Changes occurring in compositions and antioxidant properties of healthy soybean seeds [Glycine max (L.) Merr.] and soybean seeds diseased by Phomopsis longicolla and Cercospora kikuchii fungal pathogens

Food Chemistry 185 (2015) 205–211

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Changes occurring in compositions and antioxidant properties of healthy soybean seeds [Glycine max (L.) Merr.] and soybean seeds diseased by Phomopsis longicolla and Cercospora kikuchii fungal pathogens Jin Hwan Lee a, Seung-Ryul Hwang a, Yeon-Hee Lee a, Kyun Kim a, Kye Man Cho b,⇑, Yong Bok Lee c,⇑ a b c

Division of Research Development and Education, National Institute of Chemical Safety (NICS), Ministry of Environment, Daejeon 305-343, Republic of Korea Department of Food Science, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea

a r t i c l e

i n f o

Article history: Received 6 December 2014 Received in revised form 24 March 2015 Accepted 25 March 2015 Available online 4 April 2015 Chemical compounds studied in this article: Daidzin (PubChem CID: 107971) Glycitin (PubChem CID: 187808) Genistin (PubChem CID: 5281377) Malonyldaidzin (PubChem CID: 9913968) Malonylglycitin (PubChem CID: 23724657) Acetyldaidzin (PubChem CID: 156155) Acetylglycitin (PubChem CID: 53398650) Malonylgenistin (PubChem CID: 90658001) Daidzein (PubChem CID: 5281708) Glycitein (PubChem CID: 5317750) Acetylgenistin (PubChem CID: 22288010) Genistein (PubChem CID: 5280961)

a b s t r a c t Changes in the compositions (isoflavone, protein, oil, and fatty acid) and antioxidant properties were evaluated in healthy soybeans and soybeans diseased by Phomopsis longicolla and Cercospora kikuchii. The total isoflavone content (1491.3 lg/g) of healthy seeds was observed to be considerably different than that of diseased seeds (P. longicolla: 292.6, C. kikuchii: 727.2 lg/g), with malonlygenistin exhibiting the greatest decrease (726.1 ? 57.1, 351.9 lg/g). Significantly, three isoflavones exhibited a slight increase, and their structures were confirmed as daidzein, glycitein, and genistein, based on their molecular ions at m/z 253.1, 283.0, and 269.1 using the negative mode of HPLC-DAD-ESI/MS. The remaining compositions showed slight variations. The effects against 2,2-diphenyl-1-picrylhydrazyl and 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radicals in healthy seeds were stronger than the diseased soybeans, depending upon the isoflavone level. Our results may be useful in evaluating the relationship between composition and antioxidant activity as a result of changes caused by soybean fungal pathogens. Ó 2015 Elsevier Ltd. All rights reserved.

Keywords: Soybean seed Phomopsis longicolla Cercospora kikuchii Isoflavone Composition Antioxidant activity

1. Introduction In recent years, the consumption of phytochemical-rich foods, including crops, fruits, vegetables, and edible plants, has been associated with the prevention of various chronic diseases (Amin, Kucuk, Khuri, & Shin, 2009; Salma Khanam, Oba, Yanase, & Murakami, 2012). Among numerous sources, the soybean [Glycine max (L.) Merr.] is one of the most important crops because of its ⇑ Corresponding authors. Tel.: +82 55 751 3272; fax: +82 55 751 3279 (K.M. Cho). Tel.: +82 55 772 1967; fax: +82 55 772 1969 (Y.B. Lee). E-mail addresses: [email protected] (K.M. Cho), [email protected] (Y.B. Lee). http://dx.doi.org/10.1016/j.foodchem.2015.03.139 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

beneficial effects on human health, including the prevention of cancer, coronary heart disease, and osteoporosis and its antioxidant properties (Kumar, Rani, Dixit, Pratap, & Bhatnagar, 2010; Scheiber, Liu, Subbiah, Rebar, & Setchell, 2001). Moreover, this species has been widely used in food and industrial applications, due to its high protein and oil concentrations. Many studies have described that the pharmacological activities of soybean are related to its contents of phytochemicals (isoflavone, anthocyanin, phenolic acid, and saponin), protein, and oil (Astadi, Astuti, Santoso, & Nugraheni, 2009; Kalogeropoulos et al., 2010). Specifically, the major phytochemicals, isoflavones, play essential roles in preventing human diseases, due to their antiatherosclerotic, antioxidant,

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and anticancer properties (Antony, Clarkson, Hughes, Morgan, & Burke, 1996). It is well-established that soybean isoflavones have aglycone (daidzein, genistein, and glycitein), glucoside, glucoside malonate, and glucoside acetylate groups (Cho et al., 2013). The content and distribution of twelve individual isoflavones in four forms exhibit remarkable differences based on the cultivar, geographic region, and environmental factors (Hoeck, Fehr, Murphy, & Welke, 2000; Lee, Yan, Ahn, & Chung, 2003). We have recently reported that the isoflavone contents differed significantly according to the storage condition and soybean seed coat colour (Cho et al., 2013; Lee & Cho, 2012). Protein, oil, and fatty acid are also considered valuable precious nutritional components owing to their potential health-promoting effects (Lin, Meijer, Vermeer, & Trautwein, 2004). Based on the above background, researchers have persistently focused on this species for the development of valuable dietary supplements, functional foods, and pharmaceuticals. Soybean is influenced by various factors including planting date, irrigation, year, germination, harvest time, and environment effects (Mengistu & Heatherly, 2006; Mengistu, Smith, Bellaloui, Paris, & Wrather, 2010). Unfortunately, this crop is usually attacked by fungal infections during cultivation, and post-harvest (Mengistu & Heatherly, 2006; Svetaz et al., 2004; Upchurch & Ramirez, 2010). Of the various pathogens, Phomopsis seed infection and purple seed stain were the cause of the most serious soybean diseases due to the reduction of the quality and yield of seeds (Pioli, Benavídez, Morandi, & Bodrero, 2000). These above symptoms are well known as infections of Phomopsis longicolla and Cercospora kikuchii pathogens (Mengistu & Heatherly, 2006; Upchurch & Ramirez, 2010). Soybean seeds infected by P. longicolla appear shriveled, elongated, cracked, and often chalky white and seeds infected by C. kikuchii are discoloured with pink, pale purple, and dark purple (Chen, Lyda, & Halliwell, 1979; Kulik & Sinclair, 1999; Spilker, Schmitthenner, & Ellett, 1981). In particular, it is well documented that P. longicolla was the major fungal pathogen species with the highest isolation in pods, stems, leaves, seeds, and roots of natural plants from hot and humid environments (Bellaloui, Mengistu, Fisher, & Abel, 2012). Furthermore, the previous research evaluated that soybean seeds infected by P. longicolla had lower phenolic compounds than healthy seeds (Bellaloui, Mengistu, & Zobiole, 2012). Although many studies have evaluated the isoflavone, protein, oil, and fatty acid contents, as well as the beneficial health properties of the soybean, there are few reports examining the variation of the composition and the antioxidant activities by fungal pathogens. Thus, our research was designed to evaluate the components and radical scavenging capacities of healthy and diseased seeds as important information on the nutritional quality of the soybean. The primary purpose of the present work was to investigate and compare the contents of four different components (isoflavone, protein, oil, and fatty acid) of healthy soybean seeds and soybean seeds diseased by P. longicolla and C. kikuchii pathogens. In addition, we evaluated for the first time the variations of antioxidant properties against DPPH and ABTS radicals caused by fungal pathogens.

P. longicolla and C. kikuchii pathogens were also collected in the same region (Fig. 1). Analytical grade water, acetonitrile, and acetic acid were purchased for HPLC analysis from J.T. Baker (Phillipsburg, NJ, USA). For quantitative analysis, isoflavone aglycone and glucoside standards were isolated from soybean seeds, as reported in our earlier study (Lee & Cho, 2012). The remaining isoflavones, acetyl and malonyl glucosides, were purchased from Fujicco Co. (Ltd. Nacalai Tesque Inc., Kobe, Japan). Fatty acid standards including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) methyl esters were obtained from Merck (Darmstadt, Germany). 2,2-Diphenyl-1picrylhydrazyl (DPPH), 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), butylated hydroxytoluene (BHT), 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and potassium persulphate were obtained from Sigma Chemical Co. (St. Louis, MO, USA). A genomic DNA Extraction Kit was purchased from Intron Biotechnology (Suwon, Korea). All other reagents were of analytical grade and were purchased from Sigma Chemical. 2.2. Instruments To measure antioxidant activity, UV–vis absorption spectra were measured on a Beckman DU650 spectrophotometer (Beckman Coulter, Fullerton, USA). Isoflavone contents were analysed using an Agilent 1100 series (Boeblingen, Germany) with a quaternary pump, Agilent 1100 series diode-array detector, and 1100 well plate autosampler. The mass data of isoflavone aglycones were obtained using an Equire 4000 LC/MS system (Bruker Daltonick GmbH, Bremen, Germany). The protein and oil contents were determined using a B-339 Auto Kieldahl analyser (Buchi, Schweiz) and a BUCHI B-811 Extraction System (Buchi, Schweiz). Fatty acid content was analysed using a gas chromatograph (Agilent 7890A series, Boeblingen, Germany). 2.3. Isolation and 26S rRNA sequence analysis of fungal isolates Two fungi strains were isolated from soybean seeds in the experimental field of the NICS, Milyang, Gyeongnam (Fig. 1). For isolation of the total genomic DNA, two isolates were prepared by inoculating a loop of fungal mass into a 250 ml Erlenmeyer flask containing 50 ml of PDB and then incubating at 25 °C with shaking at 150 rpm for 5 days. Filtered mycelia were freeze-dried and were then subjected to DNA extraction using a Genomic DNA Extraction Kit. The extracted DNA was then used as a template for PCR to amplify 26S rRNA. The PCR amplification, ligation, transformation, plasmid purification, and nucleotide sequence were confirmed as

2. Materials and methods 2.1. Plant material and chemicals The soybean cultivar (cv. Daemangkong) was developed by the National Institute of Crop Science (NICS), Rural Development Administration (RDA), Korea. This cultivar was grown in the experimental field of the NICS, Milyang, Gyeongnam, in 2011. After harvesting, the healthy seeds were dried under natural light and stored at 40 °C until analysis. The seeds diseased by

A

B

C

Fig. 1. Healthy and infected soybean seeds (cv. Daemangkong): (A) healthy seeds; (B) diseased seeds (P. longicolla); (C) diseased seeds (C. kikuchii).

J.H. Lee et al. / Food Chemistry 185 (2015) 205–211

described previously (Cho et al., 2009). The 26S rRNA sequence identity searches were performed using the BLSATN and PSIBLAST tools on the NCBI website.

2.4. Preparation of calibration curve and sample for isoflavone quantification To prepare the calibration curves, the peak areas of the isoflavone standards on the HPLC chromatogram at 254 nm were integrated and plotted against the concentration to create a linear curve. The standard stock solutions were prepared in DMSO to obtain a concentration of 1000 lg/ml. Calibration curves were prepared by dilution of the individual stock solutions in DMSO to nine different concentrations (1, 2, 5, 10, 20, 40, 60, 80, and 100 lg/ml), and their correlation coefficients (r2) were higher than 0.998. For the sample preparation, the soybean seeds were grounded for 5 min using an HR 2860 coffee grinder (Philips, Drachten, Netherlands). The powdered sample (1.0 g, 60 mesh) was extracted using 20 ml of 80% methanol in a shaking incubator at room temperature for 6 h. The supernatant was centrifuged at 3000g for 5 min and filtered through a 0.45 lm syringe filter (Whatman Inc., Maidstone, UK) prior to the HPLC analysis.

2.5. HPLC and HPLC-DAD-ESI/MS analyses For the chromatographic separations of the individual isoflavones, a sample (20 ll) of the 80% methanol extract was injected into an analytical C18 column, (Lichrophore 100 RP-18e, 5 lm, 125 mm  4 mm, Merck KGaA, Darmstadt, Germany), and the column temperature was set to 25 °C. A gradient of the mobile phase was performed with 0.1% acetic acid (v/v) in water (elution A) and acetonitrile (elution B). The gradient was set as follows: 020 min, 20% B; 30 min, 25% B; 40 min, 35% B, and then held for 10 min before returning to the initial conditions. The flow rate was 1 ml/ min and the detection was made at 254 nm. The mass spectrometry of the isoflavone aglycones was performed as follows: capillary voltage 300 V; fragmentation voltage, 80 V; drying gas temperature, 300 °C; gas flow (N2), 2 l/min; and nebuliser pressure, 50 psi. The instrument was operated in the negative ion mode scanning from m/z 100 to m/z 350 at a scan rate of 1.5 s/cycle. The separation methods for mass analysis were conducted using the above mentioned HPLC conditions.

2.6. Protein, oil, and fatty acid analyses Protein, oil, and fatty acid were analysed by the method of Cho et al. (2013). To measure the protein content, the pulverised seeds (0.2 g) were digested by a Buchi B-435 digestion system and a Buchi B-412 scrubber with H2SO4 (20 ml) and catalyst (3.0 g, CuSO4:K2SO4 = 1:9, w/w). This content was analysed using Kjeldahl nitrogen in an automated distillation unit (Büchi 339) and was calculated as the percentage of nitrogen, multiplied by 6.25. Oil content was determined by a Buchi B-811 extraction system. In short, the crushed seeds (2.0 g) were added to n-hexane (200 ml) in an extraction thimble and boiled at 105 °C for 2 h. The mixture was cooled to room temperature in a desiccator, and then weighed. The total oil content was calculated on a soybean seed dry mass basis. Fatty acid compositions were measured by methylation of the extracted fat using a H2O:MeOH:toluene mixture (1:20:10, v/v/v) (Cho et al., 2013). Individual fatty acid methyl esters were analysed using the gas chromatography conditions in our previous study (Cho et al., 2013). The contents were calculated as the percentage of the total fatty acids.

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2.7. Antioxidant activity The powdered seeds (1.0 g, 60 mesh) were extracted with 80% methanol (20 ml) for 24 h at room temperature. The crude extract was filtered through a Whatman No. 42 filter paper to remove the sediment, and the supernatant was instantly assayed for radical scavenging activity. The DPPH radical scavenging activity was analysed using the method described by Cho et al. (2013). Shortly, samples (0.1 ml) of various concentrations were mixed with methanol (0.49 ml) and 1 mM DPPH methanolic solution (0.39 ml). The mixture was vortexed and incubated for 30 min at 25 °C in darkness. The absorbance was measured at 517 nm by a spectrophotometer, and BHT was used as a positive control. This activity was expressed as a percentage using the following formula: DPPH radical scavenging effect (%) = (1  absorbance of sample/absorbance control)  100. The scavenging effect against ABTS radical was defined as the ability of different substances to scavenge the ABTS+ radical cation (Cho et al., 2013) in comparison with Trolox (standard material). ABTS+ was dissolved in ethanol to a concentration of 7 mM. This radical cation was generated by reacting the ABTS+ stock solution with 2.45 mM potassium persulfate, and the mixture was maintained for 10–14 h in darkness. The ABTS+ stock solution was melted with ethanol to an absorbance of 0.70 at 734 nm. The ABTS+ solution (0.9 ml) was mixed with the sample (0.1 ml) and was then measured using a spectrophotometer. This activity was determined as a percentage by the following formula: ABTS+ scavenging effect (%) = [(absorbance of control  absorbance of sample)/absorbance of control]  100. 2.8. Statistical analysis All the measurements were made in triplicate. The results were subject to variance analysis using Sigma Plot 2001 (Systat Software Inc., Chicago, IL, USA) and the contents of the nutritional components were expressed as the mean ± SD (standard deviations).

3. Results and discussion 3.1. Identification of P. longicolla and C. kikuchii The highest 26S rRNA sequence similarities (100%) are observed between two strains (SCL01 and SCL02), P. longicolla STAM 64 and C. kikuchii CK35. The phylogenetic study clearly establishes that the strains SCL01 and SCL02 are closely related to P. longicolla and C. kikuchii, respectively (data not shown). 3.2. Identification of isoflavone in healthy and diseased soybean seeds As presented in Fig. 2, soybean seeds have twelve isoflavones of four groups: aglycone, glucoside, malonylglucoside, and acetylglucoside (Cho et al., 2013; Lee, Yan, et al., 2003). On the basis of individual standards, a representative HPLC chromatogram of soybean isoflavones was produced within 35 min at a wavelength of 254 nm (Fig. 4A). The identification of each peak was confirmed using published data (Cho et al., 2013; Lee, Yan, et al., 2003) and the retention time in the chromatogram. The isoflavone’ retention times are as follows: peak 1 (daidzin, tR = 9.9 min), peak 2 (glycitin, tR = 9.9 min), peak 3 (genistin, tR = 15.8 min), peak 4 (malonyldaidzin, tR = 16.5 min), peak 5 (malonylglycitin, tR = 17.6 min), peak 6 (acetyldaidzin, tR = 21.4 min), peak 7 (malonylgenistin, tR = 22.1 min), peak 8 (acetylglycitin, tR = 25.4 min), peak 9 (daidzein, tR = 25.4 min), peak 10 (glycitein, tR = 27.9 min), peak 11 (acetylgenistin, tR = 28.9 min), and peak 12 (genistein, tR = 34.5 min).

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R3

Category Aglycone

O HO

C H2 C C O

O

tyl Ace ide cos Glu

R1

R2

R3

H H OCH3

H OH H

H H H

H H OCH3

H OH H

C6O5H11 C6O5H11

Acetyldaidzin Acetylgenistin Acetylglycitin

H H

H

C6O5H11+COCH3

OH

C6O5H11+COCH3

OCH3

H

C6O5H11+COCH3

Malonyldaidzin Malonylgenistin Malonylglycitin

H H

H OH

C6O5H11+COCH2COOH

OCH3

H

C6O5H11+COCH2COOH

Glucoside Daidzin

O O OH HO OH R2

l ony Mal

Isoflavone Daidzein Genistein Glycitein Genistin Glycitin

O Acetyl

R1 O

OH Malonyl

Aglycone

C6O5H11

C6O5H11+COCH2COOH

In general, the HPLC system coupled with mass spectrometry was an excellent method for the peak assignment and determination of compounds (Lee & Cho, 2012). To identify the exact molecular weights, the increased isoflavones in seeds diseased by P. longicolla and C. kikuchii pathogens were analysed using the negative ionisation mode of HPLC-DAD-ESI/MS analysis. Their molecular ions were observed at m/z 253.1 (Fig. 3A), 283.0 (Fig. 3B), and 269.1 (Fig. 3C) and were tentatively identified as isoflavone aglycones, including daidzein, glycitein, and genistein. Moreover, these peaks were confirmed by comparison with previously reported research (Lee & Cho, 2012).

Relative intensity (%)

Fig. 2. Classes and chemical structures of isoflavones in soybean.

3.3. Comparison of isoflavone contents in healthy and diseased soybean seeds

Relative intensity (%)

m/z

-

[M-H]

[M-{(CH3)-H}]

-

m/z

Relative intensity (%)

The soybean has many valuable phytochemicals, including isoflavones, saponins, flavonoids, triterpenes, and phenolic acids (Astadi et al., 2009; Kalogeropoulos et al., 2010). For this reason, numerous researchers have focused on the phytochemical composition that leads to the beneficial properties of the soybean. However, previous studies have not extensively demonstrated the variation in the isoflavone content caused by fungal pathogens. The present work was the first to evaluate the changes of isoflavones by P. longicolla and C. kikuchii pathogens as the most serious soybean diseases. A typical HPLC chromatogram of isoflavones (1491.3 lg/g) in Daemangkong (healthy seeds), a Korean soybean cultivar, is shown in Fig. 4B. Among the twelve isoflavones, malonylgenistin (7) had the highest content with 726.1 ± 3.6 lg/ g, followed by malonyldaidzin (4) (551.8 lg/g), daidzein (1) (104.3 lg/g), genistin (3) (82.7 lg/g), and malonylglycitin (5) (24.3 lg/g), while acetylisoflavone derivatives exhibited the lowest contents (ND: not detected) (Table 1). In more detail, the malonylglucoside form was the predominant compound, representing approximately 87% of the total isoflavone content, and the remaining isoflavone groups exhibited the following order: glucoside form > aglycone form > acetylglucose form. The soybean isoflavones generally present in the order of malonylglucoside (70– 80%), glucoside (25%), acetylglucoside (5%), and aglycone groups (2%) (Yamabe, Kobayashi-Hattori, Kaneko, Endo, & Takita, 2007). According to the results shown above, the soybean isoflavone content may be positively correlated with environmental factors (light, temperature, agronomic condition, year, region, moisture) and cultivars (Hoeck et al., 2000; Lee, Yan, et al., 2003). The HPLC chromatogram of the diseased seeds by P. longicolla is presented in Fig. 4C, and the individual isoflavones show remarkable variation upon comparison with the healthy seeds. The total isoflavone content (292.6 lg/g) was 5 times lower than that of healthy seeds. In particular, the malonylgenistin (726.1 ? 57.1 lg/g) and malonyldaidzin (551.8 ? 43.1 lg/g) contents decreased

-

[M-H]

-

[M-H]

m/z Fig. 3. Mass fragmentation patterns of identified isoflavone aglycones, (A) daidzein; (B) glycitein; (C) genistein.

significantly by 12.7 and 12.8 times, respectively (Table 1). Interestingly, the isoflavone aglycones, of daidzein, genistein, and glycitein, showed slight increases of 2.1 ? 16.8, trace ? 22.5, and

209

Relative abundance (mAU)

Relative abundance (mAU)

J.H. Lee et al. / Food Chemistry 185 (2015) 205–211

8

Retention time (min)

Relative abundance (mAU)

Relative abundance (mAU)

Retention time (min)

8

8

Retention time (min)

Retention time (min)

Magnified chromatograms B

C

D

Fig. 4. HPLC chromatograms of twelve isoflavone derivatives in soybean seeds, 1. daidzin, 2. glycitin, 3. genistin, 4. malonyldaidzin, 5. malonylglycitin, 6. acetyldaidzin, 7. acetylglycitin, 8. malonylgenistin, 9. daidzein, 10. glycitein, 11. acetylgenistin, and 12. genistein. (A) Isoflavone standard mixture; (B) healthy seeds; (C) diseased seeds by P. longicolla; (D) diseased seeds by C. kikuchii.

ND ? 6.1 lg/g, respectively. This phenomenon is similar to the results of previous research on phytochemicals in infected wheat (Ghassempour, Mollayi, Farzaneh, Sharifi-Tehrani, & Aboul-Enein, 2011). The isoflavone contents and the HPLC chromatogram of seeds diseased by the C. kikuchii pathogen are presented in Fig. 4D and Table 1. Although the total isoflavone content primarily decreased after the infection of this pathogen (1491.3 ? 727.2 lg/ g), a slight increase was also observed in three isoflavone aglycones (daidzein: 2.1 ? 7.6 lg/g; genistein: trace ? 0.7 lg/g; glycitein: ND ? trace), as was also the case for the P. longicolla pathogen. Malonlygenistin showed the most remarkable difference (726.1 ? 351.9 lg/g) among the 12 isoflavones, and malonyldaidzin exhibited the second most significant variation with 551.8 ? 222.0 lg/g. As a result, malonylglucoside type was observed the highest variation in P. longicolla and C. kikuchii pathogens. It is commonly demonstrated that malonlyglucoside type is the highest isoflavone contents in soybean seeds and concentrations of acetylglucoside, glucoside, aglycone types tend to increase during heating, processing, cooking, and fermentation (Coward, Smith, Kirk, & Barnes, 1998; Shao et al., 2009). Briefly, malonylglucoside could be converted to glucoside or aglycone types from various phenomenon, such as innate instability (Coward et al., 1998; Lee, Ahn, et al., 2003; Shao et al., 2009). Also, the decrease ration of malonylglucoside in our results may be caused by sample condition and storage period until analysis. Therefore, changes in

soybean isoflavone contents may not necessarily be due to the influence of the fungal pathogen attack. Several studies exhibited that the compositions and contents of isoflavones showed significant differences according to the soybean parts, including the seed, seed coat, embryo, and cotyledon (Kim et al., 2007; Lee & Cho, 2012). In addition, many researchers have suggested that the phytochemical compositions and contents in various crops were observed remarkable variations as a result of fungal pathogens (Bruno & Sparapano, 2006; Svetaz et al., 2004). Therefore, our research provides important information on isoflavone fluctuations and fungal pathogens in soybean seeds. Furthermore, isoflavone may be a key factor and a useful ingredient in determining the quality of diseased soybean seeds. We demonstrate for the first time the variations of isoflavone content in healthy and diseased soybean seeds by comparison with previously reported results. 3.4. Comparisons of protein, oil, and fatty acid contents in healthy and diseased soybean seeds It has been established that protein, oil, and fatty acid are important nutritional components responsible for the various health benefits of the soybean (Chatterrjee & Bhattacharjee, 2013; Rayaprolu, Hettiarachchy, Chen, Kannan, & Mauromostakos, 2013; Yuan, Ren, Zhao, Luo, & Gu, 2012).

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Table 1 Comparison of isoflavone contents in healthy and diseased seeds. Soybean seeds

Isoflavone content (lg/g)a Glucoside

Healthy seeds Diseased seeds (P. longicolla) Diseased seeds (C. kikuchii)

Malonylglucoside

Acetylglucoside

Aglycone

Din

Gly

Gin

MDin

MGly

MGin

AcDin

AcGly

AcGin

Dein

Glein

Gein

104.3 ± 1.6 75.9 ± 18 69.7 ± 1.3

NDb ND ND

82.7 ± 1.6 71.1 ± 0.9 66.1 ± 0.8

551.8 ± 3.1 43.1 ± 0.3 222.0 ± 2.3

24.3 ± 0 .5 tr 9.2 ± 0.7

726.1 ± 3.6 57.1 ± 0.6 351.9 ± 2.2

ND ND ND

ND ND ND

2.1 ± 0.2 16.8 ± 0.4 7.6 ± 0.5

ND 6.1 ± 0.6 tr

ND ND ND

trc 22.5 ± 0.7 0.7 ± 0.1

Di, daidzin; Gly, glycitin; Gi, genistin; MDi, malonyldaidzin; MGly, malonylglycitin; AcDi, acetyldaidzin; MGi, malonylgenistin; AcGly, acetylglycitin; De, daidzein; Gle: glycitein; AcGi: acethylgenistin; Ge: genistein. a All values are expressed as mean ± SD of triplicate experiments on dry weight basis. b ND, not detected. c tr, trace.

Table 2 Comparisons of protein, oil, and fatty acid composition in healthy and diseased seeds.

a

Soybean seeds

Protein (%)a

Oil (%)a

Healthy seeds Diseased seeds (P. longicolla) Diseased seeds (C. kikuchii)

43.2 ± 2.3 40.5 ± 0.7 43.0 ± 1.1

20.4 ± 1.8 17.4 ± 1.2 20.3 ± 0.9

Fatty acid composition (%)a C16:0

C18:0

C18:1

C18:2

C18:3

10.9 ± 0.6 10.1 ± 0.3 9.9 ± 0.5

3.6 ± 0.3 3.8 ± 0.6 3.6 ± 0.3

24.7 ± 1.2 25.0 ± 1.5 25.2 ± 0.7

53.1 ± 2.3 51.9 ± 2.7 52.7 ± 1.8

5.9 ± 0.2 6.4 ± 0.8 6.0 ± 0.7

All values are expressed as mean ± SD of triplicate experiments on dry weight basis.

Moreover, this crop is renowned as a rich source of primary metabolites, including average values of approximately 30–42% protein and 18–20% oil (Brummer, Graef, Orf, Wilcox, & Shoemaker, 1997). Although earlier works showed variations in the contents of the components (Kim et al., 2007; Lee & Cho, 2012) of the soybean, little data have been reported on the effect on their content of fungal pathogens. As illustrated in Table 2, the protein and oil contents exhibited slight fluctuations, but showed no considerable differences between healthy and diseased soybean seeds. In healthy seeds, protein and oil were measured as 43.2% and 20.4%, and their contents were similar to previously reported data (Cho et al., 2013; Lee & Cho, 2012). The contents of protein and oil in seeds diseased by the P. longicolla pathogen showed slight decreases (protein: 43.2 ? 40.5%; oil: 20.4 ? 17.4%) relative to healthy seeds. The soybean seeds infected by C. kikuchii also exhibited little differences in protein (43.2 ? 43.0%) and oil (20.4 ? 20.3%) contents (Table 2). These results indicate that protein and oil may be not essential factors for the evaluation of the quality of soybean seeds by fungal pathogens. In the fatty acid analysis, the individual and total contents showed slight variations between the healthy and diseased seeds (Table 2). The most abundant individual composition was detected for C18:2 (51.9–53.1%), followed by C18:1 (24.7–25.2%), C16:0 (9.9–10.9%), and C18:3 (5.9–6.4%), whereas C18:0 showed the lowest content with a range of 3.6–3.8%. Furthermore, the total unsaturated fatty acid content (>75%) was considerably higher than that of the saturated fatty acid (<25%), as shown in earlier work (Lee & Cho, 2012). As a result, our research did not exhibit important differences in the protein, oil, and fatty acid contents between healthy and diseased soybean seeds. Protein, oil, and fatty acid also exhibited no remarkable differences between cultivars and environmental conditions, as previous literatures (Cho et al., 2013; Lee & Cho, 2012). However, the present work was the first to investigate protein, oil, and fatty acid contents in soybean seeds infected with fungal pathogens. 3.5. Comparisons of antioxidant activities on DPPH and ABTS radicals of healthy and diseased soybean seeds The scavenging activities against DPPH and ABTS radicals have been commonly used to evaluate the levels of antioxidants in crops, vegetables, and fruits (Cho et al., 2013; Lee & Cho, 2012).

Many researchers have focused on the antioxidant capacities of the soybean due to its therapeutic and preventative properties with respect to human health. Unfortunately, the earlier works have not investigated the variation of the antioxidant activities upon infection by soybean fungal pathogens. Thus, this study examined the antioxidant capacities using DPPH and ABTS radical scavenging methods on soybean seeds infected by P. longicolla and C. kikuchii pathogens. To measure the antioxidant properties, we used the percentage inhibition of DPPH and ABTS radicals from a 80% methanol extract of soybean seeds, as described in previous study (Cho et al., 2013). The DPPH radical scavenging effects of BHT (positive control) as well as the extracts of healthy and diseased seeds increased with increasing concentration. BHT showed a higher radical scavenging effect than did the healthy and diseased seeds. The antioxidant abilities exhibited significant differences, and the inhibition percentages of potent free radical scavengers were 87 (healthy seeds), 60 (diseased seeds by C. kikuchii), and 47% (diseased seeds by P. longicolla), respectively, at a concentration of 1.0 mg/ml. This phenomenon suggests that the isoflavone contents in the extracts of healthy and diseased seeds may be responsible for the major portion of antioxidant properties through radical scavenging activities, as described in previous research (Akitha Devi et al., 2009). Other phenolic compounds of soybean seeds may also be important factors in determining the DPPH radical scavenging abilities (Akitha Devi et al., 2009; Alu’datt, Rababah, Ereifej, & Alli, 2013). The scavenging activity against ABTS radical was compared with the Trolox (positive control) value (Cho et al., 2013; Lee & Cho, 2012). The 80% methanol extracts of all samples had lower effects than Trolox, and their activities increased with increasing concentration, as was also observed in the results obtained from the DPPH assay. Furthermore, all sample extracts exhibited slightly higher ABTS radical abilities than the results on DPPH radical. This suggests that the DPPH radical inhibition may be attributed to the scavenging activities of hydrogen donating antioxidants in phenolic compounds of soybean seeds, while ABTS radical inhibition may be correlated with the scavenging capacities of hydrogen donating as well as chain breaking antioxidants (Choi, Jeong, & Lee, 2007). The healthy and diseased seeds exhibited remarkable differences in ABTS radical scavenging activities, and their effects were as follows: healthy seeds (93%) > seeds infected by C. kikuchii (69%) > seeds infected by P. longicolla (54%), at a concentration of

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1.0 mg/ml. The loss of the ABTS radical scavenging effects by fungal pathogens may be correlated with the decrease in isoflavones, as was reported in the data on DPPH radical (Akitha Devi et al., 2009; Alu’datt, Rababah, Ereifej, & Alli, 2013; Cho et al., 2013). Consequently, the soybean seeds infected by fungal pathogens were observed to have lower antioxidant properties against DPPH and ABTS radicals owing to their lower isoflavone contents in comparison with healthy seeds. 4. Conclusion This research has evaluated for the first time the nutritional components, including isoflavone, protein, oil, and fatty acid, in healthy soybean seeds and soybean seeds infected by P. longicolla and C. kikuchii fungal pathogens. The total isoflavone content (1491.3 lg/g) in healthy seeds markedly decreased in comparison with seeds diseased by P. longicolla (292.6 lg/g) and C. kikuchii (727.2 lg/g), whereas the protein, oil, and fatty acid showed only slight variations. Isoflavone aglycones, daidzein, glycitein, and genistein, showed slight increase, and the main isoflavone, malonylgenistin (726.1 lg/g), exhibited the greatest decreases of 57.1 and 351.9 lg/g for P. longicolla and C. kikuchii pathogens, respectively. In addition, the antioxidant capacities against DPPH and ABTS radicals of diseased seeds were significantly lower than those of healthy seeds. Based on the above data, we believe that fungal pathogens, such as P. longicolla and C. kikuchii, may lead to a great decrease of isoflavone content in soybeans, which is an important component of their quality and antioxidant properties. Future studies are needed to investigate other phytochemicals that provide potential antioxidant properties that may be affected by fungal pathogens in the soybean. Acknowledgement This study was supported by Rural Development Administration, Repulic of Korea (Project No. PJ906961). References Akitha Devi, M. K., Gondi, M., Sakthivelu, G., Giridhar, P., Rajasekaran, T., & Ravishankar, G. A. (2009). Functional attributes of soybean seeds and products, with reference to isoflavone content and antioxidant activity. Food Chemistry, 114, 771–776. Alu’datt, M. H., Rababah, T., Ereifej, K., & Alli, I. (2013). Distribution, antioxidant and characterisation of phenolic compounds in soybeans, flaxseed and olives. Food Chemistry, 139, 93–99. Amin, A. R. M., Kucuk, O., Khuri, F. R., & Shin, D. M. (2009). Perspectives for cancer prevention with natural compounds. Journal of Clinical Oncology, 27, 2712–2725. Antony, M. S., Clarkson, T. B., Hughes, C. L., Morgan, T. M., & Burke, G. L. (1996). Soybean isoflavones improve cardiovascular risk factors without affecting the reproductive system of peripubertal rhesus monkeys. Journal of Nutrition, 126, 43–50. Astadi, I. R., Astuti, M., Santoso, U., & Nugraheni, P. S. (2009). In vitro antioxidant activity of anthocyanins of black soybean seed coat in human low density lipoprotein (LDL). Food Chemistry, 112, 659–663. Bellaloui, N., Mengistu, A., Fisher, D. K., & Abel, C. A. (2012). Soybean seed composition as affected by drought and Phomopsis in Phomopsis susceptible and resistant genotypes. Journal of Crop Improvement, 26, 428–453. Bellaloui, N., Mengistu, A., & Zobiole, L. H. S. (2012). Phomopsis seed infection effects on soybean seed phenol, lignin, and isoflavones in maturity group V genotypes differing in Phomopsis resistance. Journal of Crop Improvement, 26, 693–710. Brummer, E. C., Graef, G. L., Orf, K., Wilcox, J. R., & Shoemaker, R. C. (1997). Mapping QTL for seed protein and oil content in eight soybean populations. Crop Science, 37, 370–378. Bruno, G., & Sparapano, L. (2006). Effects of three esca-associated fungi on Vitis vinifera L.: II. Characterization of biomolecules in xylem sap and leaves of healthy and diseased vines. Physiological and Molecular Plant Pathology, 69, 195–208. Chatterrjee, D., & Bhattacharjee, P. (2013). Comparative evaluation of the antioxidant efficacy of encapsulated and un-encapsulated eugenol-rich clove extracts in soybean oil: Shelf-life and frying stability of soybean oil. Journal of Food Engineering, 117, 545–550. Chen, M. D., Lyda, S. D., & Halliwell, R. S. (1979). Infection of soybeans with conidia of Cercospora kikuchii. Mycologia, 71, 1158–1165.

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