adding 0.75 mL methanol to 1.5 mL of DPPH (80 µg/mL solution). The absorbance of blank, positive control, and samples were recorded.
The percentage of inhibition can be calculated using the formula: Where AB is the absorbance of the control and AA is the absorbance of the test. EC 50 (µg/mL) was defined as the half-maximal effective concentration of the amount of sample necessary to decrease the absorbance of DPPH by 50%. It was obtained by interpolation from the linear regression analysis.

Statistical analysis
The results were expressed as mean ± standard deviation of at least triplicate measurements. Analysis of variance (ANOVA) and Duncan's multiple range test were used for determining the significant differences at P < 0.05. All statistical analyses were carried out using the statistical program are SAS version 8.0 and Microsoft Excel 2010 software.

Total phenolic and flavonoid contents
The content of total phenols in different extracts was presented in Table 1. The highest phenolic content was found in methanol extracts of pods (95.41 ± 1.18 mg GAE/g). Whereas methanol extracts of seeds contained considerably least content of phenols (6.87 ± 1.45 mg GAE/g).
The results of the total flavonoids contents determination of the examined plant extract are presented in Table 1. The highest flavonoid content was found in methanol extracts of leaves (44.59 ± 2.15 mg RE/g). Meanwhile, the methanol extract of seeds and pods contained less flavonoid content (9.29 ± 1.65 mg RE/g and 3.64 ± 0.87 mg RE/g, respectively). All data are mean ± SD of triplicate (n = 3) analyses. Values with a different superscript in the same column differ significantly (P < 0.01).

GC-MS analysis
The chemical components in methanol extract from the leaves, seed, and pods of common bean were successfully analyzed using GC-MS in Figure 2. In total, 76 compounds were detected and presented in Table 2. Of these, the presence of 29, 18, and 29 various phytocompounds in methanol extract from the leaves, seed, and pods respectively. The methanol extract from pods presented the highest amount of phytocomponents compared to methanol extract from leaves and seed. The present study successfully identified the bioactive components present in methanol extract from leaves, pods, and seeds of the common bean by GC-MS included phenolics, flavonoids, fatty acids, amino acid, terpenoids, sterols, carbohydrates, alcohols, volatile oils, fatty acid ester, ester, amines, and others.

Antioxidant properties by DPPH
In this study, the DPPH radical scavenging potential of methanol extracts from the leaves, seed, and pods of common bean and ascorbic acid were represented in Table 3 and EC 50 values result of different extracts were calculated by using concentration with mean percent inhibition of the DPPH radical curve of each different extracts also were presented in Table 3. From Table 3, all the extracts showed an inhibitory potential against DPPH free radical. The inhibitory percentages varied from 5.12 ± 0.35% for the seeds extract to 98.88 ± 0.03% for the vitamin C. The EC 50 values of the antioxidant capacity varied significantly (P < 0.01) from 23.31 μg/mL for the vitamin C to 486.2 μg/mL for the seeds extract. As it was known, the lower the EC 50 value the higher the antioxidant capacity of the plant extract. As can be seen from Table 3, the methanol extracts of leaves have the highest antioxidant capacity with inhibitory percentages are 48.74 ± 0.32% at a concentration of 100 μg/mL and EC 50 value was 137.4 μg/mL compared to the other extracts and 6 times lower than vitamin C. The methanol extracts of seed had the lowest antioxidant capacity with inhibitory percentages was 13.99 ± 1.22% at a concentration of 100 μg/mL and EC 50 value was 486.2 μg/mL compared to the other extracts and 21 times lower than vitamin C.

Discussion
The consumption of common bean (Phaseolus vulgaris L.) has been greatly connected with many physiological and health-promoting effects such as the prevention of cardiovascular diseases, obesity, diabetes mellitus, and cancers [10]. The antioxidant properties of phenolic compounds lie in their ability to neutralize free radicals and the chelation of transition metals, thus they counteract the initiation and propagation of oxidative processes [24]. In the present study, total phenolic acid and total flavonoid contents and antioxidant activity in vitro were determined for methanol extracts of leaves, seeds, and pods of common bean.
In the present study, the total phenolic contents of seeds were 6.87 ± 1.45 mg GAE/g (Table 1) whereas according to Yao et al. [13] reported that common bean contained 8.59 mg GAE/g total phenols, besides, according to Ombra et al. [25] reported that total phenolic content of the common bean in the range of 0.14-1.29 mg GAE/g. The total flavonoid content of seeds bean was 9.29 mg RE/g (Table 1) and was higher than the previously reported by Oomah et al. [26] for selected common bean in the range of 0.41-1.02 mg RE/g. The difference in the total contents of phenolic acid and flavonoid may be due to differences in the geographical region, environmental, climatic condition, and storage, and processing methods [27]. In addition, in the current study, the total contents of flavonoid and phenolic acid were different among leaves, pods, and seeds bean. These results showed that different levels of phenolic acids and flavonoids were influenced by the interaction between parts of plants. This finding is in agreement with that of Males et al. [28] who reported that I. candida contains higher phenolic compounds in leaves (1.031-1.423%) compared to stem (0.411-0.516%). Ghasemzadeh et al. [29] also recorded the total flavonoid and phenolic acid contents in the leaves were more than in the rhizomes, followed by contents in the stems. Elkhamlichia et al. [30] also confirmed that Calycotome villosa subsp. Intermedia had the total flavonoids contents in seeds. Previous studies by Ferry et al. [31] and Elattar and Virji [32] have shown that some flavonoids components such as quercetin, rutin had anticancer activities and were able to inhibit cancer cell growth. Therefore, the results of this study showed that flavonoids are important components of this plant.
Among the compounds discovered, several are reported as potential therapeutic agents. For instance, 9, 12-octadecadienoic acid, methyl ester is effective antihistamines, anti-coronary, insectifuge, and antieczemic [33]. Terpenoids have also been reported to exhibit antiplasmodial, antineoplastic, and antiviral activities [34]. Van Acker et al. [35] found other molecular parameters related to electron distribution and structure, which correlate with the antioxidant action of vitamin E and its derivatives. Besides, flavonoids and phenolics are polyphenols that have been reported to possess great antioxidant properties due to the reducing ability of flavonoids when they play an important role in neutralizing free radicals and scavenging radicals or suppressing lipid peroxidation [26]. Vitamin P (Rutin) is a flavonol, it has demonstrated a number of pharmacological activities, including antioxidant, cytoprotective, vasoprotective, anticarcinogenic, neuroprotective, and cardioprotective activities [36][37][38][39], whereas showdomycin and clindamycin were known as an antibiotic [38].
Phenolic compounds restrain the formation of superoxide anion as well as the production of reactive oxygen species by inhibiting key enzymes such as protein kinase, xanthine oxidase, lipoxygenase, cyclooxygenase, S-transferase, glutathione, and NADH oxidase [26]. Moreover, in aqueous and lipophilic phases, these compounds also serve as hydrogen donating radical scavengers. The ability of flavonoids to complex with metal ions plays an important role in their antioxidant activity [24]. There is a specific relationship between flavonoid structures and their antioxidant activity as the larger the number of hydroxyl groups in the flavonoid nucleus, the greater would be the antioxidant activity [40].
According to [25] reported the common bean to have EC 50 value in the range of 1570-55200 μg/mL. In this study, the EC 50 value of seeds was 486.2 μg/mL whereas, the EC 50 value of leaves was 137.4 μg/mL showed that the antioxidant activity of leaves bean higher than seeds (Table 3). Moreover, the total flavonoid contents of leaves bean also higher than seeds (Table 1). Hence, the different antioxidant activity might as well be due to the presence of phenolic compounds, especially the flavonoid contents in this plant. The antioxidant activity of phenolic compounds was based on several different mechanisms. It has the ability to scavenging of free radicals by single electron transfer and the hydrogen atoms in their hydroxyl groups, chelation of metal ions such as iron and copper, or inhibition of enzymes responsible for a free radical generation [26,41].
From the determination of total phenolic contents, total flavonoid contents, and antioxidant activity in this study observed that the extracts of the pod bean showed the highest content of total phenolic; however, leaves bean although containing slightly fewer content of phenolic acid, exhibited higher total flavonoid content, and its highest the antioxidant capacity. The antioxidant capacity of phenolic compounds depends on the number and position of free OH groups [42], which means, the many free hydroxyl groups present in polyphenols, the higher their radical scavenging capacity. This reinforced the idea that the antioxidant potential could be linked strongly to the content of flavonoids in this plant.

Conclusions
Common bean could be a good source of natural antioxidants. In this study showed the methanol extracts from leaves, pods, and seeds of common bean exhibit good antioxidant ability on the DPPH radical scavenging potential, in which, the extracts of the leaves showed higher scavenging activities than the pods and seeds. Moreover, the total flavonoid content in extracts from leaves also higher than the pods and seed although the total phenolic acid content was found in extracts from extracts of pods higher than the leaves and seeds. Therefore, the results of this study showed that the positive relationship between total flavonoids content and antioxidant activities in this plant.