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ISSN : 1225-8504(Print)
ISSN : 2287-8165(Online)
Journal of the Korean Society of International Agricultue Vol.30 No.4 pp.357-369
DOI : https://doi.org/10.12719/KSIA.2018.30.4.357

Evaluation of Phytochemical econtents and antioxidant activity of Korean common bean (Phaseolus vulgaris L) landraces

Kyung Jun Lee*, Myoung-Jae Shin*, Gyu-Taek Cho*, Gi-An Lee**, Kyung-Ho Ma***, Jong-Wook Chung*****, Jung-Ro Lee*
*National Agrobiodiversity Center, National Institute of Agricultural Sciences (NAS), RDA, Jeonju-Si 54874, Korea
**Research Policy Bureau, RDA, Jeonju-Si 54875, Korea
***Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science (NIHHS), RDA, Jeonju-Si 54874, Korea
****Department of Industrial Plant Science & Technology, Chungbuk National University, Cheongju, 28644, Korea
Corresponding author : (Phone) +82-43-261-2524 (E-mail) jwchung73@chungbuk.ac.kr (Phone) +82-63-238-4880 (E-mail) jrmail@korea.kr
August 17, 2018 December 13, 2018 December 14, 2018

Abstract


The Korean common bean (Phaseolus vulgaris L.) has been receiving increased attention as a functional food. The objective of this study was to reveal the phytochemicals genetic variation and antioxidant activity of 209 Korean common bean landraces. Antioxidant activity was evaluated with the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate), ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), ferric reducing antioxidant power (FRAP), and superoxide dismutase (SOD) assay. Antioxidant activities among common bean accessions showed wide variation. Four flavonoids (kaempferol, myricetin, quercetin, and naringenin) of the 209 Korean common bean landraces were measured using HPLC. Among them, kaempferol had the highest phytochemicals compared to the other three flavonoids. Using the relative antioxidant capacity index (RACI), it was found out that the IT104587 had the highest antioxidant activity. Meanwhile, in clustering analysis, the Korean common bean landraces were classified into three clusters. Among them, cluster II contained 64 landraces with higher antioxidant activities and phytochemicals than the other clusters, except DPPH. The results could provide information on the valuable Korean common bean landraces for the development of new common bean varieties.



한국 재래종 강낭콩 유전자원의 phytochemical 및 항산화 활성 평가

이 경준*, 신 명재*, 조 규택*, 이 기안**, 마 경호***, 정 종욱*****, 이 정로*
*농촌진흥청 국립농업과학원 농업유전자원센터
**농촌진흥청 연구정책과
***농촌진흥청 국립원예특작과학원 인삼특작부
****충북대학교 농업생명환경대학 특용식물학과

초록


    Rural Development Administration
    PJ010883

    Introduction

    The Korean common bean (Phaseolus vulgaris L.), an important food crop from an economic and nutritional point of view, is cultivated and consumed worldwide (Lopez et al,. 2013). It is a rich source of protein, complex carbohydrates, dietary fiber, and minerals and italso contains biologically active phytochemicals important for human health (Rochfort & Panozzo, 2007). Previous studies identified several phenolic compounds such as flavones, flavonols, isoflavones, anthocyanins, flavanols, and phenolic acids in common bean (Akillioglu & Karakaya, 2010;Beninger et al., 2005;de Lima et al., 2014;Lopez et al., 2013;Xu & Chang, 2008). Their distribution is qualitatively and quantitatively different on the two main parts of seeds which are cotyledon and testa. The phenolic acids and non-flavonoid phenolic compounds are mainly found in cotyledons, while proanthocyanidins and anthocyanins are only contained in the testa (Rochfort & Panozzo, 2007).

    Oxidative stress caused by free radicals disturbs the normal redox state within the human body causing chronic diseases including cardiovascular disease, obesity, and certain types of cancer (Del Pino-García et al., 2016). The natural antioxidants of plant origins are purported to be capable of controlling such disturbances and contribute to preventing oxidative stress-related diseases (Chen et al., 2016). Flavonoids are well-studied natural antioxidants. Many studies provide evidence that the consumption of flavonoid-rich foods is closely associated with a lower incidence of certain chronic diseases (Santhakumar et al., 2015;Zhang et al., 2016).

    The functional effects of common bean consumption may be due to the presence of abundant phytochemicals including polyphenolics, which possesses both anticarcinogenic and antioxidant properties (Cardador-Martinez et al., 2002). The antioxidant activity of polyphenols is directly related to their chemical structures, such as degree of glycosylation and number and position of hydroxyl groups attached in relation to the carboxyl functional group (Aguilera et al., 2010;Balasundram et al., 2006). Meanwhile, the antioxidant activity of phenolic compounds present in Phaseolus vulgaris is of interest and reported in many recent studies (Chen et al., 2015;Kan et al., 2016;Wang et al., 2016).

    Evaluation of antioxidant activity and bioactive polyphenols in a diverse group of common beans is important in improving the knowledge of the functional potential of common beans. The objective of this study was to evaluate 209 Korean common bean landraces preserved in Korean gene bank for phytochemical distribution and antioxidant activity.

    Materials and Methods

    Plant materials

    Two-hundred-nine Korean common bean landraces were obtained from the National Agro-biodiversity Center (NAS) of the Rural Development Administration (RDA), Korea (http://genebank.rda.go.kr). The common bean accessions were harvested at the experimental field in 2015 using conventional cultural practices implemented by NAS.

    Common bean extraction

    One-hundred milligrams of each ground sample was added to 1 ml of 75% EtOH and sonicated for 1 hour. The suspension was then centrifuged at 13,000 rpm for 10 minutes. The clear supernatant was moved in a new tube and used for the phytochemical and antioxidant activity assays.

    DPPH assay

    DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) radical- scavenging activities were measured using Lee et al., 2017 method with slight modifications. Briefly, 100 μl of sample was added to DPPH solution (150 μl; 150 μM in anhydrous EtOH). The mixture was incubated at 25°C in the dark for 30 minutes. Absorbance at 517 nm was measured using a spectrophotometer (Epoch; Bio-Tek, Winooski, VT, USA). The results were expressed as IC50 values.

    ABTS assay

    ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical-scavenging activity was estimated using also the Lee et al., 2017 method with some changes(). Briefly, 190 μl diluted ABTS radical-scavenging solution (7 mM ABTS + 2.45 mM potassium persulfate) was added to 10 μl of sample solution. After six minutes of incubation at RT, absorbance at 735 nm was determined using a spectrophotometer. The results were expressed as ascorbic acid equivalents (AAE) per gram of dry weight (mg AAE/g).

    Ferric reducing antioxidant power assay (FRAP)

    Ferric reducing antioxidant power (FRAP) was also determined using the method published by Lee et al. (2017) with minor modifications. Briefly, 300 μl of FRAP reagent (0.1 M acetate buffer at pH 3.6, 10 mM tripydyltriazine (TPTZ) solution in 40 mM HCl, 20 mM ferric chloride solution (FeCl3)) was mixed with ten microliters of sample and incubated at 37 °C for 30 minutes. The mixture was measured at 593 nm. The results were expressed as ascorbic acid equivalents (AAE) per gram of dry weight (mg AAE/g).

    SOD assay

    The superoxide anion scavenging activity was also measured as described by Lee et al. (2017) with some modifications. The superoxide anion radicals were generated in 100 μl of Tris-HCl buffer (16 mM, pH 8.0) containing 0.3 mM nitroblue tetrazolium (NBT), 50 μl NADH (0.936 mM) solution, and 100 μl sample + 50 ul Tris-HCl buffer (16 mM, pH 8.0). The reaction was initiated by adding 50 μl phenazine methosulfate (PMS) solution to the mixture and incubated at 25°C for five minutes, then the absorbance was measured at 560 nm. The results were expressed as IC50 values.

    Total polyphenol content assay (TPC)

    Total polyphenol content was measured using a modified Folin–Ciocalteu method (Waterhouse, 2003). 100 μl of sample solution was added to 100 ul Folin–Ciocalteu reagent and incubated at room temperature for three minutes. After that, 100 μl of 2% sodium carbonate was added and the mixture was incubated at room temperature for 30 minutes. Absorbance was measured at 750 nm on a spectrophotometer using distilled water as a blank. Total phenolic content was reported as milligrams of gallic acid equivalents (GAE) per gram of dry weight sample (mg GAE/g).

    Total flavonoid content assay (TFC)

    Total flavonoid content was measured with the aluminum chloride colorimetric assay by Lee et al., 2017. First, 100 μl of sample was measured then 100 μl of 2% AlCl3·6H2O in methanol was added. The absorbance was measured after 10 minutes against a blank of methanol at 430 nm. Flavonoid concentration was expressed in ug/g dry weight as quercetin equivalents (ug QE/g).

    Analysis of flavonol content in Korean common bean landraces

    One gram of each sample was transferred into 5 ml polypropylene tubes and mixed with 2.5 ml of 80% methanol containing 1.2M hydrochloric acid for hydrolysis. The mixture was vortexed briefly and then incubated at 80? for 2 hours with tube inversion for mixing the samples with the extract solution at 15-minute intervals. After incubation, the samples were centrifuged at 14,000 rpm for 3 minutes and the supernatant was transferred into 2 ml Eppendorf tubes. The supernatant was collected and filtered using a 0.45 μm syringe filter prior to analysis with an Agilent 1260 Infinity HPLC system (Agilent Technol., CA, USA). HPLC conditions were as follows: solvent A, 0.1% TFA/H2O; solvent B, CH3CN; gradient, 20% (B), 20-50% (B) in 5 minutes, 50-100% (B) in 6 minutes, hold at 100% (B) for 1 minute, reequilibrate at 20% (B) for 3 minutes; column temperature, 30°C; and flow rate, 0.40 ml/min. The filter detector was set at 370 nm.

    Data analysis

    Using SPSS Statistics 20 (SPSS Inc., Chicago, IL, USA), the least significant difference and correlation analysis determine the differences among the 209 accessions of Korean common bean landraces. Principal component analyses (PCA) was performed using the R statistical software environment (http://www.r-project.org). Statistical significance was defined when the P value was less than 0.05.

    Results

    Phytochemicals contents in 209 Korean common bean landraces

    There was variation phytochemicals contents among the Korean common bean landraces as shown in Table 1 and Additional Table 1. Total polyphenol content (TPC) ranged from 0.55 to 2.23 mg gallic acid equivalent (GAE)/ g with an average 1.3 mg GAE/g among the accessions evaluated. The maximum value obtained was almost fourtimes that of the accession with the lowest value. Among the 209 accessions, IT108863 (2.23 ± 0.03 mg GAE/g) and IT168095 (2.22 ± 0.13 mg GAE/g) had the highest while IT100888 (0.55 ± 0.03 mg GAE/g) had the lowest. Total flavonoid content (TFC) ranged from 0.2 to 0.86 mg quercetin equivalent (QE)/g. TFC in the accessions with the highest values was 4.3-times the value of the accessions with the lowest. IT105167 (0.86 ± 0.01 mg QE/g) had the highest TFC, while IT209349 (0.20 ± 0.01 mg QE/g) was the lowest. Kaempferol content of 209 Korean common bean landraces ranged from 0.0 to 104.3 mg/100 g dried seed with an average 10.0 mg. Among them, IT026294 showed the highest kaempferol content (104.3 ± 0.1 mg/ 100 g dried seed), while IT103601, IT103630, IT104302, IT104587 had no kaempferol. Myricetin showed a range of 0.0 to 6.7 mg/100 g dried seed with an average of 0.2 mg. IT168117 had the highest myricetin content (6.7 ± 0.0 mg/ 100 g dried seed). Myricetin was not detected in 38 Korean common bean landraces. Quercetin ranged from 0.0 to 23.6 mg/100 g dried seed with an average of 4.8 mg. Among 209 Korean common bean landraces, IT103849 showed the highest levels of quercetin (23.6 ± 0.2 mg/100 g dried seed), while 12 landraces did not have any quercetin. Naringenin ranged from 0.27 to 3.5 mg/100g dried seed) with an average of 0.8 mg. The highest naringenin level (3.5 ± 0.0 mg/100 g dried seed) was detected in IT138156 while the lowest (0.27 ± 0.01 mg/100 g dried seed) in IT103883.

    Antioxidant activities of 209 Korean common bean landraces

    DPPH radical-scavenging activity ranged from 62.3 to 643.9 (IC50) with an average of 105.0 among the accessions evaluated (Table 1 and Additional Table 1). Among them, IT180804 had the highest DPPH radical-scavenging activity (62.3 ± 0.1, IC50) and IT103971 had the lowest (643.9 ± 72.5, IC50). The ABTS antioxidant activities were 0.28 to 1.49 mg ascorbic acid equivalent (AAE)/g. IT110958 and IT112955 had the highest activity (1.49 ± 0.01 mg AAE/g and 1.49 ± 0.02 mg AAE/g, respectively) and IT102849 had the lowest (0.28 ± 0.02 mg AAE/g). FRAP was found at its highest level in IT189598 (2.76 ± 0.02 mg AAE/g) and lowest level in IT102849 (0.32 ± 0.03 mg AAE/g). SOD assay was 50.4 to 299.8 (IC50). IT102849 (50.4 ± 2.4, IC50) had the highest and IT180465 (299.8 ± 39.4, IC50) was the lowest.

    The integration of antioxidant capacity results derived from different chemical methods was used to calculate the Relative Antioxidant Capacity Index (RACI) (Sun and Tanumihardjo 2007). It was found out that IT104587 had the highest RACI (1.72), followed by IT109102 (1.64), IT104342 (1.63), and lasty, the IT189598 with the lowest value (-1.37).

    Correlation among the phytochemicals and antioxidant activities of the 209 Korean common bean landraces

    The correlations among TPC, TFC, and antioxidant activities are shown in Table 2. DPPH (IC50) was negatively correlated with quercetin (r=-0.146, p<0.05) and TFC (r=-0.616, p<0.01). ABTS had positive correlations with TPC (r=0.827, p<0.01), FRAP (r=0.848, p<0.01), SOD (r=0.538, p<0.01), and quercetin (r=0.362, p<0.05) and negative correlation with naringenin (r=-0.189, p<0.01). TPC showed positive correlations with FRAP (r=0.887, p<0.01), SOD (r=0.521, p<0.01), and quercetin (r=0.348, p<0.01) and negative correlation with naringenin (r=-0.178, p<0.05). FRAP was positively correlated with SOD (r=0.497, p<0.01) and quercetin (r=0.429, p<0.01). SOD was negatively correlated with myricetin (r=-0.142, p<0.05) and naringenin (r=-0.142, p<0.05). TFC showed negative corrections with myricetin (r=-0.190, p<0.01) and naringenin (r=-0.154, p<0.05) and positive correlation with kaempferol (r=0.165, p<0.05), respectively.

    Principal Component Analysis (PCA)

    Principal component analysis (PCA) using the phyto-chemicals and antioxidant activities of Korean common bean landraces indicated that three principal components (PCs) with Eigen values >1 (Table 3) explained the 62.1% of the variances. The first PC with Eigen value of 1.83 explained 33.6% of the total variance. FRAP was the variable that had the largest positive loadings and ABTS and TPC also had positive variances in the first PC. The second PC with Eigen values of 1.32 explained an additional 17.4% of the total variance, wherein DPPH was the variable that had the largest positive loading while TFC had the largest negative loading. The third PC with Eigen values of 1.06 explained an additional 11.1% of the total variance wherein naringenin had the highest positive variance.

    Fig. 2 plots the first two PCs and divides the 209 Korean common bean landraces into three clusters according to their phytochemicals and antioxidant activities. In phytochemicals and antioxidant activities, kaempferol and myricetin did not show significant difference in the three clusters (Table 4). Cluster I, II, and III contained 99, 64, and 46 accessions, respectively. Among them, Cluster III had the lowest antioxidant activities than the other clusters, except for DPPH. Cluster I showed the lowest TFC. Cluster II showed the lowest DPPH and highest TFC, and Cluster III the highest naringenin contents.Fig. 1

    Discussion

    For centuries, farming communities have contributed to the evolution, enrichment, and maintenance of landrace diversity on farms (Brush, 1995;FAO, 2010;Jarvis et al., 2008). If landraces sufficiently offer natural variation in the population, these can be improved by simple trait selection effectively (Sthapit & Rao, 2009). However, few researches had been done to understand the landrace diversity or to improve these landraces. The results in this study revealed that 209 Korean common bean landraces had different phytochemicals and antioxidant activity (Table 1). These data could contribute to more efficient conservation and utilization of Korean common bean landraces in broadening the genetic base of commercially grown varieties. Phenolic compounds are considered the major components that contribute to the total antioxidant activities of crops (Yao et al., 2010). They can act as hydrogen or electron donors, when they react with oxidants. Also, they are also stable against the antioxidant-derived radicals and show metal chelating properties. With these attributes, phenolic compounds present strong free-radical scavenging activity as well as antioxidant activity (RiceEvans et al., 1997). In this study, the mean value for TPC of Korean common bean landraces was 1.3 mg GAE/g (0.55 to 2.23 mg GAE/g). Orak et al. (2016) reported that TPC of Phaseolus vulgaris in Turkey ranged from 0.33 to 0.36 mg GAE/g. Golam Masum Akond et al. (2011) also reported that TPC of 29 common beans ranged from 5.87 to 14.14 mg GAE/g. These differences of TPC were likely caused by the differences in the sources of common bean samples and the different environmental conditions.

    The seeds of Phaseolus vulgaris contain various flavonoids including quercetin, myricetin, cynidine, procyanidin, naringenin, catechin, hesperetin and kaempferol (Lopez et al., 2013). Among various flavonoids, kaempferol, quercetin, myricetin, and naringenin were observed predominantly in different Phaseolus vulgaris (Lin et al., 2008). In this study, the content of four flavonoids: kaempThe seeds of Phaseolus vulgaris contain various flavonoids including quercetin, myricetin, cynidine, procyanidin, naringenin, catechin, hesperetin and kaempferol (Lopez et al., 2013). Among various flavonoids, kaempferol, quercetin, myricetin, and naringenin were observed predominantly in different Phaseolus vulgaris (Lin et al., 2008). In this study, the content of four flavonoids: kaempferol, myricetin, naringenin, and quercetin, were evaluated in 209 Korean common bean landraces. Among four flavonoids, kaempferol had the highest levels and myricetin had the lowest (Table 1). USDA (2013) reported that the content of kaempferol, myricetin, and quercetin in Phaseolus vulgaris ranged from 0.45 to 52.8, 0.13 to 0.33, and 0.01 to 6.82 mg/100 g, respectively. Results showed similar ranges of flavonoid contents to the USDA report.

    Flavonoids and total polyphenols are important secondary plant metabolites present at high levels in plants under stress (Koh et al., 2009;Stanojevic et al., 2009), as they play a role in reducing the oxidative stress caused by reactive oxygen species (ROS) (Patil & Jadhav 2013). In this study, myricetin, quercetin, naringenin, and TFC were positively correlated with antioxidant activities, whereas kaempferol did not show the correlations with antioxidant assay. Gee & Johnson (2001) and Malaveille et al., (1996) reported that flavonoids exert their effects by controlling the mechanisms of ROS production, which may be a positive effect considering the importance of antioxidant agents during oxidative stress. Our results show similar correlations of flavonoids with antioxidant activities, with the exception of kaempferol. The correlation between kaempferol and antioxidant activity was different from other studies which may be related to the complex mixtures in the extracts having distinct activities (Hou et al., 2003;Mensor et al., 2001).

    The Korean common beans landraces antioxidant activity was evaluated using four different methods (Table 1 and additional Table 1). The results showed that each antioxidant method showed a difference in the ranks of antioxidant activities among 209 Korean common bean landraces. Additionally, each method was significantly affected by different phytochemicals (Table 2). Different antioxidants showed substantially varying antioxidative effectiveness in different systems because of a difference in molecular structure (Moharram & Youssef, 2014). Because it is difficult to measure each antioxidant component separately, and because there are interactions among these different antioxidant components in network with each other, several methods were developed to assess the total antioxidant capacity of all the non-enzymatic antioxidant components (Singh & Singh, 2008).

    Sun and Tanumihardjo (2007) reported that each method of measuring antioxidant capacity has its own limitations because multiple reaction mechanisms and different phase locations can affect the measurements. Our results also showed that the rankings of antioxidant activity in the 209 Korean common bean landraces were different when different measurement methods were used. Sun and Tanumihardjo (2007) proposed the relative antioxidant capacity index (RACI). The key advantage of the RACI is that it is a numerical scale that can integrate multiple chemical methods, thus allowing comparison of the antioxidant capacity of a large number of samples. To compare data obtained by different chemical methods to evaluate antioxidant activity, RACI was used. The results of the RACI can be used to select common bean landraces with high antioxidant activity in order to develop new breeding materials.

    The success of crop improvement depends on the genetic variability and the collection of germplasms as raw material for plant breeding (Ferguson, 2007;Upadhyaya et al., 2010). However, germplasm collections are not only to be used by breeders who have different interests and possibly different requirements (van Treuren and van Hintum, 2003). While breeders will focus on characters of immediate perceived value, other biologists may be interested in the potential variation or use of the the collections in understanding the properties and behavior of the plant (Ferguson, 2007). In this study, 209 Korean common bean landraces were evaluated and results showed significant differences in phytochemical compositions and antioxidant activities. The results will contribute to an improved use of these legumes as a functional food crop and/or as a new ingredient in a food diet.

    적 요

    1. 본 연구는 한국 재래종 강낭콩 209자원의 phytochemical 및 항산화활성을 평가하였다.

    2. 항산화활성은 DPPH, ABTS, FRAP, SOD를 분석하였으 며 phytochemical은 kaempferol, myricetin, quercetin, naringenin 함량을 각각 분석하였다.

    3. 항산화활성은 강낭콩 자원 간 다양한 분포를 보였으며 DPPH의 경우 62.3~643.9 (IC50), ABTS의 경우 0.28~1.49 mgAAE/g, FRAP의 경우 0.41~5.44 mgAAE/g, SOD의 경우 50.4 ~ 299.8 (IC50)로 나타났다.

    4. Relative antioxidant capacity index (RACI)로 강낭콩 자원의 항산화활성을 비교한 결과 IT104587이 가장 높은 항 산화활성을 보였으며 IT189598이 가장 낮은 항산화활성을 보 였다.

    5. 분석된 Phytochemical 중에서 한국 재래종 강낭콩에서는 Kaempferol이 가장 높은 함량을 나타냈다.

    6. PCA 분석 결과 209자원은 3개의 그룹으로 나뉘었으며 이중 그룹 III에 속한 46자원의 강낭콩이 낮은 항산화활성 및 phytochemical 함량을 보였다.

    7. 본 연구 결과는 한국 재래종 강낭콩의 항산화활성 및 phytochemical 정보를 제공하며 이 정보는 강낭콩 품종 개발 을 위한 기초 정보로 사용될 수 있을 것이다.

    ACKNOWLEDGMENTS

    This study was carried out with the support of the “Research Program for Agricultural Science & Technology Development (Project No. PJ010883)”. It was also supported by the 2017 Postdoctoral Fellowship Program of the National Institute of Agricultural Science funded by the Rural Development Administration, Republic of Korea.

    Figure

    KSIA-30-357_F1.gif

    The 2D scatter diagram of principal component analysis (PCA) of 209 Korean common bean landraces based on phytochemicals and antioxidant activities.

    Table

    Analysis of variance (ANOVA) and descriptive statistics of 209 Korean common bean landraces for total polyphenol content, total flavonoid content, and antioxidant activities.

    Phytochemical contents and antioxidant acitivities of 209 Korean common bean landraces

    Correlations among total polyphenol content, total phenolic acid content, total flavonoid content, and antioxidant activities of 209 Korean common bean landraces.

    Principal component analysis for the phytochemicals and antioxidant activities of 209 Korean common bean landraces, Eigen values, and percentage variability explained by the first three components.

    Cluster average values for the TPC, TFC, antioxidant activities, and number of accessions per groups.

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