Neuroprotective Secondary Metabolite Produced by an Endophytic

7 days ago - Sunghee Bang‡ , Ji Hoon Song§ , Dahae Lee§ , Changyeol Lee‡ , Soonok ... Roots of Glehnia littoralis have been used to heal stroke ...
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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Neuroprotective Secondary Metabolite Produced by an Endophytic Fungus, Neosartorya fischeri JS0553, Isolated from Glehnia littoralis Sunghee Bang,†,‡ Ji Hoon Song,†,§ Dahae Lee,§ Changyeol Lee,‡ Soonok Kim,∥ Ki Sung Kang,*,§ Jong Hun Lee,*,⊥ and Sang Hee Shim*,‡ ‡

College of Pharmacy and Innovative Drug Center, Duksung Women’s University, Seoul 01369, Republic of Korea College of Korean Medicine, Gachon University, Seongnam 13120, Republic of Korea ∥ Biological Resources Assessment Division, National Institute of Biological Resources, Incheon 22689, Republic of Korea ⊥ Department of Food Science and Biotechnology, College of Life Science, CHA University, Pocheon 13488, Republic of Korea

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ABSTRACT: Roots of Glehnia littoralis have been used to heal stroke as a traditional medicine. Even though many studies on this plant have been conducted, the secondary metabolites produced by its endophytes and their bioactivities have not been investigated thus far. Therefore, a new meroditerpenoid named sartorypyrone E (1) and eight known compounds (2−9) were isolated from extracts of cultured Neosartorya fischeri JS0553, an endophyte of G. littoralis. The isolated metabolites were identified using spectroscopic methods and chemical reaction, based on a comparison to literature data. Relative and absolute stereochemistries of compound 1 were also elucidated. To identify the protective effects of isolated compounds (1−9) in HT22 cells against glutamate-induced cytotoxicity, we assessed inhibition of cell death, intracellular reactive oxygen species (ROS) accumulation, and calcium ion (Ca2+) influx. Among the isolates, compound 8, identified as fischerin, showed significant neuroprotective activity on glutamate-mediated HT22 cell death through inhibition of ROS, Ca2+ influx, and phosphorylation of mitogen-activated protein kinase, including c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38. The results suggested that the metabolites produced by the endophyte N. fischeri JS0553 might be related to the neuroprotective activity of its host plant, G. littoralis. KEYWORDS: endophyte, Neosartorya f ischeri, neuroprotection, sartorypyrone E

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INTRODUCTION Currently, antioxidant-rich fruits and vegetables are increasingly being studied to alleviate health problems caused by oxidative stress. Thus, it is reasonable to exploit effective functional foods based on natural products with antioxidant properties for treating oxidative damage. One of the illnesses caused by oxidative cellular damage is stroke. Many adult disabilities and death have been caused by stroke in the world.1 Neuroprotective natural compounds have been considered as one of the most potential strategies to treat stroke.2−4 Lately, many studies have been made on developing new therapeutic agents for neuroprotection.5 However, the only agent approved by the U.S. Food and Drug Administration (FDA) as a clinical remedy of stroke is a recombinant tissue plasminogen activator.6 Owing to the extremely small time frame in which a patient is eligible to receive a recombinant tissue plasminogen activator, there is a need for novel approaches to develop effective treatment of stroke. Food therapy could be a strategy to treat neuronal diseases, such as stroke. Since Glehnia, as a food material, has long been used to treat stroke, it could be a strategy to treat neuronal diseases. Glehnia littoralis F. Schmidt ex Miq. (family Apiaceae), also commonly known as beach silvertop, American silvertop, or “bangpung”, is a halophyte that survives in a high-salt environment and known as a wind-resistant herb.7 The leaf of this plant has been consumed as a vegetable in East Asian countries, including China, Japan, and Korea, since ancient times because © XXXX American Chemical Society

this herb has been known to wash away yellow dust, detoxify heavy metals, and be beneficial against respiratory diseases, such as rhinitis and asthma.8 On the other hand, the root parts of the plant have been applied as an oriental medicine for remedy of stroke.9 Previous reports on the phytochemistry of G. littoralis revealed that it contains various bioactive compounds, such as furanocoumarin, polyacetylene, flavonoid, lignan, monoterpenoid glycoside, and essential oil.10−12 Nevertheless, the bioactive compound responsible for its effect against stroke has not been identified from the plant itself, which turned our attention from the plant to the microbe associated with it. Plantassociated microorganisms are commonly called as endophytes, which are defined as microorganisms that, in their life cycle, colonize intra- and/or intercellularly within tissues of a host plant but cause no negative effects. They are known to interact with their host plant, protecting the host from pathogenic invasion and obtaining nutrition from the host. Moreover, endophytes have been regarded as a great source for bioactive and chemically novel compounds of potential use in the fields of medicine, agriculture, and industry.13 An endophytic fungus, Neosartorya fischeri JS0553, was isolated from G. littoralis plant. Several studies reported that Received: October 7, 2018 Revised: January 20, 2019 Accepted: January 22, 2019

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DOI: 10.1021/acs.jafc.8b05481 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry N. fischeri could produce secondary metabolites, such as aszonalenins, fischerin, neosartorin, fiscalins, helvolic acid, and fumitremorgins.14−16 None of these metabolites have been evaluated for their neuroprotective activities. Therefore, we aimed to elucidate neuroprotective activities of metabolites by endophytes isolated from the plant G. littoralis, which might elucidate the relationship between plants and endophytes in terms of biological activities. During stroke, glutamate is excessively accumulated in the brain, causing neuronal cell death in the brain.17 Oxidative stress and elevation of intracellular calcium ion [Ca2+]i are major causative factors of neuronal cell death induced by glutamate.18 It suggests that the prevention of oxidative stress and an increase in the [Ca2+]i level are useful targets in the development of drugs for neuropathological conditions, especially stroke.19

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HT22 cells were retained in the humidified incubator maintaining 37 °C and 5% carbon dioxide. Extraction and Isolation. Cultural media were extracted with ethyl acetate 3 times. Sodium sulfate was put into the ethyl acetate layer to dry residual water and then evaporated under reflux at 35 °C to yield extract (1.7 g). Crude extract was subjected to vacuum liquid column chromatography over silica gel, and 9 fractions (fractions A−I) were obtained by eluting crude extracts with n-hexane/acetone/MeOH (from 19:1:0, 9:1:0, 6.2:1:0, 5.6:1:0, 4:1:0.1, 3:1:0.1, to 0:0:1; 1 L each). Fraction E was loaded on silica gel column chromatography with n-hexane/EtOAc (100:0 → 75:25, v/v; 300 mL each) to isolate compounds 5 (10.5 mg) and 6 (8.5 mg). Fraction F was separated into 10 fractions using open column chromatography with n-hexane/ acetone/MeOH (from 19:1:0, 9:1:0, 7.3:1:0, 5.6:1:0, 4:1:0, 0:1:0, to 0:0:1; 700 mL each). Further column chromatography for fraction F3 was performed with elution of CHCl3/acetone/MeOH (from 1:0:0, 90:1:0, 70:1:0, 0:1:0, to 0:0:1; 300 mL each) to obtain compounds 2 (27.0 mg) and 4 (3.5 mg). Fraction F5 was further purified with silica gel column chromatography using CHCl3/MeOH (from 95:1, 90:1, 65:1, 60:1, to 0:1; 100 mL each) to obtain compound 7(13.5 mg). For fraction G, reversed-phase column chromatography was employed using solvents of H2O/MeOH (75:25 → 0:100, v/v; 300 mL each) to obtain 20 fractions. Semi-preparative HPLCs on reverse phase were conducted for fractions G7, G10, and G15. Compound 3 (1.5 mg) was obtained from G3 with gradient solvents of H2O/acetonitrile (70:30 → 50:50, v/v), while compound 9 (2.7 mg) was isolated from G10 with a gradient of H2O/acetonitrile (50:50 → 25:75, v/v). Finally, fraction G11 was injected to HPLC with a gradient of H2O/ acetonitrile (30:70 → 10:90, v/v) to afford compounds 1 (15.0 mg) and 8 (4.5 mg). Sartorypyrone E (1). Brown viscous mass; [α]23 D , −0.30 (c 0.05, MeOH); IR vmax, 3306, 292, 2830, 1021 cm−1; UV (MeOH) λmax, 289 nm; 1H NMR data (500 MHz, CD3OD), 13C NMR data (125 MHz, CD3OD), and heteronuclear multiple bond correlation (HMBC, CD3OD, H- → C-), see Table 1; (−)HRESIMS observed, m/z 431.2802 [M − H]− (calculated for C26H39O5, 431.2797). Synthesis of (S)- and (R)-MTPA Derivatives of Compound 1. Compound 1 (1.0 mg) dissolved in 600 μL of anhydrous pyridine was mixed with a slight excess of dimethylaminopyridine as a catalyst and put for 5 min at room temperature for reaction. The reaction mixtures were added by R-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (R-MTPA-Cl, 10 μL). After 2 h of reaction at room temperature, they were quenched through the addition of 50 μL of MeOH. The reaction mixtures were subjected to semi-preparative HPLC [column, Phenomenex, Luna C18 (2), 5 μm, 100 Å, 250 × 10.0 mm, room temperature; flow rate, 2.0 mL/min; UV detection, 210 and 254 nm] with a gradient of acetonitrile/H2O (from 10:90 to 100:0) over 40 min to purify S-MTPA ester (1a). Through the same method as described above, R-MTPA ester (1b) was synthesized using S-α-methoxy-α(trifluoromethyl)phenylacetyl chloride (S-MTPA-Cl). S-MTPA Ester of Compound 1 (1a). 1H NMR (CD3OD, 500 MHz), δH 6.01 (1H, s, H-5), 5.16 (2H, t, J = 7.0 Hz, H-8 and H-16), 5.07 (1H, t, J = 6.5 Hz, H-12), 5.02 (1H, d, J = 10.0 Hz, H-20), 3.06 (2H, d, J = 7.0 Hz, H2-7), 2.24 (1H, m, H2-18a), 2.19 (3H, s, H3-22), 2.04 (3H, m, H2-11a, H2-15a, and H2-15b), 2.01 (1H, m, H2-10a), 1.97 (1H, m, H2-18b), 1.95 (1H, m, H2-11b), 1.92 (3H, m, H2-10b, H2-14a, and H2-14b), 1.70 (1H, m, H2-19a), 1.72 (3H, s, H3-23), 1.60 (3H, s, H3-25), 1.57 (3H, s, H3-24), 1.34 (1H, m, H2-19b), 1.15 (3H, s, H3-27), 1.12 (3H, s, H3-26); (+)LC−ESI−MS, m/z 671.3 [M + Na]+, 649.5 [M + H]+. R-MTPA Ester of Compound 1 (1b). 1H NMR (CD3OD, 500 MHz), δH 6.01 (1H, s, H-5), 5.16 (2H, t, J = 7.5 Hz, H-8 and H-16), 5.08 (1H, t, J = 6.5 Hz, H-12), 4.99 (1H, d, J = 10.0 Hz, H-20), 3.06 (2H, d, J = 7.5 Hz, H2-7), 2.25 (1H, m, H2-18a), 2.17 (3H, s, H3-22), 2.05 (3H, m, H2-11a, H2-15a, and H2-15b), 2.02 (1H, m, H2-10a), 1.96 (1H, m, H2-11b), 1.94 (3H, m, H2-10b, H2-14a, and H2-14b), 1.72 (1H, m, H2-19a), 1.72 (3H, s, H3-23), 1.60 (3H, s, H3-25), 1.57 (3H, s, H3-24), 1.34 (1H, m, H2-19b), 1.12 (3H, s, H3-27), 1.08 (3H, s, H3-26); (+)LC−ESI−MS, m/z 671.3 [M + Na]+, 649.5 [M + H]+.

MATERIALS AND METHODS

General Experimental Procedures. A Jasco P-2000 polarimeter was used to obtain optical rotations of compounds using a 100 mm cell. An infrared spectrum was measured on an Agilent Cary 630 Fourier transform infrared (FTIR) spectrometer. A circular dichroism (CD) spectrum was obtained by a Jasco J-1100 spectrometer. High-resolution electrospray ionization time-of-flight mass spectrometry (HR-ESI− TOF−MS) data were obtained using a Q-TOF micromass spectrometer (Waters, Milford, MA, U.S.A.). Nuclear magnetic resonance (NMR) experiments were performed at room temperature on a Varian NMR spectrometer (1H, 500 MHz; 13C, 125 MHz; Varian, Palo Alto, CA, U.S.A.) with tetramethylsilane (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, U.S.A.), and chemical shifts were expressed as δ values. Preparative high-performance liquid chromatography (HPLC) was conducted on a Waters system equipped with a 600 controller, 996 photodiode array (PDA) detector, and Luna 5 μm C18(2) column (250 mm × 10 cm, Phenomenex, Torrance, CA, U.S.A.). Open column chromatography was performed using silica gel (Kieselgel 60, 70− 230 mesh, Merck, Germany). Precoated silica gel 60 F254 and RP-18 F254S plates (Merck) were used for thin-layer chromatography (TLC). TLC chromatograms were detected by ultraviolet (UV) and heating for visualization after the plates were dipped into 10% aqueous H2SO4 reagent. In this procedure, we used analytical-grade solvents for analytical experiments. Fungal Materials. The fungus JS0553 was isolated from the plant G. littoralis, which was collected in a swamp area in Suncheon, South Korea, in September 2011. Leaf tissues of this plant were cut into pieces (0.5 × 0.5 cm). The leaf tissues were then rinsed with 2% NaOCl, 70% ethanol, and sterilized distilled water in that order for 1 min to remove external microorganisms. Fungal strains derived from leaf tissues of this plant were incubated on malt extract agar medium, including yeast extract (4 g), malt extract (10 g), potato dextrose broth (4 g), and agar (18 g), to 1 L of sterilized distilled water, which were added to 50 ppm each of kanamycin, chloramphenicol, and Rose Bengal at 22 °C for 7 days. The actively growing fungal strains were transferred on fresh potato dextrose agar medium (24 g of potato dextrose broth and 18 g of agar to 1 L of sterilized distilled water). The fungal strain was identified to be N. fischeri based on its internal transcribed spacer sequences by Dr. Soonok Kim, one of the authors. The fungal strain was deposited on 20% aqueous glycerol stock in a liquid N2 tank at the Wildlife Genetic Resources Bank (NIBRGR0000118808) of the National Institute of Biological Resources (Incheon, Korea). Mass Cultivation of a Fungus. The JS0553 strain was incubated on a potato dextrose agar medium plate for 7 days at room temperature. Agar plates, including the strain, were cut into small pieces (1.0 × 1.0 cm) under aseptic conditions. Next, these pieces were inoculated in autoclaved Erlenmeyer flasks (10 × 500 mL) on a solid rice medium (80 g of rice and 120 mL of sterilized distilled water) and cultured at room temperature for 30 days. Cell Culture. The immortalized mouse hippocampal HT22 cells were grown in complement growth medium, Dulbecco’s modified Eagle’s medium, containing 10% fetal bovine serum and antibiotics. B

DOI: 10.1021/acs.jafc.8b05481 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. NMR Data of Sartorypyrone E (1) in CD3OD δH (multi, J in Hz)

number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

5.97 (s) 3.06 (d, 7.0) 5.16 (dt, 7.5, 1.5) 2.03 (m), 1.95 (m) 1.98 (m), 2.07 (m) 5.08 (dt, 7.0, 1.0) 1.95 (m) 2.07 (m) 5.16 (dt, 7.5, 1.5) 2.24 (ddd, 14.0, 9.5, 4.5), 2.00 (m) 1.70 (m), 1.34 (m) 3.23 (dd, 10.5, 1.5) 2.19 1.73 1.57 1.61 1.12 1.15

(s) (s) (s) (s) (s) (s)

Cell Viability Assay. To evaluate the neuroprotective effect of the compounds, HT22 cells were plated onto 96-well plates at a density of 1 × 104 cells per well and allowed to attach for 24 h. Viability of cells was determined using the EZ-CyTox cell viability assay kit following the instructions of the manufacturer.20 In brief, the cells were then treated with 5 mM glutamate for 24 h in the absence or presence of test compounds. The cells were treated with 10 μL of EZ-CyTox reagents and incubated for 30 min. Optical density was measured at 450 nm with an E-Max microplate reader (Molecular Devices, Sunnyvale, CA, U.S.A.). Evaluation of Intracellular Reactive Oxygen Species (ROS). Intracellular ROS was determined using cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) as a ROS detection reagent.21 HT22 cells were treated with 5 mM glutamate in the presence or absence of 2.5 and 5 μM compound 8 and incubated for 8 h. The cells were incubated with H2DCFDA for 30 min and then washed with phosphate-buffered saline (PBS) 3 times. Fluorescent intensity was then measured using a fluorescent microplate reader (SPARK 10 M, Tecan, Männedorf, Switzerland). Fluorescent images were obtained under a fluorescent microscope (IX71, Olympus, Tokyo, Japan), which was equipped with a charge-coupled device (CCD) camera. Measurement of Intracellular Ca2+. [Ca2+]i levels were determined using Fluo-4 AM (Invitrogen, Eugene, OR, U.S.A.), a membrane-permeable fluorescent indicator for Ca2+.22 The cells were treated with 5 mM glutamate and compound 8 (2.5 and 5 μM). After exposure to glutamate for 8 h, the cells were loaded with 2.5 μM Fluo4 AM, incubated for 30 min, and then washed with PBS 3 times. Fluorescent images of Fluo-4 were obtained using a fluorescent microscope (IX71) and analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.). Analysis of Apoptotic Cell Death. The cells were harvested after exposure to 5 mM glutamate with/without 2.5 and 5 μM compound 8 for 12 h. The same number of cells was washed with annexin-V-binding buffer (Invitrogen) followed by incubation with Alexa-Fluor-488-conjugaged annexin V (Invitrogen) for 20 min. The cells were stained with propidium iodide. Fluorescent images were

δC

type

168.83 103.20 168.15 102.02 161.79 22.91 122.85 136.44 41.00 27.85 125.54 136.00 41.00 27.62 125.75 136.13 38.11 31.02 79.23 73.92 19.75 16.43 16.27 16.32 25.07 25.82

C C C CH C CH2 CH C CH2 CH2 CH2 C CH2 CH2 CH C CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3 CH3

HMBC (H- → C-)

3, 6, and 22 2, 3, 8, and 9 3, 7, 10, and 23 9, 11, and 12 10 and 12 10, 14, and 24 12, 13, and 24 14 and 17 14, 18, and 25 16, 19, and 20 17, 18, and 20 18, 19, 21, and 26 5 and 6 8, 9, and 10 12, 13, and 14 16, 17, and 18 20, 21, and 27 20, 21, and 26

acquired using a Tali-Image-based cytometer (Invitrogen). AnnexinV-positive cells were analyzed with TaliPCApp (version 1.0). Western Blotting Assay.23 To determine the activation of mitogen-activated protein kinases (MAPKs), the cells were harvested after the exposure to 5 mM glutamate for 8 h with or without 2.5 and 5 μM compound 8. The cells were then lysed with radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris−HCl at pH 7.4, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.5% sodium dodecyl sulfate, and 0.5% sodium deoxycholate] containing freshly added phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF) and proteinase inhibitor cocktail. Equal amounts of proteins were used to separate by polyacrylamide gel electrophoresis and then transferred on polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). Non-fat milk solution [5% in Tris-buffered saline and Tween 20 (TBST)] was used to block non-specific binding of antibodies. After blocking for 1 h, the membranes were reacted with primary antibodies for ERK, p-ERK, p38, p-p38, JNK, p-JNK, and glyceraldehyde 3-phosphate dehydrogenase. All antibodies were purchased from Cell Signaling (Danvers, MA, U.S.A.). The membranes were incubated with horseradish-peroxidase-conjugated rabbit immunoglobulin G (IgG, Cell Signaling) and then reacted with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL, U.S.A.). Images for immunoreactive bands were acquired with a Fusion Solo chemiluminescence system (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Data were analyzed with the ImageJ software. Statistical Analysis. All experimental data were collected from three individual experiments at least and presented by the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) with the Bonferroni correction for multiple comparisons was used to determine statistical significance. Values of p < 0.05 were considered as statistically significant.

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RESULTS AND DISCUSSION Isolation and Structural Elucidation. Using a series of chromatographies, we isolated nine secondary metabolites

C

DOI: 10.1021/acs.jafc.8b05481 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Structures of compounds 1−9 isolated from N. fischeri JS0553.

from ethyl acetate extracts of the endophytic fungus N. fischeri JS0553, including one new compound (compound 1) and eight known compounds (compounds 2−9). Even though compound 1 was reported in the commercial chemical library, there is no report on either its isolation from natural sources or its spectral data. The known compounds were identified to be sartorypyrone A (2),24 cyclotryprostatin B (3),25 fumitremorgin B (4),26 fumitremorgin A (5),27 aszonalenin (6),28 acetylaszonalenin (7),29 fischerin (8),30 and pyripyropene A (9)31 (Figure 1). The structures of known compounds were elucidated by comparing the spectral data to those in the references. Compound 1 was obtained as a colorless oil. On the basis of its HRESIMS data (observed [M − H]−, m/z 431.2802; calculated for C26H39O5, 431.2797), compound 1 was found to have a molecular formula of C26H40O5. In its 1H NMR spectrum, four olefinic protons at δH 5.97 (s, H-5), 5.16 (dt, J = 7.5 and 1.5 Hz, H-8 and H-16), and 5.08 (dt, J = 7.0 and 1.0 Hz, H-12), an oxygenated methine at δH 3.23 (dd, J = 10.5 and 1.5 Hz, H-20), and six methyl groups at δH 2.19 (s, H3-22), 1.73 (s, H3-23), 1.61 (s, H3-25), 1.57 (s, H3-24), 1.15 (s, H3-27), and 1.12 (s, H3-26) could be observed. 13C NMR combined with HSQC data of compound 1 revealed the presence of one ester carbon at δC 168.83 (C-2), six non-protonated sp2 carbons at δC 168.15 (C-4), 161.76 (C-6), 136.44 (C-9), 136.13 (C-17), 136.00 (C-13), and 103.20 (C-3), four sp2 methine carbons at δC 125.75 (C-16), 125.54 (C-12), 122.85 (C-8), and 102.02 (C-5), one sp3 methine carbon at δC 79.23 (C-20), one sp3 quaternary carbon at δC 73.92 (C-21), seven sp3 methylene carbons at δC 41.00 (C-10 and C-14), 38.11 (C-18), 31.02 (C-19), 27.85 (C-11), 27.62 (C-15), and 22.91 (C-7), and six sp3 methyl carbons at δC 25.82 (C-27), 25.07 (C-26), 19.75 (C-22), 16.43 (C-23), 16.32 (C-25), and 16.27 (C-24). The presence of a 4-hydroxy-6-methyl-2H-pyran-2-one moiety was

Figure 2. Key correlations observed in the 1H−1H COSY (bold lines): (a) key HMBC (→) and (b) key ROESY (↔) correlations of compound 1.

Figure 3. Δδ values (δS − δR) obtained for the S- and R-MTPA ester (1a and 1b) derivatives of compound 1.

presumed by the chemical shift of the carbonyl carbon C-2, methyl-bearing carbon C-6, and oxygenated sp2 carbon C-4, which was additionally confirmed by HMBC from H-5 to C-3, C-4, and C-22. Interpretation of the 1H−1H correlation spectroscopy (COSY) spectrum provided five partial structures, one CH(O)−CH2−CH2 spin system corresponding to a D

DOI: 10.1021/acs.jafc.8b05481 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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C-20/C-19/C-18 unit, two C(CH3)CH−CH2−CH2 spin systems corresponding to C-17(C-25)/C-16/C-15/C-14 and C-13(C-24)/C-12/C-11/C-10 units, and one C(CH3) CH−CH2 spin system corresponding to a C-9(C-23)/C-8/ C-7 unit, as shown in Figure 2. Three trisubstituted olefinic moieties were confirmed to be linked in a linear fashion by HMBC from H-8 to C-3 and C-10, from H-12 to C-10 and C-14, and from H-16 to C-14 and C-18. Furthermore, HMBC from two singlet methyl protons H3-26 and H3-27 to the oxygenated quaternary carbon C-21 and the oxymethine carbon C-20 of the CH(O)−CH2−CH2 unit ascertained that an oxygenated isopropyl group was linked to C-20. Thus, this compound was found to have a diterpene moiety composed of four units of isoprene groups. Connection of the 4-hydroxy-6-methyl-2H-pyran-2-one moiety with the diterpene moiety was also confirmed by HMBC. Attachment of C-7 of the linear diterpene unit to C-3 of the pyranone ring was evident from strong HMBC from H2-7 to C-2, C-3, and C-4. Geometry of the olefinic groups was shown by interpretation of the rotating-frame Overhauser spectroscopy (ROESY) spectrum. Strong ROESY correlations between H-7b and H3-23, between H-11b and H3-24, and between H-15b and H3-25, in addition to those between H-8 and H2-10b, between H-12 and H2-14b, and between H-16 and H2-18b, suggested E configurations of all of the double bonds in the linear diterpene moiety. The stereogenic carbon C-20 was determined to have S configuration by measuring differences in chemical shifts of 1 H resonances for S- and R-MTPA ester (1a and 1b) derivatives using modified Mosher’s method (Figure 3). Preventive Effect of Compound 8 on HT22 Cell Death Induced by Glutamate. Glutamate is well-known to be an excitable neurotransmitter and contributes to not only various biological functions in the brain but also neuronal cell death

Figure 4. Compound 8 prevented glutamate-induced HT22 cell death. (a) Protective effects of isolated compounds in this study were tested at a concentration of 20 μM against glutamate-induced HT22 cell death [mean ± SEM; (∗∗) p < 0.001 compared to the glutamatetreated group]. (b) Viability of HT22 cells was determined after exposure to 5 mM glutamate for 24 h in the presence or absence of compound 8 [mean ± SEM; (∗∗) p < 0.001 compared to the glutamate-treated group].

Figure 5. Compound 8 blocked the elevation of intracellular ROS and Ca2+ induced by glutamate in HT22 cells. (a) HT22 cells were stained with H2DCFDA (DCF) after exposure to 5 mM glutamate (Glu) for 8 h, and fluorescent images were obtained. (b) Quantitatively analyzed data represented by a fold increase in fluorescent intensity compared to that of the control group [mean ± SEM; (∗∗) p < 0.001 compared to the glutamate-treated group]. (c) Cells were exposed to 5 mM glutamate for 8 h and stained with Fluo-4 AM. (d) Images were quantitatively analyzed, and data are represented by a fold increase in fluorescent intensity [mean ± SEM; (∗∗) p < 0.001 compared to the glutamate-treated group]. E

DOI: 10.1021/acs.jafc.8b05481 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 6. Compound 8 prevented glutamate-induced apoptosis in HT22 cells. (a) HT22 cells were exposed to 5 mM glutamate (Glu) for 12 h in the presence or absence of compound 8 and subjected to Tali-Image-based cytometric analysis. (b) Images were quantitatively analyzed, and data are represented by percentages of annexin-V-positive cells indicating apoptotic cells [mean ± SEM; (∗∗) p < 0.001 compared to the glutamatetreated group]. (c) Cells were harvested after exposure to 5 mM glutamate for 8 h, and western blotting analysis for MAPKs was performed. (d−f) Immunoreactive bands were quantitatively analyzed, and data are represented as a fold increase against that of the control group [mean ± SEM; (∗∗) p < 0.001 compared to the glutamate-treated group].

Antiapoptotic Effect of Compound 8 on GlutamateInduced HT22 Cells. Glutamate induces cell death through apoptosis and necrosis in early and later times after its administration to HT22 cells, reapectively.34 In this study, the number of annexin-V-positive cells indicating apoptotic cells markedly decreased by compound 8 compared to glutamate in HT22 cells (Figure 6a). Quantitatively analyzed data showed that the percentage of apoptotic cells in the glutamate-treated group (65.45%) was significantly reduced by 2.5 and 5 μM compound 8 to 38.43 and 36.21%, respectively (Figure 6b). These results suggested that compound 8 efficiently prevented glutamateinduced apoptotic HT22 cell death. Mechanistically, MAPKs have been reported to be key molecules in glutamate-induced oxidative stress. Typically, MAPKs play critical roles in both survival and death.31 However, during glutamate toxicity, MAPKs, including ERK, p-38, and JNK, can be activated and sustained for a prolonged period, resulting in neuronal cell death.35 Therefore, we then assessed the effect of compound 8 on the prolonged MAPK activation induced by glutamate. In our results, the phosphorylation of ERK, JNK, and p38 increased by glutamate was significantly diminished by compound 8 (panels c−f of Figure 6). These data indicated that inhibition of sustained phosphorylation of MAPKs could be a key molecular mechanism of compound-8-mediated protection against glutamate-induced HT22 cell death. In conclusion, the endophytic fungus N. fischeri JS0553 was isolated from one piece of the leaf of G. littoralis, and its mass

during acute brain insults and neurodegenerative diseases. As a part of our ongoing project to discover strong neuroprotectants from endophytic metabolites, nine compounds isolated from cultures of N. fischeri JS0553 were evaluated for their biological activities. At the concentration of 20 μM, the most effective neuroprotective compound was compound 8, fischerin (Figure 4A). Interestingly, our results showed that the viability of cells decreased by glutamate was significantly recovered by compound 8, even at lower concentrations (Figure 4B). These results suggest that compound 8 is a bioactive compound of grape seed extracts for eliminating glutamate-induced cytotoxicity. Effect of Compound 8 on Intracellular ROS in Glutamate-Induced HT22 Cells. In the cell death of a neuron during pathological conditions, oxidative stress is considered as the most crucial factor. The existence of higher glutamate significantly increased intracellular ROS in HT22 cells.32,33 Therefore, we examined to observe whether compound 8 can diminish a glutamate-induced increase in intracellular ROS. Intracellular ROS compared to glutamate-treated cells was significantly decreased by compound 8 (panels a and b of Figure 5). In addition to ROS, Ca2+ plays a role as a secondary messenger in cells, specifically neurons, involved in brain function. However, excessive levels of [Ca2+]i can contribute to neuronal cell death. This study showed that [Ca2+]i increased by glutamate was also significantly reduced by compound 8 (panels c and d of Figure 5). These results suggested that compound 8 can block intracellular ROS and Ca2+ increased by glutamate for protecting HT22 cells. F

DOI: 10.1021/acs.jafc.8b05481 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry cultures on solid rice media (10 g each × 80 g of rice) were extracted with ethyl acetate to yield 1.7 g of extracts. Its chemical investigation yielded sartorypyrone E (1, 15.0 mg), sartorypyrone A (2, 27.0 mg), cyclotryprostatin B (3, 1.5 mg), fumitremorgin B (4, 3.5 mg), fumitremorgin A (5, 10.5 mg), aszonalenin (6, 8.5 mg), acetylaszonalenin (7, 13.5 mg), fischerin (8, 4.5 mg), and pyripyropene A (9, 2.7 mg). The active compound, fischerin (8) could be detected by UV at 280 nm. Compound 8, fischerin, was not detected in the plant extracts. Nevertheless, compound 8 with its strong neuroprotective activity might have potential as a novel approach to develop neuroprotective agents. In addition, isolation of the neuroprotective compound from the endophyte of G. littoralis could be the scientific background that this plant has been used for the treatment of stroke for a long time. Furthermore, further studies investigating the interaction between this plant and its endophytic fungus could be required.

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(6) Green, A. R.; Shuaib, A. Therapeutic strategies for the treatment of stroke. Drug Discovery Today 2006, 11, 681−693. (7) Kim, S. M.; Shin, D. I.; Song, H. S.; Kim, S. K.; Yoon, S. T. Geographical distribution and habitat characteristics of Glehnia littoralis Fr. Schmidt in South Korea. Korean J. Med. Crop Sci. 2005, 13, 171−177. (8) Nam, J. Y.; Ryu, K. S. Pharmacognostical studies on Korean ‘Bang Poong’. Korean J. Pharm. 1975, 6, 151−159. (9) Park, J. H.; Ahn, J. H.; Lee, T.-K.; Kim, I. H.; Cho, J. H.; Lee, J.C.; Won, M.-H.; Yan, B.-C.; Shin, B.-N.; Hwang, I. K.; Kim, J. D.; Hong, S.; Lee, Y. J.; Kang, I. J. Pretreated Glehnia littoralis extract prevents neuronal death following transient global cerebral ischemia through increases of superoxide dismutase 1 and brain-derived neurotrophic factor expressions in the gerbil hippocampal Cornu Ammonis 1 area. Chin. Med. J. 2017, 130, 1796−1803. (10) Matsuura, H.; Saxena, G.; Farmer, S. W.; Hancock, R. E. W.; Towers, G. H. N. Antibacterial and antifungal polyine compounds from Glehnia littoralis ssp. leiocarpa. Planta Med. 1996, 62, 256−259. Masuda, T.; Takasugi, M.; Anetai, M. Psoralen and other linear furanocoumarins as phytoalexins in Glehnia littoralis. Phytochemistry 1998, 47, 13−16. (11) Hiraoka, N.; Chang, J.-I.; Bohm, L. R.; Bohm, B. A. Furanocoumarin and polyacetylenic compound composition of wild Glehnia littoralis in North America. Biochem. Syst. Ecol. 2002, 30, 321−325. (12) Kitajima, J.; Okamura, C.; Ishikawa, T.; Tanaka, Y. New glycosides and furocoumarin from the Glehnia littoralis root and rhizoma. Chem. Pharm. Bull. 1998, 46, 1595−1598. (13) Ovbiagele, B.; Kidwell, C. S.; Starkman, S.; Saver, J. L. Potential role of neuroprotective agents in the treatment of patients with acute ischemic stroke. Curr. Treat Options Cardiovasc. Med. 2003, 5, 441− 449. (14) Gomes, N. M.; Bessa, L. J.; Buttachon, S.; Costa, P. M.; Buaruang, J.; Dethoup, T.; Silva, A. M. S.; Kijjoa, A. Antibacterial and antibiofilm activities of tryptoquivalines and meroditerpenes isolated from the marine-derived fungi Neosartorya paulistensis, N. laciniosa, N. tsunodae and the soil fungi N. f ischeri and N. siamensis. Mar. Drugs 2014, 12, 822−839. (15) Zheng, Z.-Z.; Shan, W.-G.; Wang, S.-L.; Ying, Y.-M.; Ma, L.-F.; Zhan, Z.-J. Three new prenylated diketopiperazines from Neosartorya f ischeri. Helv. Chim. Acta 2014, 97, 1020−1026. (16) Shan, W.-G.; Wang, S.-L.; Lang, H.-Y.; Chen, S.-M.; Ying, Y.M.; Zhan, Z.-Z. Cottoquinazolines E and F from Neosartorya f ischeri NRRL 181. Helv. Chim. Acta 2015, 98, 552−556. (17) Mitani, A.; Andou, Y.; Kataoka, K. Selective vulnerability of hippocampal CA1 neurons cannot be explained in terms of an increase in glutamate concentration during ischemia in the gerbil: Brain microdialysis study. Neuroscience 1992, 48, 307−313. (18) Castilho, R. F.; Ward, M. W.; Nicholls, D. G. Oxidative stress, mitochondrial function, and acute glutamate excitotoxicity in cultured cerebellar granule cells. J. Neurochem. 1999, 72, 1394−1401. (19) Wojda, U.; Salinska, E.; Kuznicki, J. Calcium ions in neuronal degeneration. IUBMB Life 2008, 60, 575−590. (20) Eom, H. J.; Lee, D.; Lee, S.; Noh, H. J.; Hyun, J. W.; Yi, P. H.; Kang, K. S.; Kim, K. H. Flavonoids and a limonoid from the fruits of Citrus unshiu and their biological activity. J. Agric. Food Chem. 2016, 64, 7171−7178. (21) Okoh, V. O.; Felty, Q.; Parkash, J.; Poppiti, R.; Roy, D. Reactive oxygen species via redox signaling to PI3K/AKT pathway contribute to the malignant growth of 4-hydroxy estradiol-transformed mammary epithelial cells. PLoS One 2013, 8, No. e54206. (22) Guo, X.; Stice, S. L.; Boyd, N. L.; Chen, S. Y. A novel in vitro model system for smooth muscle differentiation from human embryonic stem cell-derived mesenchymal cells. Am. J. Physiol. Cell Physiol. 2013, 304, C289−C298. (23) Kang, S. H.; Song, J. H.; Kang, H. K.; Kang, J. H.; Kim, S. J.; Kang, H. W.; Lee, Y. K.; Park, D. B. Arsenic trioxide-induced apoptosis is independent of stress-responsive signaling pathways but

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b05481. NMR and MS spectra of compounds 1 and 8 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +82-31-750-5402. E-mail: [email protected]. *Telephone: +82-31-881-7158. E-mail: [email protected]. *Telephone: +82-2-901-8774. E-mail: sangheeshim@duksung. ac.kr. ORCID

Ki Sung Kang: 0000-0003-2050-5244 Jong Hun Lee: 0000-0002-3083-7595 Sang Hee Shim: 0000-0002-0134-0598 Author Contributions †

Sunghee Bang and Ji Hoon Song contributed equally to this work. Funding

This research was supported by the National Research Foundation of Korea (NRF-2018R1A2B6001733 and NRF2016R1A6A1A03007648) and the National Institute of Biological Resources (NIBR201921101). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.jafc.8b05481 J. Agric. Food Chem. XXXX, XXX, XXX−XXX