JOURNAL OF MEDICINAL FOOD J Med Food 17 (11) 2014, 1222–1231 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.3014

Ligularia fischeri Extract Protects Against Oxidative-Stress-Induced Neurotoxicity in Mice and PC12 Cells Soo Jung Choi,1,2 Jae Kyeom Kim,3,4 Soo Hwan Suh,2,5 Cho Rong Kim,3 Hye Kyung Kim,6 Chang-Ju Kim,7 Gwi Gun Park,8 Cheung-Seog Park,9 and Dong-Hoon Shin3 1

Functional Food Research Center, Korea University, Seoul, Korea. Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, North Carolina, USA. 3 Department of Food and Biotechnology, Korea University, Seoul, Korea. 4 Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota, USA. 5 Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Osong, Korea. 6 Department of Food Biotechnology, Hanseo University, Seosan, Korea. 7 Department of Physiology, Kyung Hee University, Seoul, Korea. 8 Department of Food Science and Biotechnology, Gachon University, Seongnam, Korea. 9 Department of Microbiology, School of Medicine, Kyung Hee University, Seoul, Korea.

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ABSTRACT Alzheimer’s disease (AD) is pathologically characterized by the presence of amyloid plaques in brain and the overproduction of amyloid beta (Ab), leading to learning and memory impairment and intense oxidative stress. In this study, the protective effect of Ligularia fischeri extract was investigated using PC12 cells. L. fischeri extract attenuated hydrogenperoxide-induced DNA fragmentation in cells. In vivo behavioral tests were performed to examine the effects of the extract on amyloid-b peptide1-42-induced impairment of learning and memory in mice. A diet containing the extract increased alternation behaviors in the Y-maze test and step-through latency of passive avoidance task. Moreover, we found that consumption of the extract decreased lipid peroxidation in a biochemical study of brain tissue in mice. High-performance liquid chromatography was used to identify the active compounds in the extract. These results suggest that L. fischeri extract could be protective against Ab-induced neurotoxicity, possibly due to the antioxidative capacity of its constituent, 3-O-caffeoylquinic acid.

KEY WORDS:  3-O-caffeoylquinic acid  amyloid beta peptide  Ligularia fischeri  oxidative stress

neurons in the brain. Many investigators surmise that deposition of amyloid beta (Ab) peptide is the crucial step in AD pathogenesis.2,3 Among the body’s organs, the brain is uniquely vulnerable to oxidative damage because of its high utilization of oxygen, increased levels of polyunsaturated fatty acids, and relatively high levels of redox transition metal ions.4,5 Overproduction of Ab leads to Abassociated free-radical-induced oxidative stress.6,7 This oxidative stress is manifested by the formation of reactive oxygen species (ROS), lipid peroxidation, DNA oxidation, and subsequent modification of proteins by the reactive lipid peroxidation products.8 Additional effects of Abassociated oxidative stress include protein oxidation, Ca2 + dyshomeostasis, mitochondrial impairment, peroxinitrite formation, inflammation, apoptosis, and other cellular responses, all of which ultimately lead to neuronal cell death.9–11 It has been reported that Ab produces hydrogen peroxide (H2O2) through metal ion reduction.12 H2O2 is one of major oxidative stress inducers. Although H2O2 is a reactive nonradical molecule, it can easily pass biological cell membranes.13

INTRODUCTION

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igularia fischeri is a member of the Compositae family, primarily distributed in damp shady regions near brooks and sloping fields in the eastern part of Korea. This plant has been consumed as a food in Korea for centuries. The leaves have been used for medicinal purposes, particularly for treating jaundice, scarlet fever, rheumatoid arthritis, and hepatic failure.1 Alzheimer’s disease (AD) is the most common cause of dementia in the geriatric population. AD was originally described in 1906 by Alois Alzheimer on the basis of the observation of amyloid plaques, neurofibrillary tangles, and vascular anomalies during an autopsy of a patient. The primary clinical features of AD are cognitive decline and mental deterioration, a consequence of progressive loss of

Manuscript received 13 August 2013. Revision accepted 2 July 2014. Address correspondence to: Dong-Hoon Shin, PhD, 612 Life Science Building, Green Campus, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-701, Republic of Korea, E-mail: [email protected]

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Antioxidants are molecules that inhibit oxidation, thereby protecting cellular components from the ravages of free radicals. A variety of antioxidants have been used to suppress the oxidative stress associated with AD, many of which protect cells from Ab-induced neurotoxicity. Recently, natural antioxidants, such as vitamins and phenolic acids, have received a great deal of attention because they are safe, and several function as chemopreventive agents against oxidative damage. Epidemiological studies suggest that dietary intake of antioxidants can affect the incidence of neurodegenerative disease such as AD.14,15 Phenolic acids are a major group of antioxidants that exhibit their antioxidant effects by quenching free radicals and promoting endogenous antioxidant capacity. However, the therapeutic capacity of most of these compounds is limited since they do not penetrate the blood brain barrier (BBB).16 The brain needs a barrier that separates it from the blood to permit the tight control of the complex neuronal signaling. The BBB is an endothelial barrier present in the capillaries that course through the brain. A few compounds have shown effects in animal models or in clinical studies. Therefore, active compounds designed to be potential neuroprotective treatments in neurological disorders should have the requirement that they can cross the BBB.17 In this study, we investigated the antioxidative effect and the protective effect of L. fischeri extracts against neuronal cell death induced by H2O2 in cultured PC12 cells. We generated an animal model of dementia with learning and memory dysfunction by injecting Ab into the ventricle of the mouse brain. Biochemical studies have demonstrated a relationship between increases in lipid peroxidation and damage to cholinergic neurons in Ab-treated mice.18,19 Antioxidant compounds were detected using high-performance liquid chromatography (HPLC), and Ab-injected mice were used to examine the effects of L. fischeri extract on memory impairment. MATERIALS AND METHODS Chemicals Ab1-42, thiobarbituric acid (TBA), phosphoric acid, tetramethoxypropane, n-butanol, H2O2, dimethyl sulfoxide (DMSO), 20 ,70 -dichlorofluorescin diacetate (DCF-DA), 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and ascorbic acid were purchased from Sigma Chemicals (St. Louis, MO, USA). Ab42-1 was purchased from Bachem (Bubendorf, Switzerland). Sample preparation Dried L. fischeri was purchased from an herb shop in the Gyeong-dong medicinal herb market (Seoul, Republic of Korea). The dried L. fischeri (0.5 kg) was ground into a powder and dissolved in ethanol (2.5 L) by shaking for 24 h at 125 rpm (1.57 g). The ethanol extract was filtered through a No. 42 filter paper (Whatman International Ltd., Middlesex, England). This extraction procedure was repeated five times. The ethanol extract was concentrated in a rotary

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evaporator (Eyela, Tokyo, Japan) under reduced pressure at 37C. Cell culture The rat pheochromocytoma (PC12) cell line (CRL-1721) was obtained from the American Type Culture Collection (Manassas, VA, USA). RPMI 1640 medium, horse serum from a donor herd, fetal bovine serum (FBS), and antibioticantimycotic were purchased from Gibco-BRL (Grand Island, NY, USA). The PC12 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) horse serum from a donor herd, 5% (v/v) FBS, and 1% (v/v) antibioticantimycotic. The cells were cultured on 100-mm-diameter tissue culture dishes (Falcon, BD Biosciences, Franklin Lakes, NJ, USA) and maintained in a 37C incubator in a humidified, 5% CO2 atmosphere. When the cultured cells were 80–90% confluent (split ratio 1:4), they were subcultured. The medium was refreshed approximately three times a week. Analysis of DNA fragmentation The PC12 cells were pretreated for 48 h with various concentrations of the L. fischeri phytochemical extract, followed by exposure to H2O2 for 24 h. The cells were harvested and washed with phosphate-buffered saline (PBS). DNA was extracted using DNAzol Reagent (Life Technologies, Carlsbad, CA, USA) and following the manufacturer’s instructions. In brief, 107 cells were lysed in 1 mL of reagent and then the DNA from the lysate was precipitated by the addition of 0.5 mL of 100% ethanol. The precipitated DNA was washed three times with 1 mL of 75% ethanol, and reconstituted in 200 lL of 8 mM NaOH solution. The concentration of DNA was measured by spectrophotometer (NanoDrop ND-1000; Thermo Scientific, Wilmington, DE, USA) and *1 lg of DNA was applied for gel electrophoresis. Each DNA sample was developed on 2% agarose gel with tris-acetate-EDTA buffer at 100 V for 1 h. Gels were subsequently stained with ethidium bromide and visualized on a Bio-Rad Universal hood imaging system (Bio-Rad, Segrate, Italy). Animals Imprinting Control Region mice (5-week-old males) were purchased from Samtako Bio Korea (Gyeonggi-do, Republic of Korea) and had ad libitum. The L. fischeri extract was mixed into the feed for 4 weeks. Subsequently, Ab1-42 was administered via intracerebroventricular (ICV) injection. The control group was injected with the nontoxic reverse fragment, Ab42-1 (410 pmol per mouse), and the Ab group was injected with Ab1-42 (410 pmol per mouse). For each mouse, the Ab peptide was dissolved in 0.85% (v/v) sodium chloride solution. Each mouse was given injections to the bregma by using a Hamilton microsyringe (depth = 2.5 mm; injection volume = 5 lL; dose = 410 pmol/mouse).20 The sample groups (Lf 400, Lf 800, and Lf 1200) were injected with Ab1-42 after their diets were supplemented with

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L. fischeri extract. The extract was mixed with the commercial diet at concentrations of 400, 800, and 1200 mg/kg of body weight per day (0.25%, 0.50%, and 0.75%, respectively). All experimental procedures were abided by the guidelines established by the Animal Care and Use Committee of Korea University. Y-maze test The Y-maze test was performed 3 days after the Ab injection. The maze was made of black plastic, and each arm of the maze was 33-cm long, 15-cm high, and 10-cm wide, and positioned at an equal angle. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8-min period. The sequence of arm entries was recorded manually. Spontaneous alternation behavior was defined as entry into all three arms by consecutive choice in overlapping triplet sets. The percentage of spontaneous alternation behaviors was calculated as the ratio of actual-to-possible alternations.21,22 Alternation behavior (%) = 100 · {possible alternations/ (total number of arm entries - 2)} Passive avoidance test The passive avoidance test was performed 7 days after the Ab injection. The apparatus consisted of an illuminated chamber connected to a dark chamber. An acquisition trial was performed on day 1. Each mouse was placed in the apparatus and left for 1 min with no light or shock, followed by a 2-min period with light and no shock to habituate the mice to the apparatus. Subsequently, the mice were individually placed in the illuminated chamber. Immediately after entering the dark chamber, an inescapable scrambled electric shock (0.5 mA, for 1 s) was delivered through the floor grid. The mice were then returned to their cages. Each mouse was again placed in the illuminated chamber 24 h later (retention trial). The interval between placement in the illuminated chamber and entry into the dark chamber was measured as the latency in both the acquisition and retention trials. The maximum testing limit for step-through latency was 300 s.21,22 Determination of lipid peroxidation Mice were sacrificed, and their brains were dissected and maintained at - 80C prior to use. The brains were homogenized in cold PBS in an ice bath. The homogenates were directly centrifuged at (33,600 g) for 10 s (twice at a 30-s interval). Aliquots of the supernatant were used to determine the malondialdehyde (MDA) levels and protein content in the brain. The MDA level was assayed for lipid peroxidation products by monitoring the formation of TBA reactive substances, as described previously.23 Briefly, 80 mL of each homogenate was mixed with 480 mL of phosphoric acid (1%, v/v) followed by addition of 160 mL of TBA solution (0.67%, w/v). The mixture was incubated at 95C in a water bath for 45 min. After cooling, the colored complex was extracted with n-butanol. The butanol phase

was separated by centrifugation, and the absorbance was measured using tetramethoxypropane as a standard at 532 nm. MDA levels are expressed as lmole per milligram of protein. DCF-DA assay Levels of cellular oxidative stress were evaluated using a DCF-DA assay. The PC12 cells were pretreated for 48 h with various concentrations of the sample, followed by exposure to H2O2 for 2 h. At the end of the treatment period, the cells were incubated with 50 lM DCF-DA for 50 min at 37C, and the dichlorofluorescein was quantified using a fluorometer (GENios; Tecan Ltd., Ma¨nnedorf, Switzerland) using 485-nm excitation and 535-nm emission filters. The results are presented as a percentage relative to the oxidative stress of the control cells (set to 100%).24 Oxidative stress (%) = 100 · (quantified DCF of sample and H2O2-treated cells/quantified DCF of untreated control cells). MTT reduction assay Cell survival was evaluated with MTT reduction. Cells were preincubated with sample for 48 h prior to H2O2 addition, followed by treatment with or without H2O2 for 2 h. Reduction of MTT was initiated by the addition of 10 lL of MTT stock solution (2.5 mg/mL) per well, followed by a 3-h incubation of the plates at 37C. Subsequently, the reaction was stopped by adding 150 lL of DMSO. The absorbance was determined at 570 nm. The reference wave was then determined at 630 nm using a microplate reader (model 550; Bio-Rad Laboratories, Hercules, CA, USA).24 Isolation of active compound The active compound was purified sequentially by solvent partition, silica gel column chromatography, and HPLC. The evaporated ethanol extract (172.68 g) was dissolved in distilled water (150 mL), and partitioned with hexane (450 mL · 3), chloroform (450 mL · 3), and ethyl acetate (450 mL · 3) sequentially for 24 h. Subsequently, each layer was separately concentrated in a rotary evaporator. The third chloroform fraction (8.59 g) was isolated by first silica-gel column chromatography and added to a chromatographic open column packed with silica gel (215 g) suspended in chloroform. The column was washed with chloroform and eluted with a stepwise gradient consisting of chloroform and ethanol (100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, v/v, sequentially; repeated three times). The solvent bed volume was 515 mL. The first fraction of the 90:10 gradient was isolated, and concentrated in a rotary evaporator. Subsequently, the evaporated sample (3.8 g) was isolated using a second silica-gel open column with silica gel (95 g) in chloroform. The column was washed with chloroform and eluted with a stepwise gradient consisting of chloroform and ethanol (100:0, 98:2, 96:4, 94:6, 92:8, 90:10, 88:12, 86:14, 84:16, 82:18, and 80:20, v/v, sequentially). The solvent bed volume was 228 mL. The

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fraction of 94:6 gradient was selected and evaporated, and the sample isolated was dissolved in ethanol and analyzed with HPLC using C18 column. Identification of active compound by HPLC HPLC was used to identify the active compound from the L. fischeri extract. The HPLC system (YL9100) was equipped with a PDA detector (YL9160), autosampler (YL9150), vacuum degasser (YL9101), binary pump (YL9111), and YL Clarity software version 3.0.4.444 (Young Lin Instruments Co. Inc., Anyang, Korea). Separation was achieved on a C18 column (4.6 · 250 mm, 5 lm Capcell Pak; Shiseido, Tokyo, Japan). The operating conditions were an injection volume of 10 lL (10 mg/mL in methanol) and an eluent flow rate of 1 mL/min. The elution solvents were labeled A (0.1% v/v formic acid in water) and B (100% methanol). Spectral data were monitored at 326 nm. The program was started with 2% B from 0 to 15 min, 2% to 10% B from 15 to 20 min, 10% to 45% B from 20 to 90 min, and 45% B from 90 to 100 min. Measurement of acetylcholinesterase inhibition activity Acetylcholinesterase (AChE) activity was determined using the modified spectrophotometric method of Ellman. Acetylcholine (ACh) iodide was used as the reaction substrate, and 5,50 -dithio-bis(2-nitro)benzoic acid (DTNB) was used to measure AChE activity. For the enzyme preparation, PC12 cells were homogenized (Glass-Col homogenizer, Terre Haute, IN, USA) with Tris-HCl buffer (20 mM TrisHCl [pH 7.5] containing 150 mM NaCl, 10 mM MgCl, and 0.5% Triton X-100); this solution was then centrifuged at 10,000 g for 15 min. The supernatant was used as the enzyme source. The AChE protein concentration was determined by a protein assay kit using bovine serum albumin as the protein standard (Bio-Rad Laboratories). Briefly, 10 lL of each sample was mixed with 10 lL of enzyme solution, added to 70 lL of reaction mixture (50 mM sodium phosphate buffer [pH 8.0] containing 0.5 mM ACh iodide and 1 mM DTNB), and incubated at 37C for 15 min. The ACh iodide and enzyme reaction was monitored at a wavelength of 405 nm using a 96-well microplate reader (GENios; Tecan Ltd.).25

FIG. 1. The effect of the Ligularia fischeri extract on hydrogen peroxide (H2O2)–induced DNA fragmentation in PC12 cells. DNA extracts from PC12 cells exposed to H2O2 with various concentrations of L. fischeri extract were subjected to agarose gel electrophoresis. Lane 1, 0.5 - 10.0 kb DNA ladder marker; lane 2, control; lane 3, 200 lM H2O2 alone; lanes 4–7, L. fischeri extract pretreatment + 200 lM H2O2.

Y-maze test The Y-maze test was performed 3 days after the Ab injection. The Ab1-42-injected mice (Ab group) exhibited significantly impaired spatial working memory (25% decrease in alternation behavior) compared with that of the control group (Fig. 2A). Mice fed with the L. fischeri extract diet displayed increased spontaneous alternation behavior after Ab injection. By contrast, the number of arm entries did not change among any of the experimental groups (Fig. 2B). Passive avoidance test The passive avoidance test was performed 7 days after the Ab injection. The Ab1-42-injected mice (Ab group) displayed a significantly shorter (186-s decrease) step-through latency time when compared with that of the control group. The L. fischeri extract diet attenuated Ab1-42-induced impairment in mice during the passive avoidance test (Fig. 3).

Statistical analysis Data were expressed as means – standard deviations. The data were analyzed by using one-way analysis of variance followed by post-hoc Tukey’s multiple-comparison test. RESULTS Effect of L. fischeri extract on DNA fragmentation Using gel electrophoresis, the protective effect of L. fischeri extract on H2O2-induced DNA fragmentation was evaluated in PC12 cells. Following 24-h treatment of PC12 cells with H2O2, a DNA fragmentation pattern was detected. Pretreatment of cells with L. fischeri extract attenuated DNA fragmentation compared with H2O2-treated cells alone (Fig. 1).

Determination of lipid peroxidation Lipid peroxidation was determined after the learning and memory tests were completed. The MDA levels increased significantly (0.5 lmole/mg protein) in the Ab1-42injected group when compared with those of the control group. The increase in MDA levels indicated an elevation of lipid peroxidation in the brain of Ab-injected mice. Diets containing 400, 800, and 1200 mg/kg of L. fischeri extract reversed the effects of Ab-induced lipid peroxidation (Fig. 4). Isolation of active compound To separate the active compound from the L. fischeri extract, solvent partition and open column chromatography

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FIG. 4. The effect of L. fischeri extract on lipid peroxidation in Abinjected mice brains. Control was injected with Ab1-42. Ab was injected with 410 pmol of Ab1-42 per mouse. Sample groups (Lf 400, Lf 800, and Lf 1200) were injected with Ab1-42 followed by feeding with L. fischeri extract (400, 800, and 1200 mg/kg per day, respectively). Values indicate the mean – SD (n = 8), *, #, x represent statistical differences between groups (P < .01).

FIG. 2. The effect of the L. fischeri extract on spontaneous alternation behavior in Y-maze test. (A) Alternation behavior, (B) total arm entries. Control was injected with Ab1-42. Amyloid beta (Ab) was injected with 410 pmol of Ab1-42 per mouse. Sample groups (Lf 400, Lf 800, and Lf 1200) were injected with Ab1-42 followed by feeding with L. fischeri extract (400, 800, and 1200 mg/kg per day, respectively). Spontaneous alternation behaviors were measured during 8 min. Values indicate the mean – standard deviation (SD, n = 8), *, # represent statistical differences between groups (P < .01). Total arm entries did not change significantly.

FIG. 3. The effect of the L. fischeri extract on step-through latency in the passive avoidance test. Control was injected with Ab1-42. Ab was injected with 410 pmol of Ab1-42 per mouse. Sample groups (Lf 400, Lf 800, and Lf 1200) were injected with Ab1-42 followed by feeding with L. fischeri extract (400, 800, and 1200 mg/kg per day, respectively). The step-through latency was determined during 300 s. *, # represent statistical differences between groups (P < .01). Values indicate the mean – SD (n = 8), P < .01.

were used. The extract was partitioned three times each using n-hexane, chloroform, and ethyl acetate, sequentially. The third chloroform fraction decreased oxidation most effectively (318.21%; Fig. 5A). In the MTT reduction assay, this fraction exhibited the highest cell viability (90.35%; Fig. 5B). On the basis of these results, the third fraction of chloroform extract was divided into 33 subfractions by first silica-gel open-column chromatography. Among these fractions, the fourth fraction (the first fraction with the chloroform to ethanol ratio of 90:10) exhibited positive activity in both the DCF-DA assay (370.85%) and the MTT reduction assay (93.87%; Table 1). In the second silica-gel opencolumn chromatography, the fourth fraction was divided into 11 subfractions. Among these fractions, the fourth fraction (with a chloroform to ethanol ratio of 94:6) exhibited positive activity in both the DCF-DA assay (119.10%) and the MTT reduction assay (102.06%; Table 2). To identify the active compound in these fractions, the ethanol extract and isolated fraction were further analyzed. The L. fischeri ethanol extract was analyzed for its composition by HPLC. Major compounds were separated on the column. Representative HPLC chromatograms of caffeoylquinic acid derivatives, including 3-O-caffeoylquinic acid (3-CQA), 5-O-caffeoylquinic acid (5-CQA), 3,4-di-O-caffeoylquinic acid (3,4-DCQA), 3,5-di-O-caffeoylquinic acid (3,5-DCQA), and 4,5-di-O-caffeoylquinic acid (4,5-DCQA) are displayed in Figure 6A. A significant peak appeared at 43 min (Fig. 6B). The active compound was ultimately identified as 3-CQA. Effect of 3-CQA on oxidative stress, cell viability, and AChE inhibition To confirm the protective effect of 3-CQA against oxidative stress, DCF-DA assay was used. H2O2-exposed PC12 cells resulted in 1800% increase of oxidative stress levels. The 48-h pretreatment of cells with 3-CQA resulted in

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Table 1. Inhibitory Effect of Ligularia fischeri Extract Separated by First Silica-Gel Open-Column Chromatography Against Oxidative Stress Fraction

FIG. 5. Protective effect of the solvent-partitioned L. fischeri extract against oxidative stress and cell death. (A) Oxidative stress, (B) cell viability. Groups: C, untreated control cultures; H, 200 lM H2O2; V, 100 lM vitamin C + 200 lM H2O2; H1–E3, 200 lM H2O2 + L. fischeri extract (H1–H3, n-hexane; C1–C3, chloroform; E1–E3, ethyl acetate [1 mg/mL]). Values indicate the mean – SD (n = 6), *, #, u, U, s, {, {, x, z represent statistical differences between groups (P < .01).

decreased oxidative stress compared with H2O2-only-exposed cells (50 lM, 1157%; 100 lM, 1145%; 150 lM, 1150%; and 200 lM, 1192% decrease compared with H2O2-only group; Fig. 7A). To evaluate oxidative-stress-induced neurotoxicity, MTT reduction assay was used. The cell viability was diminished by H2O2 exposure. As shown in Figure 7B, pretreatment of cells with 3-CQA inhibited H2O2-induced cytotoxicity in a dose-dependent manner (50 lM, 13%; 100 lM, 16%; 150 lM, 19%; and 200 lM, 23% increase compared with H2O2-only group). To confirm the AChE inhibition effect of 3-CQA, AChE inhibition was measured. As shown in Figure 7C, 3-CQA inhibited AChE activity in a dose-dependent manner (50 lM, 7%; 100 lM, 14%; 150 lM, 17%; and 200 lM, 23% increase compared with untreated group). DISCUSSION Oxidative stress is an important factor in the development of various neurodegenerative diseases. Ab peptide, the central constituent of senile plaques in the brains of patients with AD, has been shown to be a source of free radicals that may lead to neurodegenerative disorders. Intracellular ac-

Control 200 lM H2O2 100 lM vitamin C + 200 lM H2O2 100:0 (1) + 200 lM H2O2 100:0 (2) + 200 lM H2O2 100:0 (3) + 200 lM H2O2 90:10 (1)1200 lM H2O2 90:10 (2) + 200 lM H2O2 90:10 (3) + 200 lM H2O2 80:20 (1) + 200 lM H2O2 80:20 (2) + 200 lM H2O2 80:20 (3) + 200 lM H2O2 70:30 (1) + 200 lM H2O2 70:30 (2) + 200 lM H2O2 70:30 (3) + 200 lM H2O2 60:40 (1) + 200 lM H2O2 60:40 (2) + 200 lM H2O2 60:40 (3) + 200 lM H2O2 50:50 (1) + 200 lM H2O2 50:50 (2) + 200 lM H2O2 50:50 (3) + 200 lM H2O2 40:60 (1) + 200 lM H2O2 40:60 (2) + 200 lM H2O2 40:60 (3) + 200 lM H2O2 30:70 (1) + 200 lM H2O2 30:70 (2) + 200 lM H2O2 30:70 (3) + 200 lM H2O2 20:80 (1) + 200 lM H2O2 20:80 (2) + 200 lM H2O2 20:80 (3) + 200 lM H2O2 10:90 (1) + 200 lM H2O2 10:90 (2) + 200 lM H2O2 10:90 (3) + 200 lM H2O2 0:100 (1) + 200 lM H2O2 0:100 (2) + 200 lM H2O2 0:100 (3) + 200 lM H2O2

Oxidative stress (%) Cell viability (%) 100.00 – 6.48 473.48 – 7.38 136.06 – 6.02 312.15 – 2.98 453.53 – 6.50 513.46 – 6.54 170.8561.22 294.53 – 4.38 359.92 – 6.23 293.51 – 6.48 296.90 – 4.10 340.00 – 9.08 381.93 – 5.52 383.52 – 6.62 397.08 – 8.35 345.06 – 3.52 368.80 – 3.22 398.03 – 2.03 438.19 – 7.28 411.97 – 3.40 401.42 – 4.93 342.07 – 7.16 356.37 – 2.94 300.13 – 3.76 335.19 – 2.06 349.80 – 3.16 309.12 – 8.07 390.60 – 4.52 292.08 – 5.49 235.54 – 4.35 352.82 – 4.49 367.66 – 3.38 415.77 – 3.15 353.68 – 6.80 430.51 – 2.30 377.29 – 0.78

100.00 – 3.34 24.95 – 6.34 96.41 – 3.86 87.48 – 3.56 89.01 – 4.37 91.34 – 2.57 93.8762.26 87.08 – 1.95 90.12 – 1.75 92.66 – 0.54 93.92 – 2.59 92.56 – 0.10 90.02 – 1.55 92.10 – 3.50 89.31 – 5.72 90.43 – 3.00 90.73 – 0.99 87.79 – 2.28 91.34 – 4.45 87.99 – 1.09 90.22 – 4.65 89.01 – 7.37 85.86 – 8.88 87.18 – 4.66 89.27 – 3.84 88.38 – 8.74 88.31 – 2.62 87.04 – 2.01 87.49 – 7.62 87.34 – 5.82 85.93 – 12.81 87.34 – 3.87 87.49 – 5.03 86.67 – 3.90 88.45 – 5.99 84.59 – 3.74

Bold values indicate the mean – SD (n = 8), P < 0.01. H2O2, hydrogen peroxide.

cumulation of ROS is the primary cause of Ab-induced cytotoxicity, ultimately leading to the peroxidation of membrane lipids, oxidation of DNA, and cell death.26,27 ROS can cause cell death in PC12 cells, but this effect can be blocked or delayed by various antioxidants.28,29 DNA laddering analysis is routinely used as a method to identify protective mechanisms associated with antioxidant protection against H2O2-induced cell death.30,31 In this study, it was found that L. fischeri extract decreased H2O2-induced DNA laddering resulting from fragmentation. The effect of dietary administration of L. fischeri extract on behavioral abilities was examined using an AD animal model following ICV injection of Ab1-42. The peptide Ab1-42 is a primary factor in the development of learning and memory deficits resulting from intracellular accumulation of ROS. The ICV injection of Ab1-42 has been previously shown to induce memory deficits.21 Memory and learning

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Table 2. Inhibitory Effect of Ligularia fischeri Extract Separated by Second Silica-Gel Open-Column Chromatography Against Oxidative Stress Fraction Control 200 lM H2O2 100 lM vitamin C + 200 lM H2O2 100:0 + 200 lM H2O2 98:2 + 200 lM H2O2 96:4 + 200 lM H2O2 94:61200 lM H2O2 92:8 + 200 lM H2O2 90:10 + 200 lM H2O2 88:12 + 200 lM H2O2 86:14 + 200 lM H2O2 84:16 + 200 lM H2O2 82:18 + 200 lM H2O2 80:20 + 200 lM H2O2

Oxidative stress (%)

Cell viability (%)

100.00 – 2.37 406.19 – 1.69 138.89 – 1.56

100.00 – 2.88 26.61 – 3.34 98.42 – 10.03

231.39 – 10.95 203.86 – 7.04 200.80 – 1.75 149.1060.87 182.57 – 1.82 156.80 – 1.83 157.25 – 0.78 157.35 – 2.28 151.83 – 0.74 151.98 – 0.99 189.93 – 4.60

73.83 – 10.15 80.20 – 10.22 80.75 – 9.09 102.0665.82 61.26 – 1.67 71.46 – 2.71 76.65 – 2.32 88.49 – 6.52 86.12 – 12.64 97.32 – 14.97 64.99 – 8.14

Bold values indicate the mean – SD (n = 8), P < 0.01.

abilities were evaluated in a Y-maze test and a passive avoidance task. Spontaneous alternation behavior, which is recognized as a measure of spatial memory, was examined using the Y-maze test (Fig. 2A). To examine the toxicity of L. fischeri extract in mice, hepatotoxicity tests were performed. Commonly, the transaminases alanine transaminase (ALT) and aspartate transaminase (AST) are used as indicators of drug-induced liver damage. The AST and ALT did

not change among any of the experimental groups (data were not shown). Our studies suggest that a diet containing the natural extract of L. fischeri had a protective effect against learning and memory deficits without toxicity. Mice treated with the L. fischeri extract exhibited attenuated Ab1-24-induced impairment of passive avoidance performance in a dose-dependent manner (Fig. 3). Thus, L. fischeri extract displayed a significant antiamnestic effect in the mouse model. According to the oxidative stress theory of AD, Ab1-42 generates free radicals in the cell membrane, resulting in lipid peroxidation and cellular dysfunction. The hypothesis that Ab1-42 induces lipid peroxidation is a key component of the Ab-associated free-radical model of neurodegeneration in AD. Antioxidants inhibit the action of Ab1-42, which causes lipid peroxidation in brain cell membranes. The byproducts of peroxidation, such as 4-hydroxynonenal and acrolein, are generated after Ab1-42 addition to neurons, and these byproducts alter the conformation of membrane proteins and neurons.5,32 In previous studies, the MDA level was increased and catalase levels (one of antioxidant defense status) were reduced in the Ab model mice.33 All groups provided L. fischeri extracts displayed significantly lower levels of lipid peroxidation in brain tissues, possibly explaining the beneficial effects of L. fischeri against cognitive deficits that were induced via ICV injection of Ab1-42 (Fig. 4). This observation suggests that L. fischeri ethanol extract mitigated Ab1-42-induced memory impairment in a mouse model, and that a diet containing L. fischeri extract

FIG. 6. High-performance liquid chromatography (HPLC) of L. fischeri ethanol extract and 3-O-caffeoylquinic acid (3-CQA). (A) Various caffeoylquinic acid derivatives of the L. fischeri ethanol extract. (B) HPLC chromatogram of isolated 3-CQA.

PROTECTIVE EFFECTS OF LIGULARIA FISCHERI EXTRACT

FIG. 7. Protective effect of 3-CQA against oxidative stress and cell death. (A) Oxidative stress, (B) cell viability, (C) Acetylcholinesterase (AChE) inhibition. All groups of (A) and (B) were treated with 200 lM H2O2. Sample groups of (A) and (B) were preincubated with 3-CQA (50, 100, 150, and 200 lM, respectively) for 48 h before H2O2 treatment. Sample groups of (C) were treated with 3-CQA (50, 100, 150, and 200 lM, respectively). Values indicate the mean – SD (n = 6).

protected brain cells of the mice against lipid peroxidation. Our results demonstrate the protective effects of L. fischeri against Ab1-42-induced brain dysfunction. In this respect, roughly one tablespoon of L. fischeri extract (24 g/60 kg of body weight person/day) consumption may be an effective dose of bioactive compound, which may also play an important role in reducing the risk of AD. Many phenolic acids may function as chemopreventive agents. In addition, several compounds inhibit neurotoxicity

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through their antioxidative properties.34,35 In this study, five major phenolic antioxidant compounds—more precisely, caffeoylquinic acid derivatives—were isolated from L. fischeri, including 5-CQA, 3-CQA, 3,4-DCQA, 3,5-DCQA, and 4,5-DCQA. These five isomers are constituents accounting for > 10% of the dried leaves of L. fischeri.36 O-Caffeoylquinic acids are esters of polyphenolic caffeic acid and quinic acid, one of the phenolic acids found in coffee, sweet potatoes, propolis, and bamboo, as well as numerous other plants.37 By using a sequential purification process, the active compound in the L. fischeri extract was identified as 3-CQA. These compounds are antioxidants, which also attenuate intestinal glucose absorption.38 In addition, 3-CQA has been shown to possess antioxidant, anticancer, anti-inflammatory, and antiobesity properties.39,40 In our study, L. fischeri extract was shown to possess strong antiamnestic properties by decreasing oxidative stress in the brain. This protective effect may be due to the antioxidant capacity of 3-CQA. To confirm the capacity of 3-CQA, oxidative stress levels were measured. The 3-CQA was shown to protect PC12 cells from oxidative toxicity in a dose-dependent manner. To further evaluate oxidativestress-induced neurotoxicity, an MTT reduction assay was performed. The assay determines the cell viability based on the redox activity of living cells that converts MTT into a purple formazan, and a decrease in cellular MTT reduction could be an index of cell damage. Cells pretreated with the 3-CQA displayed decreased H2O2-induced cytotoxicity. Moreover, the extract dose-dependently protected PC12 cells from oxidative-stress-induced cell death. The decrease in ACh levels appears to be one of the characteristic features in AD patients. Cognitive dysfunction is closely related to a loss of the neurotransmitter ACh. AChE catalyzes the hydrolysis of the ACh to choline and acetate in the peripheral and central nervous systems. AChE inhibitors that inhibit the hydrolysis of ACh in order to increase cholinergic neurotransmitters are widely used in patients.41 Based on this purpose, the AChE inhibition effect of 3-CQA was measured. The AChE inhibition was increased by 3-CQA treatment in a dose-dependent manner. Taken together, the data indicated that 3-CQA protects against oxidative-stressinduced cytotoxicity. Also, 3-CQA mitigated the deficiency of cholinergic neurotransmitter by increasing AChE inhibition. An additional important aspect to be discussed is whether 3-CQA crosses through the BBB. There is no direct evidence that 3-CQA penetrates through the BBB. However, indirect effect of this compound on the brain, such as a protection of the BBB, has been reported. It has been reported that 3-CQA improves neuroprotective effects against H2O2-induced toxicity and transduction efficiency of one of the ribosomal proteins for crossing the BBB in ischemic animal models.42 These reports suggest that 3-CQA may enhance protection of neuron cells, thereby improving their therapeutic potential against oxidative-stress-induced cytotoxicity. In conclusion, the L. fischeri extract effectively reversed the deleterious effects of oxidative damage in both in vitro and in vivo models of AD. Moreover, biochemical experiments using brain tissues showed lowered oxidative stress

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levels. L. fischeri ethanol extract containing the active ingredient 3-CQA inhibited brain cell oxidation by lowering Ab1-42-induced oxidative stress. These results suggest that L. fischeri and 3-CQA are potentially applicable as protective agents against AD development.

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