[beta]-secretase (BACE1)-inhibiting stilbenoids from Smilax Rhizoma.

Abstract

In the course of searching for BACE1 ([beta]-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of Smilax Rhizoma (the dried rhizomes of Smilax china L.) showed potent inhibitory activity. The active compounds were identified as a trans/cis-resveratrol mixture, oxyresveratrol, veraphenol, and cis-scirpusin A. They were shown to non-competitively inhibit BACE1 with the Ki values of 5.4 x [10.sup.-6], 5.4 x [10.sup.-6], 3.4 x [10.sup.-6], and 5.4 x [10.sup.-6]M and [IC.sub.50] values of 1.5 x [10.sup.-5], 7.6 x [10.sup.-6], 4.2 x [10.sup.-6], and 1.0 x [10.sup.-5]M, respectively. The active compounds were less inhibitory to [alpha]-secretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, suggesting that they were relatively specific inhibitors of BACE1.

[c] 2006 Elsevier GmbH. All rights reserved.

Keywords: Smilax china; Smilax Rhizoma; Stilbenoids; Alzheimer's disease; BACE1 ([beta]-secretase)

Introduction

Alzheimer's disease is a neurodegenerative disorder clinically characterized by the deposition of amyloid protein (amyloid plaques) in the parenchyma of the amygdale, hippocampus, and neocortex (Sisodia and Price, 1995). In Alzheimer's disease, the major component of the amyloid plaque is the [beta]-amyloid protein (A[beta]), which is a 39-43 amino acid peptide composed of a portion of the transmembrane domain and the extracellular domain of the amyloid precursor protein (APP). APP is cleaved by three types of proteases, which are designated [alpha]-, [beta]-, and [gamma]-secretase. To initiate A[beta] formation, [beta]-secretase cleaves the APP to form the N-terminus of A[beta] at the Asp + 1 residue of the A[beta] sequence. Following [beta]-secretase cleavage, C99 is the substrate of the second protease, [gamma]-secretase, which cleaves the APP to generate the C-terminus of A[beta], and the mature peptide is secreted from the cell (Citron, 2002). Alternate pathway, a third protease, [alpha]-secretase cleaves within the A[beta] sequence, thus precluding the formation of A[beta]. Cleavage by [alpha]-secretase produces a large soluble N-terminal fragment (Vassar et al., 1999). Among the secretases, BACE is at present the most attractive target for the inhibition of amyloid production since there is strong evidence that it is the major [beta]-secretase in neurons and the absence of BACE results in the inhibition of the production of amyloid and C99 stubs or [beta]-CTF (C-Terminal Fragment) without any major side-effects (Sinha et al., 1999). This initial work has been followed by several publications describing substrate-based peptidic inhibitors of [beta]-secretase (Ghosh et al., 2000, 2001; Tung et al., 2002). Therefore, the [beta]-secretase inhibitors could be a promising target for developing anti-dementia drugs.

All drugs considered for Alzheimer's disease must cross the blood-brain-barrier (BBB) and the plasma membrane (Dewachter and Van Leuven, 2002). Enzyme inhibitors with therapeutic potential are preferably smaller than 700 Da, so large peptide-based inhibitors are not viable drug candidates. Thus, the secondary metabolites of plants and microbes which have relatively low-molecular weight and high lipophilicity might be good drug candidates for BACE1 inhibitors (Jeon et al., 2003). By this background, in the course of screening [beta]-secretase inhibitors from the 266 plant extracts, Smilax Rhizoma (dried rhizomes of Smilax china L.) showed high inhibitory activity. We will discuss the isolation and structure elucidation of the active compounds and on their inhibitory activity on BACE1.

Materials and methods

General

Fluorescence was measured with a Bio-TEK ELISA microplate fluorescence reader ELx 800 (VT, USA). Optical density was measured by a Bio-TEK ELx 808 (VT, USA). [.sup.1.H]- and [.sup.13.C]-NMR spectra were recorded on a Bruker Avance Digital 400 spectrometer (Karlsruhe, Germany) at 400 and 100 MHz, respectively. Chemical shifts are given in [delta] (ppm) from TMS. TLC was performed on a precoated silica gel plate (Kiesel gel 60F254, Merck, USA). Silica gel column chromatography was carried out using a Kiesel gel 60 (Merck, USA). Sephadex LH-20 was a product of Sigma-Adrich (St. Louis, MO, USA). (-)-Epigallocatechin gallate which was used as a positive control in BACE1 assay was purchased from Sigma-Aldrich.

Enzyme assays

A BACE1 (recombinant human BACE1) assay kit was purchased from the PanVera Co., USA The assay was carried out according to the supplied manual with modifications according to Park et al (Park et al., 2004). Briefly, a mixture of 10 [micro]L of an assay buffer (50 mM sodium acetate, pH 4.5), 10 [micro]L of BACE1 (1.0U/mL), 10 [micro]L of the substrate (750 nM Rh-EVNLDAEFK-Quencher in a 50 mM ammonium bicarbonate), and 10 [micro]L of a sample dissolved in the assay buffer was incubated for 60 min at 25 [degrees]C under dark condition. The mixture was allowed for excitation at 530 nm and the emitted light at 590 nm was collected. The inhibition ratio was obtained by the following equation: Inhibition (%) = [1 - {(S - [S.sub.0])/(C - [C.sub.0])}] x 100, where C was the fluorescence of a control (enzyme, assay buffer, and substrate) after 60min of incubation, [C.sub.0] was the fluorescence of control at zero time, S was the fluorescence of the tested samples (enzyme, sample solution, and substrate) after 60 min of incubation, and So was the fluorescence of the tested samples at zero time. All data are the mean of duplicated experiments. To check the quenching effect of the samples, the sample solution was added to the reaction mixture C, and any reduction in fluorescence by the sample was then investigated. [alpha]-Secretase activity was measured by an [alpha]-secretase assay kit with TACE according to the manual from R & D Systems, MN, USA Chymotrypsin, trypsin, and elastase were assayed according to the protocol described in the reference using N-benzoyl-L-Arg-pNA, N-benzoyl-L-Tyr-pNA, and N-succinyl-Ala-Ala-Ala-pNA as substrates, respectively (Chung et al., 1983; Hubert et al., 1992; Bieth et al., 1974). All data presented are the mean values of the duplicate experiments.

Plant material, extraction, isolation and instrumental analyses

Plant material: Smilax Rhizoma was purchased at an herbal medicine market in Daegu, Korea and identified by Dr. Jong-Hwan Kwak at Sungkyunkwan University, Suwon, Korea. A voucher specimen (no. SC-0401) has been deposited at The Innovative Research Laboratory of Natural Products Medicine, Kyungpook National University, Daegu, Korea.

Extraction and isolation: The dried Smilax Rhizoma (1.8 kg) was refluxed in MeOH (2000 mL x 3) and the extract was evaporated to dryness. The MeOH extract (100.0g) was suspended in water and the suspension was consecutively partitioned with methylene chloride (C[H.sub.2][Cl.sub.2], 700 mL x 3) and ethyl acetate (EtOAc, 700 mL x 3). The EtOAc layer showed high inhibitory activity on the BACE1. The EtOAc soluble fraction (6.9 g) was chromatographed on a Sephadex LH-20 column (4.0 x 68.0 cm, stepwise gradient of 50-100% MeOH) to yield nineteen fractions (Fr.1~19). The HPLC ([mu]-Bondapak C18, 7.8 x 300mm, Waters, 1% HOAc in 35% MeOH, 1.5 mL, [min.sup.-1]) of Fr. 8 and Fr. 12 afforded 11.5mg of compound 1 and 2.0 mg of 4, respectively. Re-chromatography of Fr.10 using a silica gel column (1.4 x 40 cm, C[H.sub.2][Cl.sub.2]-MeOH-HOAc = 30:1:1 [right arrow] 1:1:1) and the resultant active fractions were applied on the HPLC (Apollo [C.sub.18], 10 x 250 mm, Alltech, 1% HOAc in 33 and 38% MeOH of Fr. 10-3 and Fr.10-5, respectively) to give compound 2 (4.0 mg) and 3 (6.4 mg).

HPLC analysis of cis- and trans-resveratrol

A Jasco (Japan) HPLC system equipped with a PU2080 pump and a MD2010 PDA detector was used for separating cis- and trans-resveratrol. Zorbax Eclipse XDB [C.sub.18] (Agilent, USA) was used as a stationary phase and 1% HOAc in a 20% aqueous MeOH was used as an initial mobile phase. The concentration of MeOH was increased up to 100% within 50min at the flow rate of 0.8mL/min. Detection was carried out under UV 254 nm. Under these conditions, trans- and cis-resveratrol appeared at 32.8 and 36.7 min, Rt respectively.

Results

Isolation and structure determination of BACE1 inhibitors

The methanolic extract of Smilax Rhizoma inhibited 76.6% of BACE 1 activity at 10 ppm. Compared to dichloromethane (32.3%) and water soluble fractions (47.5%), ethyl acetate soluble fraction (68.4%) showed stronger activity at 1 ppm, therefore, ethyl acetate soluble fraction was considered for further purification. Out of the first 19 fractions obtained by a Sephadex LH-20 chromatography of the ethyl acetate soluble fraction, Fr. 8, 10, and 12 showed potent inhibitory activity (70.1%, 72.3%, and 85.3% at 1 ppm, respectively) against BACE1. By further purification of the major compounds in these fractions, the active compounds 1-4 were isolated.

Compound 1 was obtained as a yellow powder that was positive to the Fe[Cl.sub.3]. In the FAB-Mass spectrum, a molecular ion peak [[M.sup.+] + H] was observed at m/z 229. In the [.sup.1.H]-NMR spectrum, nine aromatic signals were detected between [delta] 6.16-7.35. The stereochemistry of 1 was determined to be a trans and cis mixture from the coupling constants (16.0 Hz, each) between [delta] 6.79 and 6.95 and coupling constants (12.0 Hz, each) between [delta] 6.31 and 6.40, respectively. In the [.sup.13.C]-NMR spectrum, the 12 major and the 12 minor aromatic carbon signals were detected with two sets of two olefinic carbons. From these data, 1 was postulated as a trans/cis-resveratrol mixture, and was finally confirmed by the comparison of their NMR data with those in the references (Oleszek. et al., 2001; Solladie et al., 2003). The cis form was hardly purified since it was easily converted into trans form. The peak ratio of trans- and cis-resveratrol was ca 4:1 in the HPLC analysis.

Compound 2 was obtained as a yellow powder that was positive to Fe[Cl.sub.3], indicating that it had phenolic group(s) in its structure. In the [.sup.1.H]-NMR spectrum, an aromatic doublet ([delta] 6.43, 2H) and triplet ([delta] 6.12, 1 H) indicated that 2 had a typical resveratrol skeleton. The coupling constant (J = 16.4 Hz) between H-7 ([delta] 7.26, 1 H, d) and H-7' ([delta] 6.80, 1 H, d) implied that the stereochemistry of 2 was a trans. In addition, the coupling patterns between [delta] 7.32 and 6.30, and 6.43 and 6.12 showed the typical 1,2,4 and 1,3,5-tri-substituted benzene rings, respectively. In the [.sup.13.C]-NMR spectrum, four oxygenated aromatic carbons ([delta] 159.6 x 2, 159.2, 157.2) and ten aromatic carbons ([delta] 105.6-142.1) were shown. From these data, 2 was postulated as oxyresveratrol, and was finally confirmed by the comparison of its NMR data with those in reference (Tripetch et al., 2002).

Compound 3 was obtained as a yellow powder that was positive to Fe[Cl.sub.3]. In the EIMS spectrum, a molecular ion peak was observed at m/z 242. In the [.sup.1.H]-NMR spectrum, an aromatic doublet ([delta] 6.67, 2H) and triplet ([delta] 6.22, 1 H) indicated that 3 had an 1,3,5-tri-substituted benzene ring. The coupling patterns at [delta] 7.36, 6.94, and 6.74 were evident for 1,2,4-tri-substituted benzene ring moiety. Compared to 2, one olefinic proton having a large coupling constant (~16.0 Hz) was absent. In the [.sup.13.C]-NMR spectrum, five oxygenated aromatic carbons ([delta] 160.3 x 2, 156.7, 157.1, 157.8) were shown. From the HMBC and HMQC spectrum, [delta] 156.7 was assigned to be C-2. Accordingly, 3 was assumed to be a cyclic ether of oxyresveratrol and was finally identified as veraphenol by the comparison of its spectral data with those in the reference (Purusotam et al., 1993).

Compound 4 was obtained as a brown powder and was positive to Fe[Cl.sub.3]. In the [.sup.1.H]-NMR spectrum, aromatic doublet protons at [delta] 7.01 (2H) and [delta] 6.66 (2 H) indicated that 4 had an 1,4-di-substituted benzene ring. The coupling patterns at [delta] 6.24 and 6.02 (d, J = 12.2 Hz each) suggested the existence of cis-olefinic protons. The proton signals at [delta] 6.78, 6.77 and 6.58 showed a typical splitting pattern of a 1,3,4-trisubstituted benzene ring. From these data, 4 was postulated as a resveratrol dimer. In addition, [delta] 4.02 and 5.25 indicated that a part of the resveratrol skeleton was partially reduced. Compound 4 was finally identified as cis-scirpusin A by comparing its spectral data with those in the reference (Kulesh et al., 1999).

The structures of the isolated compounds are presented in Fig. 1.

Inhibitory activity against BACE1

All isolated compounds inhibited BACE1 in a dose-dependent manner and were non-competitive with a substrate in the Dixon plots (Figs. 2 and 3). The inhibition constant (Ki) of 1, 2, 3, and 4 were 5.4 x [10.sup.-6], 5.4 x [10.sup.-6], 3.4 x [10.sup.-6], and 5.4 x [10.sup.-6]M, and the [IC.sub.50] values were 1.5 x [10.sup.-5], 7.6 x [10.sup.-6], 4.2 x [10.sup.-6], and 1.0 x [10.sup.-5] M, respectively. The [IC.sub.50] value of the positive control (-)-epigallocatechin gallate was 1.6 x [10.sup.-6]M.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

To check the enzyme specificity, the inhibitory activities on tumor necrosis factor alpha converting enzyme (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase were compared with those of BACE1. Up to 100 [micro]M, the isolated compounds did not show significant inhibition against the above enzymes. Thus, the isolated compounds appeared to be relatively specific inhibitors of BACE1 as is the case of the other natural inhibitors we had isolated (Jeon et al., 2003; Park et al., 2004).

Discussion

Recently, many synthetic inhibitors of BACE1 have been reported including two transition-state analog inhibitors of [beta]-secretase, OM99-1 and OM99-2 with [IC.sub.50] of ~1.6nM (Ghosh et al., 2001). They were synthesized on the basis of the model on the cleavage site of the [beta]-secretase of the Swedish mutation. However, only a few articles on naturally occurring [beta]-secretase inhibitors have been reported by our group (Jeon et al., 2003; Park et al., 2004; Kwak et al., 2005).

The isolated compounds may not be directly considered as a potential drug candidate since they have relatively hydrophilic phenolic hydroxyl groups, however, this is the first report on the BACE1-inhibiting activity of stilbenoids. The isolated compounds are expected to be useful in the study of the mechanisms of Alzheimer's disease.

[FIGURE 3 OMITTED]

Acknowledgment

This work was supported by a Grant from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.

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S.-Y. Jeon (a), S.-H. Kwon (a), Y.-H. Seong (b), K. Bae (c), J.-M. Hur (a), Y.-Y. Lee (d), D.-Y. Suh (d), K.-S. Song (a,*)

(a) Division of Applied Biology and Chemistry, College of Agriculture and Life Sciences, Kyungpook National University, 1370 Sankyuk-Dong, Daegu 702-701, Republic of Korea

(b) College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea

(c) College of Pharmacy, Chungnam National University, Yusung, Daejon 305-764, Republic of Korea

(d) Yeongnam Agricultural Research Institute, NICS, RDA, Milyang, Gyeongnam 627-803, Republic of Korea

Received 7 February 2006; accepted 19 May 2006

*Corresponding author. Tel.: [phone]; fax: [phone].

E-mail address: [e-mail] (K.-S. Song).