Rapid Identification of Cholinesterase Inhibitors from the Seedcases of

Article pubs.acs.org/JAFC

Rapid Identification of Cholinesterase Inhibitors from the Seedcases of Mangosteen Using an Enzyme Affinity Assay Hyung Won Ryu,‡,∥ Sei-Ryang Oh,‡,∥ Marcus J. Curtis-Long,§ Ji Hye Lee,† Hyuk-Hwan Song,‡ and Ki Hun Park*,† †

Division of Applied Life Science (BK21 plus), IALS, Gyeongsang National University, Jinju 660-701, Republic of Korea Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, 30-Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea § Graduate Program in Biochemistry and Biophysics, Brandeis University, 415 South Street, Waltham, Massachusetts 02453, USA ‡

S Supporting Information *

ABSTRACT: Enzyme binding affinity has been recently introduced as a selective screening method to identify bioactive substances within complex mixtures. We used an assay which identified small molecule binders of acetylcholinesterase (AChE) using the following series of steps: incubation of enzyme with extract; centrifugation and filtration; identification of small molecule content in the flow through. The crude extract contained 10 peaks in the UPLC chromatogram. However, after incubation the enzyme, six peaks were reduced, indicating these compounds bound AChE. All these isolated compounds (2, 3, and 5−8) significantly inhibited human AChE with IC50s = 5.4−15.0 μM and butyrylcholinsterase (IC50s = 0.7−11.0 μM). All compounds exhibited reversible mixed kinetics. Consistent with the binding screen and fluorescence quenching, γ-mangostin 6 had a much higher affinity for AChE than 9-hydroxycalabaxanthone 9. This validates this screening protocol as a rapid method to identify inhibitors of AChE. KEYWORDS: Garcinia mangostana, cholinesterase, UPLC-PDA-QTOF-MS, fluorescence quenching, enzyme binding affinity

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anticancer activities.13 Some mangosteen juice products contain whole fruit as a formulation strategy to add phytochemical value because the peel contains more xanthonoids.14,15 The conventional activity-guided isolation process of complex extracts is limited in overall efficiency because of the tedious manipulation required. Activity guided fractionation has been developed as a way to directly identify binders of a particular enzyme without the need for purification. A method has recently been developed in which centrifugation has been used to identify active compounds from complex mixtures. In this way, binders of aromatase16 and bovine serum albumin17 have been identified. For inhibition to occur, the enzyme and inhibitor must form a complex, and thus, should an enzyme be mixed with biological extracts, any small molecules that bind the enzyme will be lost from the mixture in chromatography. In this study, we demonstrate an efficient approach to identify AChE inhibitors from mangosteen seedcases using an analogous centrifugation followed by phase liquid chromatography fitted with a photodiode array quantitative time-of-flight mass spectrometer (UPLC-PDA-QTOF-MS). AChE binding affinity examination by UPLC identified six compounds (2, 3, and 5−8) that appeared to interact strongly with AChE. These six promising compounds were isolated and their inhibitory and binding characteristics were examined, all of which were consistent with significant inhibition.

INTRODUCTION The purpose of this manuscript is to describe a new rapid method to identify acetylcholinesterase (AChE) inhibitors from complex mixtures of natural products. AChE (EC 3.1.1.7) is a hydrolase that plays a key role in cholinergic transmission by catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh).1 This enzyme is linked to pathogenesis through the cholinergic hypothesis of Alzheimer’s disease (AD), which states that low levels of acetylcholine (ACh) directly leads to brain deficiency because of signaling dysfunction in synaptic transmission.2 Low concentrations of ACh can arise from the accumulation of beta amyloid (Aβ) protein fragments that form hard plaques which are toxic to neuronal cells and can interfere with ability of ACh.3 Thus, an optimum level of ACh should be maintained to sustain healthy brain function. On the other hand, butyrylcholinesterase (BChE, EC 3.1.1.8) is a nonspecific cholinesterase that hydrolyzes many different choline esters including acetylcholine.4 Therefore, BChE has been implicated in the onset of neurodegenerative disease.5 Thus, it is attractive to explore leading medicinal plants or structures that may mediate AD through inhibition of AChE-BChE.6 With the growing number of incidences of AD as the average age of the population increases, the need to rapidly screen for natural products active against this disease only becomes stronger. Garcinia mangostana, known as mangosteen, belongs to the family of Clusiaceas and grows mainly in Southeast Asia. Various parts of this species have been used in traditional medicine to treat skin and urinary tract infections. Mangosteen seedcases and fruits yield a variety of xanthonoids with antioxidant,7,8 antibacterial,9 anti-inflammatory,10 antiglycosidase,11,12 and © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1338

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Figure 1. (A) UPLC-PDA chromatograms of the chloroform extract of G. mangostana seedcases: (black) not exposed to enzyme, (blue) after the incubation with hAChE, (red) after incubation with esBChE. Peaks numbered as 1−10 were isolated from G. mangostana seedcases in our study. (B) Chemical structures of isolated xanthonoids (2, 3, and 5−8) from the G. mangostana.

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°C at a flow rate of 500 L/h and source temperature of 100 °C. The capillary and cone voltages were set to 2300 and 35 V, respectively. The Q-TOF premier was operated in the more sensitive “V” mode with 9000 mass resolving power. The data were collected for each test sample from 100 to 1500 Da with a 0.25 s scan time and a 0.01 s interscan delay over a 15 min analysis time. Leucine-enkephalin was used as the reference compound (m/z 554.2615 in the negative mode) and an infusion flow rate of 1 μL/min. Enzyme Binding Affinity of Cholinesterase. To 75 μL of 100 mM sodium phosphate buffer was added 5 μL cholinesterase (100 U/ mL in 100 mM sodium phosphate buffer) and 20 μL of 5 mg/mL CHCl3 extract in MeOH subsequently. The final concentration of extract was adjusted to 1000 ppm. The mixture was incubated for 30 min at 37 °C on a plate shaker. Then, the sample was centrifuged at 12 000 rpm for 10 min and the supernatant was collected for analysis. The supernatant solution was filtered through a membrane (PTFE 0.2 μm, hydrophobic, Advantec, Japan), and 2 μL solutions were injected for UPLC-PDAQTOF-MS analysis. Control experiment was carried out with the same procedure without enzyme.16,18 Cholinesterase Inhibitory Activity. The in vitro inhibition assays of BChE (EC 3.1.1.8, 0.05 U/mL) from equine serum, AChE (EC 3.1.1.7, 0.05 U/mL) from electric eel, and human AChE (0.25 U/mL) were run in sodium phosphate buffer 100 mM, pH 8.0 (reaction buffer). AtCh and BtCh were used, respectively, as substrates, and DTNB was used as the chromophoric reagent.19 Inhibition assays were carried out on a SpectraMax M2Multi-Mode Microplate Reader (Molecular Devices, CA, U.S.A.) equipped with a 10.0 mm quartz cell (or plate) and a thermostatted bath. Solutions of tested compounds were prepared starting from 10 mM stock solutions in DMSO, which were diluted into aqueous assay medium to give a final content of organic solvent lower than 1%. hAChE inhibitory activity was determined in a reaction mixture containing 2 μL of a solution of hAChE (0.25 U/mL in 100 mM sodium phosphate buffer, pH 8.0), 30 μL of a 3.3 mM solution of DTNB in 100 mM sodium phosphate buffer (pH 8.0), 10 μL of a solution of the inhibitor (5−10 concentrations ranging from 0.1 to 200 μM), and 138 μL of reaction buffer. After incubation for 10 min at 37 °C, ACh (20 μL of 1 mM aqueous solution) was added as the substrate, and hAChEcatalyzed hydrolysis was followed by measuring the increase of

MATERIALS AND METHODS

General Apparatus and Chemicals. NMR spectra were recorded on a Bruker AM500 instrument (1H NMR at 500 MHz, 13C NMR at 125 MHz, Billerica, Massachusetts, U.S.A.). Electron ionization (EI) and EIhigh resolution (HR) mass spectra were obtained on a JEOL JMS-700 instrument (JEOL Ltd., Tokyo, Japan). Optical rotations were measured on a Perkin-Elmer 343 polarimeter (Perkin-Elmer, Bridgeport, U.S.A.). Melting points were measured on a Thomas Scientific Capillary Melting Point Apparatus and are uncorrected. Column chromatography was performed on RP-18 (ODS-AQ, 25 and 10 μm, YMC, Kyoto, Japan). Thin layer chromatography (TLC) was done on precoated TLC plates with silica gel 60 F254 (0.25 mm, normal phase, Darmstadt, Merck). These were visualized using a UVGL-58 254 nm Hand-held UV lamp (UVP, Cambridge, U.K.) or by spraying with 10% H2SO4 in ethanol followed by heating. CD3OD, acetone-d6, and CDCl3 were purchased from Cambridge Isotope Lab. Inc., (MA, U.S.A.). CH3CN (HPLC grade) and water were purchased from J.T. Baker (U.S.A.). Equine serum BChE (esBChE), Electrophorus electricus AChE (eeAChE), human erythrocyte AChE (hAChE), acetylthiocholine iodide (AtCh), butyrylthiocholine iodide (BtCh), 5-5′-dithiobis(2-nitrobenzoic acid) (DTNB), NaH2PO4, Na2HPO4, and DMSO were purchased from Sigma-Aldrich Co., U.S.A. All solvents were distilled before use. Plant Materials. G. mangostana (Clusiaceae) [imported from Vietnam, as permitted by Korea Food and Drug Administration (KFDA)] was purchased from LanSea Food Co., Ltd. (www.lansea.co. kr, Seoul, Korea). UPLC-PDA-QTOF-MS Analysis. The chloroform extracts were analyzed by UPLC PDA QTOF-MS. Chromatographic separations were performed on a 2.1 × 100 mm, 1.7 μm ACQUITY BEH C18 chromatography column. The column temperature was maintained at 35 °C, and the mobile phases A and B were water with 0.1% formic acid and acetonitrile with 0.1% formic acid, respectively. The gradient duration program was as follows: 0−1 min, 10% B; 1−12 min, 10−98% B; wash to 13.5 min with 98% B; and a 1.5 min recycle time. The flow rate was 0.4 mL/min. The mass spectrometer was operated in positive ion mode. N2 was used as the desolvation gas. The desolvation temperature was set to 350 1339

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Table 1. Peak Numbers, Name, Analytical Parameters, and Xanthonoids Identified in G. mangostana Seedcases peak

UPLC tR (min)

HRESIMS [M+H]+

ppm

molecular fomular

UV−vis maxima (nm)

control (area)

after AChE (area)

after BChE

xanthonoids assignation

1 2 3 4 5 6 7 8 9 10

8.62 8.77 8.94 9.16 9.53 9.79 9.93 10.51 11.40 11.81

425.1594 427.1758 425.1590 409.1657 327.1221 397.1637 381.1729 411.1791 409.1676 425.1971

−2.7 1.4 −1.6 1.5 −4.3 1.3 7.1 −7.1 6.1 1.6

C24H24O7 C24H26O7 C24H24O7 C24H24O6 C19H18O5 C23H24O7 C23H24O5 C24H26O6 C24H24O6 C25H28O6

219, 316 219, 321 221, 319 221, 317 221, 320 221, 317 221, 319 242, 317 222, 289 222, 316

37053 67077 24780 26375 7885 20732 10124 500597 9617 11640

58700 2057 3574 12553 trace trace trace 158012 2377 2057

49789 1125 2698 9534 trace trace trace 80803 1631 1975

mangostingone mangostanol allanxanthone E mangostenone F gudraxanthone γ-mangostin 8-deoxygartanin α-mangostin 9-hydroxycalabaxanthone β-mangostin

Table 2. Inhibitory Effects of Extracts and Xanthonoids (2, 3, and 5−9) on Cholinesterase Activities cholinesterase compound

IC50a (μM) human erythrocytes AChE

type of inhibition Ki (μM)b

IC50a (μM) electrophor s electricus AChE

type of inhibition Ki (μM)b

IC50a (μM) equine serum BChE

type of inhibition Ki (μM)b

CHCl3 EtOH 50% EtOH water 2 3 5 6 7 8 9 eserine

2.7 μg/mL 7.9 μg/mL 34.2 μg/mL NDc 14.6 ± 0.7 15.0 ± 1.2 11.7 ± 0.7 5.4 ± 0.3 6.2 ± 0.3 8.0 ± 0.5 >100 0.043 ± 0.002

NTd NT NT NT mixed (6.4 ± 0.2) mixed (11.5 ± 0.9) mixed (7.7 ± 0.5) mixed (5.1 ± 0.4) mixed (4.8 ± 0.3) mixed (3.7 ± 0.2) NT NT

4.8 μg/mL 45.6 μg/mL ND ND 6.3 ± 5.4 67.4 ± 0.3 18.9 ± 1.7 2.5 ± 3.3 11.0 ± 0.6 6.3 ± 0.6 >100 0.049 ± 0.003

NT NT NT NT mixed (4.3 ± 0.6) mixed (75.7 ± 0.9) mixed (12.6 ± 1.2) mixed (2.8 ± 0.2) mixed (10.9 ± 0.2) mixed (8.0 ± 0.5) NT NT

6.5 μg/mL 31.8 μg/mL ND ND 6.0 ± 0.2 11.0 ± 0.4 9.0 ± 1.2 0.7 ± 0.03 9.2 ± 0.5 2.9 ± 0.7 86.3 ± 2.4 0.073 ± 0.006

NT NT NT NT mixed(12.8 ± 0.4) mixed (12.4 ± 0.2) mixed (6.5 ± 1.3) mixed (0.4 ± 1.3) mixed (12.0 ± 0.1) mixed (3.0 ± 0.5) NT NT

a All compounds were examined in a set of experiments repeated five times; IC50 values of compounds represent the concentration that caused 50% enzyme activity loss. bValues of inhibition constant. cND is not detected. dNT is not tested.

absorbance at 412 nm for 5.0 min at 37 °C. The concentration of compound that caused 50% inhibition of the hAChE activity (IC50) was calculated by nonlinear regression of the response/log (concentration) curve, using Sigma Plot, version 10.0 (SPCC Inc., Chicago, IL). eeAChE and esBChE inhibitory activities were assessed similarly using acetylthiocholine iodide as the substrate. Kinetic parameters were determined using the Lineweaver−Burk double-reciprocal-plot method at increasing concentrations of substrates and inhibitors.20 The inhibitor, eserine (Sigma-Aldrich, St. Louis, MO), was used in the assays for comparison. Fluorescence Quenching Measurements. All fluorescence spectra were measured on a SpectraMax M2Multi-Mode Microplate Reader (Molecular Devices, CA, U.S.A.) equipped with a 10.0 mm quartz cell and a thermostatted bath. In a typical fluorescence measurement, 2 μL of hAChE solution (100 mM sodium phosphate buffer, pH 8.0), 0.25 U/mL, was added to the quartz cell and addition of inhibitor solution (10 μL) to attain final concentrations. Fluorescence spectra of hAChE-xanthone mixture were recorded in the range from 300 to 400 nm. Both slits of excitation and emission were 2 nm with an excitation wavelength at 276 nm.21−23 Extraction and Isolation of the Inhibitors from G. mangostana. The seedcases of G. mangostana (1 kg) were extracted by chloroform for 3 days at room temperature to give crude extract (40 g). Crude extract (10 g) was submitted to MPLC (LC-Forte/R, YMC Korea Co. LTD) reversed phase silica gel (YMC ODS-AQ, 25 μm, 120 g) using a stepwise MeOH−H2O gradient (10−100% methanol, 10 mL/min, 120 min) to give 60 fractions. This MPLC procedure was repeated four times using the same conditions before further isolation. The bioactive constituents from each MPLC column were eluted as a single, UV-absorbing band and pooled. Fractions 18−25 (8.0 g) were chromatographed on a Sephadex LH-20 column eluted with MeOH and isolated by preparative (prep)-HPLC using a reversed-phase column

(1.9 × 25 cm, YMC ODS-AQ, 10 μm particle size), eluted with ACN− H2O gradient (5−70% ACN, 14 mL/min, 60 min) by repeated injection of 10 mg/mL MeOH dilutions to yield pure 2 (82 mg) and 3 (65 mg). Fractions 26−37 (980 mg) were chromatographed on a Sephadex LH20 column eluted with MeOH and isolated by prep-HPLC using a reversed-phase column (2.0 × 25 cm, YMC ODS-AQ, 10 μm, 120 g), eluted with MeOH−H2O gradient (10−80% methanol, 10 mL/min, 60 min) by repeated injection of 10 mg/mL MeOH dilutions to yield pure 5 (11 mg) and a mixture of 6 and 7 (Fr. 26−37-B). Fractions 26−37-B (118 mg) were chromatographed by prep-HPLC using a reversed-phase column [Waters Atlantis T3 column (1.9 × 25 cm, 5 μm particle size), eluted with MeOH−H2O gradient (5−70% methanol, 60 min) by repeated injection of 10 mg/mL MeOH dilutions to yield pure 6 (48 mg) and 7 (38 mg). Fractions 39−49 (8.7 g), enriched with compound 8, were purified over a silica gel (10 × 50 cm, 230−400 mesh, 300 g) column using the solvent system hexane−acetone (10−90% acetone, 10 mL/min, 60 min) to afford 8 (1730 mg). All isolated compounds were identified on the basis of the following spectroscopic data (Supporting Information). Statistical Analysis. All the measurements were made in triplicate. The results were subject to variance analysis using Sigma plot. Differences were considered significant at p < 0.05.

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RESULTS AND DISCUSSION The extracts from different polar solvents (CHCl3, EtOH, and H2O) were tested for enzymatic inhibitory activities against ChEs. The high potency of the chloroform extract (IC50 = 2.7 μg/μL, hAChE) encouraged us to identify the compounds responsible for its ChEs inhibition. Protein binding affinity can be used to identify inhibitors of a particular enzyme. This is because, for inhibition to occur, an enzyme inhibitor complex 1340

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Figure 2. (A) Effect of xanthonoids (2, 3, and 5−8) on the hAChE catalyzed hydrolysis of acetylthiocholine iodide. (B) Hydrolytic activity of hAChE as a function of enzyme concentration at different concentrations of compound 6. Concentrations of compound 6 for curves from top to bottom were 0, 1.25, 2.5, and 5.0 μM, respectively. (C, D) Lineweaver−Burk plot showing inhibition of compounds 6 and 7 on the hydrolytic activity of hAChE. The plot is expressed as 1/velocity versus 1/thiocholine (mM−1) without or with inhibitor. (Inset) Insets I and II represent the secondary plot of the slope and the intercept of the straight lines versus concentration of compounds (6 and 7), respectively.

Figure 3. (A, B) Lineweaver−Burk plot for the inhibition of esBChE by compounds 6 and 7. The plot is expressed as 1/velocity versus 1/thiocholine (mM−1) with or without inhibitor. (Inset) Insets I and II represent the secondary plot of the slope and the intercept of the straight lines versus concentration of compounds (6 and 7), respectively.

must form. Herein small molecules within the extracts of mangosteen seedcases that bind to cholinesterase were identified by assessing which compounds were lost from the crude extract upon incubation with the enzyme. The binding event was

assessed using an experimental set up involving an initial incubation step (1000 ppm extracts with 0.5 U enzyme), followed by centrifugation, and finally analysis of the flow through (i.e., fraction not bound by the enzyme) using UPLC1341

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xanthonoids in comparison with previously published data.11,12,24 Thus, peaks (1−10) were identified as mangostingone (1, peak 1), mangostanol (2, peak 2), allznxanthone E (3, peak 3), mangostenone F (4, peak 4), gudraxanthone (5, peak 5), γ-mangostin (6, peak 6), 8-deoxygartanin (7, peak 7), αmangostin (8, peak 8), 9-hydroxycalabaxanthone (9, peak 9), and β-mangostin (10, peak 10). We next isolated the six active compounds using chromatography over octadecyl-functionalized silica gel. The isolated compounds were fully characterized with spectroscopic data including 2D-NMR. The representative xanthone 6 was obtained as a yellowish powder having molecular formula C23H24O7 and 12 degrees of unsaturation, as established by high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z 397.1637 [M+H]+, calcd for C23H25O6, 397.1573). 1H and 13C NMR data in combination with molecular formula indicated a tricyclic skeleton with two aromatic rings, corresponding to a xanthone. Two prenyl appendages were identified by successive connectivities in the COSY spectrum. HMBC correlations of H-11 (δH 3.21) with C-2 (δC 112.6) and of H-16 (δH 4.03) with C-8 (δC 129.5) unveiled that these prenyl chemotypes were situated on C-2 and C-8 (Supporting Information). Thus, compound 6 was confirmed to be γmangostin (Figure 1B). The purified compounds were next assayed for inhibition of hAChE. Eserine, a known ChE inhibitor, was used as a positive control.25 Xanthonoids (2, 3, and 5−8) showed dose-dependent inhibitory effects against hAChE, with IC50 values ranging between 5.4 and 15.0 μM (Table 2). Consistent with the amount of reduction of intensity in the UPLC assay, there was a significant difference in inhibitory potencies of the strong binding compound set (2, 3, and 5−8, IC50s = 5.4−15.0 μM) and the weaker binding compound 9 (peak 9, IC50 > 100). This indicates that hAChE binding affinity facilitates ranking of inhibitors of hAChE in a complex mixture. Our compounds feature at least one prenyl group. According to a previous study, parent compound, 1,3,5,6-tetrahydroxyxanthone, showed very low efficacy (IC50 > 200 μM) toward AChE.26 Thus, prenyl appendages within the isolated active compounds appear to play a pivotal role in the inhibition of cholinesterase. All inhibitors showed a similar relationship between enzyme activity and concentration. The inhibition of hAChE (Km = 0.1 mM) by compound 6, the most potent inhibitor (Ki = 5.1 μM) is illustrated in Figure 2, representatively. Increasing the inhibitor concentration resulted in lowering of the slope of the line (Figure 2A). The plots of residual enzyme activity versus enzyme concentration at different concentrations of compound 6 gave a family of straight lines with a y-axis intercept indicating that 6 is a reversible inhibitor (Figure 2B). The enzyme inhibitory properties of target compound 6 were modeled using double reciprocal plots (Lineweaver−Burk and Dixon analysis). The results show a family of lines with common intercept on the left of the vertical axis and above the horizontal axis (Figure 2C and D). This implies that increasing inhibitor concentration leads to a decrease in Vmax and an increase in Km, indicating that compound 6 was a mixed-type inhibitor.20 The Ki values of all compounds were measured by Dixon plot (Table 2). On the other hand, as illustrated in Table 2, the isolated six xanthones (2, 3, and 5−8) were found to exhibit a significant degree of esBChE inhibition with IC50 values ranging between 0.7−11.0 μM. These compounds were also mixed inhibitors of this enzyme (Figure 2C and D, and Figure 3). The binding affinities of the purified inhibitors to ChEs were measured by monitoring quenching of intrinsic protein

Figure 4. (A, B) Effect of compounds 6 and 9 on intrinsic fluorescence of hAChE. Measurement conditions: xanthonoids (6 and 9) concentrations were at 0, 6.25, 12.5, 25, 50, and 100 μM, respectively. [hAChE] = 0.25 units, [esBChE] = 0.05 units, pH 8.0, at 37 °C, λex = 276 nm, λem = 305 nm.

PDA-QTOF-MS. By comparing the UPLC traces from experiments carried out in the presence and absence of enzyme, peaks that were reduced when the enzyme was present were correlated with compounds that bind the enzyme. Representative UPLCPDA chromatograms of extracts are shown in Figure 1A. The peak areas of six compounds (2, 3, and 5−8) within the chloroform extract of mangosteen were significantly reduced upon treatment with hAChE (blue) and esBChE (red). This result indicates that six compounds, corresponding to peaks (2, 3, and 5−8) have significant interaction with both ChEs. Importantly, the extent to which the peak areas were reduced did not correlate with peak size (for instance 1 and 2 have similar sizes but 2 was reduced by approximately 100% whereas 1 had a very small depletion). This indicates binding is dictated by affinity rather than bulk concentration. UPLC-PDA-QTOF-MS analyses were carried out using a C18 column with a linear gradient of acetonitile/water. As presented in Figure 1A, complete chromatographic separation of xanthonoids (1−10) was achieved within 15 min. Each peak was characterized using MS. The exact molecular ions [M+H]+ of peaks (1−10) were 425, 427, 425, 409, 327, 397, 381, 411, 409, and 425. Table 1 shows UV−vis absorption maxima, retention times, and mass spectral data of molecular ions of 1342

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fluorescence. hAChE has high intrinsic fluorescence arising from 14 Trp, 17 Tyr, and 30 Phe.21 The quenching of the intrinsic fluorescence of hAChE by compounds 6 and 9 is displayed in Figure 4. A dramatic difference in fluorescence quenching was observed between 6 and 9: hAChE 6 (IC50 = 5.4 μM) versus 9 (IC50 > 100 μM); esBChE 6 (IC50 = 0.7 μM) versus 9 (IC50 = 86.3 μM). This is in agreement with the affinity difference between both compounds observed in the UPLC-PDA chromatogram (Figure 1, peaks 6 and 9).

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(7) Jung, H. A.; Su, B. N.; Keller, W. J.; Mehta, R. G.; Kinghorn, A. D. Antioxidant xanthones from the pericarp of Garcinia mangostana (Mangosteen). J. Agric. Food Chem. 2006, 54, 2077−2082. (8) Naczk, M.; Towsend, M.; Zadernowski, R.; Shahidi, F. Proteinbinding and antioxidant potential of phenolics of mangosteen fruit (Garcinia mangostana). Food Chem. 2011, 128, 292−298. (9) Chomnawang, M. T.; Surassmo, S.; Wongsariya, K.; Bunyapraphatsara, N. Antibaceterial activity of Thai medicinal plants against Methicillin-resistant Staphylococcus aureus. Fitoterapia 2009, 80, 102−104. (10) Chen, L. G.; Yang, L. L.; Wang, C. C. Anti-inflammatory activity of mangostins from Garcinia mangostana. Food Chem. Toxicol. 2008, 46, 688−693. (11) Ryu, H. W.; Cho, J. K.; Curtis-Long, M. J.; Yuk, H. J.; Kim, Y. S.; Jung, S. I.; Kim, Y. S.; Lee, B. W.; Park, K. H. α-Glucosidase inhibition and antihyperglycemic activity of prenylated xanthones from Garcinia mangostana. Phytochemistry 2011, 72, 2148−2154. (12) Ryu, H. W.; Curtis-Long, M. J.; Jung, S. I.; Jin, Y. M.; Cho, J. K.; Ryu, Y. B.; Lee, W. S.; Park, K. H. Xanthones with neuraminidase inhibitory activity from the seedcases of Garcinia mangostana. Bioorg. Med. Chem. 2010, 18, 6258−6264. (13) Shan, T.; Ma, Q.; Guo, K.; Liu, J.; Li, W.; Wang, F.; Wu, E. Xanthones from mangosteen extracts as natural chemopreventive agents: Potential anticancer drugs. Curr. Mol. Med. 2011, 12, 666−677. (14) Pedraza-Chaverri, J.; Cárdenas-Rodríguez, N.; Orozco-Ibarra, M.; Pérez-Rojas, J. M. Medicinal properties of mangosteen (Garcinia mangostana). Food Chem. Toxicol. 2008, 46, 3227−3239. (15) Chin, Y. W.; Kinghorn, A. D. Structural characterization, biological effects, and synthetic studies on xanthones from Mangosteen (Garcinia mangostana), a popular botanical dietary supplement. MiniRev. Org. Chem. 2008, 5, 355−364. (16) Luo, L.; Shen, L.; Sun, F.; Ma, Z. Immunoprecipitation coupled with HPLC−MS/MS to discover the aromatase ligands from Glycyrrhiza uralensis. Food Chem. 2013, 138, 315−320. (17) Zhang, Y.; Peng, M.; Liu, L.; Shi, S.; Peng, S. Screening, identification, and potential interaction of active compounds from Eucommia ulmodies leaves binding with bovine serum albumin. J. Agric. Food Chem. 2012, 60, 3119−3125. (18) Sun, F.; Shen, L.; Ma, Z. Screening for ligands of human aromatase from mulberry (Mori alba L.) leaf by using high-performance liquid chromatography/tandem mass spectrometry. Food Chem. 2011, 126, 1337−1343. (19) Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88−95. (20) Bowden, A. C. A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochem. J. 1974, 137, 143−144. (21) Ryu, H. W.; Curtis-Long, M. J.; Jung, S. I.; Jeong, I. Y.; Kim, D. S.; Kang, K. Y.; Park, K. H. Anticholinesterase potential of flavonols from paper mulberry (Broussonetia papyrifera) and their kinetic studies. Food Chem. 2012, 132, 1244−1250. (22) Radic, Z.; Kalisiak, J.; Fokin, V. V.; Sharpless, K. B.; Taylor, P. Interaction kinetics of oximes with native, phosphylated, and aged human acetylcholinesterase. Chemico-Biol. Interact. 2010, 187, 163−166. (23) Radic, Z.; Taylor, P. Interaction kinetics of reversible inhibitors and substrates with acetylcholinesterase and its fasciculin 2 complex. J. Biol. Chem. 2001, 276, 4622−4633. (24) Nilar, L. J. H. Xanthones from the heartwood of Garcinia mangostana. Phytochemistry 2002, 60, 541−548. (25) Chemnitius, J. M.; Haselmeyer, K. H.; Gonska, B. D.; Kreuzer, H.; Aech, R. Mipafox differential inhibition assay for heart muscle cholinesterases: Substrate specificity and inhibition of three isoenzymes by physostigmine and quinidine. Gen. Pharmac. 1977, 28, 567−575. (26) Sabphon, C.; Sermboonpaisarn, T.; Sawasdee, P. Cholinesterase inhibitory activities of xanthones from Anaxagorea luzonensis A. Gray. J. Med. Plants Res. 2012, 6, 3781−3785.

ASSOCIATED CONTENT

* Supporting Information S

Characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +82-55-772-1965. Fax +82-55-772-1969. E-mail khpark@ gnu.ac.kr. Author Contributions ∥

H.W.R. and S.-R.O. equally contributed this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by National Research Foundation Grant founded by Korea government (MEST) (2013M3A9A6003180), Rural Development Administration, Republic of Korea, and a grant from the Next-Generation BioGreen 21 Program (SSAC, PJ009571), Rural Development Administration, Republic of Korea.

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ABBREVIATIONS: AD, Alzheimer’s disease; IC50, The inhibitor concentration leading to 50% activity loss; AChE, acetylcholinesterase; BChE, butyrylcholinesterase; ACh, acetylcholine; HRESIMS, Highresolution electrospray ionization mass spectrometry

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REFERENCES

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dx.doi.org/10.1021/jf405072e | J. Agric. Food Chem. 2014, 62, 1338−1343