Antioxidative activities of caffeoyl-proline dipeptides

Food Chemistry 130 (2012) 847–852

Contents lists available at SciVerse ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Antioxidative activities of caffeoyl-proline dipeptides Seon-Yeong Kwak, Song Lee, Jin-Kyoung Yang, Yoon-Sik Lee ⇑ Yoon-Sik Lee, School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea

a r t i c l e

i n f o

Article history: Received 16 March 2011 Received in revised form 14 July 2011 Accepted 23 July 2011 Available online 9 August 2011 Keywords: Antioxidant Caffeoyl-proline dipeptides DPPH radical scavenging activity Lipid peroxidation inhibition

a b s t r a c t The benefits of antioxidants in treating degenerative diseases, delaying ageing and preserving food, have been extensively studied. In our previous study, we found that caffeoyl-Pro-His dipeptide (CA-Pro-HisNH2) showed the highest antioxidative activity among the histidine- containing caffeic acid dipeptides (CA-His-Xaa-NH2 and CA-Xaa-His-NH2). We assumed that the addition of proline to the CA-Pro-HisNH2 structure may synergistically enhance its antioxidative activity. Therefore, we synthesised twenty different caffeoyl-proline dipeptides (CA-Pro-Xaa-NH2), and evaluated their antioxidative activities with 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and lipid peroxidation inhibition tests. Some of the CA-Pro-Xaa-NH2 showed higher free radical scavenging activities than CA, and even CA- CA-Pro-HisNH2. However, CA-Pro-His-NH2 still exhibited the highest antioxidative activity in reducing lipid peroxidation. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Oxidation is a highly conserved and unavoidable process that occurs in almost every living organism. However, several oxidants disturb the balance of living cells with adverse consequences. One of the most important elements in the process of oxidation is oxygen. While oxygen is an essential element for life, unconsumed oxygen molecules can break the balance of living systems by being converted into free radicals such as hydroxyl (OH), peroxyl (ROOH) and superoxide (O2) radicals. These reactive oxygen species, which are by-products from aerobic cellular metabolic pathways, can cause oxidative stress, and lead to DNA and RNA damage and the oxidation of unsaturated fatty or amino acids, resulting in deformation of proteins through radical chain reaction (Apel & Hirt, 2004; Halliwell, 1996). In addition, the accumulation of oxidative stress can accelerate the ageing process, and causes human degenerative diseases including Alzheimer’s disease (Benzi & Moretti, 1995; Perry, Castellani, Hirai, & Smith, 1998; Zhao, 2009). For these reasons, there has been continued interest to develop antioxidants exhibiting good performance to suppress the activity of reactive oxygen species (ROS). Many kinds of antioxidants exist in nature, including vegetables, fruits, grain cereals, nuts, legumes, meat, wine and green tea (Moure et al., 2001). However, these natural antioxidants are unsuitable for practical applications, due to their low concentration, insufficient activity, lack of long-term stability and poor resistance to high temperatures (Pokorny, 2007).

⇑ Corresponding author. Tel.: +82 2 880 7073; fax: +82 2 876 9625. E-mail address: [email protected] (Y.-S. Lee). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.07.096

Therefore, many synthetic antioxidants, such as tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and bisphenol (BP), have been developed and utilised in the food industry to prevent food deterioration (Chen, Chan, Kwan, & Zhang, 1997; Shahidi, 2000), in the cosmetic industry to delay the ageing process (Guan, Chu, Fu, & Ye, 2005) and in the pharmaceutical industry to treat degenerative diseases related to the over-production of free radical (Rayner, Duong, Myers, & Witting, 2006). However, their use is limited because of their possible toxicity at high concentrations (Branen, 1975). Caffeic acid (CA), which belongs to the hydroxycinnamic acid family, is a well known antioxidant. It can exist as a stable phenoxy radical because of its fully conjugated structure after quenching free radicals by donating a hydrogen radical (Cheng, Ren, Li, Chang, & Chen, 2002; Shahidi, Janitha, & Wanasundara, 1992). It has been previously reported that CA shows enhanced antioxidative activity when a peptide or amino acid is conjugated to it, most likely due to structural stabilisation (Kwak, Seo, & Lee, 2009; Pekkarinen, Stockmann, Schwarz, Heinonen, & Hopia, 1999; Seo, Kwak, & Lee, 2010; Spasova et al., 2006; Stankova et al., 2009). In a previous study, we synthesised histidine-containing caffeic acid dipeptides (CA-His-Xaa-NH2 and CA-Xaa-His-NH2) and observed that CA-Pro-His-NH2 showed the highest antioxidative activity (Seo et al., 2010). We hypothesised that the addition of proline to the CA-Pro-His-NH2 structure might synergistically enhance its antioxidant activity because CA-His-Pro-NH2 showed much less antioxidant activity than CA-Pro-His-NH2, even though they both have the same amino acid compositions. Therefore, to explain the exceptionally enhanced antioxidant activity of CA-Pro-His-NH2 and the effect of proline, we synthesised 20 kinds of caffeoyl-proline dipeptides (CA-Pro-Xaa-NH2)

848

S.-Y. Kwak et al. / Food Chemistry 130 (2012) 847–852

and evaluated their antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and emulsified-lipid peroxidation tests (Chen, Muramoto, & Yamauchi, 1995; Mitsuda, Yasumodo, & Iwami, 1966). 2. Materials and methods 2.1. Chemicals Fmoc–Rink amide linker coupled aminomethyl surface-layered polystyrene (Rink amide AM) (100–200 mesh, 0.82 mmol/g) resin, 4-hydroxymethyl-2(5H)-furanone twenty-millilitre filtered reactors (Libra tube RT-20M), benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), (2-(1H-benztri azole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU), hydroxybenzotriazole (HOBt), and Fmoc-protected amino acids were obtained from BeadTech (Seoul, Korea). Diisopropylethylamine (DIPEA) was bought from Alfa Aesar (Ward Hill, MA). Caffeic acid (CA), ninhydrin, linoleic acid (99%) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were bought from Sigma (St. Louis, MO). Butylated hydroxyanisole (BHA), ammonium thiocyanate (NH4[+]SCN[]), ferrous chloride (FeCl2) and polyoxyethylenesorbitan monolaurate (Tween 20), triisopropylsilane (TIPS) and 3,6dioxa-1,8-octanedithiol (DODT) were bought from Aldrich (St. Louis, MO). N-Methyl-2-pyrrolidone (NMP), piperidine, dichloromethane (DCM), diethyl ether, ethanol and methanol were bought from Dae–Jung Chemicals (Shiheung city, Korea). Trifluoroacetic acid (TFA) was bought from Acros Organics (Morris Plains, NJ). 2.2. Apparatus CA-Pro-Xaa-NH2 were analysed by high- performance liquid chromatography (HPLC, Thermo Scientific Spectra System AS3000; Thermo-Fisher, Waltham, MA), using an AAPPTec Spirit Peptide C18 reverse phase column (120 Å, 5 lm, 4.6  250 mm; AAPPTec, Louisville, KY). Also, the derivatives were also characterised by a QUATTRO triple quadrupole tandem mass spectrometer (Waters Micromass, Milford, MA,). The colour reactions such as DPPH and linoleic acid peroxidation test were followed by UV/Visible spectrophotometry (Optizen 2120 UV, Mecasys Co. Ltd., Daejeon, Korea). The NMR spectra were acquired by a Bruker AVANCE-600 spectrometer (Bruker, Rheinstetten, Germany) operating at 600 MHz. 2.3. Synthetic procedure for CA-Pro-Xaa-NH2 Peptide derivatives were manually synthesised through SPPS (Merrifield, 1963) with the Fmoc-strategy on Rink amide AM resin by using 4-hydroxymethyl-2(5H)-furanone twenty-millilitre filtered reactors (Libra tube RT-20M, Beadtech Inc. Co., Ltd., Seoul, Korea). Each reaction step was monitored by ninhydrin colour test (Kaiser, Colescott, Bossinger, & Cook, 1970). Fmoc-amino acid (2 eq.), BOP (2 eq.) or HBTU (2 eq.), and HOBt (2 eq.) were dissolved in NMP, and this mixture was added to the resin with DIPEA (4 eq.). Each coupling reaction was performed for 1.5 h at 25 °C, and each Fmoc deprotection was carried out using 20% piperidine/ NMP for 30 min. After peptide coupling and Fmoc deprotection, CA (2 eq.), BOP (2 eq.) or HBTU (2 eq.), and HOBt (2 eq.) in NMP were added to the peptide anchored resin with DIPEA (4 eq.). CA coupling reaction was performed for 5 h at 25 °C, and the product was separated from the resin by treating the resin with 31.3% TFA / 0.83% DODT / 0.83% H2O / 0.3% TIPS in dry DCM for 1 h. The resin was filtered, and the filtrate was concentrated under high vacuum, then, precipitated with cold diethyl ether. The resulting CA-Pro-Xaa-NH2 were identified by a quadrupole tandem mass spectrometer, and their

purities were analysed by RP-HPLC using the following conditions: gradient elution with A: 0.1% TFA/water, B: 0.1% TFA/acetonitrile; from 0% to 100% B over 35 min, at a flow rate of 1.0 mL/min; detection: UV, 260 nm. Six kinds of selected CA-Pro-Xaa-NH2 were purified by a preparative RP-HPLC column using an A to B gradient (A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile; from 10% to 90% B over 30 min, at a flow rate of 4.0 mL/min) and freeze-dried for the lipid peroxidation test. 2.4. Measurement of free radical scavenging activity The percentage of radical scavenging activity (% RSA) was determined by measuring the degree of decrease in the absorbance of the DPPH solution after adding the antioxidant. Methanolic DPPH solution (0.1 mM, 1480 lL) was mixed with 20 lL of 1.85 mM CA-Pro-Xaa-NH2 or CA dissolved in methanol in a 1.5-mL Eppendorf tube. The absorbance was measured at 516 nm after 10 min. The percentage of RSA was calculated through the following equation:

%RSA ¼ ½Abs516nm ðt ¼ 0Þ  Abs516nm ðt ¼ t0 Þ=Abs516nm ðt ¼ 0Þ  100 Control was a mixture of 0.1 mM methanolic DPPH solution (1480 lL) and 20 lL of methanol instead of the antioxidant. Each experiment was performed in triplicate and repeated three times. 2.5. Measurement of anti-oxidative activity Linoleic acid emulsion (50 mM) was prepared by mixing 0.284 g of linoleic acid, 0.284 g of Tween 20 and 50 mL of 0.1 M sodium phosphate buffer (pH 7.0). For the oxidation test, we prepared a reaction mixture containing 0.5 mL of water, 2.5 mL of the linoleic acid emulsion, 2.0 mL of 0.1 M sodium phosphate buffer (pH 7.0) and 0.5 mL of methanolic test samples in a 10-mL glass vial. The total reaction volume was fixed uniformly to 5.5 mL, and the final concentration of antioxidant was 90 lM. The glass vials containing reaction mixture were capped with rubber septa, and kept at 50 °C under dark conditions for 48 h. As a negative control, methanol was added instead of the antioxidant. Part of the reaction mixture was withdrawn at specific intervals, and analysed using a modified ferric thiocyanate method (Chen, Muramoto, Yamauchi, & Nokihara, 1996) as follows to evaluate antioxidative activities: reaction mixture (25 lL) was mixed with 1.175 mL of 75% ethanol, 25 lL of 20 mM FeCl2 in 3.5% HCl and 25 lL of 30% ammonium thiocyanate in a 1.5-mL Eppendorf tube. After exactly 3 min, the absorbance was determined at 500 nm, when the colour was developed by FeCl2 and thiocyanate and reached a maximum. Each assay was performed in triplicate and repeated three times. 2.6. 1H NMR studies Samples for NMR experiments were approximately in 3 mM/ MeOD. The samples were purified by RP-HPLC and lyophilised before dissolving in MeOD. The NMR spectra were acquired by a Bruker AVANCE-600 spectrometer at 297 K. The fraction of cis-proline conformer was determined by integrating well-resolved peaks in the one-dimensional 1H NMR spectra. 3. Results and discussion 3.1. Synthesis of CA-Pro-Xaa-NH2 Twenty different CA-Pro-Xaa-NH2 were synthesised through the solid phase method using Fmoc strategy on Rink amide AM

849

S.-Y. Kwak et al. / Food Chemistry 130 (2012) 847–852

H2N (a) (1) Rink Amide AM resin (0.82 mmol/g)

O

H N

O O

R

(b) - (c)

N H

(2) O

O O

O

H N

N O

R

N N H

(d) - (e)

R N H

H N O

O OH (4)

(3) OH O

N (f)

R N H

NH2 O (5) CA-Pro-Xaa-NH 2 Derivatives

O OH OH

Fig. 1. Solid-phase synthesis of CA-Pro-Xaa-NH2. Reagent and conditions: (a) Fmoc-L-amino acids (2 eq.), BOP (2 eq.), HOBt (2 eq.) and DIPEA (4 eq.) in NMP for 1.5 h, (b) 20% Piperidine/NMP (v/v) for 30 min, (c) Fmoc-L-Pro-OH (2 eq.), BOP (2 eq.), HOBt (2 eq.) and DIPEA (4 eq.) in NMP for 1.5 h, (d) repeat (b), (e) CA (2 eq.), BOP (2 eq.), HOBt (2 eq.) and DIPEA (4 eq.) in NMP for 5 h, (f) cleavage cocktail: 31.3% TFA / 8.3% DODT / 8.3% H2O / 0.3% TIPS in dry DCM for 1 h, and diethyl ether precipitation. R: side chain of amino acid.

Table 1 Characterisation of CA-Pro-Xaa-NH2. Compounds

1 2 3 4 5 6 7 8 9 10

ESI-MS

CA-PA-NH2 CA-PC-NH2 CA-PD-NH2 CA-PE-NH2 CA-PF-NH2 CA-PG-NH2 CA-PH-NH2 CA-PI-NH2 CA-PK-NH2 CA-PL-NH2

Compounds

calculated [M + H]+

Found

348.4 380.4 392.4 406.4 424.5 334.3 414.2 390.5 405.5 390.5

348.2 380.1 392.1 406.1 424.2 334.1 414.2 390.1 405.2 390.1

resin (Fig. 1). The resin was treated with 31.3% TFA / 0.8% DODT / 0.8% H2O / 0.3% TIPS in dry DCM for 1 h at 25 °C to separate the product. CA-Pro-Xaa-NH2 derivatives were obtained as white powder by diethyl ether precipitation with 60–85% purity and characterised by ESI–MS (Table 1). Six kinds of selected CA-Pro-Xaa-NH2, which were purified by a preparative RP-HPLC column, were obtained with >95% purity. 3.2. Free radical-scavenging activities of CA-Pro-Xaa-NH2 DPPH radical-scavenging test was performed to evaluate the free radical-scavenging activity (RSA) of CA-Pro-Xaa-NH2. Most CA-Pro-Xaa-NH2 derivatives showed higher % RSA than CA, and few derivatives such as 16, 1 and 3 maintained the % RSA of CA (Fig. 2). These results show that % RSA of CA was influenced by the type of proline dipeptides. Among them, 20, 11, 2, 15, 10, 8 and 9 showed more than 70% RSA. Compound 20 (CA-Pro-TyrNH2) had a phenolic hydroxyl group, 11 (CA-Pro-Met-NH2) and 2 (CA-Pro-Cys-NH2) had sulphur, 15 (CA-Pro-Arg-NH2) and 9 (CAPro-Lys-NH2) had a positively charged group, 10 (CA-Pro-LeuNH2) and 8 (CA-Pro-Ile-NH2) had neutral aliphatic chains.

11 12 13 14 15 16 17 18 19 20

ESI-MS

CA-PM-NH2 CA-PN-NH2 CA-PP-NH2 CA-PQ-NH2 CA-PR-NH2 CA-PS-NH2 CA-PT-NH2 CA-PV-NH2 CA-PW-NH2 CA-PY-NH2

calculated [M + H]+

Found

408.5 391.4 374.4 405.4 433.5 364.4 378.4 376.4 463.5 440.5

408.1 391.1 374.1 405.1 433.3 364.2 378.1 376.2 463.1 440.1

Interestingly, compound 20 (CA-Pro-Tyr-NH2), 19 (CA-Pro-TrpNH2), 7 (CA-Pro-His-NH2) and 5 (CA-Pro-Phe-NH2) exhibited different % RSA, even though all of these had aromatic side chains and showed higher % RSA than CA. The percentage of RSA decreased in the following order: 20 (78.7 ± 3.9) > 19 (66.3 ± 1.2) > 7 (60.6 ± 0.7), 5 (58.5 ± 6.8). It was previously reported that CA showed enhanced antioxidative activity when conjugated with aromatic amino acids (Son & Lewis, 2002; Spasova et al., 2006). Furthermore, the % RSA of these CA-Pro-Xaa-NH2 derivatives is affected by the kinds of aromatic side chain next to proline (Rice-Evans, Miller, & Paganga, 1996). We found that some CA-Pro-Xaa-NH2 derivatives revealed higher % RSA than 7, while 7 showed the highest% RSA among histidinecontaining CA dipeptide conjugates. 3.3. Antioxidative activities of CA-Pro-Xaa-NH2 The lipid peroxidation inhibition test was performed using Tween 20-emulsified linoleic acid (>99%) to measure the antioxidative activities of CA-Pro-Xaa-NH2. The lipid peroxyl radicals induced by air spontaneously cause lipid peroxidation for the

850

S.-Y. Kwak et al. / Food Chemistry 130 (2012) 847–852

% Radical Scavenging Activity

100 90

77.2

76.4 77.8

76.2

80

78.7

76.8

71.2

70

60.6

60 50 40 30 20 10 0 CA

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Fig. 2. DPPH radical scavenging activity of CA-Pro-Xaa-NH2. [Antioxidant] / [DPPH] (mol/mol) = 0.25. Each experiment was performed in triplicate and repeated three times. The values are given as the mean ± standard error.

2.0 1.8

Absorbance at 500 nm

1.6 1.4

Control

1.2

CA-PC CA-PM

1.0

CA-PL CA-PY

0.8

CA-PR 0.6

CA-PH CA

0.4

BHA 0.2 0.0 0

10

20

30

40

50

Time (hours) Fig. 3. Antioxidative activities of CA-Pro-Xaa-NH2 on the basis of time in emulsified lipid peroxidation system. The absorbance was determined at 500 nm by ferric thiocyanate method. Conditions: the final concentration of antioxidant was 90 lM; the reaction was performed at 50 °C under dark conditions. Each experiment was performed in triplicate and repeated three times. The values are given as the mean ± standard error.

experimental period. We selected six kinds of CA-Pro-Xaa-NH2 having more than 70% RSA, i.e. 20, 11, 2, 15 10 and 7. We excluded 8 and 9 from the experimental group because % RSA of 8 and 9 were too close to the standard boundary, although both have % RSA of above 70%. On the other hand, we included 7 into the experimental group, to compare the degree of antioxidative activities with selected compounds, since 7 showed excellent lipid peroxidation inhibitory activity in our previous work. We evaluated the antioxidative activities by measuring UV absorbance for 48 h at specified intervals (Fig. 3) and evaluated the percent of lipid peroxidation inhibition (% Pi). The percentage of Pi was calculated when the absorbance of the control reached approximately 1 (0.9–1.1). As shown in Fig. 4, CA-Pro-His-NH2 (% Pi = 88.8 ± 0.9) inhibited lipid peroxidation as much as BHA (% Pi = 89.2 ± 0.5), and other CAPro-Xaa-NH2 showed inferior lipid peroxidation inhibitory activity (% Pi = 36.4–51.7). In addition, all of the CA-Pro-Xaa-NH2 had enhanced antioxidant activity compared to CA alone (% Pi = 9.7 ± 2.5); this is probably due to increased accessibility to the emulsified lipid phase and thermal stability caused by peptide conjugation (Taneja & Ahmad, 1994).

As shown in Figs. 2 and 4, compounds 11, 15, 10, 2 and 20 showed a similar tendency in % Pi and % RSA. However, 7 showed different type of antioxidant activity in % Pi and % RSA. When 7 was observed in Figs. 2–4, it showed enhanced % RSA and % Pi compared to CA, but exhibited inferior% RSA than 11, 15, 10, 2 and 20 in the DPPH radical-scavenging test. On the other hand, compound 7 showed noticeably higher antioxidative activity than other CA-ProXaa-NH2 derivatives in the lipid peroxidation inhibition test. This can be explained by the two different environments for measuring antioxidative activity. The DPPH radical-scavenging test was performed using the hydrophilic solvent, methanol, which can generate hydrogen bonds, whereas the lipid peroxidation inhibition test was carried out in a biphasic system which consisted of emulsified linoleic acid and sodium phosphate buffer. We concluded that caffeoyl–peptide conjugates may have better accessibility to the emulsified lipid than CA itself because they have both hydrophobic and hydrophilic parts (Kwak et al., 2009). However, the excellent lipid oxidation inhibitory activity of 7 could not be explained by accessibility only, but by structural benefit. We assumed that only 7 could have optimised structure for maintaining CA’s antioxidant

S.-Y. Kwak et al. / Food Chemistry 130 (2012) 847–852

851

Fig. 4. The percent of lipid peroxidation inhibition of CA-Pro-Xaa-NH2 in emulsified lipid peroxidation system, which was determined when the absorbance value of control reached approximately to 1 at 500 nm. Conditions: the final concentration of antioxidant was 90 lM; reaction was performed at 50 °C under dark conditions. Each experiment was performed in triplicate and repeated three times. The values are given as the mean ± standard error.

activity at high temperature by stabilising its phenoxyl radical after quenching free radicals. 3.4. Conformation analysis of CA-Pro-His-NH2 We focused on hydrogen bonding effects of CH = proton. CH = proton without hydrogen bonding in trans proline configuration was observed at 6.43 ppm, which was downfield shifted to 6.70 ppm upon hydrogen bonding in cis-proline configuration. NMR experiment on CA-Pro-NH2 (Supporting Fig. 1) revealed that the ratio of cis/trans conformer was 70%. This follows a similar trend to the previous report that cis-proline conformer is more stabilised by favourable interaction between the aromatic ring and the proline residue (We & Raleigh, 1998). NMR analysis of CAPro-His-NH2 (Supporting Fig. 2) showed that the ratio of cis/trans conformer was >90%, which was determined by CH = proton and His Ce1–H proton. The chemical shift of His Ce1–H proton appeared at 8.40 ppm, when histidine existed as imidazolium ion (Ash et al., 2000). However, His Ce1–H proton of CA-Pro-His-NH2 appeared at 8.80 ppm, in a more downfield position, probably due to the hydrogen bonding effect (Ash et al., 2000). To compare the ratio of cis/ trans between CA-Pro-His-NH2 and other CA-Pro-Xaa-NH2, we performed an NMR experiment on CA-Pro-Leu-NH2 as well (Supporting Fig. 3). The ratio of cis conformer of CA-Pro-Leu-NH2 was approximately 70%, which was determined by CH = proton and CH3 proton. Therefore, we conclude that CA-Pro-His-NH2 mostly exists as cis-conformer, which was stabilised by interaction between the aromatic ring and the proline, and hydrogen bonding effect. In addition, when CA-Pro-His-NH2 exists as cis-conformer, hydroxyl group of CA can be stabilised by imidazolium ion, after hydrogen radical abstraction to quench free radicals. This mechanism strongly supports the exceptionally enhanced antioxidant activity of CA-Pro-His-NH2. CA-Pro-NH2, 1H NMR (600 MHz, MeOD, 297 K): cis-conformer: d/ppm = 1.92–2.05 (m, 2H, cH), 2.08–2.24 (m, 2H, bH), 3.86; 3.74 (m, 2H, dH), 4.49 (dd, 1H, aH), 6.70 (d, 1H, CH=), 6.77 (d, 1H, ArH), 6.97 (dd, 1H, Ar-H), 7.05 (d, 1H, Ar-H), 7.45 (d, 1H, CH=). trans-conformer: d/ppm = 1.92–2.05 (m, 2H, cH), 2.08–2.24 (m, 2H, bH), 3.86; 3.74 (m, 2H, dH), 4.58 (m, 1H, aH), 6.43 (d, 1H, CH=), 6.77 (d, 1H, Ar-H), 6.90 (dd, 1H, Ar-H), 7.05 (d, 1H, Ar-H), 7.46 (d, 1H, CH=). CA-Pro-His-NH2, 1H NMR (600 MHz, MeOD, 297 K): cis-conformer: d/ppm = 1.91–2.05 (m, 2H, cH), 2.05–2.28 (m, 2H, bH),

3.35; 3.12 (dd, 2H, CH2), 3.76–3.88 (m, 2H, dH), 4.47 (dd, 1H, aH), 4.74 (dd, 1H, CH), 6.72 (d, 1H, CH=), 6.77 (d, 1H, Ar-H), 6.97 (dd, 1H, Ar-H), 7.07 (d, 1H, Ar-H), 7.37 (s, 1H, His Cd2-H), 7.51 (d, 1H, CH=), 8.80 (s, 1H, His Ce1–H). trans conformer: d/ppm = 1.91– 2.05 (m, 2H, cH), 2.05–2.28 (m, 2H, bH), 3.35; 3.12 (dd, 2H, CH2), 3.76–3.88 (m, 2H, dH), 4.47 (dd, 1H, aH), 4.74 (dd, 1H, CH), 6.38 (d, 1H, CH=), 6.77 (d, 1H, Ar-H), 6.90 (dd, 1H, Ar-H), 7.07 (d, 1H, Ar-H), 7.37 (s, 1H, His Cd2-H), 7.51 (d, 1H, CH=), 8.40 (s, 1H, His Ce1–H). CA-Pro-Leu-NH2, 1H NMR (600 MHz, MeOD, 297 K): cis-conformer: d/ppm = 0.92 (d, 3H, CH3), 0.95 (d, 3H, CH3), 1.64–1.69 (m, 3H), 1.91–2.05 (m, 2H, cH), 2.05–2.27 (m, 2H, bH), 3.88; 3.75 (m, 2H, dH), 4.40 (dd, 1H, aH), 4.50 (dd, 1H, CH), 6.70 (d, 1H, CH=), 6.77 (d, 1H, Ar-H), 6.97 (dd, 1H, Ar-H), 7.05 (d, 1H, Ar-H), 7.46 (d, 1H, CH=). trans conformer: d/ppm = 0.78 (d, 3H, CH3), 0.85 (d, 3H, CH3), 1.64–1.69 (m, 3H), 1.91–2.05 (m, 2H, cH), 2.05–2.27 (m, 2H, bH), 3.86; 3.74 (m, 2H, dH), 4.47 (dd, 1H, aH), 4.74 (dd, 1H, CH), 6.43 (d, 1H, CH=), 6.77 (d, 1H, Ar-H), 6.91 (dd, 1H, Ar-H), 7.05 (d, 1H, Ar-H), 7.42 (d, 1H, CH=). 4. Conclusion We prepared 20 kinds of CA-Pro-Xaa-NH2 to show that proline had an important role in providing excellent antioxidative activity in histidine-containing CA dipeptides. Five kinds of CA-Pro-XaaNH2, 11, 15, 10, 2 and 20, showed slightly higher % RSA than 7 (CA-Pro-His-NH2). However, considering the results from the DPPH radical-scavenging test and lipid peroxidation inhibition test, 7 (CA-Pro-His-NH2) was the best antioxidant. From this study, we demonstrated that proline gives a critical effect on the enhanced antioxidant activity of CA-Pro-His-NH2. In addition, we found that the imidazole ring of histidine could have an optimised structure with proline to exert synergistic antioxidative activity. Further study on the structural benefits of CA-Pro-His-NH2 is under way. Acknowledgement This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A050432) and WCU (World Class University) program, Korea Science and Engineering Foundation, Ministry of Education, Science and Technology (R32-2010-00010213-0).

852

S.-Y. Kwak et al. / Food Chemistry 130 (2012) 847–852

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2011.07.096. References Apel, K., & Hirt, H. (2004). Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology, 55, 373–399. Ash, E. L., Sudmeier, J. L., Day, R. M., Vincent, M., Torchilin, E. V., Haddad, K. C., et al. (2000). Unusual 1H-NMR chemical shifts support (His) Ce1–HO=C H-bond: Proposal for reaction driven ring flip mechanism in serine protease catalysis. Proceedings of the National Academy of Sciences, 97, 10371–10376. Benzi, G., & Moretti, A. (1995). Are reactive oxygen species involved in alzheimer’s disease? Neurobiology of Aging, 16(4), 661–674. Branen, A. L. (1975). Toxicology and biochemistry of butylated hydroxyanisole and butylated hydroxytoluene. Journal of the American Oil Chemists’ Society, 52(2), 59–63. Chen, H. M., Muramoto, K., & Yamauchi, F. (1995). Structural-analysis of antioxidative peptides from soybean beta-conglycinin. Journal of Agricultural and Food Chemistry, 43(3), 574–578. Chen, H. M., Muramoto, K., Yamauchi, F., & Nokihara, K. (1996). Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. Journal of Agricultural and Food Chemistry, 44(9), 2619–2623. Chen, Z. Y., Chan, P. T., Kwan, K. Y., & Zhang, A. (1997). Reassessment of the antioxidant activity of conjugated linoleic acids. Journal of the American Oil Chemists Society, 74(6), 749–753. Cheng, Z. Y., Ren, J., Li, Y. Z., Chang, W. B., & Chen, Z. D. (2002). Study on the multiple mechanisms underlying the reaction between hydroxyl radical and phenolic compounds by qualitative structure and activity relationship. Bioorganic & Medicinal Chemistry, 10(12), 4067–4073. Guan, Y. Q., Chu, Q. C., Fu, L. A., & Ye, J. N. (2005). Determination of antioxidants in cosmetics by micellar electrokinetic capillary chromatography with electrochemical detection. Journal of Chromatography A, 1074(1–2), 201–204. Halliwell, B. (1996). Antioxidants in human health and disease. Annual Review of Nutrition, 16, 33–50. Kaiser, E., Colescott, R. L., Bossinger, C. D., & Cook, P. I. (1970). Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Analytical Biochemistry, 34(2), 595–598. Kwak, S. Y., Seo, H. S., & Lee, Y. S. (2009). Synergistic antioxidative activities of hydroxycinnamoyl-peptides. Journal of Peptide Science, 15(10), 634–641. Merrifield, R. B. (1963). Solid phase peptide synthesis I. The synthesis of a tetrapeptide. Journal of the American Chemical Society, 85(14), 2149–2154.

Mitsuda, H., Yasumodo, K., & Iwami, F. (1966). Antioxidative action of indole compounds during the autoxidation of linoleic acid. Eiyo to Shokuryo, 19, 210–214. Moure, A., Cruz, J. M., Franco, D., Dominguez, J. M., Sineiro, J., Dominguez, H., et al. (2001). Natural antioxidants from residual sources. Food Chemistry, 72(2), 145–171. Pekkarinen, S. S., Stockmann, H., Schwarz, K., Heinonen, I. M., & Hopia, A. I. (1999). Antioxidant activity and partitioning of phenolic acids in bulk and emulsified methyl linoleate. Journal of Agricultural and Food Chemistry, 47(8), 3036–3043. Perry, G., Castellani, R. J., Hirai, K., & Smith, M. A. (1998). Reactive oxygen species mediate cellular damage in alzheimer disease. Journal of Alzheimer’s Disease, 1, 45–55. Pokorny, J. (2007). Are natural antioxidants better – and safer – than synthetic antioxidants? European Journal of Lipid Science and Technology, 109(6), 629–642. Rayner, B. S., Duong, T. T. H., Myers, S. J., & Witting, P. K. (2006). Protective effect of a synthetic anti-oxidant on neuronal cell apoptosis resulting from experimental hypoxia re-oxygenation injury. Journal of Neurochemistry, 97(1), 211–221. Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20(7), 933–956. Seo, H. S., Kwak, S. Y., & Lee, Y. S. (2010). Antioxidative activities of histidine containing caffeic acid-dipeptides. Bioorganic & Medicinal Chemistry Letters, 20(14), 4266–4272. Shahidi, F. (2000). Antioxidants in food and food antioxidants. Nahrung-Food, 44(3), 158–163. Shahidi, F., Janitha, P. K., & Wanasundara, P. D. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition, 32(1), 67–103. Son, S., & Lewis, B. A. (2002). Free radical scavenging and antioxidative activity of caffeic acid amide and ester analogues: Structure–activity relationship. Journal of Agricultural and Food Chemistry, 50(3), 468–472. Spasova, M., Kortenska-Kancheva, V., Totseva, I., Ivanova, G., Georgiev, L., & Milkova, T. (2006). Synthesis of cinnamoyl and hydroxycinnamoyl amino acid conjugates and evaluation of their antioxidant activity. Journal of Peptide Science, 12(5), 369–375. Stankova, I., Chuchkov, K., Shishkov, S., Kostova, K., Mukova, L., & Galabov, A. S. (2009). Synthesis, antioxidative and antiviral activity of hydroxycinnamic acid amides of thiazole containing amino acid. Amino Acids, 37(2), 383–388. Taneja, S., & Ahmad, F. (1994). Increased thermal-stability of proteins in the presence of amino acids. Biochemical Journal, 303, 147–153. We, W. J., & Raleigh, D. P. (1998). Local control of peptide conformation: Stabilization of cis proline peptide bonds by aromatic proline interactions. Biopolymer, 45, 381–394. Zhao, B. (2009). Natural antioxidants protect neurons in Alzheimer’s disease and Parkinson’s disease. Neurochemical Research, 34, 630–638.