Food Science and Technology Research
Online ISSN : 1881-3984
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Original papers
Effect of Bean Extract of Yabumame (Amphicarpaea bracteata (L.) Fernald subsp. edgeworthii (Benth.) H.Ohashi) on Low-Density Lipoprotein Oxidation In Vitro
Lifeng YangJyunichi KirikoshiShogo SekimotoMikako TakasugiKenji FukunagaRyota HosomiAtsuyuki HishidaNobuo KawaharaTakashi YamagishiHirofumi Arai
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2015 Volume 21 Issue 4 Pages 589-596

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Abstract

Yabumame, a legume family member, similar to hog-peanut, is an important traditional food for the Ainu people of Japan. However, its nutritional components and bioactivities are unknown. It has been suggested that low-density lipoprotein (LDL) oxidation by reactive oxygen species (ROS) may be a key factor in the onset of atherosclerosis. In this study, we evaluated the antioxidant activity of yabumame extract (YE) against LDL oxidation in vitro. Total polyphenol content in YE was 1.9%. YE showed 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity in solution. YE suppressed lipid peroxidation in LDL induced by free radicals or transition metal ions. YE also inhibited the oxidative modification of apolipoprotein B-100 in the oxidized LDL. The results suggest that YE can suppress LDL oxidation by ROS that leads to atherosclerosis.

Introduction

Low-density lipoprotein (LDL) is the particle which consists of apolipoprotein B-100 (apo B-100) and lipids such as phospholipids, cholesterol, cholesterol esters, and triacylglycerol. Apo B-100 contributes to the solubility of LDL and lipid metabolism in the circulatory system. Inappropriate dietary habits or genetic predispositions can induce excess LDL levels in the blood and vascular intima. It has been suggested that LDL oxidation in the vessel wall by reactive oxygen species (ROS), such as superoxide anions, hydroxyl radicals, peroxyl radicals, and peroxynitrite may be an early event in the development of atherosclerosis; however, the mechanisms of LDL oxidation in vivo remain unclear (Halliwell and Gutteridge, 2007; Peluso et al., 2012). The major targets of ROS in LDL are polyunsaturated fatty acid residues of phospholipids, cholesterol esters, and triacylglycerol (Arai, 2014). In lipid peroxidation of LDL induced by ROS, lipid hydroperoxides are accumulated through radical chain reactions. The lipid hydroperoxides are decomposed in the presence of transition metal ions such as copper, and various aldehydes are generated as secondary products. The lipid hydroperoxides and aldehydes can modify apo B-100, leading to dysfunction. Oxidized LDL (oxLDL) is scavenged by monocyte-derived macrophages through scavenger receptors, which become foam cells (Moore and Tabas, 2011). The foam cells develop into fatty streaks and then fibrous plaques, which are involved in the pathogenesis of atherosclerosis.

The intake of plant polyphenols from food and its mitigating effect on the oxidative stress associated with many kinds of diseases, including atherosclerosis, has been investigated (Riegsecker et al., 2013; Terao et al., 2008; Uysal et al., 2013). Soybeans, a popular and widely used edible bean cultivated worldwide, contain abundant phenolic compounds, including isoflavones such as daidzin and genistin. Several research groups have reported that soybean extract exerts antioxidant activity and that phenolic compounds may contribute to this activity (Georgetti et al., 2006; Malencic et al., 2008). Soybean isoflavones such as daidzein and genistein can inhibit LDL oxidation by breaking the chain reaction of lipid oxidation through scavenging ROS (Kerry and Abbey, 1998; Lee et al., 2005). In addition, the antioxidant activity of black soybean against LDL oxidation was stronger than that of yellow soybean (Astadi et al., 2009; Takahashi et al., 2005). Yabumame (Amphicarpaea bracteata (L.) Fernald subsp. edgeworthii (Benth.) H.Ohashi) is an annual vine in the legume family, similar to hog-peanut (American wild peanut), and is mainly distributed in Japan (Figure 1A) (Iwatsuki et al., 2001). The edible portion of yabumame is an elliptic underground bean with dark spots (Figure 1B). Yabumame bean has been consumed as an important traditional food for the Ainu people; however, its nutritional components and bioactivities are unknown. In the present study, we analyzed the nutritional composition of yabumame beans and investigated the antioxidant effect of yabumame bean extract (YE) on lipid peroxidation and apo B-100 modification in LDL oxidized by ROS in vitro.

Fig. 1.

The gross plant morphology (A) and edible underground seeds (B) of yabumame.

Materials and Methods

Chemicals    2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), and 2,4-dinitrophenylhydrazine were purchased from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals were of reagent grade.

Nutritional composition analysis    All analytical methods were based on the Japanese Agricultural Standards. The moisture and crude ash contents of freeze-dried yabumame were analyzed by the atmospheric heat drying method and the burning method, respectively. The amounts of crude protein and crude fat in the samples were determined by the Kjeldahl method and the Soxhlet method, respectively.

Preparation of YE    Yabumame (Amphicarpaea bracteata (L.) Fernald subsp. edgeworthii (Benth.) H.Ohashi) was cultivated at the Division of Hokkaido, Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation (accession number: 14865-03HK), and the underground beans were obtained. The powder of freeze-dried beans (250 g) was mixed with 2 L of 80% methanol and incubated at 65°C for 2 h with continuous shaking. The suspension was centrifuged at 3,400 × g for 5 min at room temperature, and the supernatant was collected. A second extraction from the precipitate was carried out by the same procedure, and the supernatants were combined and filtered. Finally, the sample solvent was evaporated and then freeze-dried, and employed as YE. YE was resuspended in 70% ethanol and stored at −20°C until use.

Determination of total polyphenols    Total polyphenol content of YE was determined by the Folin-Denis method (Folin and Denis, 1915) with a slight modification. Briefly, YE diluted in 1 mL of water was mixed with 1 mL of Folin reagent (Kanto Chemical Co. Inc., Tokyo, Japan). The mixture was incubated at room temperature for 3 min, and then 1 mL of 10% Na2CO3 was added. After 30 min-incubation at 30°C, absorbance at 760 nm was measured. The total polyphenol content was expressed as gallic acid equivalent.

Radical scavenging assay    Free radical scavenging activity of YE in organic solvent was analyzed using DPPH (Blois, 1958). DPPH (200 µM) dissolved in 400 µL of methanol was mixed with 400 µL of YE methanol solution. The reaction mixture was incubated at room temperature for 30 min in the dark, and absorbance at 517 nm was then measured.

Preparation of human LDL    Human blood (200 mL) was freshly obtained, with 0.4% citric acid as the anti-coagulant, from a healthy volunteer fasted for more than 12 h; informed consent was obtained from the participant. Plasma was isolated by centrifugation at 1,200 × g for 20 min at 4°C. LDL was obtained from the plasma using the discontinuous density-gradient ultracentrifugation method (Hatch, 1968). Eight milliliters of KBr solution (1.019 g/mL) was layered on 16 mL of the plasma in the centrifuge tube. After centrifugation at 100,000 × g for 16 h at 16°C, the supernatant including chylomicrons, very-low-density lipoprotein, and intermediate-density lipoprotein was removed. The density of the lower layer was adjusted to 1.063 g/mL by adding KBr. Then, 21 mL of the lower layer was covered with 3.5 mL of KBr solution (1.063 g/mL) and centrifuged at 100,000 × g for 20 h at 16°C. The LDL fraction (density 1.019 – 1.063 g/mL) was obtained in the supernatant and was ultrafiltered (MWCO 100 kDa) to remove albumin at 1,200 ×g for 1 h at 4°C. Thereafter, LDL was dialyzed against phosphate buffered saline (PBS, pH 7.4) treated with chelating resin (Chelex 100; Sigma-Aldrich Corporation, St. Louis, MO, USA) at 4°C for 24 h. Protein concentration of the LDL fraction was determined by Lowry's method (Lowry et al., 1951). The LDL fraction was stored at 4°C under nitrogen until use.

Peroxyl radical-mediated oxidation of LDL    LDL (200 µg protein/mL) was pre-incubated with YE (0.1, 0.2, and 0.5 mg/mL) in PBS containing 10 mM diethylene triamine pentaacetic acid at 37°C for 5 min. LDL oxidation was induced by adding 5 mM AAPH, and the reaction mixture was incubated at 37°C. Aliquots (50 µL) were withdrawn from the reaction mixture every two hours and mixed with 0.9 mL methanol containing 2 mM butyl hydroxyl anisole, followed by 1.5 mL n-hexane with vigorous vortexing. After centrifugation at 1,200 × g for 5 min at 4°C, the upper layer was collected. Extraction from the lower layer was conducted with 1.5 mL n-hexane again in a similar manner. Then, the combined n-hexane phase was evaporated and the residual neutral lipids were dissolved in 100 µL isopropanol, and cholesteryl ester hydroperoxides (CE-OOH) were determined by reversed-phase high-performance liquid chromatography (HPLC) with UV detection at 235 nm, as described previously (Arai, et al., 1996). The samples were eluted on an InertSustain C8 column (150 × 4.6 mm i.d.; GL Sciences Inc., Tokyo, Japan) at 40°C with methanol at a flow rate of 1.0 mL/min, and cholesteryl linoleate hydroperoxide (Cayman Chemical Company, Ann Arbor, MI, USA) was used as a standard compound.

Metal ion-catalyzed oxidation of LDL    LDL (200 µg protein/mL) was pre-incubated with YE (0.1, 0.2, and 0.5 mg/mL) in PBS at 37°C for 5 min. LDL oxidation was induced by adding 10 µM CuCl2, and the reaction mixture was incubated at 37°C. Aliquots (50 µL) were taken from the reaction mixture every two hours and mixed with 450 µL of 0.2% 2-thiobarbituric acid. Samples were incubated at 95°C for 5 min, and centrifuged at 20,000 × g for 5 min at 4°C. 2-Thiobarbituric acid reactive substances (TBARS) were determined by reversed-phase HPLC with fluorescence detection monitoring at 515 nm for excitation and 553 nm for emission, and 1,1,3,3-tetramethoxypropane was used as a standard compound (Fukunaga et al., 1998). HPLC was performed using an InertSustain C18 column (150 × 4.6 mm i.d.; GL Sciences Inc.) at 40°C with acetonitrile/water (30:70, v/v) at a flow rate of 0.7 mL/min.

Apo B-100 analysis in oxLDL    LDL was oxidized by AAPH or CuCl2 as described above. OxLDL was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using an acrylamide slab gel system (AnykD TGX gel; Bio-Rad Laboratories, Hercules, CA, USA) according to the method of Laemmli (Laemmli, 1970). The migrated proteins in the gel were blotted onto a polyvinylidene difluoride membrane (Arai, et al., 1999). The membrane was treated with serum-free blocking buffer (Thermo Fisher Scientific Inc., Rockford, IL, USA) and then incubated with primary antibody, anti-human apo B-100 monoclonal (6H12) antibody (MP Biomedicals, Santa Ana, CA, USA) for 1 h. The membrane was incubated with secondary antibody, goat anti-mouse IgG-Fc fragment conjugated with horseradish peroxidase (Bethyl Laboratories, Inc., Montgomery, TX, USA) for 1 h after washing. Immunoreactive species were then visualized using enhanced chemiluminescence reagent (GE Healthcare, Little Chalfont, UK).

Protein carbonyl analysis of oxLDL    LDL oxidized by AAPH or CuCl2 as described above was derivatized with 2,4-dinitrophenylhydrazine (Levine et al., 1994) and similarly analyzed by SDS-PAGE and western blotting with rabbit anti-dinitrophenyl antisera (Dako Cytomation, Glostrup, Denmark) as a primary antibody and goat anti-rabbit IgG-Fc conjugated with horseradish peroxidase (Bethyl Laboratories Inc.) as a secondary antibody, followed by the visualization with the enhanced chemiluminescence technique.

Heparin-binding activity of apo B-100 in oxLDL    OxLDL (20 µg protein/100 µL) by AAPH or CuCl2 as described above was incubated with 100 µL of heparin Sepharose 6 Fast Flow (GE Healthcare) for 3 h at 4°C. The heparin Sepharose beads were washed 10 times with 1 mL of PBS containing 0.05% Tween 20. SDS-PAGE sample buffer (100 µL) was added to the heparin Sepharose and incubated at 95°C for 5 min. The supernatant containing the extracted heparin Sepharose-binding fraction in oxLDL was analyzed by the SDS–PAGE with Coomassie Brilliant Blue staining.

Results and Discussion

While yabumame is not a well recognized food in general, it is a traditional food of the Ainu people. We initially determined the nutritional contents of yabumame; moisture, crude protein, crude fat, carbohydrate, and crude ash contents of freeze-dried yabumame are shown in Table 1.

Table 1. Nutritional composition of yabumame (%).
moisture protein crude fat carbohydrate crude ash
12.3 28.0 9.1 45.3 5.3

The extraction yield of YE prepared using 80% methanol was 16.4%. Total polyphenol content in YE was 1.93 ± 0.02 g gallic acid equivalent/100 g of dried extract (mean ± SD, n = 3), which was almost the same as that of soybean extract, 2.10 ± 0.02 g gallic acid equivalent/100 g of dried extract (mean ± SD, n = 3). This indicates that yabumame may contain similar levels of polyphenols as soybean; however, the active substances remain to be identified. There are reports suggesting a variety of phenolic compounds in soybeans and soybean products may contribute to their antioxidant activities (Liu et al., 2005). Hence, we investigated the in vitro antioxidant effects of YE, in particular LDL oxidation, as one of its biological functions, with the aim to utilize yabumame as a functional food in the future.

The radical scavenging activity of YE was measured by DPPH assay, which is widely used to evaluate the antioxidant activity of food components in organic solvent. DPPH has an absorption maximum at 517 nm in the radical state. When DPPH radicals are scavenged by antioxidants, the absorbance at 517 nm should decrease. Malencic et al. suggested that there is a positive correlation between the DPPH radical scavenging activity of soybean seed extract and its polyphenol content (Malencic et al., 2008). Liang et al. reported that genistein and daidzein exert DPPH radical scavenging activity (Liang et al., 2010). As shown in Figure 2, absorbance at 517 nm was dose-dependently reduced in the presence of YE, indicating that YE may contain antioxidants such as phenolic substances, capable of scavenging free radicals.

Fig. 2.

DPPH radical scavenging activity of YE.

Free radical scavenging activity of YE was analyzed using the DPPH assay. DPPH (200 µM) was mixed with an equal amount of YE in methanol and incubated at room temperature for 30 min in the dark; absorbance of the solution at 517 nm was then measured. Data are presented as means ± SD (n = 3).

LDL oxidation induced by ROS is thought to be responsible for the onset of atherosclerosis; however, the underlying mechanisms have not been fully clarified (Itabe, 2009). The primary target of ROS in LDL is the esterified polyunsaturated fatty acids such as linoleic acid (Hevonoja et al., 2000). Free radicals are major ROS and induce lipid peroxidation in LDL by chain reactions, resulting in the accumulation of lipid hydroperoxides. To induce free radical mediated-lipid peroxidation of LDL in vitro, AAPH is commonly used as a water-soluble peroxyl radical generator (Pinchuk and Lichtenberg, 2014). It is reported that phospholipid hydroperoxides and CE-OOH are major products of lipid peroxidation at the early stage of LDL oxidation (Vance and Vance, 2008). Frei et al. suggested that CE-OOH is a reliable index of lipid peroxidation of human plasma mediated by free radicals in vitro (Frei et al., 1988). To evaluate the antioxidant activity of YE against lipid peroxidation of LDL oxidation in vitro, we employed AAPH and measured CE-OOH by HPLC according to our previous method (Arai et al., 1996). In the absence of YE, CE-OOH accumulated time-dependently, as shown in Fig. 3. On the other hand, CE-OOH formation was suppressed by YE in a dose-dependent manner; in particular, 0.5 mg/mL of YE inhibited CE-OOH production almost completely until 6 h incubation. This indicates that YE is an effective inhibitor of peroxyl radical-mediated lipid peroxidation of LDL.

Fig. 3.

Effect of YE on peroxyl radical-mediated lipid peroxidation of LDL.

LDL (200 µg protein/mL) was oxidized by 5 mM AAPH at 37°C in the presence of YE. CE-OOH was determined by HPLC with UV detection. Data are presented as means ± SD (n = 3). Control (●), 0.1 mg/mL YE (■), 0.2 mg/mL YE (◆), 0.5 mg/mL YE (▵).

Several epidemiological and histochemical studies suggest that metal ions might be associated with LDL oxidation (Kruszewski, 2004; Stocker and Keaney, 2004; Yuan and Li, 2003). Our previous study also indicates that iron and copper can generate ROS that induce LDL oxidation (Arai et al., 2005). In lipid peroxidation mediated by transition metal ions, the radical chain reactions are promoted by lipid alkoxy and peroxyl radicals, which are generated from the redox decomposition of lipid hydroperoxides by Fenton-type reaction (Gutteridge, 1995), and reactive aldehydes including malondialdehyde and 4-hydroxy-2-nonenal (HNE) are generated as end products (Frankel, 1998). The aldehydes can be determined as TBARS, a marker of metal ion-catalyzed lipid peroxidation of LDL. Figure 4 illustrates the effect of YE on TBARS production in Cu2+-oxidized LDL. TBARS increased time-dependently in the absence of YE, while YE dose-dependently prevented TBARS formation; in particular, 0.5 mg/mL of YE exhibited quite strong inhibition. This indicates that YE is an efficient antioxidant of lipid peroxidation in LDL mediated by transition metal ions.

Fig. 4.

Effect of YE on transition metal ion-catalyzed lipid peroxidation of LDL.

LDL (200 µg protein/mL) was oxidized by 10 µM Cu2+ at 37°C in the presence of YE. TBARS was determined by HPLC with fluorescence detection. Data are presented as means ± SD (n = 3). Control (●), 0.1 mg/mL YE (■), 0.2 mg/mL YE (◆), 0.5 mg/mL YE (▲).

Another target of ROS in LDL is apo B-100, which is located on the surface of LDL. Each LDL particle contains a single molecule of apo B-100, the primary apolipoprotein of LDL (Segrest et al., 2001). Moreover, apo B-100 is a 515 kDa protein that plays an important role in lipid metabolism. Oxidative modification of apo B-100 might result in the loss of its function. We analyzed apo B-100 in LDL by SDS-PAGE and subsequent western blotting using anti-human apo B-100 antibody. Apo B-100 was detected as a single band at a molecular weight of more than 250 kDa in native LDL (Figure 5, lane 1). OxLDL by AAPH for 4 h (lane 2) and Cu2+ for 2 h (lane 4) were detected as ladder bands in the molecular weight range from about 50 to 250 kDa. In addition, weak bands were also observed at a very high molecular weight position in lanes 2 and 4 as compared to native LDL. It has been suggested that ROS lead to cleavage of polypeptide chains and formation of intermolecular cross-linked proteins (Stadtman and Levine, 2003). The result indicates that the oxidation of LDL by AAPH and Cu2+ led to both the fragmentation and polymerization of apo B-100. Meanwhile, 0.5 mg/mL of YE suppressed apo B-100 fragmentation by AAPH (lane 3) and Cu2+ (lane 5). This might be due to YE scavenging of ROS derived from AAPH and Cu2+.

Fig. 5.

Effect of YE on apo B-100 fragmentation.

LDL was oxidized by AAPH for 4 h or by Cu2+ for 2 h as described in the legends of Figs. 3 and 4, respectively. OxLDL was subjected to SDS-PAGE and western blotting using anti-human apo B-100 monoclonal antibody as the primary antibody and goat anti-mouse IgG-Fc conjugated with horseradish peroxidase as the secondary antibody. Immunoreactive species were visualized by the enhanced chemiluminescence method. Lane 1, native LDL; lane 2, LDL oxidized by AAPH; lane 3, LDL oxidized by AAPH in the presence of 0.5 mg/mL YE; lane 4, LDL oxidized by Cu2+, and lane 5, LDL oxidized by Cu2+ in the presence of 0.5 mg/mL YE. A representative result of three independent experiments is shown.

It has been proposed that the oxidative modification of apo B-100 is induced by the reaction with lipid hydroperoxides (Kato et al., 1997) and aldehydes such as HNE (Uchida et al., 1994), in which carbonylated proteins are generated. Protein carbonyl analysis is widely utilized to detect oxidized proteins and is a good marker of oxidative stress, as carbonylated proteins are relatively stable (Dalle-Donne et al., 2003). The carbonyl groups in oxidized protein can be derivatized with 2,4-dinitrophenylhydrazine, forming dinitrophenyl hydrazone products that are detected by western blotting using anti-dinitrophenyl antibody (Levine et al., 2000; Requena et al., 2003). As shown in Figure 6, while protein carbonyls were mostly unobserved in native LDL (lane 1), protein carbonyls were detected in LDL oxidized by AAPH for 4 h (lane 2) or Cu2+ for 2 h (lane 6) as smear bands in the molecular range of about 50 kDa to 250 kDa. It is postulated that the fragmented and polymerized apo B-100 observed in Figure 5 may be carbonylated, considering the observed molecular weight range of 50 – 250 kDa. The levels of CE-OOH (Figure 3) and TBARS (Figure 4) at these incubation periods increased in the absence of YE, suggesting that apo B-100 was oxidatively modified by lipid hydroperoxides or aldehydes. In contrast, YE suppressed protein carbonyl formation in LDL oxidized by AAPH (lane 3, 4, 5) or Cu2+ (lane 7, 8, 9) in a dose-dependent manner. YE might protect apo B-100 from oxidative modification by lipid hydroperoxides and aldehydes during LDL oxidation.

Fig. 6.

Effect of YE on protein carbonyl formation in oxLDL.

LDL was oxidized by AAPH for 4 h or by Cu2+ for 2 h as described above. Protein carbonyls in oxLDL were derivatized with 2,4-dinitrophenylhydrazine, and were analyzed by SDS-PAGE and western blotting using rabbit anti-dinitrophenyl antisera as the primary antibody and goat anti-rabbit IgG-Fc conjugated with horseradish peroxidase as the secondary antibody. Immunoreactive species were visualized by the enhanced chemiluminescence method. Lane 1, native LDL; lanes 2–5, LDL oxidized by AAPH; lanes 6–9, LDL oxidized by Cu2+; lanes 3 and 7, 0.1 mg/mL of YE; lanes 4 and 8, 0.2 mg/mL of YE; lanes 5 and 9, 0.5 mg/mL of YE. A representative result of three independent experiments is shown.

Apo B-100 has LDL receptor binding sites and heparin-binding sites that contribute to LDL metabolism in the circulatory system (Boren et al., 1998; Weisgraber and Rall, 1987), which are abundant in basic amino acid residues including lysine. Kato et al. suggests that lipid hydroperoxides such as 13-hydroperoxyoctadecadienoic acid can react with the lysine residues of proteins (Kato and Osawa, 2010) and the recognition moieties of the adducts were confirmed as Nε-(hexanonyl)lysine (Kato et al., 1999). Nε-(Hexanonyl)lysine was observed in LDL oxidized by peroxyl radicals or copper ions in vitro (Kato et al., 1997). Uchida et al. has proposed that HNE, a degradation product of lipid hydroperoxides, can also modify the protein-based lysine ε-amino group and form Michael addition-type adducts (Szweda et al., 1993; Uchida and Stadtman, 1992). In fact, HNE adducts were found in oxLDL and atherosclerotic lesions (Uchida et al., 1994). These oxidative modifications of apo B-100 lead to a decrease in its positive charge. Oorni et al. indicated that oxidative modification of the lysine residues of apo B-100 in LDL decreases their binding to negatively charged proteoglycans (Oorni et al., 1997). Here, we examined the heparin-binding activity of apo B-100 in LDL oxidized by AAPH or CuCl2 using SDS-PAGE as previously described (Arai et al., 1999). A single high molecular weight band was detected for native LDL, meaning that apo B-100 has heparin-binding activity (Figure 7, lane 1). In contrast, the single band was very weak in oxLDL by AAPH for 4 h (lane 2) or Cu2+ for 2 h (lane 4), indicating that the heparin-binding activity of apo B-100 in LDL was considerably reduced by the oxidation. In the presence of YE at 0.5 mg/mL, the single band remained in LDL oxidized by AAPH (lane 3) or Cu2+ (lane 5). The results suggest that the lysine residues of apo B-100 are modified by lipid hydroperoxides and aldehydes during LDL oxidation by ROS, and can be blocked by YE.

Fig. 7.

Effect of YE on heparin-binding activity of apo B-100 in oxLDL.

OxLDL (20 µg protein/100 µL) prepared in the same manner as above was incubated with 100 µL of heparin Sepharose 6 Fast Flow for 3 h at 4°C. The heparin Sepharose beads were washed 10 times with 1 mL of PBS containing 0.05% Tween 20 and treated with 100 µL of SDS-PAGE sample buffer at 95°C for 5 min. The supernatant containing the extracted heparin Sepharose-binding fraction in oxLDL was analyzed by the SDS–PAGE and stained with Coomassie Brilliant Blue. Lane 1, native LDL; lane 2, LDL oxidized by AAPH; lane 3, LDL oxidized by AAPH in the presence of 0.5 mg/mL YE; lane 4, LDL oxidized by Cu2+, and lane 5, LDL oxidized by Cu2+ in the presence of 0.5 mg/mL YE. A representative result of three independent experiments is shown.

Conclusion

YE exerted inhibitory effects on both lipid peroxidation and apo B-100 oxidation, which are indices of LDL oxidation induced by major ROS, free radicals and transition metal ions. Although the mechanism of the antioxidant effect of YE is not clear, it might be attributable to the radical scavenging polyphenols in YE. In future studies, the structure and amounts of antioxidants in yabumame should be clarified. The present results suggest that yabumame might contribute to preventing LDL oxidation by ROS that leads to atherosclerosis.

Acknowledgment    We appreciate Dr. Kanda, S., Kansai Medical University for preparing human blood plasma. This work has been supported by the Tojuro lijima Foundation for Food Science and Technology.

References
 
© 2015 by Japanese Society for Food Science and Technology
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