Effect of Lycoris chejuensis and Its Active Components on

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Effect of Lycoris chejuensis and Its Active Components on Experimental Models of Alzheimer’s Disease Joonki Kim,†,⊥ Yurim Park,‡,⊥ Yoon Sun Chun,‡ Jin Wook Cha,† Hak Cheol Kwon,† Myung Sook Oh,§ Sungkwon Chung,*,‡ and Hyun Ok Yang*,†,∥ †

Natural Products Research Center, Korea Institute of Science and Technology, 290 Daejeon-dong, Gangneung, Gangwon-do 210-340, Republic of Korea ‡ Department of Physiology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi-do 440-746, Republic of Korea § Department of Life and Nanopharmaceutical Science & Kyung Hee East−West Pharmaceutical Research Institute, Kyung Hee University, Seoul 130-701, Republic of Korea ∥ Department of Biological Chemistry, University of Science & Technology (UST), Daejeon 305-333, Republic of Korea S Supporting Information *

ABSTRACT: We found that an extract of Lycoris chejuensis and its three isolated active components, narciclasine, 7deoxynarciclasine, and 7-deoxy-trans-dihydronarciclasine, each significantly reduced the formation of amyloid-β peptides in HeLa cells transfected with an amyloid precursor protein carrying the Swedish mutation up to 45 ± 3.6%. The extract down-regulated amyloid precursor protein, especially the mature form by up to 88%, and reduced the ability of secretases to generate toxic amyloid-β. Double-transgenic mice treated with the extract for 4 months also showed significantly reduced levels of amyloid-β and plaques while exhibiting improved memory functions in the Morris water maze and novel object recognition tests. In conclusion, the extract and isolated active components of L. chejuensis decreased the production of amyloid-β by attenuating amyloid precursor protein levels. Furthermore, the extract improved the disrupted memory functions in animals while inhibiting amyloid plaque formation. Thus, this extract, as well as its active components, could prove beneficial in the treatment of Alzheimer’s disease. KEYWORDS: Lycoris chejuensis, Alzheimer’s disease, Amyloid-beta, Amyloid-beta precursor protein, Narciclasine

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INTRODUCTION The pathological hallmarks of Alzheimer’s disease (AD) include accumulation of extracellular senile plaques and aggregations composed of hyperphosphorylated tau protein filaments. It has been suggested that the major component of senile plaques, the neurotoxic amyloid-β (Aβ) peptide, may play a central role in the development of AD.1−3 Aβ is produced by proteolysis of amyloid precursor protein (APP), a type I transmembrane protein.4 APP undergoes Nglycosylation in the endoplasmic reticulum to become immature APP (imAPP). It is then transported to the Golgi apparatus, where the completed, mature form of APP (maAPP) is produced by O-glycosylation.5,6 Cleavage of APP can be mediated by two different pathways. In the amyloidogenic pathway, APP is sequentially cleaved by β-secretase 1 (BACE1), producing a soluble ectodomain fragment termed sAPPβ and a C-terminal fragment termed C99. C99 can be further cleaved by γ-secretase to produce Aβ. In the nonamyloidogenic pathway, APP is successively cleaved by αsecretase within the Aβ sequence, producing sAPPα and another C-terminal fragment termed C83. C83 can be further cleaved by γ-secretase to produce the p3 peptide and the APP intracellular domain, thereby precluding the formation of Aβ.7 Finally, the α-secretase-mediated ectodomain shedding of APP involves the A disintegrin and metalloproteinase (ADAM) family, including ADAM9, ADAM10, and ADAM17.8,9 © 2015 American Chemical Society

Following their characterization, the secretases became targets in research aiming to decrease Aβ levels. More specifically, it was observed that either inhibiting β- or γsecretases or stimulating α-secretase could decrease Aβ level.1,4 However, inhibition of γ-secretase may disrupt Notch signaling as a side effect, which is essential for development and differentiation.10 Moreover, potent β-secretase inhibitors not only pose a challenge in terms of their ability to cross the blood−brain barrier, but also present many other obstacles in clinical trials.11,12 Research has also focused on enhancing αsecretase as a novel strategy for the treatment of AD.13 However, ADAM10 and ADAM17 contribute to the proteolytic shedding of not only APP but also of other targets that are involved in inflammation and cancer such as tumor necrosis factor-α and epidermal growth factor receptor ligands.14 Therefore, it is desirable to find other therapeutic agents that can decrease the formation of toxic Aβ products without modulating β-, γ-, or α-secretases. Recent searches for new drug candidates for AD have included intensive investigation of plants, including Lycoris species and their products.15−18 Galantamine, isolated from the Received: Revised: Accepted: Published: 6979

February 24, 2015 July 11, 2015 July 13, 2015 July 28, 2015 DOI: 10.1021/acs.jafc.5b00889 J. Agric. Food Chem. 2015, 63, 6979−6988

Article

Journal of Agricultural and Food Chemistry

with a stepwise elution of aqueous MeCN that was further divided to eight fractions (CJ-3-F1-8). For isolation of active compounds, preparative HPLC was conducted. To isolate 7-deoxynarciclasin and 7-deoxy-trans-dihydroxynarciclasine, fraction CJ-3-F4 (500 mg, 40% MeCN in water eluate) was subjected to reversed phase chromatography [C18, 250 mm × 21.20 mm, flow rate 10 mL/min, gradient elution of 10−20% aqueous MeCN containing 0.02% trifluoroacetic acid for 40 min]. Narciclasine was isolated from the CJ-3-F6 fraction (400 mg, 60% MeCN in water eluate) by reversed-phase chromatography [C18, 250 mm × 21.20 mm, flow rate 10 mL/min, gradient elution of 10−50% aqueous MeCN containing 0.02% trifluoroacetic acid for 40 min]. The EtOH extract of CJ (20 mg) was dissolved in 1 mL of water, and 10 μL of the resulting sample solution was injected for HPLC analysis. The mobile phase used for analysis started at 10% MeCN in water (v/v) and was then changed to 20% MeCN in water (v/v) for 30 min. The flow rate was 1 mL/min. Narciclasine and 7-deoxynarciclasine were detected at 254 nm wavelength, and wavelength of 223 nm was used for 7-deoxy-transdihydronarciclasine detection. Cell Culture. HeLa cells stably transfected with APP751 carrying the Swedish mutation (APPsw) were cultured at 37 °C with 5% CO2. The culture media was Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum containing 100 units/mL penicillin, 100 μg/mL streptomycin, 260 μg/mL Zeocin, and 400 μg/mL G418. sAPPα, sAPPβ, and Aβ Peptide Assay. Cells at 80% confluence in a 35 mm dish were cultured for the times indicated in DMEM culture medium without serum but with CJ extract or active compounds solubilized in DMSO. Control cells were treated similarly and incubated with serum-free DMEM solution containing DMSO. For Aβ detection, the conditioned media were analyzed by a sandwich enzyme-linked immune-sorbent assay (ELISA; Invitrogen, CA, USA) according to the supplier’s instructions. Similarly, sAPPα, sAPPβ-WT, and sAPPβ-sw were analyzed by ELISA (IBL, Hamburg, Germany). Western Blotting. Cells were incubated at 37 °C with CJ extract for the times indicated. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and homogenized with lysis buffer (10 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.25% Nonidet P-40, 2 mM EDTA) using a cell scraper. Cell lysates were complemented with a protease inhibitor mixture (Calbiochem, MA, USA). The lysed cells were centrifuged at 12000g for 10 min at 4 °C, and the amount of protein in the supernatant was determined by Bradford assay (Biorad, CA, USA). A total of 40 μg of protein from each sample was subjected to SDS-PAGE using 4−20% gradient Tris/ glycine gels (Invitrogen, CA, USA). Next, the proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk powder in Tris-buffered saline/Tween 20 for 1 h at room temperature. Membranes were then incubated with primary antibodies and rabbit anti β-tubulin (Sigma, MO, USA) overnight at 4 °C. After washing, membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat antirabbit IgG antibody (1:2000 dilution; Invitrogen, USA) and washed. Peroxidase activity was visualized with enhanced chemiluminescence. Blots were quantified with Multi Gauge software using a LAS-3000 system (Fujifilm, Tokyo, Japan). The following antibodies were used: AntiAPP, C-terminal (Sigma-Aldrich, MO, USA), anti-Aβ, 6E10 (Covance, NJ, USA), anti-β-site APP cleaving enzyme, BACE1 (Chemicon, MA, USA), anti-ADAM9 (Cell Signaling, MA, USA), anti-ADAM10 (Calbiochem, MA, USA), and anti-TACE, ADAM17 (Chemicon, MA, USA). Secretase Activity Assay. The activities of α- and β-secretases were measured using the InnoZyme TACE activity kit (Calbiochem, MA, USA) and the β-secretase activity assay kit (BioVision, CA, USA). Each of the α- and β-secretase activity assays were conducted according to the manufacturer’s protocol. Subsequently, lysates, which were lysed with protein extraction reagent (Novagen, MA, USA), plated onto precoated plates, and the activities of bound secretases measured using the fluorescence of the substrates. Animals and Drug Administration. Male APP/Presenilin-1 (PS1) double transgenic (TG) mice (Strain name, B6C3-Tg(APPswe/

Chinese medicinal herb Lycoris radiata, is currently licensed for use in Europe as an acetylcholinesterase inhibitor for AD treatment.19 Recent research has also reported anticancer20 and anti-inflammatory21 effects of plant extracts or active compounds isolated from Lycoris species. However, there has been no report on whether Lycoris directly affects AD pathological pathways, such as the formation of Aβ. More specifically, no biological effects have been reported for Lycoris chejuensis K. Tae et S. Ko (CJ), which originates from Jeju Island in Korea.22,23 Because of their rich starch contents, Lycoris species plants, including CJ, have been traditionally used for hunger crops.24,25 In this study, we found that the CJ extract potently lowered Aβ levels in vitro and in vivo. CJ was also observed to decrease several byproducts of secretase activity. We assumed that the action of the CJ extract was related to decreased level of both immature and mature APPs, which is one of the novel mechanisms in APP processing. Furthermore, we were able to elucidate active components of the CJ extract that play a causal role in its effects. Taken together, we have revealed that CJ extract can reduce Aβ levels via the attenuation of APP while minimally altering the activity of β-, γ-, and α-secretases. Furthermore, we suggest that the effect of CJ extract is mediated by its active components, narciclasine, 7-deoxynarciclasine, and 7-deoxy-trans-dihydronarciclasine.

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MATERIALS AND METHODS

Chemicals. Plant extraction and partition used an extra pure grade solvent (DUKSAN, Gyeonggi-do, Republic of Korea). All HPLC analyses used HPLC grade CHROMASOLV solvents (Sigma-Aldrich, MO, USA). Methanol-d4 (99.8%, Cambridge Isotope Laboratories, MA, USA) and dimethyl sulfoxide (DMSO)-d6 (99.9%, Cambridge Isotope Laboratories, MA, USA) were used for nuclear magnetic resonance (NMR) measurements. Diaion HP-20 (SUPELCO, MO, USA) was used as an adsorbent for flash column chromatography. Deionized (DI) water (18 MΩ-cm) was generated using a Barnstead Water Purification Systems (Thermo Fisher Scientific, MA, USA). Plant Materials. CJ plants were obtained by collecting samples at the Hantaek Botanical Garden in Yongin, Gyunggi-do, Republic of Korea, from April to June 2010. The plant was identified by Jung Hwa Kang from Hantaek Botanical Garden. A voucher specimen was deposited in the herbarium of the Korea Institute of Science and Technology (Gangneung, Gangwon-do, Republic of Korea) under the number KNB00065. Samples were immediately stored at −20 °C until they were analyzed. Spectroscopic Analyses. NMR spectra were recorded on a Varian VNMRS 500 MHz NMR spectrometer (Agilent, CA, USA) or a Bruker AVANCE III 400 MHz NMR spectrometer (Bruker, MA, USA). Low-resolution electrospray ionization mass spectrometry data were acquired by an Agilent 1200 series HPLC system/6120 quadrupole MSD (Agilent, CA, USA). A Waters 1525 binary pump/ 2996 photodiode array detector (Waters, MA, USA) with a Phenomenex Luna column (Phenomenex, CA, USA) [C18, 5 μm, 150 mm × 4.6 mm] was used for HPLC analysis. Preparative HPLC were performed with a Gilson 321 HPLC system with a Phenomenex Luna column [C18, 10 μm, 250 mm × 21.20 mm]. Preparation of CJ Extract. Air-dried bulbs (590 g) of CJ were extracted with 3 L of EtOH (50% v/v) by reflux for 3 h. The extract was then cooled at room temperature and filtrated. Finally, the filtrate was evaporated under reduced pressure at 40 °C to result in 200 g of CJ EtOH extract (yield 34%). Isolation and Analysis of Active Compounds. First, 200 g of CJ EtOH extract was suspended in 1 L of water and then subjected to successive liquid partition chromatography. Using the same amount of n-hexane, CH2Cl2 and n-BuOH solvent gave 1, 1, and 5.5 g of organic soluble fractions, respectively. Then the n-BuOH soluble fraction (CJ3) was refractionated by a Diaion-HP20 flash column chromatography 6980

DOI: 10.1021/acs.jafc.5b00889 J. Agric. Food Chem. 2015, 63, 6979−6988

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In Vivo Soluble Aβ42 Detection. For in vivo detection of brain soluble Aβ42 peptides, after undergoing behavior tests, mice were anesthetized and decapitated at the age of 9 months. The cerebral cortex was dissected from the exposed brains, and the tissue samples were stored at −80 °C until use. The cerebral cortex samples (100 mg) were homogenized in Tris-buffered saline solution (20 mM Tris; 137 mM NaCl; pH 7.4) containing a complete protease inhibitors tablet (Sigma, MO, USA). The extraction ratio (brain tissue:Tris-buffered saline) was 1:5 or 1:10 (w/v). Tissue homogenates were centrifuged at 100000g for 1 h at 4 °C. Aβ42 levels in the resulting supernatants, but not in the pellet, were determined using the same sandwich ELISA kit used for in vitro detection. Immunohistochemistry. At biopsy, three randomly selected mice from each group were killed and whole brains were removed. These brains were immersion-fixed in 10% neutral buffered formalin and processed in paraffin wax. Brains in paraffin blocks were sagitally sectioned (50 μm) across the hippocampus and were immunostained using 4G8, which recognizes the 17−24 amino acid segment within Aβ as the primary antibody. After washing, a goat antimouse secondary antibody was incubated for an additional 30 min at room temperature, and sections were visualized with avidin−biotin−horse radish peroxidase complex according to the manufacturer’s instruction. The number of dark-stained plaques was counted and the groups were compared. Statistical Analysis. All results are presented as the mean ± SEM. The overall significance of experimental results was examined by Levene’s test for variance homogeneity. A One-Way analysis of variance (ANOVA) was then conducted along with a two-tailed Dunnett t-test. The threshold for statistical significance was p < 0.05 with the appropriate Bonferroni correction for multiple comparisons.

PSEN 1dE9)85Dbo/J; stock number no. 004462) used in this study were obtained from Jackson Laboratory (ME, USA) at the age of 4 months. Mice expressed a chimeric mouse/human APP containing the K595N/M596L Swedish mutations as well as a mutant human PS1 carrying the exon 9-deleted variant under the control of mouse prion promoter elements. These promotor elements predominantly directed transgene expression to central nervous system neurons. All animals were housed in solid bottom cages with pellet food and water available ad libitum. The mice were also maintained on a 12/12 h light−dark cycle in a temperature and humidity-controlled room (23 °C, 50%). Starting at the age of 5 months, the TG mice were randomly distributed into groups of seven mice. One group of mice was administered CJ extract in doses of 50 or 150 mg/kg/10 mL daily per os until the end of all experiments. The other groups of mice served as controls and were administered saline during the drug administration period. The animal protocols used in this study were in accordance with and granted (grant no. AP-2010KB001) by the Korea Institute of Science and Technology Animal Care and Ethics Committee. Morris Water Maze Test. At the age of 8 months, the mice were subjected to the Morris water maze (MWM) test. The MWM consists of a circular pool (120 cm in diameter and 60 cm in depth) filled with 24−26 °C water to a depth of 40 cm. The water was made opaque (white in color) with water-soluble nontoxic paint. A nonvisible escape platform, 8 cm in diameter, was submerged approximately 1 cm below the water surface in the center of the designated target quadrant of the pool. The two phases of the MWM tests, acquisition and retention, were conducted for six consecutive days. During the first five days, the acquisition phase was conducted by placing each mouse in the MWM to swim for a total of four trials per day, with a 1 h intertrial interval. During each trial, mice were given a maximum of 90 s to find the platform and had to remain on the platform for at least 5 s. If the mouse was unable to find the platform within the 90 s time frame, it was placed directly on the platform for 5 s and then returned to its cage. For each trial, the mouse was randomly placed under one of the four visual cues. During the acquisition phase, the amount of time it took for a mouse to find the hidden platform (escape latency) was measured. For those mice that did not find the hidden platform in the allotted time, a score of 90 s was given. The retention phase of the MWM occurred on the day immediately following the last day of the acquisition phase. During the retention phase, the platform was removed and mice were given 120 s to freely explore the pool. The total duration of time spent in the target quadrant, which had contained the escape platform during the acquisition phase, was measured. After the test, mice were removed from the pool and returned to their cages. Novel Object Recognition Test. After the MWM test, mice were left to rest in their home cages for 1 week. After this period, a novel object recognition test (NORT) was conducted to test the spontaneous tendency of mice to explore a novel object more often than a familiar one.26 A plastic chamber (50 cm × 50 cm × 50 cm) with 10 cm spaced grids at the bottom was used. The NORT procedure consisted of three different phases: three days of habituation, one day of acquisition, and one day of retention. During the habituation phase, each day mice were individually subjected to a single 10 min habituation session during which they were introduced to an empty arena. On the fourth day (acquisition phase), mice were subjected to a single 10 min exploring session, during which two floorfixed objects (A and B) were placed in a symmetric position from the center of the arena with enough space between each other and from the walls. Both plastic objects were the same in color, smell, shape, and size. Mice were allowed to explore the objects freely in the arena. On the fifth day (retention phase), mice were subjected to a single 5 min exploration session. In this session, mice were placed in the arena, which contained two objects, A and C. Object C was a novel object with a different color, smell, and shape than object B. The memory index for each mouse was expressed as the ratio of the amount of time spent exploring the novel object C (TC × 100)/(TA + TC), where TA and TC were the time spent exploring objects A and C during the 5 min, respectively.

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RESULTS AND DISCUSSION

CJ Extract Decreased Levels of Aβ40 and Aβ42 Secreted from Cells. We tested whether CJ extract can alter Aβ40 and Aβ42 production in HeLa cells stably transfected with APPsw. Transfected HeLa cells showed very reproducible results with Aβ production. Cells were incubated with 1, 5, 10, and 25 μg/mL of CJ extract for 8 h, and Aβ40 and Aβ42 levels from the conditioned media were measured using specific ELISA kits (Figure 1). Aβ40 and Aβ42 levels were

Figure 1. Effect of CJ extract on Aβ40 and Aβ42 secretion in cells. HeLa−APPsw cells were incubated with the indicated concentration of CJ extract for 8 h. The culture media were collected and analyzed for Aβ levels by using sandwich ELISA. Both forms of Aβ levels were significantly decreased by CJ extract treatment at 5, 10, and 25 μg/mL (n = 6). The results were quantified and expressed as mean ± SEM. (∗∗,∗∗∗) Significantly different (p < 0.01, p < 0.001) from the control group. 6981

DOI: 10.1021/acs.jafc.5b00889 J. Agric. Food Chem. 2015, 63, 6979−6988

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Journal of Agricultural and Food Chemistry decreased in cells cultured with CJ extract in a dose-dependent manner. At 5 μg/mL, CJ extract significantly decreased the levels of Aβ40 and Aβ42 by 53.5 ± 2.1% and 45 ± 3.6%, respectively. Furthermore, incubating cells with CJ extract even at a concentration of 25 μg/mL for 8 h had no effect on cell viability according to the MTT-based assay (data not shown). These results suggest that the CJ extract significantly decreases levels of both Aβ40 and Aβ42 without inducing cell toxicity. CJ Extract Decreased sAPPα, sAPPβ, and C-Terminal Fragments (CTFs) Levels. Aβ is produced through the sequential proteolysis of APP by β- and γ-secretases. Alternatively, the cleavage of APP can be mediated by α- and γ-secretases, precluding the production of Aβ. Thus, Aβ levels are determined by which secretase processes APP. If the effect of CJ extract on Aβ production occurs via modulation of β- or α-secretase, the levels of APP proteolytic products would have been altered accordingly. Therefore, we examined the effects of CJ extract on the levels of the APP proteolytic products sAPPα, sAPPβ, CTFα, and CTFβ. First, the levels of sAPPα were measured from the conditioned media after cells were incubated with 1, 5, 10, and 25 μg/mL CJ extract for 8 h. CJ extract at 5 μg/mL significantly reduced the levels of sAPPα by 39.8 ± 2.0% (Figure 2A). Therefore, the CJ extract was able to reduce levels of the α-secretase product. We next measured levels of sAPPβwt (the product resulting from cleavage of the endogenous APP) and sAPPβ-sw (the product from the transfected APPsw) using specific ELISA kits. Both sAPPβ-wt and sAPPβ-sw levels were significantly decreased by CJ extract in a dose-dependent manner (Figure 2A). After culture with 5 μg/mL CJ extract, the levels of sAPPβ-wt and sAPPβ-sw were decreased by 25.6 ± 2.0% and 28.6 ± 2.6%, respectively. These results indicated that β-secretase products were also reduced by the CJ extract. We also examined the levels of C-terminal stubs of APP, CTFβ, and CTFα. CTFβ and CTFα are produced from APP by β- and αsecretases, respectively. Figure 2B shows a typical Western blot derived from cell lysates in which the band densities are expressed relative to β-tubulin density. At the 5, 10, and 25 μg/ mL of CJ extract, the levels of CTFβ were decreased in a dosedependent manner by 37, 70, and 87%, respectively. The CJ extract concentrations of 10 and 25 μg/mL were also effective in decreasing CTFα levels by 29 and 32%, respectively. These results demonstrate that CJ extract was effective in decreasing the products of APP cleavage from both pathways. Therefore, we can hypothesize that the inhibitory effect of CJ extract on Aβ production is unlikely to be mediated via the modulation of β- or α-secretase activities. CJ Extract Decreased Levels of both ADAM10 and ADAM17. To confirm our hypothesis, we examined the effects of CJ extract on the expression of BACE1 and ADAMs in HeLa cells. BACE1 has been identified as the major β-secretase and is the rate-limiting factor in Aβ production.24 The ADAM family, more specifically ADAM9, ADAM10, and ADAM17, are known to function as α-secretases that catalyze the shedding of the APP ectodomain. Among them, ADAM10 is accountable for the constitutive activity as it is the major ADAM family member, while ADAM9 and ADAM17 are responsible for the regulation of cleavage.27,28 In the current study, we treated cells with 1, 5, 10, and 25 μg/mL of CJ extract for 8 h. We then measured the expression of BACE1, ADAM9, ADAM10, and ADAM17 from cell lysates using Western blot analysis. Figure 3A shows typical results obtained from four different experiments. The CJ extract did

Figure 2. Effect of CJ extract on sAPPα, sAPPβ and CTFα, CTFβ formation. HeLa−APPsw cells were incubated for 8 h with 1, 5, 10, and 25 μg/mL CJ extract. (A) sAPPα, sAPPβ-wt, and sAPPβ-sw levels were quantified using sandwich ELISA. CJ extract significantly reduced the levels of sAPPα (n = 5), sAPPβ-wt (n = 4), and sAPPβ-sw (n = 5). (B) Representative Western blot result showed that the levels of CTFα and CTFβ decreased by CJ extract treatment. Proteins in the cell lysate were analyzed by the immunoblotting and immunoblotting results were quantified from 5 different experiments. The levels of CTFα and CTFβ were significantly decreased by CJ extract treatment. All of the results were quantified and expressed as mean ± SEM (∗,∗∗,∗∗∗) Significantly different (p < 0.05, p < 0.01, p < 0.001) from the control group.

not affect the expression of BACE1, and densitometry analysis of the bands corresponding to BACE1 clearly supports this conclusion (Figure 3B). ADAM9 exists as pro-ADAM9 and is converted to mature ADAM9. CJ extract did not change the level of pro-ADAM9, but it did show a trend toward increasing the level of mature ADAM9 as shown in Figure 3A,C. ADAM10 is present in the Golgi apparatus as a pro-enzyme at steady state, but its mature form is found in the plasma 6982

DOI: 10.1021/acs.jafc.5b00889 J. Agric. Food Chem. 2015, 63, 6979−6988

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Journal of Agricultural and Food Chemistry

Figure 3. Effect of CJ extract on protein expressions of α-secretases and β-secretase. HeLa−APPsw cells were incubated for 8 h with 1, 5, 10, and 25 μg/mL CJ extract. Representative Western blot result showed that the expression levels of BACE1 (n = 5; (A,B) and ADAM9 (n = 4; (A,C)) were not significantly changed by CJ extract treatment. However, CJ extract reduced the expression of ADAM10 (n = 4; (A,D)) and ADAM17 (n = 4; (A,E)). The results were quantified and expressed as mean ± SEM. (∗,∗∗) Significantly different (p < 0.05, p < 0.01) from the control group.

Figure 4. Effect of CJ extract on levels of mature and immature APP. (A) HeLa−APPsw cells were incubated for 8 h with 1, 5, 10, and 25 μg/mL CJ extract. A representative Western blot result showed that both maAPP and imAPP levels were significantly reduced by the CJ extract in a dosedependent manner. (B) HeLa−APPsw cells were incubated with 25 μg/mL CJ extract for 1, 2, 5, and 8 h, respectively. A representative Western blot result showed that both maAPP and imAPP levels were significantly decreased in a time-dependent manner. The results were quantified and expressed as mean ± SEM (n = 5). (∗,∗∗,∗∗∗) Significantly different (p < 0.05, p < 0.01, p < 0.001) from the control group.

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DOI: 10.1021/acs.jafc.5b00889 J. Agric. Food Chem. 2015, 63, 6979−6988

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Journal of Agricultural and Food Chemistry membrane.8 We found that the level of the mature form of ADAM10 was slightly decreased by 15% with 25 μg/mL of CJ extract treatment (Figure 3A,D). Moreover, both precursor and active forms of ADAM17 were significantly decreased as shown in Figure 3A,E. Quantitative analysis showed that CJ extract at 25 μg/mL decreased precursor and active forms of ADAM17 by 54% and 33%, respectively. These data suggest that CJ extract did not affect the levels of BACE1 and ADAM9. Furthermore, CJ extract decreased ADAM10, which may underlie the constitutive α-secretase activity. However, the effect of CJ extract on ADAM10 is not large enough to fully account for the inhibitory effect of the extract on Aβ production. CJ Extract Did Not Affect α- and β-Secretase Activities. Compared to the minimal effects of CJ extract on ADAM 9 and ADAM10, ADAM17 was significantly decreased by treatment with CJ extract (Figure 3E). To confirm if this reduction corresponded with changes in ADAM17 activity, we further investigated whether CJ extract would influence secretase activities. As a way to test the effect of CJ extract on α-secretase activity by ADAM17 in cell lysates, we utilized an ADAM17 activity assay kit. Briefly, an ADAM17-specific fluorogenic substrate was incubated in the presence of 1, 5, 10, and 25 μg/mL CJ extract, and then the quenched fluorescence substrate was measured. Using this approach, we found that CJ extract did not show any effect on ADAM17 activity (Supporting Information Figure S1). We also tested the effect of CJ extract on BACE1 activity in cell lysates using a BACE1 assay kit and found that CJ extract also did not show any effects on the BACE1-specific substrate (Supporting Information Figure S1). Both results are included as Figure S1 of the Supporting Information. These results show that CJ extract did not affect β- and α-secretase activity even though the extract slightly decreased ADAM10 and ADAM17 protein levels. CJ Extract Decreased Levels of Both Mature and Immature APP. We observed that CJ extract decreased the levels of products generated by both β- and α-secretases while failing to change their activities. Therefore, we examined whether CJ extract affected the level of their common substrate, APP. APP undergoes N-glycosylation in the endoplasmic reticulum, where it is referred to as imAPP. This form of APP then transits to the Golgi apparatus, where it undergoes Oglycosylation to become maAPP. We measured the timedependent effects of CJ extract on APP levels by incubating cells with 25 μg/mL of CJ extract for 1, 2, 5, and 8 h. A typical Western blot result shows that the levels of APP were decreased by CJ extract in a time-dependent manner. Quantitative analysis showed that after 2 h of treatment, CJ extract reduced the totalAPP, maAPP, and imAPP by 62%, 83%, and 49%, respectively (Figure 4A). We then measured the level of these APPs from cell lysates using Western blot analysis after cells were treated with 1, 5, 10, and 25 μg/mL of CJ extract for 8 h. By using this technique, we found that CJ extract decreased both maAPP and imAPP in a dose-dependent manner (Figure 4B). Moreover, the amount of maAPP was decreased by 32%, 58%, and 88% with 5, 10, and 25 μg/mL of CJ extract, respectively. However, imAPP levels were less sensitive to CJ extract when compared to those of maAPP. These results demonstrate that CJ extract may attenuate Aβ production via decreasing levels of both maAPP and imAPP. CJ Extract Improved Performance in the Morris Water Maze Test. To confirm that the effect of CJ extract on Aβ reduction resulted in improvements in cognitive performance,

we assessed the spatial memory of mice in the MWM. As shown in Figure 5A, the non-TG background mouse group

Figure 5. Effect of CJ extract on mouse performance in the Morris water maze test. CJ extract (50 or 150 mg/kg/day) or saline was administered orally to 5-month-old transgenic (TG) mice for 3 months and 1 h before the first trial of each acquisition day. The acquisition phase (A) consisted of five consecutive days in which each TG mouse underwent a total of four trials with a 1 h intertrial interval. During the acquisition phase, the amount of time taken for the mouse to find the hidden platform (escape latency) was measured. The retention phase (B) occurred the day immediately following the last day of the acquisition phase. During the retention phase, the platform was removed and the mice were allowed 120 s to explore the pool. The time spent in the target quadrant, which had contained the escape platform during the acquisition phase, was recorded. The data are shown as mean ± SEM (n = 7/group). (∗,∗∗) Significantly different (p < 0.05, p < 0.01) from the background group. (#) Significantly different (p < 0.05) from the TG control group.

showed a significant decrease in the escape latency from the first day (48.8 ± 6.8 s) to the fifth day (16.4 ± 3.2 s) of acquisition. In contrast, TG mice showed no progress in finding the escape platform from the first day (75.1 ± 8.0 s) to the fifth day (66.8 ± 7.2 s). Treating TG mice with the CJ extract (150 mg/kg) induced a significant improvement in learning, with the escape latency starting at 73.0 ± 4.7 s on the first day and progressing to 36.3 ± 5.0 s on the fifth day. Following the acquisition phase, we tested the retention of spatial memory on 6984

DOI: 10.1021/acs.jafc.5b00889 J. Agric. Food Chem. 2015, 63, 6979−6988

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Journal of Agricultural and Food Chemistry the sixth day. As shown in Figure 5B, the time spent in the target quadrant, which had contained the escape platform, was significantly lower in the TG mouse group (29.1 ± 1.5 s) than the non-TG mouse group (38.5 ± 2.2 s). However, 150 mg/kg of CJ extract lead to a significant increase in the time mice spent in the target quadrant, increasing it to 40.4 ± 4.8 s. TG mice treated with 50 mg/kg of CJ extract also showed a trend toward spending more time in the target quadrant relative to the untreated TG mouse group. CJ Extract Improved Object Memory Function in the Novel Object Recognition Test. In the NORT, non-TG mice spent over 80% of the total exploration time interacting with a novel object, whereas TG mice only spent 60% of their time with the same object. Both 50 and 150 mg/kg of CJ extract significantly increased the ratio of the time spent with the novel object, up to 79.2 ± 3.8 and 82.0 ± 4.5%, respectively (Figure 6). Taken together with the MWM test results, this

Figure 7. Effect of CJ extract on the level of soluble Aβ42 protein in mouse cerebral cortex. Transgenic (TG) mouse brain tissue was collected after behavioral tests at 9 months of age. The cerebral cortex samples (100 mg) were homogenized in Tris-buffered saline solution (20 mM Tris; 137 mM NaCl; pH 7.4) containing complete protease inhibitors tablets. The extraction ratio (brain tissue:Tris-buffered saline) was 1:5 or 1:10 (w/v). The tissue homogenates were centrifuged at 100000g for 1 h at 4 °C. The soluble Aβ42 level in the resulting supernatants was measured using the sandwich ELISA kit according to the manufacturer’s protocol. The data represent the mean ± SEM (n = 4). * Significantly different (p < 0.05) from the TG control group.

amount of the plaque depends on the rates of its production, secretion, aggregation, and clearance.29 We performed an immunohistochemical examination of TG and non-TG mouse brain sections and found that although non-TG mice did not exhibit significant Aβ plaques, TG mice showed significant accumulation of Aβ plaques in the cerebral cortex and hippocampal regions. Importantly, treatment with both 50 and 150 mg/kg of CJ extract for four months significantly reduced the amount of plaque in the cerebral cortex to 50.9 ± 4.5% and 44.9 ± 1.9%, and in the hippocampus to 64.8 ± 13.9% and 42.5 ± 9.2%, respectively (Figure 8). Taken together with the obvious enhancement of cognitive function, the decreased levels of Aβ42 and the changes in the amount of plaque in mouse brains clearly demonstrates that CJ extract has beneficial effects on the pathology and symptoms of these mouse models of AD. Isolation and Analysis of Active Compounds. With its profound effect on both in vitro and in vivo AD models, we decided to determine which active components account for the effects of the CJ extract. To accomplish this, hydroethanolic extracts of CJ bulbs were further partitioned with n-hexane, CH2Cl2, and n-BuOH successively, then each organic soluble fraction was obtained as 1, 1, and 5.5 g, respectively. Flash column chromatography of the n-BuOH soluble fraction resulted in eight subfractions [CJ-3-F1−8]. 7-Deoxynarciclasine (40 mg) and 7-deoxy-trans-dihydronarciclasine (10 mg) were yielded from fraction CJ-3-F4. Narciclasine (80 mg) was isolated from another fraction, CJ-3-F6, by reversed-phase chromatography. The structures of the isolated compounds were confirmed by comparing previously reported spectral data.30,31 To confirm the composition of the three active components in the CJ extract, a 50% EtOH extract of CJ (20 mg) was analyzed using HPLC. The results of this analysis are included as Figure S2 of the Supporting Information.

Figure 6. Effect of CJ extract on novel object recognition test. CJ extract (50 or 150 mg/kg/day) or saline was administered orally to 5month-old transgenic (TG) mice for 3 months and 1 h before the acquisition phase test. During the acquisition phase test, mice were placed in a square-shaped arena and allowed to habituate to two identical objects for 10 min. On the following day, mice were placed in the same arena and and familiar object and one novel object were placed in the arena while the mice explored freely for 5 min. The total time spent exploring each object was measured and the memory index was calculated according to the following equation: Memory index (%) = (exploring time on novel object/total exploring time for both objects) × 100. The data represent the mean ± SEM (n = 7/group). (∗∗) Significantly different (p < 0.01) from the background group. (#,##) Significantly different (p < 0.05, p < 0.01) from the TG control group.

suggests that the CJ extract significantly improved the cognitive performance of TG mice, which are a model for the major symptoms of AD in mice and humans. CJ Extract Decreased Aβ42 Level in TG Mouse Brains. Aβ42 usually stays at undetectable levels in non-TG background mice. However, it is dramatically increased (up to 106.9 ± 9.7 pg/mL) in the cerebral cortex of TG mice. 150 mg/kg of CJ extract significantly reduced Aβ42, down to 81.0 ± 2.9 pg/ mL. On the other hand, 50 mg/kg of CJ extract showed no change relative to the TG mouse group (Figure 7). CJ Extract Reduced Aβ Plaques Detected by Immunohistochemistry. The extracellular accumulation of Aβ in the form of plaques is a pathological hallmark of AD. The 6985

DOI: 10.1021/acs.jafc.5b00889 J. Agric. Food Chem. 2015, 63, 6979−6988

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concentrations for 8 h. The levels of Aβ40 and Aβ42 in the conditioned media were determined using specific ELISA kits (Figure 9). Levels of both Aβ40 and Aβ42 were significantly decreased by all three active compounds in a dose-dependent manner without any cytotoxicity. Among them, 7-deoxynarci-

Figure 8. Effect of CJ extract on plaque deposition in the brains of transgenic mice. Mouse brain tissue was collected after behavioral tests at 9 months of age. Representative photomicrographs show the results of anti-Aβ antibody (4G8) staining in the cerebral cortex (A−D) and hippocampus (E−H) of mice from background group (A,E), transgenic (TG) control group (B,F), and TG mice treated with the CJ extract at 50 (C,G) and 150 (D,H) mg/kg. The numbers of Aβ plaques in each slice were counted and compared with the TG control group (I). The data represent the mean ± SEM (n = 3). (∗,∗∗) Significantly different (p < 0.05, p < 0.01) from the TG control group.

Figure 9. Effect of the three active compounds from CJ on Aβ40 and Aβ42 secretions in cells. HeLa−APPsw cells were incubated with the indicated concentrations of narciclasine, 7-deoxynarciclasine, and 7deoxy-trans-narciclasine, the three active compounds from CJ, for 8 h. The culture media were collected and analyzed for Aβ levels by using sandwich ELISA. Both forms of Aβ levels were significantly decreased by three of the active components from CJ (n = 6). The results were quantified and expressed as mean ± SEM. *** Significantly different (p < 0.001) from the control group.

Active Compounds from CJ Extract Also Decreased Levels of Aβ40 and Aβ42 in Cells. Isolated narciclasine, 7deoxynarciclasine, and 7-deoxy-trans-dihydronarciclasine were used to treat APPsw-transfected HeLa cells at various 6986

DOI: 10.1021/acs.jafc.5b00889 J. Agric. Food Chem. 2015, 63, 6979−6988

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Foundation, Republic of Korea to H.O.Y. This work was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A1A2006475) to S.C.

clasine and 7-deoxy-trans-dihydronarciclasine showed stronger Aβ reduction at lower concentrations. As such, the inhibitory effect of CJ extract on Aβ production is most likely due to these three compounds. The effect of CJ on the reduction of APP levels is very interesting. This extract preferentially decreased maAPP over imAPP, which in turn induced a reduction of Aβ levels. We propose that CJ may delay the exit of imAPP from the ER and thereby decrease transport to the Golgi apparatus. This would lead to attenuation of maAPP formation. This reduction of APP maturation could impair APP trafficking to the plasma membrane and disrupt its cleavage by α-secretase.5,6 Our data showed that CJ significantly reduced both sAPPα and CTFα levels. Once mature APP reaches the plasma membrane, it can undergo clathrin-dependent endocytosis. APP can also be cleaved by β-secretase in the early endosome.32−35 Therefore, CJ may also affect the endocytosis of APP from the plasma membrane, which can explain the decreased levels of sAPPβ and CTFβ and subsequently the levels of Aβ40 and Aβ42. CJ extract also decreased the production of Aβ42 as well as the number of Aβ plaques in the brains of TG mice. Treatment with CJ extract recovered cognitive dysfunction observed in the MWM and NORT, consistent with decreases in Aβ42 and the number of plaques in the cerebral cortex and hippocampus. Such modulatory functions have not been previously reported in natural products. As such, this extract could prove beneficial as a treatment for AD. Moreover, CJ would not produce the various side effects that come from interfering with secretase activities. In conclusion, we found a novel function of CJ that decreases Aβ levels via the attenuation of APP. This showed beneficial effects on cognitive function and plaque formation in animal models of AD. We also revealed that the active components of CJ extract were narciclasine, 7-deoxynarciclasine, and 7-deoxytrans-dihydronarciclasine. Further study on these three active components should be conducted in vivo to elucidate the specific action of their mechanisms toward the goal of treating patients with AD, and additional research using human islet amyloid polypeptides may contribute to better understanding in interactions with Aβ proteins.36

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Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED Aβ, β-amyloid; AD, Alzheimer’s disease; ADAM, A disintegrin and metalloproteinase; APP, Amyloid β precursor protein; BACE1, β secretase 1; CJ, Lycoris chejuensis K. Tae et S. Ko; imAPP, immature amyloid beta precursor protein; maAPP, mature amyloid beta precursor protein; MWM, Morris water maze; NORT, novel object recognition test; PS1, presenilin-1; TG, transgenic

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ASSOCIATED CONTENT

S Supporting Information *

The effect of CJ extract on the activities of ADAM17 and BACE1. HPLC analysis of CJ extract. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b00889.

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AUTHOR INFORMATION

Corresponding Authors

*For H.O.Y.: phone, +82 33 650 3501; fax, +82 33 650 3529; E-mail, [email protected]. *For S.C.: phone, +82 31 299 6103; fax, +82 31 299 6129; Email, [email protected]. Author Contributions ⊥

J.K. and Y.P. contributed equally to this work.

Funding

This work was funded and supported by the institutional programs of the Korea Institute of Science and Technology (grant no. 2Z04381) and the Bio-Synergy Research Project (NRF-2012M3A9C4048793) of the Ministry of Science, ICT and Future Planning through the National Research 6987

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