Anticholinesterase potential of flavonols from paper mulberry (Broussonetia papyrifera) and their kinetic studies

Food Chemistry 132 (2012) 1244–1250

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Anticholinesterase potential of flavonols from paper mulberry (Broussonetia papyrifera) and their kinetic studies Hyung Won Ryu a, Marcus J. Curtis-Long b, Sunin Jung a, Il Yun Jeong c, Dong Sub Kim c, Kyu Young Kang a, Ki Hun Park a,⇑ a b c

Division of Applied Life Science (BK21 Program), IALS, GyeongSang National University, Jinju 660-701, Republic of Korea Graduate Program in Biochemistry and Biophysics, Brandeis University, 415 South Street, Waltham, MA 02453, USA Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, Jeonbuk 580-185, Republic of Korea

a r t i c l e

i n f o

Article history: Received 27 August 2011 Received in revised form 23 October 2011 Accepted 16 November 2011 Available online 25 November 2011 Keywords: Human acetylcholinesterase Butylcholinesterase Time-dependent inhibitor Fluorescence quenching Broussonetia papyrifera

a b s t r a c t It is necessary to develop food additives to help treat chronic disorders like neurodegenerative diseases from medicinal plants. Ethanol extracts of paper mulberry were found to display significant inhibition against cholinesterases, enzymes that are strongly linked with Alzheimer’s disease (AD). The active components were identified as prenylated flavonols (2–4) that inhibited two related human cholinesterases in a dose-dependent manner, with IC50’s ranging between 0.8 and 3.1 lM and between 0.5 and 24.7 lM against human acetylcholinesterase (hAChE) and butylcholinesterase (BChE), respectively. Prenyl groups within these flavonols were found to play a critical role for inhibition because the parent compound 1, quercetin, was inactive (IC50 > 500 lM) towards the target enzymes. Flavonols (2–4) showed mixed inhibition kinetics as well as slow and time-dependent reversible inhibition toward hAChE. The affinity between protein and inhibitors was investigated using fluorescence quenching. The affinity constants (KSA) of inhibitors increased in proportion to their inhibitory potencies. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction With the increase in life expectancy associated with the modern lifestyle, neuronal degenerative diseases are currently increasing rapidly. For instance, Alzheimer’s disease (AD) is predicted to affect one in 85 people globally by 2050 (Brookmeyer, Johnson, Ziegler-Graham, & Arrighi, 2007). At the cellular level, AD is characterised by the formation of amyloid peptide fibrils that eventually lead to cell death. Acetylcholinesterase (AChE. EC 3.1.1.7) is a hydrolase that plays a key role in cholinergic transmission by catalysing the rapid hydrolysis of the neurotransmitter acetylcholine (ACh) (Gabrovska et al., 2008). Neurons in AD affected brains have low levels of Ach. This, and other corroborating data (Perry et al., 1978), led to the ‘‘cholinergic hypothesis’’ of AD, which states that low levels of ACh directly leads to brain deficiency (Bartus, Dean, Beer, & Lippa, 1982). In AD, low ACh levels can arise from the accumulation of beta amyloid (bA) protein fragments that form hard plaques which can interfere with the ability of ACh to effect synaptic transmission (Wollen, 2010). The use of acetylcholinesterase inhibitors (AChEIs) elicits numerous responses which mediate the symptoms of AD (Tabet, 2006), including an increase of neural ACh levels through the inhibition of AChE-catalysed

⇑ Corresponding author. Tel.: +82 55 772 1965; fax: +82 55 772 1969. E-mail address: [email protected] (K.H. Park). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.11.093

ACh hydrolysis (Giacobini, 2004). Although there are diverse results and viewpoints, butylcholinesterases (BChEs) have been implicated in the onset of neurodegenerative diseases (Fallarero et al., 2008). Thus, dual action AChE–BChE inhibitors might facilitate cognitive improvement in AD (Decker, Kraus, & Heilmann, 2008; Kamal et al., 2008). However, for any inhibitor to be effective in vivo, it must exhibit sufficient lipophilicity to traverse two lipid bilayers to reach AChE, as it is localised in the trans-Golgi network (TGN)/endosomal lumen (Li et al., 2004). Given the huge potential medical benefits, it is very attractive to explore leading structures or scaffolds that may moderate AD due to AChE–BChE and are sufficiently lipophilic to reach the target enzymes (Utsuki et al., 2006). Broussonetia papyrifera, known as paper mulberry, belongs to the family of Moraceae and grows naturally in Asia and pacific countries. The roots, bark and fruits are all used in traditional Chinese medicine and the fruits have been used for treatment of impotence and ophthalmic disorders in China (Lee et al., 2001). The constituents of this plant inhibit lipid peroxidation (Ko, Yu, Ko, Teng, & Lin, 1997) and PTP 1B enzyme (Chen, Hu, An, Li, & Shen, 2002). This species is also proven to have potent inhibitory activity against tyrosinase (Zheng, Cheng, Chao, Wu, & Wang, 2008). We also recently reported a-glucosidase inhibition of paper mulberry’s polyphenols (Ryu, Curtis-Long et al., 2010; Ryu, Lee et al., 2010). In this study, we demonstrate that the ethanol extract of the root of B. papyrifera displays dual AChE–BChE inhibition. The most

H.W. Ryu et al. / Food Chemistry 132 (2012) 1244–1250

potent compounds (2–4) were examined for their cholinesterase inhibitory activities and their inhibition mechanisms. We additionally investigated the affinity between inhibitors (2–4) and hAChE by fluorescence quenching. 2. Materials and methods 2.1. Materials The roots of B. papyrifera were collected in July 2008, from a hillside (200 m altitude) in Gyeongnam province in the south-east of Korea. They were identified by Prof. Myong Gi Chung. A voucher specimen (KHPark 210709) of this raw material was deposited at the Herbarium of Gyeongsang National University (GNUC). The bark was peeled from roots and stored at 4 °C before extraction. All chemicals used were of reagent grade and were purchased from Sigma Chemical Co. (St. Louis, Mo, USA), unless otherwise stated. All solvents were distilled before use. 2.2. General procedure NMR spectra were recorded on a Bruker AM500 instrument (1H NMR at 500 MHz, 13C NMR at 125 MHz). Electron ionisation (EI) and EI-high 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, USA). Melting points were measured on a Thomas Scientific Capillary Melting Point Apparatus and are uncorrected. Column chromatography was performed on silica gel (230–400 mesh, Merck), RP-18 (ODS-A, 12 nm, S-150 lM, YMC) and Sephadex LH-20 (Amersham Bioscience). Thin layer chromatography was performed on precoated TLC plates on silica gel 60 F254 (Merck, 0.25 mm, normal phase). These were visualised by UV illumination (254 nm) or by spraying with 10% H2SO4 in ethanol followed by heating. 2.3. Cholinesterase inhibitory activity Cholinesterase was assayed according to standard procedures by following the hydrolysis of acetylthiocholine iodide (AtCh) or butyrylthiocholine iodide (BtCh) by monitoring the formation of 5thio-2-nitrobenzoate spectrometrically at 412 nm (Ellman, Courtney, Andres, & Featherstone, 1961). The reaction mixture contained 100 mM sodium phosphate buffer (pH 8.0), 20 ll of test sample solution and either 2 ll of human erythrocyte AChE (0.25 U/ml), 20 ll of Electrophorus electricus AChE (0.03 U/ml) or 2 ll of equine serum BChE (0.05 U/ml) solution, which were mixed and incubated for 10 min at room temperature. Assays were then initiated with the addition of 30 ll of 5,50 -dithiobis-(2-nitrobenzoic acid) (3.3 mM) and 20 ll of AtCh (human erythrocyte, 1 mM), 10 ll of AtCh (E. electricus, 1 mM) and 7 ll of BtCh (1 mM), respectively. All assays were performed in triplicate. Compounds showing the highest inhibitory activities were further characterised by determining the concentration required to inhibit 50% of the enzyme activity under the assay conditions (defined as the IC50 value). Kinetic parameters were determined using the Lineweaver–Burk double–reciprocal-plot method at increasing concentration of substrates and inhibitors. The inhibitors quercetin and eserine (Sigma–Aldrich, St. Louis, MO) were used in the assays for comparison.

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zyme activities were continuously measured spectrophotometrically for 300 s. To determine the kinetic parameters associated with time dependent inhibition of acetylcholinesterase, progress curves for 300 s were obtained at one inhibitor concentration using fixed substrate concentrations (Ha & Kubo, 2007; Ryu, Curtis-Long et al., 2010; Ryu, Lee et al., 2010). The data were analysed using the nonlinear regression program [Sigma Plot (SPCC Inc., Chicago, IL)]. 2.5. Fluorescence quenching measurements All fluorescence spectra were measured on a SpectraMax M2 Multi-Mode Microplate Reader (Molecular Devices, CA, USA) equipped with a 10.0 mm quartz cell and a thermostat bath. In a typical fluorescence measurement, 30 ll of hAChE solution (pH 8.0) with the concentration of 5 U/ml was added accurately to the quartz cell and then titrated by successive additions of inhibitor. Titrations were operated manually and mixed moderately. The fluorescence emission spectra were measured at 18 and 37 °C with the width of the excitation and emission slit both adjusted at 2.0 nm. The excitation wavelength was 276 nm, and the emission spectrum was recorded from 300 to 400 nm (Radic, Kalisiak, Fokin, Sharpless, & Taylor, 2010; Radic & Taylor, 2001). All experiments were performed in triplicate, and the mean values were calculated. 2.6. Extraction and isolation of the inhibitors from B. papyrifera The root bark of B. papyrifera was extracted in separate flasks (100 g dry bark each) with 0.5 l of chloroform, 50% ethanol in water, ethanol, or distilled water at room temperature for 3 days to examine the enzymatic inhibitory activities against ChE as a function of solvent used (Table 1). The ethanol extract was determined as the target extract for isolation of ChE inhibitors as it gave the strongest ChE inhibition. Dried bark of B. papyrifera (1.0 kg) was extracted with (10 l  3) for 3 days at room temperature, and then filtered and the clarified solvent was evaporated under reduced pressure to afford the EtOH extract (126 g, 12.6%). This extract was fractionated by column chromatography (CC) on silica gel (10  30 cm, 230–400 mesh, 700 g) and eluted using chloroform/acetone [100:1 (1.5 l), 50:1 (1.5 l), 25:1 (1.5 l), 10:1 (3 l), 5:1 (2.5 l), 2:1 (2 l), 1:1 (1 l) and only acetone (2 l)] mixtures to give five pooled fractions F1–F5 based on the comparison of TLC profiles. Fraction F2 (5.2 g) was fractionated by silica gel flash column chromatography (CC) employing a gradient of hexane to EtOAc resulting in 11 subfractions (F2.1–F2.11). Subfractions F2.8–F2.9, enriched with 5 and 6, were combined (460 mg) and further purified by silica gel flash CC to yield compounds 5 (25 mg) and 6 (15 mg). Fraction F3 (1.8 g) was subjected to flash CC employing a gradient CHCl3 to acetone giving 12 subfractions (F3.1–F3.12). Fraction F3.3 (153 mg) was purified by Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) with CH3OH as the eluant to yield compound 4 (27 mg). Fraction F4 (18.0 g) was subjected to flash CC employing a gradient of CHCl3 to acetone giving 12 subfractions (F4.1–F4.12). Subfraction F4.3 and F4.4, enriched with compounds 2 and 3 were combined (1.2 g) and further purified by reversedphase CC (ODS-A, 12 nm, S-150 lM) eluting with CH3OH:H2O (4:1) to afford compounds 2 (57 mg) and 3 (8 mg). All isolated compounds were identified on the basis of the following spectroscopic data. The structures of compounds 1–6 are shown in Fig. 1. 2.7. 8-(1,1-Dimethylallyl)-50 -(3-methylbut-2-enyl)-30 ,40 ,5,7-tetra hydroxyflanvonol (2)

2.4. Slow and time-dependent inhibitory activity Slow and time-dependent assays as well as the associated progress curves were carried out using 0.25 U/ml hAChE, and 1 mM AtCh in 100 mM sodium phosphate buffer (pH 8.0) at 37 °C. En-

Amorphous yellow powder; mp 73–74 °C; EIMS m/z 438 [M]+; HREIMS m/z 438.1648 (calcd for C25H26O7, 438.1679); 13C NMR (125 MHz) d 18.4 (C-4000 ), 26.6 (C-5000 ), 29.5 (C-1000 ), 31.3 (C-200 ), 31.3 (C-300 ), 42.7 (C-100 ), 100.7 (C-6), 105.9 (C-4a), 109.9 (C-500 ), 112.7

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Table 1 Inhibitory effects of extracts and compounds (1–6) on cholinesterase activities. Compound

Cholinesterase IC50a (lM) equine serum BChE

a b c

c

20.5 lg/ml 10.8 lg/ml 23.2 lg/ml >200 >500 0.52 ± 0.02 1.5 ± 0.1 24.7 ± 2.3 31.9 ± 3.2 24.2 ± 1.7 3.7 ± 0.6

CHCl3 EtOH 50% EtOH Water 1 2 3 4 5 6 Eserine

Type of inhibition Ki (lM)b NT NT NT NT NT Mixed Mixed Mixed Mixed Mixed NT

(0.5 ± 0.1) (1.2 ± 0.2) (11.5 ± 0.5) (33.9 ± 0.4) (20.1 ± 1.3)

IC50a (lM) human erythrocytes AChE

Type of inhibition Ki (lM)b

22.7 lg/ml 12.1 lg/ml 25.3 lg/ml >200 >500 0.82 ± 0.06 3.1 ± 0.3 2.7 ± 0.6 108.5 ± 5.4 80.1 ± 3.3 0.15 ± 0.03

NT NT NT NT NT Mixed (0.9 ± 0.2) Mixed (2.3 ± 0.9) Mixed (2.8 ± 0.2) NT NT NT

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. Values of inhibition constant. NT is not tested.

OH O 6

A HO

OH O 3

8

OH

C

B 6'

1

HO

O

OH

O

OH

2

OH

OH

3

OH

OH O

HO

O

O

O

O

HO

O

O

OH OH

OH

OH O O

OCH3

4

HO

4'

OH O

HO

OH

2'

O 1

OH O OH

5

OH O

6

Fig. 1. Chemical structures of isolated compounds (2–6) from the B. papyrifera and parent compound 1.

(C-8), 115.2 (C-60 ), 122.7 (C-20 ), 123.5 (C-10 ), 124.3 (C-2000 ), 129.8 (C30 ), 133.7 (C-3000 ), 137.2 (C-3), 145.8 (C-2), 147.0 (C-40 ), 149.8 (C-50 ), 151.9 (C-400 ), 157.1 (C-8a), 160.7 (C-5), 164.6 (C-7), 177.9 (C-4, C@O).

(C-1000 ), 123.8 (C-10 ), 124.1 (C-100 ), 133.2 (C-3000 ), 133.5 (C-300 ), 139.8 (C-3), 146.9 (C-40 ), 150.3 (C-50 ), 154.1 (C-8a), 158.0 (C-5), 158.3 (C-2), 161.1 (C-7), 180.7 (C-4, C@O).

2.8. Papyriflavonol A (3)

2.10. Brossoflurenone A (5)

Amorphous yellow powder; mp 202–204 °C; EIMS m/z 438 [M]+; HREIMS m/z 438.1647 (calcd for C25H26O7, 438.1679); 13C NMR (125 MHz) d 18.3 (C-400 ), 18.3 (C-5000 ), 22.4 (C-1000 ), 26.3 (C-4000 ), 26.3 (C-500 ), 29.4 (C-100 ), 94.2 (C-8), 104.4 (C-4a), 112.0 (C6), 114.1 (C-20 ), 121.8 (C-60 ), 123.3 (C-10 ), 123.6 (C-2000 ), 123.8 (C-2000 ), 129.5 (C-50 ), 132.1 (C-300 ), 133.3 (C-3000 ), 137.0 (C-3), 145.4 (C-2), 146.7 (C-40 ), 147.2 (C-30 ), 156.0 (C-8a), 159.3 (C-5), 163.0 (C-7), 176.8 (C-4, C@O).

Yellowish plate; mp 206–208 °C; EIMS m/z 502 [M]+; HREIMS m/z 502.1995 (calcd for C30H30O7, 502.1992); 13C NMR (125 MHz) d 18.7 (C-4000 ), 26.2 (C-5000 ), 28.5 (C-200 ), 28.5 (C-300 ), 28.7 (C-40000 ), 28.7 (C-50000 ), 41.9 (C-100 ), 67.9 (C-10000 ), 78.6 (C-3000 ), 102.1 (C-6), 104.3 (C-60 ), 106.9 (C-4a), 108.5 (C-30 ), 109.7 (C-10 ), 111.0 (C-8), 113.7 (C-500 ), 115.5 (C-1000 ), 120.6 (C-20000 ), 131.6 (C-2000 ),135.0 (C-3), 138.3 (C-30000 ), 146.8 (C-50 ), 147.4 (C-20 ), 148.0 (C-40 ), 149.6 (C-400 ), 150.0 (C-2), 156.1 (C-8a), 161.4 (C-7), 161.9 (C-5), 171.0 (C-4, C@O).

2.9. Broussoflavonol B (4) 2.11. Brossoflurenone B (6) Amorphous yellow powder; mp 178–179 °C; EIMS m/z 452 [M]+; HREIMS m/z 452.1830 (calcd for C26H23O7, 452.1835); 13C NMR (125 MHz) d 18.4 (C-400 ), 18.7 (C-500 ), 23.0 (C-2000 ), 23.3 (C200 ), 26.3 (C-4000 ), 26.4 (C-5000 ), 60.9 (3-OCH3), 106.5 (C-4a), 108.3 (C-6), 113.3 (C-8), 116.7 (C-30 ), 117.2 (C-60 ), 122.7 (C-20 ), 123.6

Amorphous yellow powder; mp 186–187 °C; EIMS m/z 504 [M]+; HREIMS m/z 504.2152 (calcd for C30H32O7, 504.2148); 13C NMR (125 MHz) d 18.3 (C-40000 ), 18.7 (C-50000 ), 23.0 (C-1000 ), 26.1 (C-4000 ), 26.2 (C-5000 ), 28.3 (C-200 ), 28.3 (C-300 ), 41.9 (C-100 ), 66.9

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H.W. Ryu et al. / Food Chemistry 132 (2012) 1244–1250

B

100

0.7 0.6

80

0.5 60

Rate

Enzyme activity (%)

A

40

6 5 4 3 2

20 0

0

20

40

60

80

100

0.4 0.3 0.2 0.1 0.0

120

0.2

[I, μM]

C

140

100

140

0.6

0.8

45 μM 90 μM 180 μM

120 100

80

1/V

1/V

D

1.56 μM 0.78 μM 0.39 μM 0 μM

120

0.4

E [Unit/ml]

80

60

60

40

40

20

20 0

0

-0.020 -0.010 0.000 0.010 0.020 0.030

-2

-1

1/S

0

[I, μM]

1

2

E 1.2 1.0

0.10

control 0 min 10 min 15 min 20 min 25 min

0.05

0.00

0

50

100

150

200

250

300

V/V0

A415 nm

0.15

0.8 0.6 0.4 0 μM 0.39 μM

0.2 0.0 0

5 10 15 20 25 30 t preinc /min

Time (sec)

Fig. 2. Effect of compounds on the activity of acetylcholinesterase for the hydrolysis of acetylthiocholine iodide at 37 °C. (A) The activities of hAChE in the production of thiocholine, which reacts with 5,50 -dithiobis-(2-nitrobenzoic acid) (DTNB). (B) Relationship of the hydrolytic activity of hAChE with enzyme concentrations at different concentrations of 2 (d, 0 lM; s, 0.39 lM; ., 0.78 lM; 4, 1.56 lM). (C) Lineweaver–Burk plots for the inhibition of compound 2 on the hydrolytic activity of hAChE. (D) Dixon plot for compound 2 determining the inhibition constant Ki. (E) Time-dependent inhibition of hAChE in the presence of 0.39 lM compound 2. (Inset) Decrease in slopes of the lines of panel E as a function of time (s, 0 lM; d, 0.39 lM).

(C-10000 ), 98.2 (C-60 ), 102.0 (C-6), 196.9 (C-4a), 108.8 (C-10 ), 110.9 (C8), 113.5 (C-30 ), 113.5 (C-500 ), 119.2 (C-2000 ), 120.9 (C-20000 ), 133.8 (C-3000 ), 134.8 (C-3), 140.0 (C-30000 ), 145.0 (C-50 ), 148.1 (C-40 ), 149.7 (C-400 ), 150.3 (C-2), 151.2 (C-20 ), 156.1 (C-8a), 161.3 (C-7), 161.9 (C-5), 170.9 (C-4, C@O).

2.12. 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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3. Results and discussion 3.1. Structural identification of cholinesterase inhibitors The extracts from four different polar solvents were tested for enzymatic inhibitory activities against ChE. The high potency of the ethanol extract encouraged us to identify the compounds responsible for its ChE inhibition (Table 1). The prenylated flavones (2–6) were isolated from the ethanol extract of B. papyrifera by chromatography over silicagel, Sephadex LH-20 and octadecylfunctionalised silicagel. Compounds (2–6) were identified as 8(1,1-dimethylallyl)-50 -(3-methylbut-2-enyl)-30 ,40 ,5,7-tetrahydroxyflanvonol (2), papyriflavonol A (3), broussoflavonol B (4), broussoflurenone A (5), broussoflurenone B (6), by comparing their spectroscopic data of those previously reported (Ryu, Curtis-Long et al., 2010; Ryu, Lee et al., 2010; Takasugi et al., 1984; Zhang, Wang, Wu, Chen, & Yu, 2001). The representative flavonol 2 was obtained as a yellowish powder having molecular formula C25H26O7 and 14 degrees of unsaturation, as established by HREIMS (m/z 438.1648 [M+], calcd 438.1679). 1H and 13C NMR data in combination with molecular formula indicated a tricyclic skeleton with two aromatic rings for a flavonol. The 3,3-dimethylally and 1,1-dimethylallyl groups were confirmed and their position identified by COSY and HMBC correlations. Thus, compound 2 was identified as 8-(1,1-dimethylallyl)-50 -(3-methylbut-2-enyl)30 ,40 ,5,7-tetrahydroxyflanvonol. 3.2. Cholinesterase inhibitory activity The prenylated flavonols (2–6) and their parent compound, quercetin 1 were screened for their in vitro AChE and BChE inhibitory activities at different concentrations using a UV assay developed by Ellman. All prenylated flavonols (2–6) were found to inhibit both enzymes with IC50’s ranging between 0.8 and 108.5 lM against human AChE and between 0.5 and 31.9 lM against BChE (Fig. 1). It is promising that the most potent flavonols (2–4) could fulfill a dual role, inhibiting both AChE and BChE (Table 1). For example, compound 2 inhibited AChE (IC50 = 0.8 lM) and BChE (IC50 = 4.5 lM), respectively. The parent compound 1, quercetin, was inactive (IC50 > 500 lM) against both enzymes. Given quercetin’s low efficacy, the prenyl appendages within the isolated active compounds appear to play pivotal roles in the inhibition of cholinesterase. The relative effect of these groups was weakly affected by connectivity (1,1-dimethylallyl vs 3,3-dimethylallyl) and position on the ring (compare 2 and 3). The presence of a free hydroxyl group at C-3 is particularly important for inhibitory potency: i.e. compare 2 and 4 (IC50 = 0.8 vs 2.7 lM). However, oxidative cyclisation between C(3)OH and C(20 ) diminished inhibition potency significantly: for example, 2 (IC50 = 0.8 lM for hAChE) vs 6 (IC50 = 80.1 lM for hAChE). This could possibly be due to a change in conformation to a more planar structure upon cyclisation. 3.3. Kinetics of enzyme inhibition All inhibitors manifested a similar relationship between enzyme activity and concentration. The inhibition of hAChE by compound 2, the most potent inhibitor (IC50 = 0.8 lM) is illustrated in Fig. 2A. Increasing the concentration of the inhibitor drastically lowered residual enzyme activity. The plots of the remaining enzyme activity vs the concentrations of enzyme at different inhibitor concentrations gave a family of straight lines, which all passed through the origin (Fig. 2B). Increase of inhibitor concentration resulted in a decrease of the slope of the line, indicating that the presence of an inhibitor did not reduce the amount of enzyme,

but just resulted in the inhibition of enzyme activity. This indicates that 2 is a reversible inhibitor. We then further characterised the inhibitory mechanism of 2 using both Lineweaver–Burk and Dixon plots. As shown in Fig. 2C, the kinetic plot shows that compound 2 is a mixed inhibitor. This is because increasing the concentration of 2 resulted in a family of lines with a common intercept on the left of the vertical axis and above the horizontal axis (Bowden, 1974). The Ki value of 2 was determined to be 0.9 lM by Dixon plot (Table 1 and Fig. 2D). To further investigate the inhibition mechanism, time dependence of the inhibition of the hydrolysis of acetylcholine by compound 2 was subsequently probed. The enzyme was preincubated with substrate (between 0 and 25 min) and the residual enzyme activity was measured by calculating the initial velocity of the hydrolysis (Fig. 2E). The enzyme showed no loss in activity over this time range. As a decrease in residual activity was seen as a function of preincubation time, compound 2 emerged to be a slow-binding inhibitor. Increasing concentrations of compound 2 led to a decrease in both the initial velocity (vi) and the steadystate rate (vs). Hence, compound 2 acted as a time-dependent, slow reversible inhibitor of hAChE similar to other compounds reported in a previous study (Upadhyay, Chompoo, Kishimoto, Makise, & Tawata, 2011). 3.4. Fluorescence quenching spectra of hAChE Proteins have intrinsic fluorescence mainly originating from tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues. When a protein interacts with another compound, its intrinsic fluorescence often changes as a function of ligand concentration (Papadopoulou, Green, & Frazier, 2005). We investigated the interaction between prenylated flavonols (2–4) and hAChE that has three fluorescent residues, Trp 12, Tyr 22, and Phe 27. Under our measurement conditions (i.e. emission from 300 to 400 nm), there was no significant emission from any of the other components of the assay mixture (including the inhibitor). The experimental data were restricted to analysis of KSV and KA, since the s0 for tryptophan quenching in hAChE is not known. KSV was analysed using the Stern–Volmer equation (Li, Zhou, Gao, Bian, & Shan, 2009)

F 0  F ¼ 1 þ K SV ½Q

ð1Þ

F0 and F are the fluorescence intensities in the absence and presence of quencher (Q). KSV is the Stern–Volmer quenching constant [LM1]. Fig. 3A–C shows a typical Stern–Volmer plot. A dramatic decrease in the fluorescence intensity, caused by quenching, was observed for all three inhibitors in proportion to increasing concentrations. The binding constants of the inhibitors could be ranked in the following order 2 > 3 > 4, which is in agreement with the order of their inhibition constants (Ki) (Tables 1 and 2). The quenching fluorescence spectra of hAChE by flavonols were recorded at two temperatures (18 and 37 °C). As seen from Fig. 3D, the values of KSV decreased with an increase of temperature. For static quenching, the relationship between the change in the fluorescence intensity and the concentration of quencher for the set of reaction can be described by the following equation

log½ðF 0  FÞ=F ¼ log K A þ n log½Q f

ð2Þ

where F0 and F are fluorescence intensities in the absence and the presence of quencher; Qf is the concentration of free flavonol; n is the number of binding sites; KA is the binding constant (Zhang, Wang, Yan, & Xiang, 2011). From the plots of linear Eq. (2) obtained by log [(F0  F)/F] vs log [Q]f, one can calculate the values of KA and n as shown in Table 2. The values of n approximates

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500 400

400 200 0 0

300

20

40

60

80

[Compound 2, μM]

500 400

600 400 200 0 0

300

200

200

100

100

20

40

60

80

[Compound 3, μM]

0

320

340

360

380

λ(nm)

700

400

D

800 FS intensity

600 500 400

300

600

3.5

320

340

360

λ(nm)

380

400

18 oC 37 oC

3.0

400

2.5

200 0 0

300

20

40

60

80

[Compound 4, μM]

F0/F

300

FS intensity

800

600

600

0

C

700 FS intensity

600

FS intensity

B

800

FS intensity

700 FS intensity

A

2.0 1.5

200 1.0 100 0.5

0

300

320

340

360

λ(nm)

380

400

0

10

20

Compound 2 (μM)

30

Fig. 3. (A–C) The effect of flavonols 2–4 on fluorescence spectra of hAChE after they were added into the enzyme solution. (Inset) Normalised fluorescence intensity of hAChe with flavonols 2–4. Measurement condition: flavonols (2–4) concentrations were at 0, 4.1, 8.3, 16.6, 33.3, and 66.6 lM, respectively. [hAChE] = 0.25 units, pH 8.0, at 37 °C, kex = 276 nm, kem = 305 nm. (D) Stern–Volmer plots for flavonols 2–4 with hAChE at 18 and 37 °C (pH 8.0).

Table 2 Binding and quenching constants and binding sites for the tested compounds (2–4) from B. papyrifera. Compound

Ksv (105 LM1)

R2

KA (104 LM1)

n

R2

2 3 4

0.5356 0.4529 0.4245

0.9992 0.9983 0.9959

4.642 3.436 0.953

0.9737 0.9607 0.8357

0.9896 0.9993 0.9935

Acknowledgement This work was supported by the Bio & Medical Technology development program of the National Research Foundation of Korea (NRF 20090081751). Appendix A. Supplementary data

to one, indicating that only a single binding site exists in hAChE for flavonols.

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2011.11.093.

4. Conclusion

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