Ameliorating effects of ethyl acetate fraction from onion (Allium cepa L.) flesh and peel in mice following trimethyltin-induced learning and memory impairment

Food Research International 75 (2015) 53–60

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Ameliorating effects of ethyl acetate fraction from onion (Allium cepa L.) flesh and peel in mice following trimethyltin-induced learning and memory impairment Seon Kyeong Park a, Dong Eun Jin a, Chang Hyeon Park a, Tae Wan Seung a, Tian Jiao Guo a, Jong Wook Song b, Jong Hwan Kim b, Dae Ok Kim c, Ho Jin Heo a,⁎ a b c

Division of Applied Life Science, Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 660701, Republic of Korea Department of Environmental Toxicology and Chemistry, Korea Institute of toxicology, Jinju 660844, Republic of Korea Department of Food Science and Biotechnology, Kyung Hee University, Yongin 446701, Republic of Korea

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Article history: Received 15 January 2015 Received in revised form 22 April 2015 Accepted 20 May 2015 Available online 24 May 2015 Keywords: Onion Allium cepa L. Cognitive function Quercetin Quercetin-4′-glucoside Isorhamnetin-4′-glucoside

a b s t r a c t The anti-amnesic effects of onion (Allium cepa L.) flesh (OF)1 and peel (OP)2 on trimethyltin (TMT)3-induced learning and memory dysfunction were investigated to confirm learning and memory function. The inhibitory effect against cellular acetylcholinesterase (AChE)4 showed that the EtOAc fraction of OP (EOP5, IC50 value = 37.11 μg/mL) was higher than the EtOAc fraction of OF (EOF6, IC50 value = 433.34 μg/mL). The cognitive effects in ICR mice were also evaluated using Y-maze, passive avoidance, and Morris water maze tests. After the behavioral tests, AChE activity (control = 100%, TMT = 128%, EOF 20 = 108%, EOP 10 = 104%, and EOP 20 = 98%), superoxide dismutase (SOD)7 activity, oxidized glutathione (GSSG)8/total glutathione (GSH)9 and malondialdehyde (MDA)10 production were examined. These results indicate that both EOF and EOP improved learning and memory function. The main compounds of the EOF and EOP were analyzed by Q-TOF UPLC/MS, and the results were as follows: The EOF (quercetin and quercetin-4′-glucoside) and the EOP (quercetin-4′-glucoside and isorhamnetin-4′-glucoside). Consequently, our results suggest that both EOF and EOP could be efficacious in improving cognitive function through AChE inhibition and antioxidant activity in mice brains. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Alzheimer's disease (AD)11 is a typical age-related neurodegenerative disorder, and is characterized by the progressive loss of cognitive function with an accompanying impairment the ability to carry out daily activities (Choi et al., 2013). In AD patients, the cholinergic function called the “cholinergic hypothesis” was important in the brain for ⁎ Corresponding author. E-mail addresses: [email protected] (S.K. Park), [email protected] (D.E. Jin), [email protected] (C.H. Park), [email protected] (T.W. Seung), [email protected] (T.J. Guo), [email protected] (J.W. Song), [email protected] (J.H. Kim), [email protected] (D.O. Kim), [email protected] (H.J. Heo). 1 OF: onion flesh. 2 OP: onion peel. 3 TMT: trimethyltin. 4 AChE: acetylcholinesterase. 5 EOP: EtOAc fraction of onion peel. 6 EOF: EtOAc fraction of onion flesh. 7 SOD: superoxide dismutase. 8 GSSG: oxidized glutathione. 9 GSH: glutathione. 10 MDA: malondialdehyde. 11 AD: Alzheimer's disease.

http://dx.doi.org/10.1016/j.foodres.2015.05.038 0963-9969/© 2015 Elsevier Ltd. All rights reserved.

cognition as learning and memory (Bohnen et al., 2005; Terry & Buccafusco, 2003). To date, acetylcholinesterase (AChE) inhibitors (e.g. tacrine, donepezil, rivastigmine, and galantamine) have been used as treatments for their ability to increase acetylcholine (ACh)12 content in the synapses of neurons by inhibiting endogenous levels of AChE and enhancing cholinergic neurotransmission in the brain. However, these agents have reported the burden of side effects (Terry & Buccafusco, 2003). In addition, another role of AChE present in AD brains was found to be associated with neuritic plaques by secretion as a soluble form, may be present with soluble amyloid-β (Aβ)13, and was associated with free radical oxidative stress and neurotoxicity implications for AD at a very early stage of amyloid plaque formation (Butterfield & Lauderback, 2002; Talesa, 2001). Increased oxidative stress (e.g. Aβ, reactive oxygen species (ROS)14, etc.) and increased levels of oxidatively-modified proteins and lipids in the brain of AD patients have also been reported to be a risk factor of AD (Vina, Lloret, Orti, & Alonso, 2004).

12 13 14

Ach: acetylcholine. Aβ: amyloid β. ROS: reactive oxidative species.

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Trimethyltin (TMT) is a neurotoxin that induces neuronal damage, including the temporal elevation of plasma corticosterone levels, bodyweight loss, and behavioral changes such as whole body tremor (Morita et al., 2006). TMT causes oxidative stress in astrocytes by involving a variety of oxidative species (O− 2 , H2O2, NO, etc.) (Gunasekar et al., 2001). In addition, TMT causes selective neuronal damage and apoptosis in the dentate gyrus in both the human and rodent hippocampal region (CA1 and CA3 subfields) of their respective central nervous systems (CNS)15, and induced σ1 receptor dysfunction, which is associated with cholinergic neurotransmission (Shin et al., 2007; Ogita et al., 2005). That is, TMT could induce neuronal damage in the cholinergic systems of animal models, which causes neuronal cell death in the hippocampus (Morita et al., 2006). Therefore, TMT-induced mice were a suitable in vivo model for investigating the study of cognitive dysfunction. The onion (Allium cepa L.) is one of the most widely consumed vegetables (Kim & Kim, 2006), and is rich in two chemical groups that have perceived health benefits for humans: Flavonoids and alk(en)yl cysteine sulfoxides (Park, Kim, & Kim, 2007). In particular, onions are a rich source of flavonols such as quercetin 3,4′-O-diglucoside and 4′-O-glucoside (Bonaccorsi, Caristi, Gargiulli, & Leuzzi, 2008). Onion extracts are also reportedly effective in many other biological activities (e.g. antimicrobial, antioxidant, anticarcinogenic, antimutagenic, antiasthmatic, immunomodulatory, prebiotic activity, and cardiovascular disease) (Martinez, Corzo, & Villamiel, 2007). Meanwhile, research into cognitive function was relatively insufficient, and in particular, the learning and memory effect of onion flesh has not yet been reported. Therefore, the beneficial effects of ethyl acetate fraction of onion flesh (EOF) and peel (EOP) on TMTinduced cognition deficits in Institute of Cancer Research (ICR)16 mice were evaluated and compared.

2. Materials and methods 2.1. Materials Acetylthiocholine, 5,5-dithio-bis(2-nitro)benzoic acid (DTNB)17, TMT, thiobarbituric acid (TBA)18, metaphosphoric acid, dimethylsulphoxide (DMSO)19, SOD determination kit, and all other chemicals used were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The total glutathione kit was purchased from Enzo life and science (Lausen, Switzerland).

sample dissolved 10% DMSO in in vitro, and sonicate for 40 min in in vivo experiments. 2.3. Inhibition of AChE The AChE assay was performed according to the colorimetric method of (Ellman, Courtney, Andres, & Featherstone, 1961) using acetylthiocholine iodide as a substrate. For the enzyme source, PC12 cells were homogenized in a Glass-Col homogenizer (Terre Haute, IN, USA) with 5 volumes of a homogenization buffer [10 mM Tris–HCl (pH 7.2), containing 1 M NaCl, 50 mM MgCl2, and 1% Triton X-100], then centrifuged 10,000 ×g for 30 min to obtain the supernatant. Sample (10 μL) were mixed with enzymes (10 μL) and incubated at 37 °C for 15 min. Absorbance at 405 nm was read immediately after adding 70 μL Ellman's reaction mixture [0.5 mM acetylthiocholine and 1 mM 5,5′-dithio-bis(2-nitrobenzoic acid) in a 50 mM sodium phosphate buffer (pH 8.0)]. Reading was repeated for 10 min at 2 min intervals to verify that the reaction occurred linearly. Tacrine as a positive control was used in this experiment because of its inhibitory effect of AChE, butyrylcholinesterase (BuChE), and modulating effect of the nicotinic receptor (Aarsland, Mosimann, & McKeith, 2004). 2.4. Animals and in vivo experimental design All animal experimental procedures were approved at the Institutional Animal Care and Use Committee of Gyeongsang National University (certificate: GNU-131105-M0067), and performed in accordance with the Policy of the Ethical Committee of Ministry of Health and Welfare, Republic of Korea. Institute of cancer research (ICR)20 mice (male, 4 weeks old) were obtained from Samtako (Osan, Korea). The mice were housed two per cage in a room maintained with a 12 h light & dark cycle, 55% humidity, and 22 ± 2 °C. The EOF and EOP were orally fed at concentrations of 10 and 20 mg/kg of body weight (designated by EOF 10, EOF 20, EOP 10, and EOP 20, respectively) once a day for 3 weeks. Sample dosage for mice was determined on the basis of previous research (Choi et al., 2012). After 3 weeks, TMT (2.5 mg/kg [7.6 μg/kg of body weight]) was dissolved in 0.85% sodium chloride solution (w/v), and was subcutaneously injected (100 μL) except control group (Choi et al., 2012). TMT group as a negative control means that mouse was only injected with TMT without feeding the any samples.

2.2. Sample preparation

2.5. Behavioral test

Onion (A. cepa L.) was prepared from a local market of Changnyeong (Korea), in July, 2013, and was authenticated by Institute of Agriculture & Life Sciences, Gyeongsang National University. A voucher specimen was deposited at the Herbarium of the Department of Agronomy, Gyeongsang National University. Divided into onion flesh (OF) and onion peel (OP) were washed, and lyophilized using a vacuum-tray freeze dryer (IlShin Lab Co., Ltd., Yangju, Korea). Lyophilized samples were ground to a powder, and stored at −20 °C. Powdered OF and OP were respectively extracted with 50-fold volumes of 70% ethanol at 40 °C for 2 h, and were filtered through Whatman No. 2 filter paper (Whatman International Limited, Kent, UK). Extracted OF and OP were consecutively solvent fractionation in a separating funnel using solvents (n-hexane, chloroform, ethyl acetate, butanol, and distilled water). The solvents were concentrated using a rotary vacuum evaporator (N-1000; EYELA Co., Tokyo, Japan), lyophilized, and stored at − 20 °C. Each

The Y-maze test was performed 3 days after the TMT injection. The maze was black-painted plastic, and each arm of the maze was 33 cm long, 15 cm high, and 10 cm wide, and was positioned at a constant angle. Each mouse was placed at the end of one arm, and allowed to move freely for 8 min. The mouse movement of arm entries was recorded to a smart 3.0 video tracking system (Panlab, Barcelona, Spain), and arm entry have been completed only when the hind foots of the mouse were placed completely. Alternation is defined as entries into the three arms in an overlapping triplet set. The alternation behavior was calculated as the ratio of actual to possible alternation (the total number of arm entries—2), multiplied by 100 (Heo et al., 2004). The passive avoidance test box was divided into two zones, lighted zone and dark zone. The mice were allowed to move freely through a circular tunnel between the two zones. Each mouse was placed in the lighted zone; as soon as it entered the dark zone, an electric shock was provided (0.5 mA, 3 s). After 24 h, the mouse was again placed in the lighted zone, and the step-through latency time was measured to dark zone (maximum limit time: 300 s) (Heo et al., 2004).

15 16 17 18 19

CNS: central nervous systems. ICR: Institute of Cancer Research. DTNB: 5,5-dithio-bis(2-nitro)benzoic acid. TBA: thiobarbituric acid. DMSO: dimethylsulphoxide.

20

ICR: institute of cancer research.

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The Morris water maze (MWM)21 test was conducted according to Morris (1984) with some modifications. The equipment consisted of a stainless steel circular pool (90 cm in diameter) that was randomly divided into quadrants (E, W, S, and N zones) with visual cues on the walls for navigation. Squid ink (Cebesa, Valencia, Spain) was added to the pool water (22 ± 2 °C) to make it opaque. A platform (6 cm in diameter) was placed in the middle of the W zone, the position of which was unchanged during the training session. The mice were allowed to swim and the latency time until they escaped from the water onto the submerged platform up to a maximum of 60 s was recorded, and they were allowed to stay on the platform for 15 s. In the training sessions (days 1–4), the mice were subjected to four trials per day for four consecutive days. In a probe test (day 5), the mice swam freely without the platform for 60 s, and the time spent in the W zone was recorded using smart 3.0 video tracking system. 2.6. Biochemical assay 2.6.1. Preparation of tissue samples After the behavioral tests, the mice were sacrificed by CO2 inhalation for biochemical studies. Brain tissues were immediately collected from the mice and minced into small pieces with surgical scissors. 2.6.2. Measurement of SOD activity, level of oxidized glutathione (GSSG)22/ total GSH, and MDA production The preparation for SOD activity involves homogenizing small pieces of brain with 40 volumes of ice-cold phosphate buffered saline (PBS)23. Homogenates were directly centrifuged at 400 ×g for 10 min at 4 °C to obtain the pellets. The pellets in 5–10 volumes of ice-cold 1 × Cell Extraction Buffer [10% SOD buffer, 0.4% (v/v) Triton X-100, and 200 μM Phenylmethane sulfonylfluoride in distilled water] were incubated on ice for 30 min, and centrifuged at 10,000 ×g for 10 min at 4 °C to obtain the supernatant. The preparation for the determination of GSH and GSSG level required homogenizing small pieces of brain with 20 volumes of 5% metaphosphoric acid, and direct centrifugation at 14,000 ×g for 15 min at 4 °C to obtain the supernatant. To determine GSSG, the supernatant was treated to 2 M 4-vinylpyridine, and incubated for 1 h at room temperature. The measurement of SOD and GSH were carried out using commercial kits. Finally, small pieces of brain were homogenized with 10 volumes of ice-cold PBS. Homogenates were directly centrifuged to obtain the supernatant to determine MDA levels (6000 ×g for 10 min at 4 °C) and AChE activity (14,000 ×g for 30 min at 4 °C). The MDA products were examined by monitoring thiobarbituric acid reactive substance formation (Lu et al., 2007). Each homogenate (160 μL) was mixed with 1% phosphoric acid (960 μL) and 0.67% thiobarbituric acid (320 μL). The mixture was incubated at 95 °C in a water bath for 1 h. After cooling, the colored complex was read absorbance at 405 nm. The protein concentration was determined using the Quant-iT™ protein assay kit (Invitrogen, Carlsbad, CA, USA). 2.7. Identification of main phenolics with Q-TOF UPLC/MS The main compounds in the EOF and EOP were qualitatively analyzed by using an ultra-performance liquid chromatography (UPLC)24 Accurate-Mass Quadrupole Time of Flight (Q-TOF)25 (Agilent Technologies, Santa Clara, CA, USA). That was operated with an electrospray source in negative ion mode to obtain MS and MS/MS data. Separation of phenolics was performed on an ACQUITY UPLC BEH C18 column 21 22 23 24 25

MWM: Morris water maze. GSSG: oxidized glutathione. PBS: phosphate buffered saline. UPLC: ultra-performance liquid chromatography. Q-TOF: Quadrupole Time of Flight.

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(2.1 × 100 mm, 1.7 μm particle size; Waters Corp, Milford, MA, USA) with a flow rate of 0.3 mL/min, and oven temperature of 40 °C. A linear solvent gradient of binary mobile phase (solvent A: 0.1% formic acid in distilled water; solvent B: 0.1% formic acid in acetonitrile) during analysis was applied as follows: 99% A/1% B at 0–2 min, 50% A/50% B at 8–10 min, and 99% A/1% B at 12 min. The conditions for MS analyses included the drying gas (N2) temperature at 350 °C, drying gas flow at 10 L/min, nebulizer pressure at 45 psi, fragmentor voltage at 175 V, capillary voltage at 4000 V and mass range from m/z 100 to 1000. 2.8. Statistical analysis All data were expressed as the mean ± SD. The statistical significance of differences among groups was calculated by a one-way analysis of variance (ANOVA). Significant differences were determined using the Duncan's new multiple-range test (p b 0.05) of SAS ver. 9.1 (SAS Institute Inc., Cary, NC, USA). 3. Results and discussion 3.1. Inhibition of AChE The level of acetylcholine, a neurotransmitter involved in the control of cognitive function, is dramatically decreased in the cortex and hippocampus in AD patients. Therefore, AChE inhibitors can be used to restore acetylcholine levels and the cholinergic activity of brain (Phan, David, Naidu, Wong, & Sabaratnam, in press). In this experiment, EOF and EOP were evaluated for their AChE inhibitory effects using Ellman's colorimetric method. AChE inhibitory activity of both EOF and EOP showed slightly lower than 1 μM tacrine (80.57%) as a positive control. The inhibitory effects of AChE in both EOF and EOP were shown in a dosedependent manner (r2 = 0.9909 and 0.9993, respectively), and indicated that EOP had a more significant inhibitory effect with lower range of concentration compared to EOF. In the inhibitory activity, which was more than 50% at different concentrations, the IC50 value of EOP (IC50 value: 37.11 μg/mL) was relatively lower, by about 10 times that of the EOF (IC50 value: 433.34 μg/mL) (Fig. 1). Onion peel has quercetin and its derivatives, which are known to be a major compound in onion, compared to the quercetin content of onion flesh (Park et al., 2007). Jung and Park (2007) reported the AChE inhibitory activity of the various phenolics from the EtOAc fraction of Agrimonia pilosa, showing that quercetin had the highest inhibitory effect among those phenolics (3-methoxy quercetin IC50 = 37.9, quercitrin IC50 = 66.9, and quercetin IC50 = 19.8 μM). Therefore, our results suggest that quercetin and the isomers of onion fractions are able to exert an inhibitory effect on cellular AChE. 3.2. Behavioral tests To confirm the attenuation effect of EOF and EOP against TMTinduced learning and memory impairment, a Y-maze test, passive avoidance test, and a MWM test were conducted. TMT-treated mice produced a selective hippocampal neuronal degeneration in the CNS, this damage is associated with cognitive dysfunction, and this cognitive dysfunction will induce behavioral changes such as hyperactivity and aggressiveness associated with Attention Deficit and Hyperactivity Disorder (ADHD)26 (Tamburella, Micale, Mazzola, Salomone, & Drago, 2012; Terry & Buccafusco, 2003). The result of the Y-maze test was shown to impair the spatial cognitive function in the alternation behavior of the TMT group (48%) compared to that of the control group (65%). All groups pretreated with EOF and EOP increased their spontaneous alternation behavior in the TMT-injected mice (Fig. 2A). In Fig. 2B, the black line in the Y-maze means the path tracing of the mice in each 26

ADHD: Attention Deficit and Hyperactivity Disorder.

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(A)

(A)

(B)

(B)

Fig. 1. Effect of EtOAc fraction of onion flesh (A) and onion peel (B) against AChE. The AChE inhibitory effect was expressed as a percentage of enzyme activity inhibited compared to the control value. The results shown are means ± SD (n = 3). Data were statistically considered at p b 0.05 versus control group, and different small letters represent statistical differences.

arm, and this changes thin to thick in accordance with increased movement. In particular, the TMT group showed many more movement than the control group. This result presents evidence of hyperactivity by causing TMT-induced cognitive dysfunction. Therefore, the number of total arm entries was similar for all groups (Fig. 2A), but the movement rate in the total zone was increased in the TMT group (12.45 cm/s, 120% increase) compared to the control group (10.46 cm/s, 100%). On the other hand, sample groups showed a lower movement rate (EOF 10 = 11.60, EOF 20 = 10.62, EOP 10 = 10.37, and EOP 20 = 9.91 cm/ s) than the TMT group (Fig. 2C). Short-term learning and memory function was evaluated by passive avoidance test (Fig. 3). The TMT group significantly shortened their steps through latency in the retention trial (40%) compared to that of the control group (100%). However, the EOF 20 (98.78%), EOP 10 (104.62%) and EOP 20 group (103.45%) showed statistically similar short-term learning memory improvements compared to TMT group. Koda, Kurodab, and Imai (2008) reported that TMT-induced mice produced severe rat's performances deficits, along with the selective loss of pyramidal neurons, triangular-shaped neurons in the cerebral cortex that transmit impulses in the hippocampal CA3 region during the MWM test. Long-term learning and memory functions were examined using the MWM test, and in the training session, all groups had decreased escape latency time at four days, while the TMT group showed a low learning and memory (39.44 s) function compared to that of control

(C)

Fig. 2. Effect of EtOAc fraction of onion flesh and onion peel on spatial working memory in Y-maze test. The spontaneous alternation behavior and number of arm entries (A), path tracing of each groups (B), and rate of total arms (C) were measured. Results shown are means ± SD (n = 8). Data were statistically considered at p b 0.05 versus control group, and different small letters represent statistical differences.

group (23.10 s) in the final training day (Fig. 4A). All groups of EOF and EOP decreased their escape latency time (EOF 10 = 27.08, EOF 20 = 25.08, EOP 10 = 23.85, and EOP 20 = 22.28 s). In the probe test, the path tracing of the mice showed that the mice of the control group

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(A)

Fig. 3. Effect of EtOAc fraction of onion flesh and onion peel on short-term learning and memory in passive avoidance test. Step-through latency (300 s) in the retention trial test was measured. Results shown are means ± SD (n = 8). Data were statistically considered at p b 0.05 versus control group, and different small letters represent statistical differences.

(B)

were relatively more circled around the W zone including the platform, whereas mice of the TMT group circled around all zones (Fig. 4B). These features explain learning and memory ability regarding platforms in the W zone. That is, the TMT group showed learning and memory impairment (22.08% stay percentage in the W zone) compared with the control group (32.71%), and both EOP and EOF showed beneficial effects in the long-term learning and memory function (Fig. 4C). The above in vivo results suggest that both EOP and EOF had excellent enhancing effects on spatial cognitive function and learning and memory functions, and protected against TMT-induced cognitive dysfunction. 3.3. AChE activity Previous research has reported that AChE was significantly increased and choline acetyltransferase (ChAT) was decreased by TMT treatment (Woodruff & Baisden, 1990). The TMT treatment group in our experiment also showed an increase of AChE activity, whereas the EOF and EOP groups effectively inhibited AChE in the brains of mice after TMT exposure. AChE activity was significantly increased in the TMT group (128%) compared to the control group (100%), whereas the AChE activity of EOF 20 group (108%), EOP 10 group (104%), and EOP 20 group (98%) was reduced (Fig. 5). Oxidative stress via Aβ and TMT induced cholinergic system dysfunction (e.g. decreasing the ACh level, choline acetyltransferase activity and increasing the AChE activity) by injury and death of neuronal cells. However, various flavonoids with efficient anti-oxidant activity showed strong protective effect by removing the basic neurotoxicity (Spencer, 2008; Woodruff & Baisden, 1990).

(C)

3.4. SOD activity, level of GSSG/total GSH and MDA production Several studies have reported that TMT causes neuronal oxidative damage (10% increase in brain lipid peroxidation 24 h after TMT administration) and the prolonged elevation of ROS levels, which may cause apoptotic cell death (Ali, Lebel, & Bondy, 1992; Thompson et al., 1996). However, SOD scavenged reactive free radicals by catalyzing the dismutation of superoxide anions into molecular oxygen and hydrogen peroxide in the neocortex and hippocampus of AD and Down's syndrome patients (Furuta et al., 1995). The TMT group had decreased SOD activity (2.32 U/mg of protein and 37% decreased in SOD activity) compared to that of control group (3.69 U/mg of protein). All EOF and EOP were significantly increased (EOF 10 = 2.64, EOF 20 = 2.86, EOP 10 = 2.97, and EOP20 = 3.60 U/mg of protein) compared to that of the TMT group (Fig. 6A). The oxidation of mitochondrial glutathione

Fig. 4. Effect of EtOAc fraction of onion flesh and onion peel on long-term learning and memory in Morris water maze (MWM) test. Escape latency in the training trial (A), path tracing in the probe trial (B) and probe trial session (C) were examined. Results shown are means ± SD (n = 8). Data were statistically considered at p b 0.05 versus control group, and different small letters represent statistical differences.

in the development of aging and apoptosis correlated with the oxidative damage to mitochondrial DNA. The GSSG/GSH ratio has been used as a useful marker of oxidative stress in all tissues, and several physiological and patho-physiological situations (Vina, Lloret, Orti, & Alonso, 2004). The results show that the GSSG/total GSH ratio was increased in the TMT group mice brain (55.95%) compared to that of the control group

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(A)

Fig. 5. AChE activity of EtOAc fraction of onion flesh and onion peel on TMT-induced mice brain homogenate. The results shown are means ± SD (n = 8). Data were statistically considered at p b 0.05 versus control group, and different small letters represent statistical differences.

(28%). In contrast, EOF 20, EOP 10 and EOP 20 groups showed significantly decreased GSSG/total GSH levels by as much as the control group (Fig. 6B). MDA is a critical marker of lipid peroxidation in erythrocytes, liver, kidneys, and ovaries of various experimental animals. In particular, brain tissue consisting of high polyunsaturated fatty acids and iron content was highly vulnerable to ROS-mediated oxidative damage (Shivarajashankara, Shivashankara, Bhat, & Rao, 2002). There was a noticeable increase in MDA production in the TMT group (1.46 nmol/mg protein, 116%) compared to the MDA production of the control group (1.25 nmol/mg protein, 100%). EOF and EOP groups had significantly reduced (EOF 10 = 6.18%, EOF 20 = 16%, EOP 10 = 26% and EOP 20 = 29% decreases) MDA production compared to that of the TMT group (Fig. 6C). Our results present that the OF and OP fractions will defend against drug-induced oxidative stress in mice brain tissues. OF and OP ethanolic extracts enhanced antioxidant status, and decreased the lipid peroxide level in aged rats (Park et al., 2007). Agrawal, Tyagi, Shukla, and Nath (2009) also reported that AChE activity and the oxidative stress (GSH, MDA levels) of donepezil in the rat model of streptozotocin (STZ)-induced dementia have been investigated, and their results present that it only reduces AChE activity without blocking oxidative stress in brain (i.e. just single-targeted therapeutic agent). In contrast, the OF and OP fractions are possibly effective as multi-targeting therapeutic agents by controlling both antioxidant activity and AChE inhibition.

(B)

(C)

3.5. Identification of main phenolics with Q-TOF UPLC/MS The main phenolics of EOF and EOP were qualitatively identified by the Q-TOF UPLC/MS analysis for MS2 fragmentation, compared to the MS2 fragmentation data obtained by Q-TOF LC/MS (MS2) from the previous literature reports (Lee & Mitchell, 2011; Park & Lee, 1996). The main phenolics were analyzed as follows: EOF (Compound A and B) and EOP (Compound B and C), and the base peak of these phenolics respectively showed molecular ions M− m/z values of 477.27 (A), 463.26 (B), and 301.17 (C) (Fig. 7). In addition, MS2 scan chromatograms were fragmented at compound A (m/z 314.18, 299.15, and 271.1510), compound B (m/z 301.16, 179.09, 151.09, and 107.09), and compound C (m/z 179.09, 151.09. 121.10, and 107.08). Consequently, these compounds were identified as isorhamnetin-mono-glucoside (probably isorhamnetin-4′-glucoside, compound A, PubChem CID: 12442954), quercetin-4′-glucoside (Q4G, compound B, PubChem CID: 12442954), and quercetin (Q, compound C, PubChem CID: 5280343). This is based on the comparison of the MS2 spectrum obtained from LC (ESI)-MS/MS to fragmentation data from the main fragments of the

Fig. 6. SOD activity (A), level of oxidized GSH/total GSH (B) and MDA production (C) of EtOAc fraction of onion flesh (OF) and onion peel (OP) on TMT-induced mice brain homogenate. The results shown are means ± SD (n = 8). Data were statistically considered at p b 0.05 versus control group, and different small letters represent statistical differences.

previous literature reports (Lee & Mitchell, 2011). Park and Lee (1996) reported that the major flavonoid compounds in onions were identified as isorhamnetin-4′-glucoside using HPLC and NMR. Those compounds consisted of quercetin as their basic structure. Quercetin is also known to be a major compound in onion peel, and for its antioxidants, free radical scavenging power, and its capability to protect against cardiovascular disease (Bonaccorsi et al., 2008). These functions of quercetin were

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(A)

(B)

(C)

Fig. 7. Q-TOF UPLC/MS spectra in negative ion mode, and chemical structures of isorhamnetin-4′-glucoside (A), quercetin-4′-glucoside (B) and quercetin (C).

presented by changing its chemical structure, that is, hydroxyl (-OH) substitution and catechol-type B-ring changing to the ortho-quinone structure of powerful antioxidant activity. In addition, it has an expected protective effect in brain neuronal cells by passing through the blood– brain-barrier (BBB)27 in in situ models (Wang et al., 2014). Thus, the long-term treatment with quercetin improved cognitive function in APPswe/PS1dE9 transgenic mouse model by protecting against Aβinduced mitochondrial dysfunction (Wang et al., 2014). However, some structural changes were needed for absorption of quercetin in the human intestine such as conjugated form with glucose and 3′-Omethylation (e.g. isorhamnetin) in B-ring (Lee & Mitchell, 2011). Aziz, Edwards, Lean, and Crozier (1998) also reported a proportionally higher

27

BBB: blood–brain-barrier.

accumulation of isorhamnetin-4′-glucoside than quercetin conjugates (e.g. Quercetin-3,4′-diglucoside and quercetin-4′-glucoside) in the plasma and urine of humans. That is, the accumulation could be induced either by the preferential absorption of isorhamnetin-4′-glucoside or by post-absorption after the conversion of quercetin-4′-glucoside to isorhamnetin-4′-glucoside via 3′-O-methylation. Isorhamnetin, which is an O-methylated metabolite formed in the small intestine and liver, and increased in the human brain, has a higher BBB permeability than aglycone by its apparent lipophilicity (Spencer, 2008). The O-methylated metabolites of derived flavonoids have the potential to improve human memory and neuro-cognitive performance via their ability to protect vulnerable neurons and long-term potentiation, which is considered a major mechanism of memory in the brain. It also allows the control on the molecular level through the activation of a number of neuronal signaling pathways such as phosphatidylinositol-3 kinase/protein kinase B/Akt,

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protein kinase C, protein kinase A, Ca-calmodulin kinase, and mitogenactivated protein kinase pathways (Spencer, 2008). Thus, isorhamnetin, one of the sixty-five flavonoids from Ginkgo biloba L. has the best effect on the potentiation of NGF-induced neurite outgrowth and neurofilament expression by inducing the expression of neurofilaments (Xu et al., 2012). These reports mean that isorhamnetin-4′-glucoside and quercetin-4′-glucoside of EOF were better absorption than quercetin and quercetin-4′glucoside of EOP. Accordingly, EOF 20 group has similar results to EOP 10 group in in vivo and ex vivo tests contrary to the results of in vitro test. Consequently, our in vivo and ex vivo biochemical tests show improvement effects for both EOP and EOF groups. Therefore, it might be considered that the anti-amnesic effect of EOF and EOP may be due in part to the presence of isorhamnetin-4′-glucoside, quercetin-4′-glucoside, and quercetin, which contribute to anticholinesterase and antioxidant activity. 4. Conclusions The anti-amnesic effects of onion (A. cepa L.) flesh and onion peel fractions on TMT-induced learning and memory impairment were evaluated for the in vivo mouse model. Both the EOF and the EOP effectively ameliorated drug-induced cognitive deficits by AChE inhibition and antioxidant activity. Isorhamnetin-4′-glucoside, quercetin-4′-glucoside, and quercetin may be considered major contributors for in vitro and in vivo tests of onions. Therefore, OF and OP, including various physiological phenolics, may be used as natural potential resources for mitigating learning and memory dysfunction caused by aging and neurodegenerative diseases. Acknowledgments This work was supported by a grant from the 2013 High Valueadded Food Technology Development Program (113023-3), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. This work was also supported by the National Research Foundation of Korea Grant funded by the Korean Government (KRF-2011-0021664) Republic of Korea. S. K. Park, D. E. Jin, C. H. Park, and T. W. Seung were supported by the BK21 plus program (2013), Ministry of Education, Republic of Korea. References Aarsland, D., Mosimann, U.P., & McKeith, I. (2004). Role of cholinesterase inhibitors in Parkinson's disease and dementia with Lewy bodies. Journal of Geriatric Psychiatry and Neurology, 17(3), 164–171. Agrawal, R., Tyagi, E., Shukla, R., & Nath, C. (2009). A study of brain insulin receptors, AChE activity and oxidative stress in rat model of ICV STZ-induced dementia. Neuropharmacology, 56(4), 779–787. Ali, S.F., Lebel, C.P., & Bondy, S.C. (1992). Reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity. Neurotoxicology, 13(3), 637–648. Aziz, A.A., Edwards, C.A., Lean, M.E.J., & Crozier, A. (1998). Absorption and excretion of conjugated flavonols, including quercetin-4′-O-β-glucoside and isorhamnetin-4′-Oβ-glucoside by human volunteers after the consumption of onions. Free Radical Research, 29(3), 257–269. Bohnen, N.I., Kaufer, D.I., Hendrickson, R., Ivanco, L.S., Lopresti, B., Davis, J.G., et al. (2005). Cognitive correlates of alterations in acetylcholinesterase in Alzheimer's disease. Neuroscience Letters, 380(1–2), 127–132. Bonaccorsi, P., Caristi, C., Gargiulli, C., & Leuzzi, U. (2008). Flavonol glucosides in Allium species: A comparative study by means of HPLC–DAD–ESI/MS/MS. Food Chemistry, 107(4), 1668–1673. Butterfield, D.A., & Lauderback, C.M. (2002). Lipid peroxidation and protein oxidation in Alzheimer's disease brain: Potential causes and consequences involving amyloid βpeptide-associated free radical oxidative stress. Free Radical Biology and Medicine, 32(11), 1050–1060. Choi, J.Y., Cho, E.J., Lee, H.S., Lee, J.M., Yoon, Y.H., & Lee, S.H. (2013). Tartary buckwheat improves cognition and memory function in an in vivo amyloid-β-induced Alzheimer model. Food and Chemical Toxicology, 53, 105–111. Choi, G.N., Kim, J.H., Kwak, J.H., Jeong, C.H., Jeong, H.R., Lee, U., et al. (2012). Effect of quercetin on learning and memory performance in ICR mice under neurotoxic trimethyltin exposure. Food Chemistry, 132, 1019–1024.

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