Anticholinesterase and β-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitory Compounds from the Heartwood of Juniperus chinensis

Abstract

Two new compounds (2, 3) and 20 known compounds (1, 4–22) were isolated from the heartwood of Juniperus chinensis LINNE (Cupressaceae), and their structures were elucidated as 9′-methoxycalocedrin (1); α-methyl artoflavanocoumarin (2); 5,7,4′-trihydroxy-2-styrylchromone (3); cedrol (4); widdrol (5); savinin (6); calocedrin (7); 10-oxowiddrol (8); 12-hydroxywiddrol (9); (+)-naringenin (10); vanillic acid methyl ester (11); (+)-taxifolin (12); (+)-aromadendrin (13); kaempferol (14); quercetin (15); (7S,8R)-dihydro-3′-hydroxy-8- hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol (16); styraxlignolide C (17); protocatechuic acid (18); vanillic acid (19); (7R,8S)-dihydro-3′-methoxy-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol 4-O-β-D-glucopyranoside (20); (7S,8S)-dihydro-3′-hydroxy-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol 4-O-α-L-rhamnopyranoside (21); and (+)-catechin (22) on the basis of spectroscopic evidence. The new compounds (2, 3) exhibited good inhibitory activities against β-site amyloid precursor protein cleaving enzyme 1 (BACE1), with IC₅₀ values of 6.25, and 11.91 µM, respectively.

Alzheimer’s disease (AD) is a neurodegenerative disease and the predominant cause of dementia among the elderly. AD provokes progressive cognitive decline, psychobehavioral disturbances, and memory loss and is characterized by the presence of senile plaque, neurofibrillary tangles, and reduced cholinergic transmission.^1,2) Although the pathogenesis of AD has not been entirely elucidated, it is believed to be due to a deficiency of the neuromediator acetylcholine, which is inactivated by acetylcholinesterase (AChE). For this reason, inhibitors of AChE are a widely used treatment strategy³⁾ and have lately become the most prescribed drug class in AD treatment since the diagnosis of acetylcholine deficit in the brains of AD patients.⁴⁾ Butyrylcholinesterase (BChE) might also play a role in Alzheimer’s disease since inhibitors of this enzyme improve learning performance in rats and reduce β-amyloid protein level.⁵⁾ Inhibition of BChE could be very relevant since this enzyme is the principal cholinesterase in the brains of late AD patients.⁶⁾ Another enzyme of interest in treatment of AD is β-site amyloid precursor protein cleaving enzyme 1 (BACE1), which is involved in the first and rate-limiting step of Aβ formation from its amyloid precursor protein (APP). Currently, several BACE1 inhibitors are in clinical trials.⁷⁾

In a preliminary study, a methanol extract of the heartwood parts of Juniperus chinenesis LINNE (Cupressaceae) showed inhibitory effects against AChE, BChE, and BACE1. J. chinensis is a widespread ornamental tree that grows in several South-East Asian countries.⁸⁾ The dried heartwood of J. chinensis has been used as a traditional folk medicine for treatment of colds, urinary infection, dysentery, hemorrhage, leucorrhea, and rheumatic arthritis.⁹⁾ The leaves of J. chinensis contain podophyllotoxin, lignans, sesquiterpenes, and terpenes, which have anti-leukemia activity,¹⁰⁾ anti-tumor activity,¹¹⁾ anti-fungal activity,¹²⁾ and anti-malarial activity,¹³⁾ respectively. In addition, the heartwood of J. chinensis contains flavonoids, lignans, and diterpenes with strong antioxidant activity¹⁴⁾ and anti-obesity activity against high-fat-diet-induced obesity.¹⁵⁾ Previous research on the aqueous and ethanol extracts of the leaves, ripe fruits, and unripe fruits of five Juniperus species (Juniperus communis ssp. nana, Juniperus oxycedrus ssp. oxycedrus, Juniperus sabina, Juniperus foetidissima, and Juniperus excelsa) investigated AChE and BChE inhibitory activities and antioxidant activities via tests of 2,2-diphenyl-1-picryhydrazyl (DPPH) and superoxide anion radical scavenging, ferrous ion-chelating, and ferric-reducing antioxidant power (FRAP).¹⁶⁾ In our study, activity-guided fractionation lead to the isolation of two new compounds (2, 3) along with 20 known compounds (1, 4–22) (Fig. 1). The present paper reports the isolation and structural elucidation of these compounds, as well as their inhibitory activities against ChEs and BACE1.

Fig. 1. Chemical Structures of Compounds Isolated (1–22) from Juniperus chinensis

Results and Discussion

Detailed analysis of the NMR data, aided by ¹H–¹H correlation spectroscopy (COSY), nuclear overhauser effect spectroscopy (NOESY), heteronuclear multiple quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC) spectra and comparison with compounds 6 and 7, compound 1 was identified as 9′-methoxycalocedrin, which was considered to be most likely artifact produced in the course of extraction by using methanol as extracting solvent.

Compound 2 was obtained as a yellow amorphous solid, and its positive high resolution (HR)-FAB-MS ion at m/z 357.0974 [M+H]⁺ was consistent with a molecular formula of C₁₉H₁₆O₇. The ¹H-NMR spectrum displayed signals at δ: 4.80 (d, J=6.8 Hz, H-2), 4.09 (m, H-3), 2.87 (dd, J=5.1, 16.6 Hz, H-4a), 2.62 (dd, J=7.5, 16.4 Hz, H-4b) for H-2, H-3, and H-4 of the 2,3-trans-catechin core; the signals at δ: 6.82 (d, J=2.05 Hz, H-2′), 6.77 (d, J=8.20 Hz, H-5′), 6.72 (d, J=8.20, 2.05 Hz, H-6′) for an ABX system were characteristic of the catechol B-ring. The ¹³C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra showed signals at δ 83.31, 67.89, and 27.98 for C-2, C-3, and C-4, respectively, of the heterocyclic carbons of flavans with 2,3-trans stereochemistry.¹⁷⁾ The circular dichroism (CD) spectrum of 2 demonstrated a negative Cotton effect at 400 nm, indicating that the absolute configuration of 2 was 2S,3R.¹⁸⁾ The ¹H-NMR spectrum also indicated two unsubstituted proton signals at δ 7.83 (1H, s) and δ 6.31 (1H, s), which were correlated with δ 103.64 (C-6)/δ 105.96 (C-4a) and δ 155.13 (C-7)/δ 165.22 (C=O), respectively, in the HMBC spectrum, suggesting the presence of pyranone ring protons of a coumarin entity in compound 2. In addition, a methyl signal at δ 2.05 (3H, s) was observed. These data showed that 2 was very similar to artoflavanocoumarin,¹⁹⁾ except for the presence of a methyl signal at δ 2.05. The mass spectrum of 2, which had a molecular ion peak 14 mass units higher than that of artoflavanocoumarin, further supported the presence of a methyl group. Its methyl group was proposed to be bonded to C-α by an HMBC correlation between δ 2.05 (CH₃) and δ 119.36 (C-α). Thus, the structure of 2 was elucidated as α-methyl artoflavanocoumarin.

Compound 3 was obtained as a pale yellowish solid, and its positive HR-electron ionization (EI)-MS showed a [M]⁺ ion at m/z 296.0682, which established that the molecular formula is C₁₇H₁₂O₅. The IR spectrum indicated the presence of phenolic hydroxyl (3320 cm⁻¹) and pyrone carbonyl (1640 cm⁻¹) groups. In the UV spectrum, λ_max values at 295 nm and 353 nm were observed, indicating the presence of a typical chromone.²⁰⁾ A typical A₂B₂ spin system at δ 7.57 (2H, d, J=8.54 Hz, H-2′,6′) and δ 6.83 (2H, d, J=8.54 Hz, H-3′,5′) was used to identify a 1′,4′-disubstituted benzene ring moiety in the ¹H-NMR spectrum. A pair of proton signals at δ 6.42 (1H, J=2.10 Hz, H-8) and δ 6.16 (1H, J=2.10 Hz, H-6) was evidence of a meta-coupled aromatic ring. The signals of two trans-olefinic protons δ: 7.60 (1H, d, J=16.06 Hz, H-β) and δ 6.93 (1H, d, J=16.06 Hz, H-α), as well as a proton signal at δ 6.29 (1H, s, H-3), were also observed. The ¹³C-NMR and DEPT spectra of 3 showed 17 carbon signals in the molecule. Among them, a signal at δ 181.54 belonged to a carbonyl carbon, five signals appearing between δ 164.47 and 157.21 belonged to oxygenated carbons, two signals at δ 125.99 and 103.82 belonged to quaternary carbons, and the others belonged to methine carbons. These data suggest that compound 3 is a 5,7,4′-trihydroxy-2-styrylchromone. Although there is a previous report on the chemical synthesis of 3 through debenzoylation of benzoyloxy-2-styrylchromones, which were synthesized using the Baker–Venkataraman method,²¹⁾ this was the first isolation of 3 from nature. The ¹H- and ¹³C-NMR signal assignments of 3 were confirmed in the present study using the HMQC and HMBC spectra.

Although compound 21 was previously reported as being isolated from the needles of Pinus silvestris²²⁾ and Pinus massoniana,²³⁾ root bark of Picea abies,²⁴⁾ bark of Illicium difengpi,²⁵⁾ and root bark of Pseudolarix kaempferi,²⁶⁾ the absolute configurations at C-7 and C-8 had not been determined. The coupling constants of the methine signals at δ 5.54 (H-7) and δ 3.34 (H-8) were 5.81 Hz, indicating a 7,8-trans configuration.²⁷⁾ In addition, a negative Cotton effect at 330 nm and a positive Cotton effect at 270 nm in the CD spectrum were similar to those of a known compound (pterolinus C, with a negative Cotton effect at 319 nm and a positive Cotton effect at 255 nm),²⁸⁾ suggesting that the absolute configurations were 7S and 8S. Consequently, 21 was determined to be (7S,8S)-dihydro-3′-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-8-hydroxymethyl-1′-benzofuranpropanol 4-O-α-L-rhamnopyranoside, which is a diastereomeric aglycone form of 16.

The other known compounds were identified as cedrol (4),²⁹⁾ widdrol (5),³⁰⁾ savinin (6), calocedrin (7),³¹⁾ 10-oxowiddrol (8),³⁰⁾ 12-hydroxywiddrol (9),³²⁾ (+)-naringenin (10),³³⁾ vanillic acid methyl ester (11),³⁴⁾ (+)-taxifolin (12), (+)-aromadendrin (13),³³⁾ kaempferol (14), quercetin (15),³⁵⁾ (7S,8R)-dihydro-3′-hydroxy-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol (16),³⁶⁾ styraxlignolide C (17),³⁷⁾ protocatechuic acid (18), vanillic acid (19),³⁸⁾ (7R,8S)-dihydro-3′-methoxy-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol 4-O-β-D-glucopyranoside (20),²⁷⁾ and (+)-catechin (22)³⁹⁾ on the basis of spectroscopic analysis, chemical evidence, and data from the literature. Although compounds 16 and 20 were previously reported from Santalum album³⁶⁾ and Viburnum awabuki,²⁷⁾ respectively, this is the first time they were isolated from J. chinensis.

The isolated compounds were assessed for inhibitory activities against ChEs and BACE1, as shown in Table 1. All of the compounds 1–22 showed AChE inhibitory activities, with IC₅₀ values ranging from 7.57 to 139.65 µM. Among them, compounds 6, 15, and 22 exhibited significant inhibitory activities against AChE, with respective IC₅₀ values of 17.14, 9.51, and 7.57 µM, compared with berberine (IC₅₀ 0.67 µM). Additionally, compounds 2, 5, 7, 9, 10, 12, 13, 16, 17, 18, 20, and 21 showed moderate inhibitory activities, with respective IC₅₀ values of 31.57, 40.08, 27.76, 21.44, 47.21, 22.86, 35.43, 20.54, 75.22, 26.14, 21.73, and 43.18 µM. However, compounds 1, 3, 4, 8, 11, 14, and 19 exhibited weak inhibitory activities, with IC₅₀ values ranging from 71.92 to 139.65 µM. In the case of BChE inhibitory activity, compounds 3, 5, and 7 displayed good BChE inhibitory activities with respective IC₅₀ values of 28.80, 30.14, and 28.12 µM, compared to the positive control berberine (IC₅₀ 14.04 µM). Also, compounds 1, 2, 4, 6, 8, 9, 15, 16, and 20–22 exhibited moderate inhibitory activities, with respective IC₅₀ values of 186.86, 151.51, 69.13, 84.60, 65.43, 152.29, 47.14, 120.34, 199.77, 36.28, and 166.27 µM. However, compounds 10–14 and 17–19 were inactive at the concentrations tested. Compounds 1–3 exhibited significant inhibitory activities against BACE1, with respective IC₅₀ values of 16.13, 11.91, and 6.25 µM, compared with quercetin (IC₅₀ 26.94 µM). In addition, compounds 6–8, 12, 15–17, 20, and 22 showed moderate inhibitory activities with respective IC₅₀ values of 185.02, 87.73, 93.75, 142.80, 26.94, 86.31, 61.95, 107.06, and 23.23 µM. In contrast, compounds 4, 5, 9–11, 13, 14, 18, 19, and 21 were inactive at the concentrations tested.

Table 1. Inhibitory Activities of Compounds 1–22 on ChEs and BACE1

1	139.65±2.51	186.86±2.61	16.13±0.51
2	31.57±1.04	151.51±0.59	11.91±0.45
3	78.35±1.15	28.80±1.57	6.25±0.16
4	95.07±0.61	69.13±0.23	>200
5	40.08±0.24	30.14±0.37	>200
6	17.14±0.66	84.60±2.12	185.02±1.00
7	27.76±2.60	28.12±1.05	87.73±1.76
8	134.06±0.28	65.43±0.40	93.75±0.34
9	21.44±0.16	152.29±2.91	>200
10	47.21±0.80	>200	>200
11	109.46±0.49	>200	>200
12	22.86±0.38	>200	142.80±0.43
13	35.43±1.23	>200	>200
14	71.92±0.35	>200	>200
15	9.51±0.39	47.14±1.60	26.94±0.10
16	20.54±0.21	120.34±2.90	86.31±2.11
17	75.22±0.58	>200	61.95±1.86
18	26.14±0.61	>200	>200
19	113.19±0.45	>200	>200
20	21.73±1.68	199.77±1.77	107.06±0.59
21	43.18±0.89	36.28±0.78	>200
22	7.57±0.45	166.27±2.12	23.23±1.22
Berberineb)	0.67±0.02	14.04±1.46
Quercetinc)			26.94±0.10

a) The 50% inhibition concentration (µM) is expressed as mean±S.E.M. of triplicate experiments. b) Berberine. c) Quercetin were used as positive reference controls in the ChEs and BACE1 assays, respectively.

Experimental

General Procedures

Optical rotations were measured with a JASCO DIP 370 digital polarimeter. The UV spectra were obtained with an Ultraspec 2100pro UV/Visible spectrophotometer, and CD spectra were recorded on a JASCO J-715 CD spectropolarimeter. The HR-EI-MS and HR-FAB-MS were performed with a JEOL JMS-700 spectrometer (Tokyo, Japan). The IR was recorded on a PerkinElmer, Inc., spectrum GX FT-IR spectrometer (Norwalk, CT, U.S.A.). The NMR spectra were recorded in CDCl₃, DMSO-d₆, and CD₃OD on a JEOL JNM ECP-400 spectrometer (Tokyo, Japan) at 400 MHz for ¹H and 100 MHz for ¹³C. Column chromatography was conducted using silica gel 60 (70–230 mesh, Merck, Darmstadt, Germany), RP-18 (40–63 µm, Merck, Darmstadt, Germany), and Sephadex LH-20 (20–100 µm, Sigma, St. Louis, MO, U.S.A.) columns. TLC (thin layer chromatography) was conducted on precoated Merck Kiesel gel 60 F₂₅₄ plates (20×20 cm, 0.25 mm) and RP-18 F_254s plates (5×10 cm, Merck), using 50% H₂SO₄ as a spray reagent.

Chemicals and Reagents

Electric-eel acetylcholinesterase (EC 3.1.1.7), horse-serum BChE (EC 3.1.1.8), acetylthiocholine iodide (ACh), butyrylthioline chloride (BCh), 5,5′-dithiobis[2-nitrobenzoic acid] (DTNB), and berberine were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). The BACE1 fluorescence resonance energy transfer (FREF) assay kit (β-secretase) was purchased from PanVera Co. (Madison, WI, U.S.A.). All the solvents used for column chromatography were purchased from Merck, Fluka, or Sigma-Aldrich, unless otherwise stated.

Materials

The heartwood parts of J. chinensis were purchased from a medicinal market in Seoul, Korea, and were authenticated by Prof. B. W. Kim, Dong Eui University, Busan, Korea. A voucher specimen (No. 20120302) was deposited in the author’s laboratory (J. S. Choi).

Extraction and Isolation

The dried heartwood parts (3.0 kg) were extracted by reflux with methanol (MeOH) (5 L×3 times). After evaporation of the solvent under reduced pressure, the crude MeOH extract (322 g) was obtained, suspended in H₂O, and successively partitioned with methylene chloride (CH₂Cl₂), ethyl acetate (EtOAc), and butanol (BuOH) to afford CH₂Cl₂ (107 g), EtOAc (147 g), and n-BuOH (59 g) fractions, respectively, as well as an H₂O residue (9 g). According to activity-guided fractionation, the CH₂Cl₂ fraction (107 g) was separated on a silica gel column (15×100 cm, 63–200 µm, particle size, Merck) and eluted with CH₂Cl₂–MeOH (100 : 0→1 : 1, gradient) to yield 24 subfractions (C1–C24). Fraction C7 (3.7 g) was separated on a silica gel column (5×80 cm) and eluted with n-hexane–EtOAc (80 : 1) to yield compound 4 (3.2 g). Fraction C11 (250 mg) was separated on a silica gel column (3×60 cm) and eluted with n-hexane–EtOAc (20 : 1) to obtain five subfractions (C11.1–CF11.5). Among them, fraction C11.2 (42 mg) was separated on a silica gel column (2×80 cm) using n-hexane–EtOAc (20 : 1), to afford compound 5 (30 mg). Fraction C11.4 (38 mg) was separated on a silica gel column (1×60 cm) and eluted with n-hexane–EtOAc (10 : 1) to afford compound 1 (13 mg). Subfraction C11.5 (50 mg) was recrystallized in MeOH to obtain compound 6 (35 mg). Fraction C18 (1.8 g) was separated on a silica gel column (3×80 cm) and eluted with n-hexane–EtOAc (4 : 1) to yield compound 7 (28 mg). The EtOAc-soluble fraction (147 g) was also separated on a silica gel column (15×100 cm) and eluted with CH₂Cl₂–MeOH (20 : 1→1 : 1, gradient) to obtain 35 subfractions (E1–E35). Fraction E7 (350 mg) was separated on a silica gel column (3×80 cm) and eluted with n-hexane–EtOAc (15 : 1) to obtain compound 8 (5 mg). Fraction E8 (3 g) was separated on a silica gel column (5×80 cm) and eluted with n-hexane–EtOAc (10 : 1→2 : 1) to yield eight subfractions (E8.1–E8.8). Among them, subfraction E8.6 (220 mg) was recrystallized in MeOH to obtain compound 9 (170 mg). Subfraction E8.8 (750 mg) was separated on a silica gel column (3×80 cm) and eluted with n-hexane–acetone (6 : 1→4 : 1) to afford compound 10 (230 mg). Fraction E9 (3 g) was separated on a silica gel column (5×80 cm) and eluted with n-hexane–acetone (2 : 1) to yield compound 2 (8 mg). Fraction E10 (5.8 g) was separated on a silica gel column (4×80 cm) and eluted with CH₂Cl₂–MeOH–H₂O (25 : 1 : 0.1) to yield seven subfractions (E10.1–E10.7). Subfraction E10.3 (260 mg) was separated on a Sephadex LH-20 column (2×60 cm) and eluted with MeOH to yield compound 11 (4 mg). Subfraction E10.5 (1 g) was separated on a silica gel column (3×80 cm) and eluted with CH₂Cl₂–MeOH–H₂O (10 : 1 : 0.1) to yield compounds 12 (25 mg), 13 (170 mg), 14 (16 mg), and 15 (30 mg). Fraction E11 (720 mg) was separated on a silica gel column (3×80 cm) and eluted with CH₂Cl₂–MeOH (20 : 1) to yield compound 16 (8 mg). Fraction E14 (2.7 g) was subjected to a silica gel column (4×80 cm) and eluted with CH₂Cl₂–MeOH–H₂O (10 : 1 : 0.1) to yield seven subfractions (E14.1–E14.7). Subfraction E14.4 (160 mg) was separated on a silica gel column (3×80 cm) and eluted with n-hexane–EtOAc (4 : 1) to yield compound 3 (16 mg). Subfraction E14.6 (740 mg) was separated on a silica gel column (3×80 cm) and eluted with EtOAc–MeOH–H₂O (30 : 2 : 1) to obtain compound 17 (12 mg). Fraction E20 (2.1 g) was separated on a Sephadex LH-20 column (4×80 cm) and eluted with MeOH to yield nine subfractions (E20.1–E20.9). Subfraction E20.2 (1 g) was chromatographed on a silica gel column (3×80 cm) and eluted with CH₂Cl₂–MeOH–H₂O (10 : 1 : 0.1) to yield compounds 18 (30 mg) and 19 (15 mg). Fraction E27 (2.2 g) was chromatographed on a silica gel column (4×80 cm) and eluted with CH₂Cl₂–MeOH (25 : 1) to yield 10 subfractions (E27.1–E27.10). Following the filtration of fraction E27.3 (640 mg), the precipitate was recrystallized using MeOH to yield compound 20 (9 mg). Repeated chromatography of E27.6 (350 mg) on a silica gel column (3×80 cm) and elution with EtOAc–MeOH–H₂O (26 : 2 : 1) yielded compound 21 (7 mg). The n-BuOH fraction (57 g) was subjected to chromatography on a Diaion HP-20 column, eluted with a gradient of H₂O–MeOH, and separated into four subfractions: 100% H₂O (B1, 7.5 g); 30% MeOH (B2, 4.3 g); 70% MeOH (B3, 25.2 g); and 100% MeOH (B4, 19.6 g). Fraction B2 was chromatographed on a silica gel column (4×80 cm) and eluted with CH₂Cl₂–MeOH–H₂O (7 : 1 : 0.1) to yield compound 22 (50 mg).

9′-Methoxycalocedrin (1)

[α]_D²⁰ +6.0° (c=0.9, Me₂CO); UV (EtOH) λ_max (log ε) 325 (4.06), 294 (3.98), 257 (4.02); IR ν_max (KBr) 3560, 1745, 1690, 1600 cm⁻¹; HR-EI-MS m/z 382.1055 [M]⁺ (Calcd for C₂₁H₁₈O₇, 382.1053). ¹H-NMR (400 MHz, CD₃OD) δ: 7.45 (1H, d, J=1.7 Hz, H-7), 7.09 (1H, dd, J=8.2, 1.7 Hz, H-6), 7.02 (1H, d, J=1.7 Hz, H-2), 6.88 (1H, d, J=8.2 Hz, H-5), 6.66 (1H, d, J=1.7 Hz, H-2′), 6.64 (1H, dd, J=8.4, 1.7 Hz, H-5′), 6.62 (1H, d, J=8.2 Hz, H-6′), 6.02 (2H, s, H-11), 5.87 (2H, d, J=10 Hz, H-10), 5.29 (1H, d, J=5.5 Hz, H-9′), 3.65 (1H, m, H-8′), 3.41 (3H, s, OCH₃), 2.83 (1H, dd, J=5.8, 14.2 Hz, H-7′a), 2.69 (1H, dd, J=8.5, 14.2 Hz, H-7′b). ¹³C-NMR (100 MHz, CD₃OD) δ: 174.54 (C-9), 150.93 (C-4), 149.79 (C-3), 149.28 (C-3′), 148.00 (C-4′), 139.49 (C-7), 132.43 (C-1′), 129.30 (C-1), 127.43 (C-6), 126.10 (C-8), 123.37 (C-6′), 110.37 (C-2′), 109.73 (C-2), 109.65 (C-5), 109.22 (C-5′), 107.58 (C-9′), 103.25 (C-11), 102.31 (C-10), 56.58 (OCH₃), 47.87 (C-8′), 36.70 (C-7′).

α-Methyl Artoflavanocoumarin (2)

[α]_D²⁰ −137.5° (c=0.1, MeOH); UV (EtOH) λ_max (log ε) 330 (3.57), 287 (3.35), 205 (4.17); IR ν_max (KBr) 3426, 1697, 1620, 1454, 1382, 1261, 1099, 802 cm⁻¹; CD (MeOH; [mdeg]) nm 400 (−4.251); HR-FAB-MS m/z 357.0974 [M+H]⁺ (Calcd for C₁₉H₁₇O₇, 357.0977). ¹H-NMR (400 MHz, CD₃OD) δ: 7.83 (1H, s, H-β), 6.82 (1H, d, J=2.05 Hz, H-2′), 6.77 (1H, d, J=8.20 Hz, H-5′), 6.72 (1H, dd, J=8.20, 2.05 Hz, H-6′), 6.31 (1H, s, H-8), 4.80 (1H, d, J=6.8 Hz, H-2), 4.09 (1H, m, H-3), 2,87 (1H, dd, J=5.1, 16.6 Hz, H-4a), 2.62 (1H, dd, J=7.5, 16.4 Hz, H-4b), 2.05 (3H, s, CH₃). ¹³C-NMR (100 MHz, CD₃OD) δ: 165.22 (C=O), 160.69 (C-8a), 155.13 (C-7), 152.09 (C-5), 146.52 (C-4′), 146.43 (C-3′), 136.94 (C-β), 131.42 (C-1′), 119.75 (C-6′), 119.36 (C-α), 116.26 (C-5′), 114.92 (C-2′), 105.96 (C-4a), 103.64 (C-6), 95.16 (C-8), 83.31(C-2), 67.89 (C-3), 27.98 (C-4), 16.78 (CH₃).

5,7,4′-Trihydroxy-2-styrylchromone (3)

UV (MeOH) λ_max (log ε) 353 (4.05), 295 (3.98); IR ν_max (KBr) 3320, 1640, 1621, 1477, 1412, 1255, 848, 805 cm⁻¹; HR-EI-MS m/z 296.0682 [M]⁺ (Calcd for C₁₇H₁₂O₅, 296.0685). ¹H-NMR (400 MHz, DMSO-d₆) δ: 12.93 (5-OH), 7.60 (1H, d, J=16.06 Hz, H-β), 7.57 (2H, d, J=8.54 Hz, H-2′,6′), 6.93 (1H, d, J=16.06 Hz, H-α), 6.83 (2H, d, J=8.54 Hz, H-3′,5′), 6.42 (1H, d, J=2.10 Hz, H-8), 6.29 (1H, s, H-3), 6.16 (1H, d, J=2.10 Hz, H-6). ¹³C-NMR (100 MHz, DMSO-d₆) δ: 181.54 (C-4), 164.47 (C-7), 162.98 (C-2), 161.50 (C-5), 159.48 (C-4′), 157.21 (C-9), 137.23 (C-β), 129.80 (C-2′,6′), 125.99 (C-1′), 116.31 (C-α), 115.87 (C-3′,5′), 106.91 (C-3), 103.82 (C-10), 98.75 (C-6), 93.87 (C-8).

(7S,8S)-Dihydro-3′-hydroxy-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol 4-O-α-L-Rhamnopyranoside (21)

UV (MeOH) λ_max (log ε) sh 228 (5.09), 281 (4.57); CD (MeOH; [mdeg]) nm 270 (+1.87), 330 (−2.79); EI-MS m/z 492 [M]⁺; ¹H-NMR (400 MHz, CD₃OD) δ: 7.07 (1H, d, J=8.4 Hz, H-5), 7.05 (1H, d, J=2.05 Hz, H-2), 6.92 (1H, dd, J=2.05, 8.40 Hz, H-6), 6.57 (1H, s, H-6′), 6.56 (1H, s, H-2′), 5.54 (1H, d, J=5.81 Hz, H-7), 5.32 (1H, d, J=1.71 Hz, C-1″), 4.04 (1H, dd, J=1.71, 3.42 Hz, C-2″), 3.85 (2H, m, H-9″a and H-9′b), 3.81 (3H, s, 3-OCH₃), 3.75 (1H, m, C-5″), 3.54 (2H, t, J=6.49 Hz, H-9), 3.43 (1H, m, H-8), 3.43 (1H, m, C-4″), 2.53 (2H, t, J=7.52 Hz, H-7′), 1.77 (2H, m, H-8′), 1.20 (3H, d, J=6.15 Hz, C-6″). ¹³C-NMR (100 MHz, CD₃OD) δ: 152.10 (C-3), 146.44 (C-4), 146.44 (C-4′), 141.92 (C-3′), 136.90 (C-1′), 139.12 (C-1), 129.50 (C-5′), 119.60 (C-5), 119.07 (C-6), 117.07 (C-2′), 116.67 (C-6′), 111.22 (C-2), 101.42 (C-1″), 88.26 (C-7′), 73.83 (C-4″), 72.20 (C-2″), 72.05 (C-3″), 70.81 (C-5″), 65.06 (C-9), 62.29 (C-9′), 56.43 (3-OCH₃), 55.95 (C-8′), 35.79 (C-8), 32.71 (C-7), 17.94 (C-6″).

In Vitro Enzyme Assay of ChE Inhibition

The inhibitory activities of the isolated compounds on ChE were measured using the spectrophotometric method developed by Ellman et al.⁴⁰⁾ ACh and BCh were used as the substrates to assay the activities of AChE and BChE, respectively. The reaction mixtures contained 140 µL of sodium phosphate buffer (pH 8.0), 20 µL of tested sample solution (final concentration; 200 µM for both compounds), and 20 µL of either the AChE or BChE solution, which were all mixed and incubated for 15 min at room temperature. All tested samples and the positive control (berberine) were dissolved in 10% analytical-grade dimethyl sulfoxide (DMSO). Reactions were initiated upon addition of 10 µL of DTNB and 10 µL of either ACh or BCh. The hydrolysis of ACh or BCh was monitored by tracking the formation of the yellow 5-thio-2-nitrobenzoate anion at 412 nm for 15 min, resulting from the reaction of DTNB with thiocholine released by the enzyme. Each reaction was performed in triplicate, and results were measured in 96-well microplates, using a microplate spectrophotometer (Molecular Devices). Percent inhibition was calculated using the formula (1−S/E)×100, where E and S are enzyme activities with or without the test sample, respectively. The inhibitory activity of each sample on the ChEs was expressed as an IC₅₀ (µM concentration required to inhibit the hydrolysis of the substrate by 50%), as calculated using log-dose inhibition curves.

In Vitro Enzyme Assay of BACE1 Inhibition

The BACE1 enzyme assay was carried out according to the supplied manual with selected modifications. Briefly, a mixture of 10 µL of assay buffer (50 mM sodium acetate, pH 4.5), 10 µL of BACE1 (1.0 U/mL), 10 µL of substrate (750 nM Rh-EVNLDAEFK-Quencher in 50 mM, ammonium bicarbonate), and 10 µL of the tested samples (final concentration: 200 µM for compounds) dissolved in 10% DMSO was incubated for 60 min at 25°C in the dark. The proteolysis of two fluorophores (Rh-EVNLDAEFK-Quencher) by BACE1 was monitored through formation of the fluorescent donor (Rh-EVNL) at wavelengths of 530–545 nm (excitation) and 570–590 nm (emission). Fluorescence was measured with a microplate spectrofluorometer (Gemini EM, Molecular Devices, CA, U.S.A.). The mixture was irradiated at 545 nm, and the emission intensity was recorded at 585 nm. The percent inhibition was obtained using the following equation: % inhibition=[1−(S₆₀−S₀)/(C₆₀−C₀)]×100, where C₆₀ is the fluorescence of the control (enzyme, buffer, and substrate) after 60 min of incubation, C₀ is the initial fluorescence of the control, S₆₀ is the fluorescence of the tested sample (enzyme, sample solution, and substrate) after 60 min of incubation, and S₀ is the initial fluorescence of the tested sample. To allow for a quenching effect of the samples, the sample solution was added to the reaction mixture C, and any reduction in fluorescence was investigated. The BACE1 inhibitory activity of test compounds was expressed in terms of the IC₅₀ value, as calculated from the log-dose inhibition curve. Quercetin was used as the positive control.

Acknowledgment

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2012R1A6A1028677).

Conflict of Interest

The authors declare no conflict of interest.

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