Antioxidative activities of some peptides isolated from hydrolyzed potato protein extract

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Antioxidative activities of some peptides isolated from hydrolyzed potato protein extract Katsuhiro Kudoa, Shuichi Onoderab, Yasuyuki Takedab, Noureddine Benkebliac,*, Norio Shiomib a

Department of Bioresources Engineering, Okinawa National College of Technology, 905 Henoko, Nago, Okinawa 905-2192, Japan Department of Food Science, Faculty of Dairy Science Rakuno Gakuen University, Ebetsu 069-8501, Japan c Department of Life Sciences, The University of the West Indies, Mona Campus, Kingston 7, Jamaica b

A R T I C L E I N F O

A B S T R A C T

Article history:

Three peptides (5A, 5C and 6C), purified from potato protein hydrolysate fractions, pos-

Received 3 November 2008

sessed antioxidative activities. Isolation and purification were carried out using gel perme-

Accepted 14 January 2009

ation chromatography and successive reverse-phase HPLC. These three peptide fractions

Available online 27 February 2009

were sequenced and identified as Phe-Gly-Glu-Arg, Phe-Asp-Arg-Arg and Phe-Gly-GluArg-Arg, respectively. The fractions 5A, 5C and 6C inhibited linoleic acid oxidation by

Keywords:

55.3%, 58.5% and 61.7% using b-carotene decolorization assay system, while the inhibition

Antioxidative activities

ratio was 32.1%, 93.0% and 93.4% in the ferric thiocyanate assay system, respectively. The

Gastric damage

peptide fractions 5A, 5C and 6C also repressed lipid oxidation by 24.2%, 14.7% and 26.4% in

Peptides

the erythrocyte membrane ghost assay system, respectively. Oral administration of 100 mg/

Potato hydrolysate

kg of body weight of the chemically synthesized peptides, 5A, 5C and 6C to rats (male Wistar) 30 min prior to ethanol injection reduced ethanol-induced gastric mucosal damage by 67.9%, 57.0% and 60.3%, respectively. Conclusively, these peptides have shown real potent antioxidative activities and could further be investigated for potential use as food additives.  2009 Elsevier Ltd. All rights reserved.

1.

Introduction

The continuous increase of the human population over the last decades has considerably influenced the demand for food and food products. Potato (Solanum tuberosum L.) is a major world crop with annual world production of more than 300 million tonnes. Potato tuber is considered the most important vegetable in many developing and developed countries. Potatoes are used for several purposes, including human consumption as fresh or processed produce (French fries, mashed potato), industrial processing (potato starch, alcohol, etc.) and recultivation (potato seed) (Feustel, 1987; Talburt, 1987). Numerous bioactive peptides with different physiological functions have been identified. Furthermore, recent findings

in animals and humans have suggested that, in gastrointestinal digestion, peptides may mediate many of the actions of parent proteins by acting as regulatory compounds with hormone-like activities (Korhonen & Pihlanto, 2003). During enzymatic hydrolysis process, proteins are cleaved to smaller molecules, namely peptides and free amino acids. Hence, the nutritional quality and safety of products are improved. New products could be generated and alternative applications for several agricultural products could be realized (Kamnerdpetch et al., 2007). In animal organisms, about 1% of oxygen taken up by breathing is converted to reactive oxygen species (ROS) in mitochondria and superoxide radicals in microsomes. Although these free radicals and ROS are known to play

* Corresponding author: Tel.: +1 876 927 1202; fax: +1 876 977 1075. E-mail address: [email protected] (N. Benkeblia). 1756-4646/$ - see front matter  2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2009.01.006

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2.

Materials and methods

2.1.

Potato extract solution

Potato tubers were washed with water, peeled and homogenized using high-speed blender to obtain slurry containing cell walls, juice and starch. The slurry was then screened to remove cell walls, then centrifuged at 10,000g for 30 min to separate starch from water soluble materials which contained about 1% proteins. The supernatant was collected as ‘‘potato extract solution’’ and used for the preparation of the potato protein hydrolysate.

2.2.

Preparation of potato protein hydrolysate

Potato extract solution was heated at 60 C for 30 min and centrifuged at 10,000g for 30 min. The supernatant was discarded and the precipitate collected and its pH adjusted to 7.0 by adding 0.2 M NaOH solution. Two proteases were added to the solution at the concentration of 1:100 (w/v): Protease 1 (Pancreatin) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and protease 2 (Amano-P) (Amano Enzyme Inc., Nagoya,

Japan). The final mixture was incubated under low stirring at 45 C for 2 h. Afterwards the mixture was centrifuged at 10,000g for 30 min and the supernatant collected while discarding the precipitate. After ultrafiltration using an Amico Centriprep YM 10 filter (Amicon Bioseparation, Millipore, Bedford, MA, USA), the supernatant eluted using ion-exchange resins (PA308, Mitsubishi Chemical Co., Tokyo, Japan) to obtain peptide-rich fractions. The different fractions obtained were finally concentrated and dried by lyophilization for 5 days using a Bench Top Freeze Drayer (Millrock Technology Inc., Kingston, NY, USA). The powders of the different fractions were collected for analysis.

2.3. Separation and isolation of peptides from potato protein hydrolysate Potato protein hydrolysate solution (1 g/20 mL) was applied on a Toyopearl HW-40C column (4.0 · 151 cm, Tosoh Co., Tokyo, Japan), and eluted with distilled water at a flow rate of 150 mL/h. The quantitative level of peptides contained in the collected fractions after the elution were detected at 280 nm absorbance. Each fraction was concentrated using a rotavapor and lyophilized. Ten different powdered fractions named P-I  X were obtained, and this preparative chromatography was repeated ten times (Fig. 1).

2.4.

Fractions purification

PF-V showed a potent antioxidative activity and hence was further purified by reverse-phase HPLC. PF-V was dissolved in trifluoroacetic acid (TFA) (20:2, w/v) and loaded onto a reversephase HPLC fitted with Megapak Crest C18-T5 column (10 · 250 mm, Japan Spectroscopic Co., Ltd., Tokyo, Japan) using a linear gradient of isopropanol/acetonitrile (7:3, v/v) from 0% to 40% in 0.1% TFA for 2 h at a flow rate of 0.8 mL/min. The loaded fraction was separated into 17 fractions (P-1 to P-17) by collecting an eluate each 5 min during the elution time from 15 to 95 min as shown in Fig. 2. This chromatographic process was repeated 138 times and the purified fractions were concentrated under vacuum and then freeze-dried. The PF-5, PF-6 and PF-10 fractions which showed high antioxidative activities were loaded on reverse-phase HPLC as described above. The peptide fractions 5A (11.2 mg), 5B (5.7 mg), 5C (6.4 mg), 5D

2.0

Absorbance at 280 nm

important roles in protection against microbial infection they also induce the inactivation of some enzymes, accumulation of lipid peroxides, and cause injury to the host cells or tissues leading to many chronic diseases, including some cancers, brain diseases and cardiovascular diseases (CVD) (Waddington et al., 2000). To date, numerous natural antioxidants have been reported to prevent diseases such as Alzheimer (Jin et al., 2004), myocardial disease (Kannan et al., 2004), and ethanolinduced liver injury (Molina et al., 2003). They also are suspected to suppress proliferation of cancer cell lines (Hirota et al., 2000). Recently, protein such as bovine serum albumin, gelatin or casein, along with their hydrolysates or peptides, as well as polyphenols, flavonoids, vitamins C and E in plants, have also been found to possess antioxidative activity (Chen et al., 1998; Okada & Okada, 1998; Suetsuna et al., 2000; Kim et al., 2001; Saito et al., 2003; Shahidi et al., 2008). Previously, we investigated the antioxidative activity of potato protein hydrolysate in vitro and in vivo and demonstrated that protein hydrolysate would be a potent antioxidant (Kudo et al., 2003). Compared to proteins, peptides have lower molecular weight and less complex structures. Consequently, their solubility, digestibility and absorbability are expected to be higher than those of proteins. Therefore, it would be interesting to synthesize and modify enzymatically or chemically the peptides to increase these chemical and biochemical properties. To date, no studies have been reported the antioxidative activity of peptides of low degree of polymerization obtained from potato extracts except of Kudo et al. (2003), while a work reported on peptides obtained from animal proteins (Kim et al., 2001). The aims of this investigation were to isolate, purify and identify bioactive peptides from potato protein hydrolysate using gel permeation chromatography and reverse-phase HPLC and to evaluate their potential antioxidative activities using different assessment methods.

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1.5

1.0

0.5

0.0 50

100

150

Fraction number Fig. 1 – Chromatogram of potato protein hydrolysate using Toyopearl HW-40C column.

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Absorbance at 216 nm

2.7. Antioxidative activity measurement of peptides using rabbit erythrocyte membrane ghost system

20

40

60

80

Retention time (min) Fig. 2 – High performance liquid chromatogram of the protein fraction PF-5 separated by Toyopearl HW-40C column chromatography.

(8.0 mg) and 5E (2.9 mg) were isolated from P-5 (52.8 mg), the peptide fractions 6A (5.0 mg), 6B (1.4 mg), 6C (3.8 mg) and 6D (1.3 mg) from P-6 (21.9 mg), and 10A (6.6 mg), 10B (2.6 mg), 10C (3.8 mg) and 10D (8.0 mg) from P-10 (21.4 mg).

2.5. Antioxidative activity measurement of peptides using b-carotene decolorization method The method of b-carotene decolourization was carried out according to method described by Miller (1968) with slight modifications. A chloroform solution (3.4 mL) containing linoleic acid (100 mg/mL), b-carotene (1 mg/mL) and Tween 40 (200 mg/mL) was evaporated to dryness under vacuum. The dry residue was dissolved in 0.2 M phosphate buffer of pH 6.8 (17.8 mL) and distilled water (200 mL). Subsequently, 4.9 mL of the blending solution were added to 0.1 mL of test (containing the different peptides) or reference solution containing BHA). The optical density was monitored at 470 nm each 10 min from 0 to 40 min at 50 C. The inhibitory ratio (IR) was calculated as follow (Igarashi et al., 1993): IRð%Þ ¼ ½ð100  PcÞ  ð100  PsÞ=ð100  PcÞ  100 Pc = [OD470 of the control after 40 min/OD470 of the control at 0 min] · 100; Ps = [OD470 of sample after 40 min/OD470 of the sample at 0 min] · 100.

2.6. Antioxidative activity measurement of peptides by ferric thiocyanate method Antioxidative activity using the ferric thiocyanate method was measured as described by Osawa and Namiki (1981) with some modifications. The reaction mixture, composed by 2 mL of 0.05 M phosphate buffer (pH 7.0), 2 mL of ethanol containing 1.25% linoleate and 0.05% Tween 80, and 1 mL of 0.05% test peptide solution, was incubated at 50 C for 168 h, and an aliquot (0.1 mL) was taken each 24 h. To this aliquot was added 9.7 mL of 75% ethanol, 0.1 mL of 30% ammonium thiocyanate and 0.1 mL of 0.02 M iron(II) chloride solution. The optical density was measured at 500 nm. For the reference standard, in the above assay system, 1 mL of BHA (butylated hydroxyanisole) solution (0.05%) was added instead of peptide solution. The inhibition ratio (IR) was calculated as follow: IRð%Þ ¼ 100  ðOD500 of the sample=OD500 of the controlÞ  100:

The oxidation of erythrocyte membrane ghost was measured according to the method described by Osawa et al. (1987) with some modifications. Rabbit blood was diluted in equal volume of SSC (saline sodium citrate buffer solution (150 mM NaCl, 15 mM Na-Citrate, pH 7.4) and centrifuged at 1500g for 20 min. The precipitate was collected and supernatant centrifuged two times under the same conditions. The collected precipitates were combined and then lysed in 10 mM sodium phosphate buffer (pH 7.4) and subsequently centrifuged at 20,000g for 40 min. The supernatant was discarded and the pellet containing the erythrocyte membranes ghost was collected. Protein concentration of erythrocyte membranes ghost was determined by the method of Lowry et al. (1951). To 0.1 mL of sample dissolved in 80% ethanol was added a mixture containing 0.85 mL of erythrocyte membrane ghost (prepared for 100 lg/mL of protein concentration) and 0.05 mL of 24 mM t-butylhydroperoxide. The final preparation (total volume of 1.0 mL) was incubated at 37 C for 30 min. The incubated mixture was then immediately cooled in iced water, and a mixture containing 10 lL of 2% BHT (butylated hydroxytoluene) then in ethanol, 0.5 mL of 20% trichloroacetic acid solution, and 0.1 mL of 0.67% thiobarbituric acid (TBA) solution were added. This mixture was boiled at 100 C for 10 min, and centrifuged at 4000g for 15 min. The optical density at 532 nm of the pink colored supernatant was measured as TBA-reactive substance (TBARS) equivalent to malondialdehyde (MDA) formed.

2.8. Measurement of the protective effect of peptide against ethanol-induced gastritis The in vivo antioxidative activity was evaluated using animal experiment (rat) and ethanol-induced acute ulcer (Szelenyi & Brune, 1988; Terano et al., 1989). Male Wistar rats of 6 weeks age were fed normally using a 25% casein diet (mineral and vitamin premix, AIN-76 likeness) for 3 days and then starved for one day as described previously (American Institute of Nutrition, 1977; Bieri, 1980). In order to obtain sufficient quantities, the three peptide fractions (5A, 5C and 6C) were chemically synthesized. Then, each peptide sample was dissolved in 5% gum Arabic solution and orally administered at a dose of 50 mg/kg body weight (BW). Thirty minutes later, rats were supplied with absolute ethanol at a dose of 5 mL/kg BW. After one hour, the stomach was excised under anesthesia with intraperitoneal injection of 5% nembutal (1 mL/kg BW). Each excised stomach was distended, immersed in 5% formalin after removal of the content, and then opened along the line of greater curvature and spread on a filter paper. The degree of gastritis was observed by naked eye, followed by image-analysis using Adobe Photoshop LE-J and NIH-Image. The percentage of the lesion area on mucosa of the stomach was estimated, and the damage inhibition ratio (DIR) was calculated as follows: DIR ð%Þ ¼ ½ðDamage ratio of controlÞ  ðDamage ratio of test sample groupÞ =ðDamage ratio of controlÞ  100:

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2.9.

Sequence analysis of isolated peptides

The N-terminal amino acid sequences of the peptides were analyzed using a gas-phase protein sequencer (Applied Biosystems model 477A, Foster City, CA, USA).

2.10.

Synthesis of peptides

Three peptides were synthesized using a solid-phase synthesizer (Applied Biosystems model Pioneer, Foster City, CA, USA).

2.11.

Statistical analysis

The experiments were carried out in triplicate and experimental work duplicated. Data were expressed as the means ± SE (n = 6) and analyzed by two-way ANOVA at p < 0.05 using Stat View 5.0 software (SAS Institute Inc., Cary, NC, USA).

3.

Results and discussion

Potato protein hydrolysate was separated using Toyopearl HW-40C column chromatography and a total of 10 different fractions, namely P-I to P-X, were obtained as shown in Fig. 1. Afterwards a first assessment of antioxidant activity of these ten fractions was carried out using b-carotene decolourization method and the yield of separation of each fraction was also estimated for each of the ten fractions (Table 1). The inhibitory ratio (IR) of the ten fractions showed that three fractions had IR values higher than 30%. The IR of the fractions PF-3, PF-5 and PF-10 was 40.2%, 39.8% and 32.1%, respectively. On the other hand, the inhibitory ratios of the ten fractions was assessed using the ferric thiocyanate method, and we noted that the IRs of the ten fractions were higher than 95%, suggesting that these fractions have high antioxidative activities in comparison to BHA (data not shown). In order to further investigate the antioxidative activity of fraction PF-5, which showed a relatively potent and stable antioxidative activity by using different methods, i.e., b-carotene decolorization and ferric thiocyanate, this faction was

subjected to further separation and purification by reversephase HPLC using an ODS column. The PF-5 fraction was subsequently separated into 17 sub-fractions (numbered from P-1 to 17) and these fractions were obtained after every 5 min from 15 to 95 min of chromatography (Fig. 2). The peptide yield of each fraction was also estimated (Table 2). Although the yield ranged between 2.8 and 22 mg, the antioxidative activity assessed by ferric thiocyanate method of the fractions separated from this PF-5 showed that three fractions, P-5, P-6 and P-10 exhibited the highest antioxidant activity of 93.2%, 94.6% and 93.2%, respectively, suggesting that the antioxidant activities of these peptides were similar to BHA. However, other fractions, P-4, P-7, P-9, P-11 and P-12, also showed antioxidant activity high than 80%. Fractions P-5, P-6 and P-10 were further purified using reverse-phase HPLC as described above. Five peptides, 5A, 5B, 5C, 5D and 5E were obtained from fraction PF-5, five peptides 6A, 6B, 6C, 6D and 6E from fraction PF-6, and four peptides 10A, 10B, 10C and 10D from fraction PF-10. The peptide fractions 5A, 5C and 6C, which showed high yields among the 14 isolated fractions, were sequenced and their amino acid sequences were Phe-Gly-Glu-Arg (FGER), Phe-Asp-Arg-Arg (FDRR) and Phe-Gly-Glu-Arg-Arg (FGERR), respectively (Fig. 3). The antioxidative activities of these three fractions in a b-carotene system was assessed and compared to the antioxidative activity of BHA (assumed to be 100%), and these activities were 55.2%, 58.4% and 61.5% to that of BHA for 5A, 5C and 6C, respectively (Fig. 4). On the other hand, by using the ferric thiocyanate method the antioxidative activity was 32.1%, 93.0% and 93.4%, respectively (Fig. 5). Moreover, in the erythrocyte membrane ghost system, the antioxidative activity was 77%, 83% and 72%, respectively (Fig. 6). The antioxidative properties of potato proteins or their hydrolysates are poorly documented. Pihlanto et al. (2008) evaluated the angiotensin-converting enzyme (ACE) and the radical-scavenging activity of potato proteins isolated from potato tubers at different physiological states. They noted

Table 2 – Antioxidative activity of peptides separated from P-V by RP-HPLC. Fraction no.

Table 1 – Antioxidative activity of the different peptide fractions separated from potato protein hydrolysate using Toyopearl HW-40C column chromatography. Fraction no. P-I P-II P-III P-IV P-V P-VI P-VII P-VIII P-IX P-X

Antioxidative activity (%)* 21.0 13.6 27. 19.4 40.2 39.8 32.1 22.5 27.1 26.0

Yield (mg) 2982 1794 816 733 547 94 67 19 80 143

* Antioxidative activity was measured by b-carotene decolorization method.

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P-1 P-2 P-3 P-4 P-5 P-6 P-7 P-8 P-9 P-10 P-11 P-12 P-13 P-14 P-15 P-16 P-17

Antioxidative activity (%)* 5.1 5.4 71.1 83.8 93.2 94.6 80.8 60.5 83.3 93.2 89.6 84.4 38.4 65.0 39.7 0 59.4

Yield (mg) 18.0 10.8 19.6 7.0 17.8 21.9 17.3 19.9 11.6 9.7 5.7 6.5 4.2 9.6 2.8 3.3 9.9

* Antioxidative activity was measured by ferric thiocyanate method.

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5A

5C

6C

BHA

0

20

40

60

80

100

Inhibition of Peroxidation (%) Fig. 5 – Antioxidative activity of 5A, 5C and 6C of linoleic acid oxidation measured by the ferric thiocyanate assay.

5A

Fig. 3 – The sequences of the amino acids 5A, 5C and 6C.

5C

5A

5C

6C

0

6C

20

40

60

80

100

Lipid Peroxidation (%) Fig. 6 – Antioxidative activity assay of 5A, 5C and 6C measured by the erythrocyte membrane ghost system.

BHA 0

20

40

60

80

10 0

Inhibitory ratio (%) Fig. 4 – Inhibitory ratio of the fractions 5A, 5C and 6C of linoleic acid oxidation system measured by the b-carotene decolorization assay.

that the hydrolysis increased the inhibition of the isolated proteins and the ACE-inhibitory potencies of the hydrolysates were high. They also noted that all samples exhibited low rad-

ical-scavenging activity, while hydrolysis for 2 h with proteases increased the activity. It was also reported that intact and hydrolyzed potato proteins lowered the production of peroxide value (PV) in patties after 7 days of storage by 44.9% and 74.5%, respectively, while thiobarbituric acid-reactive substances (TBARS) values were reduced 40.9% and 50.3%, respectively (Wang & Xiong, 2005). Moreover, Wang and Xiong (2008) investigated the inhibition of oxidant-induced biochemical changes of pork myofibrillar protein by hydrolyzed potato protein (HPP), and found that the presence of HPP reduced the extent of myofibril or protein isolate (MPI)

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Area of damaged gastric body(%)

20

15

10

5

0 Control

PF- 3

PF - 5

PF - 10

Area of damaged gastric mucosal(%)

Fig. 7 – Effect of separated potato peptides on gastric mucosal damage induced by ethanol in rats.

12

9

6

3

Control

5A

5C

6C

Fig. 8 – Effect of the synthesized chemically peptides 5A, 5C and 6C on damaged area of the gastric mucosal induced by ethanol in rats.

oxidation in all samples. Similarly, the major root storage proteins purified from sweet potato, trypsin inhibitors (TIs), were from tuberous roots of two different cultivars showed dosedependent DPPH and hydroxyl radical-scavenging activities with glutathione as a control (Hou et al., 2005). The ability of some hydrolysate fraction to protect gastric mucosal against ethanol damage was investigated in rats. Fractions with high antioxidative activities, namely PF-I to PF-X, were administered orally to male Wistar rat at a dose of 100 mg/kg of body weight 30 min prior to ethanol injection. The area ratios of ethanol-induced gastric mucosal damage were reduced in all rats administered with protein fractions. Although seven fractions did show an inhibitory ratio of

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ethanol-induced gastric mucosal damage lower than 50% (data not shown), the inhibitory ratios of fractions PF-3, PF-5 and PF-10 were higher and at 83.3%, 81.1% and 87.7%, respectively (Fig. 7). Finally, the protective effects of synthesized 5A, 5C and 6C against ethanol-induced gastric mucosal damage were examined in vivo. These three fractions inhibited ethanol-induced gastric damage with inhibition ratios of 67.9%, 57.0% and 60.3%, respectively (Fig. 8). These three peptides have shown similar sequence of amino acids by containing phenylalanine at the N-terminus and arginine at the C-terminus. In addition, acidic amino acids were localized in these sequences between phenylalanine and arginine. Hence, it is thought that the analogous amino acid sequences of these peptides were due to the specificity of the enzyme used in protein hydrolysis. Fraction 6C has also an arginine bound to the C-terminus of 5A. Interestingly, when measuring linoleic acid oxidation, 5C and 6C showed a stronger antioxidant activity than 5A, although the inhibition of the ethanol-induced gastric damage of the three fractions was similar. This suggests that the two consecutive arginine of the C-terminus are related to potent inhibition of linoleic acid oxidation. To the best of our knowledge to date, there is no data on the potential protective role of potato proteins on the gastric damage or ethanol-induced gastric mucosal damage. However, it was reported that some specific proteins, e.g. heat shock proteins or HSPs, could have a role in gastric mucosal protection (Yanaka et al., 2007). On the other hand, it was reported that oxygen radicals would be involved in the development of the ethanol-induced gastric mucosal damage (Szelenyi & Brune, 1988). Presently, addition of antioxidants to food products is practiced in order to protect them from off-flavour development. However, chemically synthesized antioxidants are less accepted by the consumers than natural products due to their relatively toxicity in the long term. From our results, potato protein hydrolysates, e.g., 5A, 5C, and 6C, which showed significant positive results as antioxidants, might serve as potential food additives. However, further studies are needed to investigate the pharmacokinetic, resistance to digestive enzymes, and assessment of their radical-scavenging activities. From these results it would be possible to determine whether the potential antioxidant activities of these peptides remains intact or is altered in vivo. Ethic statement: The experimental work on animals was approved by Animal Experiment Committee of the University of Rakuno Gakuen, and rats were managed in accordance with the Guide for the Care and Use of Laboratory Animals.

R E F E R E N C E S

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