Antiamnesic Effect of Broccoli (Brassica oleracea var. italica) Leaves

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Antiamnesic Effect of Broccoli (Brassica oleracea var. italica) Leaves on Amyloid Beta (Aβ)1−42-Induced Learning and Memory Impairment Seon Kyeong Park,† Jeong Su Ha,† Jong Min Kim,† Jin Yong Kang,† Du Sang Lee,† Tian Jiao Guo,† Uk Lee,§ Dae-Ok Kim,# and Ho Jin Heo*,† †

Division of Applied Life Science (BK21 plus), Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea § Department of Special Purpose Trees, Korea Forest Research Institute, Suwon 16631, Republic of Korea # Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea ABSTRACT: To examine the antiamnesic effects of broccoli (Brassica oleracea var. italica) leaves, we performed in vitro and in vivo tests on amyloid beta (Aβ)-induced neurotoxicity. The chloroform fraction from broccoli leaves (CBL) showed a remarkable neuronal cell-protective effect and an inhibition against acetylcholinesterase (AChE). The ameliorating effect of CBL on Aβ1−42-induced learning and memory impairment was evaluated by Y-maze, passive avoidance, and Morris water maze tests. The results indicated improving cognitive function in the CBL group. After the behavioral tests, antioxidant effects were detected by superoxide dismutase (SOD), oxidized glutathione (GSH)/total GSH, and malondialdehyde (MDA) assays, and inhibition against AChE was also presented in the brain. Finally, oxo-dihydroxy-octadecenoic acid (oxo-DHODE) and trihydroxyoctadecenoic acid (THODE) as main compounds were identified by quadrupole time-of-flight ultraperformance liquid chromatography (Q-TOF UPLC-MS) analysis. Therefore, our studies suggest that CBL could be used as a natural resource for ameliorating Aβ1−42-induced learning and memory impairment. KEYWORDS: Brassica oleracea var. italica, broccoli leaf, learning and memory function, amyloid beta, oxo-dihydroxy-octadecenoic acid, trihydroxy-octadecenoic acid

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INTRODUCTION Alzheimer’s disease (AD) is characterized by the progressive loss of learning and memory impairment and the inability to carry out daily activities.1 The classic hallmark of AD is the accumulation of extracellular amyloid plaques composed of amyloid beta (Aβ), which is a 40−42 amino acid (AA) peptide fragment, by producing the amyloid protein precursor.2 Excessive Aβ leads to Aβ-associated oxidative stress, which induces reactive oxygen species (ROSs) and oxidatively modified proteins subsequent to the reactive lipid peroxidation products (4-hydroxy-2-trans-nonenal and acrolein). In addition, Aβ-associated oxidative stress induces free fatty acid release, Ca2+ dyshomeostasis, mitochondrial dysfunction, peroxynitrite formation, inflammatory response, and apoptosis of neuronal cells.3 Aβ production, oligomerization, or fibrillogenesis triggers a cascade of synaptic failure, synapse loss, neuronal cell death, and eventually dementia.4 Moreover, imbalances of the formation and removal of oxidative stress are thought to cause several chronic diseases, including cancer, diabetes mellitus, ischemic cardiovascular diseases, AD, and Parkinson’s disease. On the other hand, antioxidants have played important roles in the prevention of these diseases. Therefore, edible plants containing health-promoting phytochemicals such as vitamins C and E and phenolic compounds have potential value, and they are regarded as functional food substances of interest.5 Moreover, studies indicate that broccoli (Brassica oleracea var. italica, family Cruciferae) extracts have the highest antioxidant activity among the 26 common vegetables © XXXX American Chemical Society

containing high concentrations of antioxidants, such as glucosinolates, isothiocyanates, and selenium, and AAs, which have antioxidant, antimicrobial, anticarcinogenic, antiaging, and anti-heart-disease activity.6−8 However, edible parts of broccoli, including mainly the leaves and stems, are discarded >70% of the time.9 These byproducts are valueless from a commercial point of view but have valuable bioactive compounds.7,9 Therefore, broccoli leaves are high-value foodstuffs that can be used to obtain functional compounds such as fatty acids, antioxidants, AAs, and vitamins.7−9 However, physiological research studies on broccoli byproducts are lacking, and, in particular, the learning and memory effect of broccoli leaves has not yet been reported. Therefore, here, the in vitro neuronal cell-protective effects and in vivo antiamnesic effects of broccoli leaf extracts on Aβ-induced neurotoxicity in mice were examined.

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MATERIALS AND METHODS

Materials. Acetylthiocholine iodide, 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), thiobarbituric acid (TBA), phosphoric acid, dimethyl sulfoxide (DMSO), a superoxide dismutase (SOD) determination kit, and all other chemicals used were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Aβ1−42 was purchased from BACHEM (Bubendorf, Switzerland). The total GSH kit was purchased from Enzo Life Sciences (Lausen, Switzerland). Received: February 2, 2016 Revised: April 14, 2016 Accepted: April 14, 2016

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DOI: 10.1021/acs.jafc.6b00559 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Sample Preparation. Broccoli leaves were obtained from a local market (Yeoju, Korea) in October 2013. These samples were authenticated by the Institute of Agriculture & Life Sciences, Gyeongsang National University, and a voucher specimen was deposited at the herbarium of the Department of Agronomy, Gyeongsang National University. Broccoli leaves were washed, lyophilized, ground, and stored at −20 °C prior to use. The powdered broccoli leaves were extracted with 50 volumes of 95% ethanol at 40 °C for 2 h. In addition, ethanolic extracts were filtered and consecutively fractionated by solvents (n-hexane, chloroform, ethyl acetate, and distilled water) using a separating funnel. The chloroform solvent fraction, which has been shown to possess in vitro antioxidant activities and neuroprotective effects in our previous research,10 was concentrated using a rotary vacuum evaporator (N-1000; EYELA Co., Tokyo, Japan), lyophilized, and stored at −20 °C until use. Cell Culture. The pheochromocytoma cells (PC12 cell) (Korea Cell Line Bank (KCLB) 21721, Seoul, Republic of Korea) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 50 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator containing 5% CO2. Neuronal Cell-Protective Effects. Cellular oxidative stress was measured by a DCF-DA assay. The chloroform fractions from broccoli leaf (CBL) samples (5, 10, 20, 50, and 100 μg/mL) were pretreated for 3 h. After 3 h, Aβ and CBL groups were treated with Aβ1−42 (100 μg/mL) for 48 h. After 48 h, cells were added to the 10 μM DCF-DA for 1 h. After 1 h of incubation, dichlorofluorescein (DCF) production was quantified by a fluorometer (Infinite F200, TECAN, Raleigh, NC, USA) at 485 nm (excitation wave) and 535 nm (emission wave).3 The cell viability was determined using an MTT reduction assay (Sigma-Aldrich Chemical Co.). Neuronal PC12 cells were plated at a density of (0.5−1) × 104 cells/well on a 96-well plate and incubated for 24 h. After 24 h, various concentrations of CBL (5, 10, 20, 50, and 100 μg/mL) were pre-incubated for 3 h, and Aβ (100 μg/mL) was treated for 48 h. Thereafter, MTT solution (5 mg/mL in phosphatebuffered saline (PBS)) was added to each well and incubated for 3 h to allow metabolization of the MTT. The amount of violet MTT formazan crystals was measured using a microplate reader (680, BioRad, Tokyo, Japan) at 570 nm (test wavelength) and 690 nm (reference wavelength).11 To evaluate lactate dehydrogenase (LDH) release as a cell membrane protective effect, CBL-pretreated PC12 cells were centrifuged at 250g for 5 min at 4 °C. The supernatant (100 μL) was then transferred to a new 96-well plate, and LDH levels were determined using the commercial kit (Sigma-Aldrich Chemical Co.). Damage of PC12 cell membranes was evaluated by measuring the amounts released into the medium of the intracellular enzyme such as LDH. Inhibition of Acetylcholinesterase (AChE). The inhibition of AChE was evaluated by Ellman’s photometric method.12 To extract the enzyme, PC12 cells were homogenized with 5-fold 10 mM TrisHCl (pH 7.2) buffer containing 1% Triton X-100, 50 mM MgCl2, and 1 M NaCl using a Glass-Col homogenizer (Terre Haute, IN, USA) and centrifuged (10000g, 30 min), and the supernatant was used as an enzyme source. The CBL samples (10 μL) were mixed with 50 mM sodium phosphate buffer (50 μL) and enzymes (10 μL) and incubated for 10 min. The incubated mixtures were added to Ellman’s reaction mixture (70 μL) (0.5 mM acetylthiocholine and 1 mM DTNB in a 50 mM sodium phosphate buffer (pH 8.0)) and incubated for 20 min. After incubation, mixtures were read at 405 nm using the microplate reader (680, Bio-Rad). A tacrine, butyrylcholinesterase (BuChE), which is known to have the inhibitory effect of AChE, and the modulating effect of the nicotinic receptor13 was used as a positive control. Animals and in Vivo Experimental Design. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) 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. Forty Institute of Cancer Research (ICR) mice (male, 4 weeks old) were obtained from

Samtako (Osan, Korea). The mice were housed two per cage and divided into five groups (eight mice per group). The room was maintained with a 12 h light−dark cycle at 55% humidity and 22 ± 2 °C. The CBL samples were dissolved in a 0.85% sodium chloride solution (w/v), sonicated at 40 °C for 40 min, and orally fed once a day for three consecutive weeks (5, 10, and 20 mg/kg body weight for the CBL 5, CBL 10, and CBL 20 groups, respectively) (Figure 1).

Figure 1. Experimental design for Aβ-induced learning and memory impairment in mice.

After 3 weeks, Aβ1−42 was injected at bregma (410 pmol, 10 μL) using a 25-μL Hamilton microsyringe fitted with a 26-gauge needle that was inserted to a depth of 2.5 mm without anesthesia.3 The control group was injected with 0.85% sodium chloride solution only. Behavioral Test. To investigate spontaneous alternation behavior, the Y-maze test was conducted to examine immediate spatial working memory performance in the 3 days after the Aβ injection. The equipment for the Y-maze test was composed of black plastic, and each arm of the maze was 33 cm long, 15 cm high, and 10 cm wide. The mouse started at the end of one arm and moved freely (8 min). Then, the movement of each mouse was recorded by a smart 3.0 video tracking system (Panlab, Barcelona, Spain). An overlapping triplet set into each of the other three arms was considered successive alternation behavior.3 To evaluate short-term learning and memory function, the passive avoidance test was conducted over 2 days. The passive avoidance box was divided into two zones (illuminated and dark zones). Each mouse was placed in the illuminated zone for 2 min by blocking the circular tunnel between the two zones. After 2 min, the mouse was free to move to the dark zone through the circular tunnel. As soon as it entered the dark zone, the foot of the mouse was given an electric shock (0.5 mA, 3 s). After 24 h, each mouse was again placed in the illuminated zone, and the time taken to enter the dark zone was measured for 300 s (maximum time).3 To examine long-term learning and memory function, the Morris water maze test was conducted according to Morris’s method with some modifications over 5 days.14 The equipment was composed of a stainless steel circular pool (90 cm in diameter) divided into quadrants (E, W, S, and N zones) with visual clues. Squid ink (Cebesa, Valencia, Spain) was added to the water in the pool to make it opaque, and the temperature was kept at 20 ± 2 °C. A platform (6 cm in diameter) was placed in the middle of the W zone, and the position of the platform was unchanged during the test. The mice were allowed to swim and measured by a smart 3.0 video tracking system (Panlab). The latency time was measured until the mice escaped from the water to the platform (maximum time: 60 s). After escaping, the mice were allowed to stay for 15 s on the platform. This process was performed four times per day for four consecutive days. In a probe test (day 5), the mice swam freely in the water tank without the platform for 60 s, and the time was recorded in the W zone. Biochemical Assays. After the behavioral tests, the brain tissues were immediately collected from the mice for biochemical studies. The tissues were minced into small pieces with surgical scissors and kept at −80 °C until use. To extract the SOD, minced brain tissues were homogenized with 40 volumes of PBS and directly centrifuged (400g, 10 min) to obtain the pellets. The obtained pellets were incubated with 10-fold 1× cell extraction buffer (10% SOD buffer, 0.4% (v/v) Triton X-100, and 200 μM phenylmethanesulfonyl fluoride in distilled water) for 30 min in B

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Journal of Agricultural and Food Chemistry ice and centrifuged at 10000g for 10 min. The obtained supernatants were used to measure SOD levels.15 To determine the glutathione (GSH) and oxidized GSH levels, minced brain tissues with 20 volumes of 5% metaphosphoric acid were homogenized and centrifuged (14000g, 15 min), and the supernatants were obtained. The supernatants were used to measure GSH levels. To determine oxidized GSH levels, the GSH extract was treated with 2 M 4-vinylpyridine and incubated (1 h, room temperature). The incubated extracts were used to measure oxidized GSH levels.15 Finally, minced brain tissues with 10 volumes of ice-cold PBS were homogenized and directly centrifuged to obtain the supernatants to determine brain malondialdehyde (MDA) levels (6000g, 10 min) and AChE activity (14000g, 30 min). The supernatants were used to measure MDA levels and AChE activity, respectively. The MDA levels as lipid peroxidation were examined by monitoring TBA reactive substance formation.11 In brief, 160 μL of MDA extract was mixed with 960 μL of 1% phosphoric acid (v/v) and 320 μL 0.67% TBA (w/ v). These mixtures were incubated in a water bath (95 °C, 1 h). After cooling, the colored complex was centrifuged (6000g, 1 min), and the supernatants were measured by a spectrometer at 532 nm. The protein concentrations of SOD, GSH, oxidized GSH, AChE, and MDA were measured using the Quant-iT protein assay kit (Invitrogen, Carlsbad, CA, USA), and all extraction procedures were performed at 4 °C. Identification of Main Compounds with Q-TOF UPLC-MS. The main compounds in the CBL were qualitatively analyzed using accurate-mass quadrupole time-of-flight ultraperformance liquid chromatography (Q-TOF UPLC-MS) (Waters Corp, Milford, MA, USA) that was operated with an electrospray source (ESI) in negative ion mode to obtain MS and MS2 data. The concentration of CBL was 100 μg/mL (in methanol), and the injection volume was 2 μL. Separation of various compounds in CBL was carried out on an ACQUITY UPLC BEH C18 column (2.l × 100 mm, 1.7 μm particle size), and the flow rate was 0.4 mL/min. The following conditions of the linear solvent gradient were applied: 0% B (100% A) at 0−0.5 min to 75% B (25% A) at 0.5−6.0 min using solvent A (0.1% formic acid in distilled water) and solvent B (0.1% formic acid in acetonitrile). The conditions for MS analyses were applied to the drying gas (N2) temperature at 120 °C, drying gas flow at 30 L/h, nebulizer pressure at 45 psi, fragmentor voltage at 175 V, capillary voltage at 3 kV, and mass range from m/z 100 to 1200. Statistical Analysis. All results were expressed as means ± SD. The statistical significance of differences among groups was calculated by a one-way analysis of variance (ANOVA). Significant differences were determined using Duncan’s new multiple-range test (p < 0.05) of SAS ver. 9.1 (SAS Institute Inc., Cary, NC, USA).

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RESULTS AND DISCUSSION Neuroprotective Effects of CBL on Aβ-Induced Neurotoxicity. The mechanism of Aβ-induced neurotoxicity is still poorly understood.16 However, Aβ is known to induce free radicals and oxidative stress and lead to the apoptosis of neuronal cells by disrupting the function of mitochondria and lysosomes.16,17 It also causes synapse loss and cellular dysfunction by amyloid plaque production.18 To measure the neuroprotective effects of CBL on Aβ-induced neurotoxicity, we measured ROS production levels, cell viability, and cell membrane protective effects using the DCF-DA, MTT, and LDH assays, respectively. The DCF-DA assay was performed to confirm the reduction of Aβ-induced intracellular ROS production in PC12 cells (Figure 2A). The Aβ-treated group (131.35%) showed a significant increase in intracellular ROS production compared with the control group (100%). However, the CBL-pretreated groups showed remarkable intracellular ROS reductions at the 10−100 μg/mL concentrations (average = 79.36%).

Figure 2. Neuronal cell-protective effects of chloroform fraction of broccoli leaves (CBL) on ROS production levels (A), cell viability (B), and cell membrane protective effect (C) on Aβ-induced neurotoxicity in PC12 cells. PC12 cells were pretreated for 3 h with various concentrations. After pre-incubation, the cells were treated with Aβ (100 μg/mL) for 48 h. Results shown are means ± SD (n = 5). Data were statistically considered at p < 0.05 versus control group, and different letters aove the bars represent statistical differences.

To evaluate the viability of neuronal cell on Aβ-induced neurotoxicity, a 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay was used. MTT (yellow dye) was converted into a violet formazan crystal by the mitochondrial activity in viable cells.19 The viability of neuronal cell for CBL on the Aβ-induced neurotoxicity was examined C

DOI: 10.1021/acs.jafc.6b00559 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry (Figure 2B). The Aβ-treated group showed significantly decreased cell viability (69.20%) compared with the control group (100%). On the other hand, the CBL-pretreated groups showed higher cell viability at all concentrations (110.12− 121.92%) than vitamin C (94.48%) as a positive control. An increase of LDH, an intracellular enzyme that can be released into the medium through damaged membranes, in the medium indicates cell membrane damage.19 The cell membrane protective effect of CBL on the Aβ-induced neuronal cell membrane damage was also examined by an LDH release assay (Figure 2C). The Aβ-treated group showed significant neuronal cell membrane damage (36.71%) compared with the control group (11.40%), whereas the CBL-pretreated groups (24.60%) showed similar membrane protective effects at 100 μg/mL concentration compared with the vitamin C-pretreated group (28.20%) as a positive control. A previous study reported that broccoli ethanolic extract had higher protective effects and cellular proliferation than the other ethanolic extracts of various fruit and vegetables (onion, garlic, green pepper, licorice, apple, kiwi, and pineapple) against the N-nitroso compounds (Nnitrosopiperidine, N-nitrosodimethylamine, and N-nitrosodibutylamine) that induced cytotoxicity.20 Our study results also showed that pretreated CBL had a remarkable neuronal cellprotective effect by inhibiting Aβ-induced neurotoxicity. Inhibition of AChE. The cholinergic function is important to learning and memory functions in the brain.21,22 In particular, endogenous AChE inhibition enhances cholinergic neurotransmission by increasing the acetylcholine (ACh) content in the synapses of neurons.21 The inhibitory effect against AChE of CBL showed >50% inhibition at 100 μg/mL concentration. The IC50 value of CBL was shown at 75.01 μg/ mL concentration (Figure 3). Behavioral Tests. To confirm the ameliorating effect of CBL on Aβ1−42-induced learning- and memory-impaired mice, Y-maze, passive avoidance, and Morris water maze tests were conducted. In Figure 4A, the Y-maze results show the impairment of the spatial cognitive function of the Aβ group (47.46%, about a 14.05% decrease) compared to the alternation

Figure 4. Ameliorating effect of chloroform fraction of broccoli leaves (CBL) on Aβ-induced special working memory impairment mice. The spontaneous alteration behavior and number of arm entries (A) and path tracing of each group (B) in a Y-maze test were measured. Control group was injected with saline (0.85%), and other groups were injected with Aβ1−42 (410 pmol/mouse, 10 μL). CBL (5, 10, and 20 mg/kg body weight per day, respectively) groups were injected with Aβ1−42 followed by feeding for 3 weeks. Results shown are means ± SD (n = 8). Data were statistically considered at p < 0.05 versus control group, and different letters above bars represent statistical differences.

behavior of the control group (61.51%). On the other hand, increased alternation behavior was shown in the CBL 20 group (63.05%). In Figure 4B, the black line represents the path tracing of mice in the Y-maze. The path-tracing results show that the control group mice engaged in similar path tracing in each arm, whereas the line of the Aβ group shows that they tended to lean toward one arm. These results also suggest spatial cognitive impairments were caused by Aβ injection, because the innate inclination of normal mice is to explore new environments.23 The passive avoidance test reflects short-term learning and memory function. The Aβ group (37.5 s, 86.73% decrease) had significantly shorter step-through latency times compared to the control group (282.50 s). However, the CBL 5 (71.50 s), CBL 10 (91.25 s), and CBL 20 groups (228.80 s) showed incremental short-term learning memory improvements compared to the Aβ group (Figure 5). Long-term learning and memory function was measured by the Morris water maze test. In the training session, the escape latency time of all groups had a decreasing tendency over 4 days. In particular, the Aβ group (40.11 s) showed a higher escape latency time than the control group (24.93 s), and the escape latency times of all CBL-feeding groups (CBL 5 = 28.29 s, CBL 10 = 31.39 s, and CBL 20 = 25.99 s) decreased by the final training day (Figure 6A). In the probe test, the pathtracing results of the mice of the control and CBL 20 groups

Figure 3. Inhibitory effect of chloroform fraction of broccoli leaves (CBL) against AChE. The inhibitory effect against AChE was expressed as a percentage of enzyme activity compared to the control value. Results shown are means ± SD (n = 5). Data were statistically considered at p < 0.05 versus control group, and different letters above bars represent statistical differences. D

DOI: 10.1021/acs.jafc.6b00559 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. Ameliorating effect of chloroform fraction of broccoli leaves (CBL) in Aβ-induced short-term learning and memory impairment mice. The step-through latency (300 s) was measured in the passive avoidance test. Control group was injected with saline (0.85%), and other groups were injected with Aβ1−42 (410 pmol/mouse, 10 μL). Sample groups were injected with Aβ1−42 followed by feeding with CBL (5, 10, and 20 mg/kg per day, respectively). Results shown are means ± SD (n = 8). Data were statistically considered at p < 0.05 versus control group, and different letters above bars represent statistical differences.

were more in the W zone, which included the platform as a safe zone, whereas the path-tracing results of the Aβ group were in all zones (Figure 6B). Figure 6C shows the retention time in the W zone, which included the platform, in the probe test. The Aβ group (26.56%) showed lower long-term learning and memory ability compared with the control group (44.72%). However, the CBL 20 group (48.86%) showed remarkable ameliorating effects on Aβ-induced learning and memory impairment. The above behavioral test results suggest that broccoli leaves had excellent ameliorating effects on spatial cognitive function as a learning and memory ability. Bae et al.24 reported that Aβ injection in rats induced neuronal apoptosis in the hippocampal CA1 pyramidal cell layer and that these results produced severe cognitive deficits. Our above results also confirm that Aβ injection in mice induced severe behavioral dysfunction, but the administration of CBL effectively ameliorated learning and memory impairment by Aβ-induced neurotoxicity. In Vivo Antioxidant Activities. Mitochondrial damage and dysfunction affect the early pathophysiology of neurodegenerative diseases, including AD. The biogenesis of antioxidative proteins and detoxification enzymes such as SOD and catalase, GSH S-transferase, and GSH have been shown to be regulated by nuclear factor erythroid-2-related factor 2 (Nrf2).25 The Aβ group showed decreased SOD levels (0.91 U/mg of protein, 51.41% decrease) compared with the control group (1.77 U/mg of protein). Although the SOD levels of the CBL groups (CBL 5 = 0.99, CBL 10 = 1.36, and CBL 20 = 1.54 U/mg of protein) showed an increasing pattern, they did not show statistical differences compared with the Aβ group (Figure 7A). The results of the oxidized GSH/total GSH ratio increased in the Aβ group (27.68%) compared with the control group (18.56%). The oxidized GSH/total GSH ratio of CBL groups also did not show statistical significance compared with the Aβ group (Figure 7B). MDA is a crucial marker of

Figure 6. Ameliorating effect of chloroform fraction of broccoli leaves (CBL) in Aβ-induced long-term learning and memory impairment mice. Escape latency in the training trial (A), swimming pattern of mice in the probe trial (B), and probe trial session (C) in the Morris water maze test were also examined. Control group was injected with saline (0.85%), and other groups were injected with Aβ1−42 (410 pmol/mouse, 10 μL). Sample groups were injected with Aβ1−42 followed by feeding with CBL (5, 10, and 20 mg/kg per day, respectively). Results shown are means ± SD (n = 8). Data were statistically considered at p < 0.05 versus control group, and different letters above bars represent statistical differences.

lipid peroxidation in various animal tissues. In particular, brain tissue, including high polyunsaturated fatty acid and iron content, is highly vulnerable to ROS-mediated lipid peroxidation.6,26 The Aβ group (4.47 nmol/mg protein) showed a 20.16% increase of MDA content compared with the control group (3.72 nmol/mg protein). The CBL groups had significantly reduced MDA levels; in particular, the CBL 20 group (3.33 nmol/mg protein) showed remarkable inhibition E

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Bidchol et al.6 reported that broccoli ethanolic extracts led to in vitro scavenging of superoxide radicals, inhibited microsomal lipid peroxidation, and had strong power-reducing and metal ion-chelating effects. The administration of CBL enhanced antioxidant status; for example, it increased SOD levels and decreased the oxidized GSH/total GSH ratio and MDA levels. Therefore, these results suggest that the severe damage of neuronal cell membranes against ROS-induced oxidative stress can be prevented by phytochemicals including the hydroxyl groups in broccoli leaves, and the broccoli leaves could serve as excellent antioxidants against Aβ-induced oxidative stress in mouse brain tissues. AChE Activity. AChE activity in the brain tissues of mice was also checked to confirm the reason for the cognitive effects in the behavioral tests. The Aβ group (120.81%) showed an increase of AChE activity in the brain tissues of mice compared with the control group (100%). In addition, the CBL groups (CBL 5 = 100.45%, CBL 10 = 96.73%, and CBL 20 = 99.60%) showed statistically significant decreases in Aβ-induced AChE activity (Figure 8). Amyloid peptides reduce ACh synthesis

Figure 8. AChE activity of chloroform fraction of broccoli leaves (CBL) in Aβ-induced mice brain homogenates. Results shown are means ± SD (n = 8). Data were statistically considered at p < 0.05 versus control group, and different letters above bars represent statistical differences.

through the leakage of choline across cell membranes and immediately affect the surface and intracellular AChE expression around amyloid plaques in AD brain tissues.18,26 Furthermore, the formation of AChE−Aβ complexes has been shown to be more neurotoxic than Aβ alone because of the changes to the signal transduction pathway of the wingless-type mouse mammary tumor virus (MMTV) integration site family.16,18 In addition, AChE could promote Aβ fibril and plaque formation, which is caused by the interaction of Aβ peptide with AChE at a specific motif located close to the peripheral anionic binding site of the enzyme.18 That is, Aβ could induce various changes in the components of the cholinergic system family, such as ACh and AChE, and the administration of CBL could ameliorate the AChE activity in Aβ-induced cholinergic dysfunction. These results suggest that CBL is an AChE inhibitor as well as a natural source of antioxidants. Identification of Main Compounds with Q-TOF UPLCMS System. The main phytochemicals of CBL were identified

Figure 7. SOD levels (A), oxidized GSH/total GSH ratio (B), and MDA levels (C) of chloroform fraction of broccoli leaves (CBL) in Aβ-induced mice brain homogenates. Results shown are means ± SD (n = 8). Data were statistically considered at p < 0.05 versus control group, and different letters above bars represent statistical differences.

of MDA production on Aβ-injected mice brain tissues (Figure 7C). In the above results, the only MDA levels in CBL groups statistically showed antioxidant activity on Aβ-induced oxidative stress. Therefore, the antioxidant activity of CBL may be affected by antioxidant compounds in CBL rather than regulation of antioxidant enzyme activity by CBL. F

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Figure 9. Q-TOF UPLC-MS data of chloroform fraction of broccoli leaves (CBL) in negative ion mode: MS scan of base peak chromatogram mode (A); MS2 scan of oxo-dihydroxy-octadecenoic acid (B); trihydroxy-octadecenoic acid (C).

Table 1. MS2 Data of the Identified Compounds in the Chloroform Fraction of Broccoli Leaves (CBL) no.

tR (min)

[M − H]− (m/z)

1

4.267

327.21

2

4.480

329.23

m/z (% base peak) MS2 85 MS2 99

[327]: 229 (59.56), 211 (100), 183 (16.64), 171 (26.56), 97(17.68), (10.80) [329]: 229 (55.61), 211 (100),, 183 (13.10), 171 (18.20), 155 (1.23), (13.35)

assigned identification oxo-dihydroxy-octadecenoic acid (oxoDHODE) trihydroxy-octadecenoic acid (THODE)

faya, Bituminaria bituminosa, Phyllostachys nigra (Lodd.) Munro, and Sasa veitchii (Carr.) Rehder et al.).3,28,29 Oxylipins are oxygenated metabolites derived from the oxidation of polyunsaturated fatty acids such as linoleic acid (18:2), α-linolenic acid (18:3), and roughanic acid (16:3) in plants and humans.30,31 Generally, the formation of oxylipin as a result of fatty acid hydroperoxides (HPOs) occurs by chemical autoxidation or the catalysis of two distinct enzymatic reactions: the lipoxygenase (LOX) and dioxygenase reactions.32 However, the oxo-derivatives and hydroxy-derivatives as main compounds in CBL are converted via several diverging pathways of HPO.30,32 Oxylipins are known as latent inflammatory modulators, and they are associated with the prevention of cardiovascular diseases, host defense, tissue injury, and surgical intervention.3,31 A 12-oxo-phytodienoic acid (OPDA), a plant-derived oxylipin, has been shown to have cytoprotective effects on H2O2-induced oxidative stress in SHSY5Y cells by controlling the Nrf2-dependent antioxidative response, which leads to the up-regulation of genes such as

by Q-TOF UPLC-MS analysis on the comparison of mass fragmentation of the MS2 scan, and the MS2 fragmentation data were compared to the previous scientific literature.3,28,29 The two main phytochemicals of CBL were analyzed as metabolites from unsaturated fatty acids. One of the main compounds showed the base peak of the [M − H]− ion at an m/z value of 327.21 at 4.267 min, and MS2 basic fragmentation ions were shown at m/z 97.06, 183.13, 211.13, and 229.14 (Figure 9B; Table 1). The compound identified as an oxo-dihydroxyoctadecenoic acid (oxo-DHODE) including the structure of the end group was shown as HOCHCH(CH2)CH3.3,28 Another of the main compounds showed the base peak of the [M − H]− ion at an m/z value of 329.23 at 4.480 min, and MS2 basic fragmentation ions were shown at m/z 99.07, 183.13, 211.13, and 229.14 (Figure 9C; Table 1). A trihydroxyoctadecenoic acid (THODE) was identified as another main compound.3,26,27 These two compounds are called oxylipins, and they have been found in several types of leaves (e.g., Myrica G

DOI: 10.1021/acs.jafc.6b00559 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

of Education (NRF-2015R1D1A3A01015931), and the Small and Medium Business Administration (2014-S2181697), Republic of Korea. S.K.P., J.S.H., J.M.K., J.Y.K., and D.S.L. were supported by the BK21 Plus program (MEST).

GSH synthesis and phase-two antioxidant/detoxification enzymes (HO-1, NQO1, and GSH reductase).33 Moreover, methanol extracts of Phyllostachys nigra (Lodd.) Munro leaves, including oxo-DHODE, and Sasa veitchii (Carr.) Rehder leaves, including oxo-DHODE and THODE, as main phytochemicals have been found to inhibit inflammatory activity in fibrosarcoma cells of TNF-stimulated mouse by inhibiting NF-κB-induced gene expression and cyclooxygenase (COX)-1 and COX-2 enzyme activities.3 In AD brains, amyloids have been reported to induce oxidative stress production and inflammatory reactions by the mitogen-activated protein kinases (MAPK) pathways implicated in oxidative stressinduced cell death and nuclear factor-kappa B (NF-κB) activity. In particular, the c-Jun NH2-terminal kinase (JNK) signaling pathway of MAPK pathways has been shown to be activated by pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, in response to cellular oxidative stress, and cellular differentiation and apoptosis have been shown to be induced as a result.34 That is, Aβ1−42 induces JNK and NF-κB activation in the hippocampus, and numerous inflammatory cytokines, including TNF and IL-1β, are expressed in the astrocytes and microglia around amyloid plaques in brain tissue.35 In particular, NF-κB activated by Aβ induced cell death by expressing the many early oxidative stress response genes in the hippocampus. When the results of previous reports were considered, broccoli leaf extracts, including oxo-DHODE and THODE as main compounds, could be diminished via various mechanisms on Aβ-induced toxicity. Many previous studies have focused on the various effects (antioxidant, anti-inflammatory, anticancer) of broccoli, including phytochemical materials such as glucosinolates, isothiocyanates, and sulforaphanes.7−9 On the other hand, our studies showed that oxylipins (oxo-DHODE and THODE) as novel phytochemicals appear as antiamnesic compounds in broccoli leaves. Although little research on oxylipin has been reported and the biological impact of these compounds remains unclear, we clearly showed a learning and memory effect through the antioxidant activity and inhibitory effect of AChE. Consequently, broccoli leaves might be considered as new substances for multitargeted therapeutic agents such as antioxidants and AChE inhibitors in neurodegenerative diseases. The ameliorating effects of broccoli leaves on Aβ-induced learning and memory impairment were examined through in vitro neuronal protective effects, in vivo behavioral tests, ex vivo antioxidant activity, and AChE inhibition. CBL including oxylipins (oxo-DHODE and THODE) effectively improved Aβ-induced cognitive impairments by neuronal cell-protective effects, antioxidant activity, and AChE inhibitory effects. Therefore, broccoli leaves might be used as a natural potential resource for improving learning and memory deficits in Aβinduced neurodegeneration such as AD.

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Notes

The authors declare no competing financial interest.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*(H.J.H.) Phone: +82 55 772 1907. Fax: +82 55 772 1909. Email: [email protected]. Funding

This study was supported by a grant from the 2013 High Valueadded Food Technology Development Program of Ministry of Agriculture Food and Rural Affairs (113023-3), a National Research Foundation (NRF) of Korea funded by the Ministry H

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