Walnut phenolic compounds: Binding with proteins and antioxidant activities

Transcription

1 第 32 卷第 22 期农业工程学报 Vol.32 No 年 11 月 Transactions of the Chinese Society of Agricultural Engineering Nov Walnut phenolic compounds: Binding with proteins and antioxidant activities Su Shiwei 1, Wang Ruican 1, Guo Shuntang 1, Zhang Chao 2, Zhang Ting 2, Liang Ming 2 (1. College of Food Science and Nutritional Engineering, China Agricultural University, Beijing , China; 2. Infinitus (China) Company Ltd., Guangzhou , China) Abstract: Walnut (Juglans regia L.) is rich in phenolic compounds (POHs) which show profound antioxidant capacity. But the extraction of POHs could not be completed as they usually interact with walnut proteins. In this study, different amount of urea or NaCl were added to walnut protein solutions in order to analyze the interactions between the POHs and proteins. Results showed that 77.7% total phenolic compounds and 34.6% total condensed tannins (at ph 3.0), 65.4% total phenolic compounds and 59.4% condensed tannins (at ph 8.0) bonding with proteins through various non-covalent bonds, among which hydrophobic bonds were predominant. Besides, the antioxidant activities of POHs were investigated. It showed that the POHs bonding with proteins through ionic bonds could scavenge 5.7% DPPH radical and chelate 5.9% Fe 2+ at ph 3.0 while scavenge 10.9% DPPH radical and chelate 11.5% Fe 2+ at ph 8.0, demonstrating the strongest antioxidant activities. In addition, the efficiency of 70% (v/v) ethanol and 60% (v/v) methanol in extracting POHs and condensed tannins from WPI at ph 3.0 and 8.0 were tested. Results showed the above two commonly used extractants could only slightly promote the extraction of POHs and were not efficient in extracting the POHs with higher antioxidant capacity. Key words: phenolic compounds; proteins; bonding; antioxidant activities doi: /j.issn CLC number: TS201.1 Document code: A Article ID: (2016) Su Shiwei, Wang Ruican, Guo Shuntang, Zhang Chao, Zhang Ting, Liang Ming. Walnut phenolic compounds: Binding with proteins and antioxidant activities[j]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2016, 32(22): (in English with Chinese abstract) doi : /j.issn 苏世伟, 王睿粲, 郭顺堂, 张超, 张婷, 梁明. 核桃酚类物质和蛋白间的相互作用及其抗氧化活性 [J]. 农业工程 学报,2016,32(22): doi: /j.issn Introduction Walnut (Juglans regia L) has been long regarded as a highly nutritious food in many regions of the world and it is an important component of the Mediterranean diet [1]. Walnut is rich in phenolic compounds (POHs), including hydrolysable tannins, condensed tannins, flavonoids and phenolic acids [2]. POHs in walnuts were reported to have favorable effects on human health owing to their apparent anti-atherogenic and antioxidant properties [3-6]. Walnut POHs were found with the highest concentration in the pellicles surrounding the kernels. But after walnut oil extraction, most POHs remain in the meal [7]. Moreover, POHs can bind with proteins through various non-covalent Received date: Revised date: Foundation item : A Grant from Infinitus (China) Company Ltd. for the Fingerprint Establishment of Walnut Peptides ( ) Biography:Su Shiwei, female, De Zhou Shandong, majoring in Bioactive peptides. Shiwei_Su@yahoo.com Corresponding author:guo Shuntang, male, Harbin Heilongjiang, doctor degree, doctoral supervisor, deputy secretary of College of Food Science and Nutritional Engineering in China Agricultural University, majoring in food science. shuntang@cau.edu.cn bonds: hydrophobic interactions [8], hydrogen and ionic bonds [9-10] in aqueous media. Those combinations would reduce the extracting efficiency of POHs [11]. Therefore, figuring out the interactions among walnut POHs and proteins would contribute to the higher extracting efficiency of walnut POHs. Thus in this study, the non-covalent interactions between POHs and walnut proteins were elucidated under acidic and alkaline conditions. Meanwhile, the antioxidant abilities of POHs bonding with walnut proteins through different mechanisms were determined. 1 Materials and methods 1.1 Materials Defatted walnut meal (by-product of cold-rolled walnut oil) was purchased from Fine Hemp Walnut Co., Ltd (Hebei province, China), DPPH (1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl), gallic acid and (+) catechin were purchased from Sigma Chem. (St. Louis, MO, USA). All other reagents were of analytical grade. 1.2 Preparation of walnut protein isolates Walnut protein isolates (WPI) was prepared according to the process described by Chen et al. [12] with minor

2 310 农业工程学报 ( 年 modifications. Defatted meal was dispersed in distilled water (water: meal=10:1, ml/mg). Then the mixture was adjusted to ph 7.5 with 1 M NaOH, extracted by stirring for 2 h at room temperature ((25±2) ) and centrifuged at g for 20 min. The supernatant was adjusted to ph 4.5 with 6 N HCl and then centrifuged at g for 20 min. The precipitate was washed with distilled water twice, collected and lyophilized as final WPI. 1.3 Extraction of free and bound POHs from WPI The free and bound POHs from WPI were extracted and fractionated by the procedure schematically represented in Fig.1 according to the method of Xu et al. [13] with slight modifications. First, an aliquot of 30 ml WPI solution at ph 3.0 or 8.0 was ultra-filtered (molecular weight cut-off of Da) to get free POHs, which were not bonded to proteins in the WPI solutions of corresponding ph. Then the residue remaining from free phenolic extractions was subjected to extraction of the rest POHs ( bound phenolic compounds). The bound POHs were fractionated by various reagents according to their interacting bonds with WPI: the POHs connecting with WPI through ionic bonds were released by the addition of 0.05 mol/l NaCl, the POHs binding with WPI by hydrogen bonds were separated by 0.05 mol/l NaCl+1.5 mol/l urea, and finally the POHs binding with WPI via hydrophobic interactions were released by 0.05 mol/l NaCl+8 mol/l urea. After each treatment, the released POHs were separated from WPI solutions by ultrafiltration using the same membrane. Then residues were washed and re-ultrafiltrated for 5 times to remove the remaining POHs that had been released. For convenience, free POHs and POHs bonding with WPI through ionic bonds, hydrogen bonds and hydrophobic interactions were respectively named as free fraction, ionic fraction, hydrogen fraction and hydrophobic fraction. Note: UF, R, and P represented ultrafiltration, retentate and permeate. Fig.1 Fractionation system of phenolic compounds from walnut protein isolates solutions at ph 3.0 and Total phenolic compounds content (TPC) The total phenolic compounds content of WPI and phenolic extractions were determined using the Folin-Ciocalteu regent according to Vallverdú et al. [14] with minor modifications. 10 ml phenolic samples were adjusted to ph 7.0 and diluted with distilled water to 25 ml. Then 500 μl of phenolic samples (ph 7.0) or WPI solution (1 mg/ml, ph 7.0) were mixed with 1.5 ml of Folin-Ciocalteu reagent followed by addition of 1.0 ml of sodium carbonate solution (200 g/l). After holding at 60 for 1 h, the absorbance was measured at 765 nm using a spectrophotometer (UV-1800, SHIMADZU, Japan). A reference curve was constructed, using gallic acid as a standard. TPC was expressed as milligrams of gallic acid equivalents (GAE) per gram of WPI. Walnut phenolics are found with the highest concentration in the hull (the pellicle surrounds the kernel) [3], thus for the determination of TPC of WPI, proteins isolated from de-hulled walnut meal were used as blank. In addition, high performance liquid chromatography confirmed that there were no POHs in the proteins used in this study (not shown). 1.5 Total condensed tannins content (TCD) The total condensed tannins content was determined by the vanillin assay which was referred to Shahidi et al. [15] with minor modifications. 4 ml samples taken out of the supernatant was freeze-dried for condensed tannin analysis. After lyophilization the samples were dissolved in 3 ml water: methanol (1:30, v/v) solution. To 0.5 ml of the samples, 2.5 ml of 2.0 % vanillin reagent and 2.5 ml of concentrated hydrochloric acid were added and mixed well. The absorbance of (+)-catechin was used as a standard in these experiments. TCD was expressed as milligrams of catechin equivalents per gram of WPI. 1.6 DPPH radical scavenging activity DPPH radical scavenging activity of phenolic extracts was measured using the method described by Zhang et al. [16] with some modifications. 10 ml phenolic samples were adjusted to ph 7.0 and diluted with distilled water to 25.0 ml. Then 2 ml of samples were added to 2 ml of DPPH-ethanol solutions (62.5 μg/ml in 99.7% ethanol) and the reaction mixtures were shaken vigorously. After being kept in room temperature for 30 min in the dark, the absorbance was determined at 517 nm. Ethanol solution without DPPH was used as blank, while the corresponding extractant without POHs was used for the control. DPPH radical scavenging ability was calculated by following equation DPPH radical scavenging ability (%)= [1 (A sample A blank )/A control ] 100/TPC Where A sample, A blank and A control were absorbance of sample, blank and control, respectively; TPC was expressed as milligrams of gallic acid equivalents (GAE) per gram of WPI. 1.7 Fe 2+ chelating activity The Fe 2+ chelating ability of each sample was measured by the method of Zhang et al. [16]. 10 ml phenolic samples were adjusted to ph 7.0 and diluted with distilled water to 25 ml. Then an aliquot of 2 ml sample was mixed with 100 μl of 20 mmol/l FeCl 2, 2 ml distilled water and 200 μl ferrozine solution (5 mmol/l). After being mixed sufficiently, the mixture was rested for 10 minutes. The absorption was determined at 562 nm, and the Fe 2+ chelating ability was calculated as follows Fe 2+ chelating ability (%) =(1 A sample /A blank ) 100/TPC The blank was prepared in the same manner except that distilled water was used instead of the sample.

3 第 22 期苏世伟等 : 核桃酚类物质和核桃蛋白间的相互作用及其抗氧化活性 Data analysis Datas were processed and plotted using Origin pro8 (Origin-lab, Northampton, MA, USA). Each experiment was performed at least in triplicate and the analysis of variance was employed to examine the difference among treatments at 95% significance level using SPSS statistics, version 17.0 (SPSS, Chicago, IL). Results were expressed as mean ± S.D. (standard deviation). 2 Results and discussion 2.1 Fractionations of total POHs from WPI solutions at acidic and alkaline conditions This research determined the TPC of every phenolic fraction to study the specific non-covalent bonds connecting POHs and WPI at acidic and alkaline conditions (ph 3.0 and 8.0). The results are shown in Fig.2. Note: Results are presented as percentages of total phenolic compounds content (TPC) in WPI. Free fraction, ionic fraction, hydrogen fraction and hydrophobic fraction represented free POHs and bound POHs which connected with WPI through ionic bonds, hydrogen bonds and hydrophobic interactions respectively. Fig.2 TPC of phenolic fractions in walnut protein solutions at ph 3.0 and 8.0 TPC of each phenolic fraction was expressed as a percentage of the total amount of POHs (19.75 mg/g) in WPI. As can be seen from Fig.2, at ph 3.0 and 8.0, POHs of free fractions accounted for 20.5% and 31.8% of total POHs in WPI respectively while POHs of the other three fractions amounted to 77.7% and 65.4% of total POHs at the respective ph. In another word, most POHs bonded with WPI through various non-covalent bonds. Furthermore, Fig.2 shows that at ph 3.0 and 8.0, there were 43.6% and 52.6% POHs connecting with proteins through hydrophobic interaction. That means hydrophobic interactions were major non-covalent bonds between POHs and WPI. The formation of hydrophobic bonds between POHs and proteins was the predominant step in the POHs-proteins complex formation: molecules of POHs and proteins recognized each other by their hydrophobic character and adapted their structures to this recognition [17]. POHs in walnut, like hydrolysable and condensed tannin [2,7], and WPI contained hydrophobic regions-the aromatic nuclei of POHs and the aliphatic and aromatic side chains of amino acids in proteins. The massive coexisted hydrophobic regions might contribute to the large amounts of hydrophobic bonds. And the results were in agreement with what had been reported by Oh et al. [18], Siebert et al. [19] and Rawel et al. [20] who investigated interactions between phenolic compounds and proteins and found that hydrophobic interactions were dominating. In contrast, it was found in this study that only 11.6% and 5.6% POHs bonded with WPI via ionic bonds at ph 3.0 and 8.0 respectively. Le et al. [21] reported ionic interactions between positively charged groups of proteins, such as ε-amino groups of lysine, and negatively charged hydroxyl groups or carboxyl of POHs would play a minor role. Therefore, bits of ionized hydroxyl or carboxyl groups of POHs might interact with positively charged ε-amino groups of lysine of proteins to form a small amount of ionic bonds between POHs and WPI. At the same ph, the amount of POHs binding with WPI through the three kinds of non-covalent bonds, as shown above, were different. Meanwhile, the percentage of POHs bonding with WPI via the same kind of bonds varied with the ph (Fig.2). The amount of free POHs in protein solution at ph 8.0 was higher than that at ph 3.0. In contrast, 22.4% POHs connected with WPI through hydrogen bonds at ph 3.0, while much less POHs (7.2%) connected with WPI at ph 8.0 in the same way. Gilli et al. [22] reported alkaline solutions as proton receptor would be adverse to the formation of hydrogen bonds. The alkaline condition (ph 8.0) might break the hydrogen bonds between POHs and WPI to some extent, resulting in more free or unbound POHs in alkaline WPI solution. In addition, in acidic condition (ph 3.0), there were 12.3% of POHs binding with WPI through ionic bonds, whereas in alkaline condition (ph 8.0), 4.0% POHs interacted with WPI by ionic bonds. At ph 3.0, most functional groups of WPI (pi 4.5) were positively charged, while at ph 8.0, positively charged groups of proteins decreased in large numbers. In another word, the sites on proteins to which POHs could ionically bind decreased drastically, reducing the POHs that could bind to WPI via ionic bonds at ph 8.0. Meanwhile, at ph 8.0, more POHs bonded with WPI through hydrophobic interactions than that at ph 3.0. It was reported hydrophobic interactions between POHs and WPI were dependent on hydrophobic regions-the aromatic nuclei of POHs and the aliphatic and aromatic side chains of amino acids in proteins [17]. In alkaline conditions, denaturation and dissociation of proteins molecules occurred [23]. And denaturation and dissociation might expose more hydrophobic protein residues to interact with POHs hydrophobically [11]. Under the effect of 8 mol/l urea, the hydrophobic interactions were broken, releasing a larger number of POHs (52.6%). In sum, the results suggest that the alkaline condition might be conducive to breaking the ionic and hydrogen bonds while enhancing the hydrophobic interactions between WPI and POHs. 2.2 Fractionations of total condensed tannins from WPI solutions at acidic and alkaline conditions Apart from hydrolysable tannins,the main POHs in walnuts [4,24], condensed tannins, such as procyanidins were

4 312 农业工程学报 also found in walnut[2,7]. Condensed tannins were considered to be more efficient in bonding and precipitating proteins[25]. So the research used vanillin assays which could selectively detect condensed tannins in the presence of other kinds of phenolic compounds to determine the content of condensed tannins in each phenolic fraction. It was notable that at ph 3.0, free condensed tannins accounted for 61.7% of the total amount of condensed tannins in WPI (there was 2.3 mg condensed tannins per gram of WPI as results showed), much more than the content of condensed tannins bonding with WPI, while at ph 8.0, only 18.0 % condensed tannins were free. Condensed tannins could be dissolved and stabilized in acidic solvent systems, and thus acidic solvent systems are commonly used to extract condensed tannins from plant materials[26]. Therefore, the acidic condition (ph 3.0) might promote the dissociation between condensed tannins and proteins. And that might also result in the fact that the TCD of ionic, hydrogen and hydrophobic fractions at ph 3.0 were all lower than those at ph 8.0, as shown in Fig 年 the Fe2+ chelation rate of 5.9% and 11.5%, respectively. The antioxidant activities of phenolic compounds are mainly determined by their hydrogen or electron-donating abilities[27]. The POHs connecting with WPI via ionic bonds embraced hydroxyl or carboxyl groups that were easily ionized. That is to say these POHs had hydroxyl or carboxyl groups that showed strong H donating ability. When the ionic bonds were broken by NaCl, these POHs released from WPI, contributing to the excellent antioxidant activity of ionic fractions. Note: Results are expressed as DPPH radical clearance rate per milligram of total phenolic compounds content (TPC). a. DPPH radical clearance activity of each phenolic fraction Note: Results are presented as percentages of total condensed tannins content (TCD) in WPI. Free fraction, ionic fraction, hydrogen fraction and hydrophobic fraction represent free condensed tannins and bound condensed tannins which connected with WPI through ionic bonds, hydrogen bonds and hydrophobic interactions respectively. Fig.3 TCD of phenolic fractions in walnut protein solutions at ph 3.0 and 8.0 It was shown in Fig.3, the content of condensed tannins of Ionic fraction, Hydrogen fraction and Hydrophobic fraction amounted to 34.6% (at ph 3.0) and 59.4% (at ph 8.0) of the total condensed tannins. What s more, at ph 3.0 (Fig.3), the proportion of condensed tannins connecting with WPI through hydrophobic interactions was 17.9%, much higher than those connecting with WPI via ionic or hydrogen bonds. In addition, at ph 8.0, 25.8% condensed tannins interacted with WPI hydrophobically. This means hydrophobic interactions were also the main non-covalent bonds connecting condensed tannins with WPI. 2.3 Antioxidant activities of POHs interacting with WPI Antioxidant activities of each phenolic fraction were analyzed; the results were shown in Fig.4a and Fig.4b. As shown in Fig.4, the DPPH radical scavenging ability and Fe2+ chelating ability of each phenolic fraction were different. The ionic fractions exhibited the highest activities with DPPH radical clearance rate of 5.7% and 10.9%, while Note: Results are expressed as Fe2+ chelating rate per milligram TPC. Fig.4 b. Fe2+ chelating activity of each phenolic fraction Antioxidant activities of phenolic compounds bonding with WPI The POHs connecting with WPI through hydrogen bonds at ph 3.0, 8.0 demonstrated DPPH radical clearance rate of 3.4%, 8.7% and Fe2+ chelation rate of 4.1% and 8.4%, respectively. And the antioxidant activities were much higher than free POHs and POHs binding with WPI via hydrophobic interactions. POHs considerably rely on hydroxyl groups to form hydrogen bonds with proteins[17]. Meanwhile, the number of hydroxyl groups greatly determines the antioxidant activities of POHs[28]. The POHs connecting with WPI through hydrogen bonds might embrace much more hydroxyl groups so as to demonstrate excellent antioxidant activities. In contrast, as shown in Fig.4, the hydrophobic fractions showed the weakest abilities in clearing DPPH and chelating Fe2+. POHs bonding with proteins via hydrophobic interactions tend to be larger polyphenols, possessing a greater number of phenolic rings

5 第 22 期苏世伟等 : 核桃酚类物质和核桃蛋白间的相互作用及其抗氧化活性 313 with a concomitant increase in hydrophobicity and molecular weight [29]. Moilanen et al. [29] put forward an equation which showed the antioxidant capacity of ellegitannins was in inverse proportion to molecular weight. The high molecular weight and relatively less free hydroxyl groups per phenyl ring might render the weakest antioxidant abilities of hydrophobic fractions. All in all, POHs interacting with WPI via ionic bonds demonstrated the most excellent antioxidant activities. The results might suggest that breaking ionic bonds between POHs and WPI was a good way to extract POHs exhibiting prominent antioxidant capability from WPI. 2.4 Efficacy of 70% (v/v) ethanol and 60% (v/v) methanol on extracting POHs from WPI Alcohols, such as methanol, ethanols with different proportions of water are the most common solvents used for the extraction of phenolic compounds. Labuckas et al. [11] used 60% (v/v) aqueous methanol and 70% (v/v) aqueous ethanol to extract POHs from defatted walnut meals and found they were suitable extractants. Thus, after elucidating the specific non-covalent bonds connecting POHs and WPI, the study evaluated the POHs extracting efficiency of 70% (v/v) ethanol and 60% (v/v) methanol at ph 3.0 and 8.0. Meanwhile, the antioxidant activities of POHs extracted using ethanol and methanol were investigated. Results are shown in Table 1. Table 1 Phenolic extracting efficiency of 60% (v/v) aqueous methanol and 70% (v/v) aqueous ethanol at ph 3.0 and 8.0 and the antioxidant activities of these phenolic extracts Extracting efficiency/% Antioxidant activities/% TPC TCD DPPH Fe 2+ ph 3.0 Ethanol 21.3± ± ± ±0.1 Methanol 22.0± ± ± ±0.1 Ethanol 34.2± ± ± ±0.3 ph 8.0 Methanol 37.0± ± ± ±0.2 Note: Extracting efficiency was presented as percentages of total phenolic compounds content (TPC) or total condensed tannins content (TCD) in WPI. DPPH and Fe 2+ represented DPPH radical scavenging and Fe 2+ chelating ability of phenolic extracts, which were expressed as mean ± S.D (standard deviation). It is shown in Table 1 that at ph 3.0 and ph 8.0, the TPC and TCD of phenolic extracts obtained by 70% (v/v) ethanol and 60% (v/v) methanol were higher than that of free fractions from walnut proteins solutions (Fig. 2 and Fig.3). That is to say, 70% (v/v) ethanol and 60% (v/v) methanol did promote the extracting efficiency of POHs from WPI. However, comparing with the large amounts of POHs interacting with WPI via various non-covalent bonds (Fig.2 and Fig.3), the promotion was limited. 70% (v/v) ethanol and 60% (v/v) methanol could not break the non-covalent bonds completely. Besides, as shown in Table 1, ethanol and methanol extracts of POHs showed similar DPPH radical scavenging ability and Fe 2+ chelating ability with the free phenolic fractions (Fig.4). That means 60% (v/v) aqueous methanol and 70% (v/v) aqueous ethanol might not be efficient in extracting the POHs with excellent antioxidant activities, such as the POHs connecting with WPI via ionic or hydrogen bonds. 3 Conclusion This investigation demonstrates that hydrophobic interactions and ionic bonds were respectively the major and minor non-covalent bonds between POHs and WPI. In contrast, the POHs interacting with protein via hydrophobic interactions tended to show the weakest antioxidant capacity, while those bonding with WPI via ironic and hydrogen bonds exhibited stronger antioxidant activities. Thus, in order to improve phenolic extraction from WPI, the extracting methods should be aimed at breaking the hydrophobic interactions between POHs and WPI, while breaking ionic and hydrogen bonds is supposed to be considered to get the POHs with stronger antioxidant activities. [Reference] [1] Bullo M, Lamuela-Raventos R, Salas-Salvado J. 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