Galangin, a flavonol derived from Rhizoma Alpiniae Officinarum, inhibits acetylcholinesterase activity in vitro

Chemico-Biological Interactions 187 (2010) 246–248

Contents lists available at ScienceDirect

Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint

Galangin, a flavonol derived from Rhizoma Alpiniae Officinarum, inhibits acetylcholinesterase activity in vitro Ava J.Y. Guo ∗ , Heidi Q. Xie, Roy C.Y. Choi, Ken Y.Z. Zheng, Cathy W.C. Bi, Sherry L. Xu, Tina T.X. Dong, Karl W.K. Tsim Department of Biology and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong SAR, China

a r t i c l e

i n f o

Article history: Available online 7 May 2010 Keywords: Alzheimer’s disease AChE inhibitors Traditional Chinese medicines Flavonoid Galangin Rhizoma Alpiniae Officinarum

a b s t r a c t Acetylcholinesterase (AChE) inhibitors are widely used for the treatment of Alzheimer’s disease (AD). Several AChE inhibitors, e.g. rivastigmine, galantamine and huperzine are originating from plants, suggesting that herbs could potentially serve as sources for novel AChE inhibitors. Here, we searched potential AChE inhibitors from flavonoids, a group of naturally occurring compounds in plants or traditional Chinese medicines (TCM). Twenty-one flavonoids, covered different subclasses, were tested for their potential function in inhibiting AChE activity from the brain in vitro. Among all the tested flavonoids, galangin, a flavonol isolated from Rhizoma Alpiniae Officinarum, the rhizomes of Alpiniae officinarum (Hance.) showed an inhibitory effect on AChE activity with the highest inhibition by over 55% and an IC50 of 120 ␮M and an enzyme-flavonoid inhibition constant (Ki ) of 74 ␮M. The results suggest that flavonoids could be potential candidates for further development of new drugs against AD. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

2. Materials and methods

Alzheimer’s disease (AD) is a neurodegenerative disease, and it is the most common form of dementia [1,2]. Cognitive dysfunction and primary memory loss are the main symptoms associated with AD [3–5]. Recent research has indicated the beneficial effects of estrogen replacement therapy (ERT) on AD [6]. Enhanced cognitive functions were shown in women with AD after treatment with estrogen [7]. Phyto-estrogens (e.g. flavonoids), a group of naturally occurring compounds that share similar chemical structures to estrogen, are also reported to improve cognitive function [8]. Phyto-estrogens are mainly derived from plants and our daily foods. Compared to estrogen, phyto-estrogens have not been reported to have carcinogenic effects. Indeed, several AChE inhibitors, e.g. rivastigmine, galantamine and huperzine are originating from plants. In addition, Katalinic´ et al. recently determined several flavonoids as inhibitors of human butyrylcholinesterase (BChE) and the inhibition potency were attributed to the chemical structures of flavonoids [9]. Taking together, plants or herbs could potentially serve as sources for novel AChE inhibitors and phyto-estrogens, especially the flavonoids could be a more appropriate substitute for use in preventing AD. In the present study, 21 flavonoids isolated from traditional Chinese medicines (TCMs) were screened and their abilities to inhibit AChE activity were determined.

2.1. Chemicals

∗ Corresponding author. Tel.: +852 2358 7924; fax: +852 2358 7323. E-mail address: [email protected] (A.J.Y. Guo). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.05.002

1,5-Bis (4-allyldimethyl-ammoniumphenyl) pentan-3-one dibromide (BW284c51), tetraisopropylpyro-phosphoramide (Iso-OMPA), acetylthiocholine iodide (ATCh), and 5,5 -dithiobis (2-nitrobenzenoic acid) (DTNB) were purchased from Sigma (St. Louis, MO). The tested flavonoids apigenin, kaempferol, baicalin, baicalein, genistein, genistin and puerarin were purchased from Wakojunyaku (Osaka, Japan). Luteolin, quercetin, rutin, hyperin, farrerol, alpinetin, scutellarin, daidzein and daidzin were purchased from National Institute of the Control of Pharmaceutical Biological Products (NICPBP) (Beijing, China). Galangin, tiliroside, quercetin-3 -O-glucoside, formononetin and calycosin were obtained from School of Pharmaceutical Science, Peking University (Beijing, China). The purities of these compounds were over 98%, and they were dissolved in DMSO to give stock solution at concentration of 25–100 mM. 2.2. Tissue collection Rat adult brains were homogenized in 10 mL ice-cold low salt lysis buffer containing 10 mM HEPES, 150 mM NaCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X100, 1 mg/mL bacitracin, 10 ␮g/mL leupeptin, 10 ␮g/mL aprotinin, 20 ␮M pepstatin, and 5 mM benzamidine HCl. The homogenates were clarified by centrifugation at 16,000 × g for 10 min at 4 ◦ C. The final dilution of rat brain used in the assay was 1:20 (protein concentration: 0.5 mg/mL).

A.J.Y. Guo et al. / Chemico-Biological Interactions 187 (2010) 246–248

247

2.3. Acetylcholinesterase inhibition assay Inhibition of AChE activity with flavonoids was tested by Ellman assay [10] with minor modification. In brief, rat brain lysate were first incubated with 0.1 mM iso-OMPA to inhibit BChE activity. 5 ␮L samples were then added to the reaction mixture containing flavonoids (10 or 50 ␮M), 0.5 mM ATCh and 0.5 mM DTNB in 100 mM phosphate buffer (pH 7.4). The increase absorbance (A) at 410 nm was measured and the specific enzymatic activity was expressed as absorbance units/minute. AChE inhibition with BW284c51 (20 ␮M) was also assayed, as a positive control. Results were reported as percentage of inhibition to AChE activity, where the optical density measured from control (without inhibitor) was considered to be 0% of inhibition. Percentage of inhibition was calculated as follows: (Aflavonoid − Acontrol )/Acontrol × 100%. For galangin, the dose-dependent inhibitory assay was performed (6.25–400 ␮M) at room temperature using 0.5 mM ATCh, and the half maximal inhibitory concentration (IC50 ) was calculated from the Prism dose-response curve obtained by plotting the percentage of inhibition versus the concentrations of galangin. The enzymeflavonoid inhibition constant (Ki ) of galangin was evaluated by generating a Hunter-Downs plot as described in Kataliníc et al. [9]. For all the treatment, the concentration of DMSO in the control and working solution of flavonoids did not exceed 0.1%. Statistical tests were done by using one-way analysis of variance (ANOVA). Data are expressed as mean ± standard errors of the mean (SEM), where number of inhibition experiments = 3. 3. Results and discussions Selective flavonoids, isolated from traditional Chinese medicines, were chosen to screen for their inhibitory activity to AChE in vitro. The results are summarized in Table 1. Among all the tested flavonoids, galangin displayed the strongest inhibitory Table 1 AChE inhibitory activity of the selective flavonoids. Flavonoid Flavanones Alpinetin Farrerol

% inhibition 10.3 ± 0.01 8.98 ± 0.04

Flavones Luteolin Apigenin Baicalein Baicalin Scutellarin Icariin

15.56 21.54 12.5 16.25 18.67 17.34

± ± ± ± ± ±

0.02 0.01 0.01 0.01 0.02 0.01

Flavonols Galangin Tiliroside Kaempferol Quercetin Quercetin-3 -O-glucoside Rutin

56.53 13.48 14.6 25.75 19.63 17.53

± ± ± ± ± ±

0.03 0.01 0.04 0.02 0.02 0.01

Isoflavones Daidzein Daidzin Formononetin Genistein Genistin Puerarin Calycosin

14.44 15.04 17.02 16.19 7.78 12.87 22.76

± ± ± ± ± ± ±

0.01 0.01 0.04 0.01 0.02 0.01 0.01

BW284C51

99.24 ± 0.01

Rat brain lysates was used to test the inhibitory effects of 21 flavonoids using ATCh as substrate (0.5 mM). The concentration of the flavonoids was 50 ␮M except Apigenin was 10 ␮M. BW284c51 (20 ␮M) served as a positive control. Percentage of inhibition was shown, mean ± SEM, n = 6.

Fig. 1. Effect of galangin on inhibition of acetylcholinesterase activity. (A) The chemical structure of galangin. (B) AChE inhibition curve of galangin (6.25–400 ␮M). The concentration of ATCh used was at 0.5 mM. Galangin IC50 value was calculated from log ␮M back to ␮M. (C) The real enzyme-flavonoid inhibition constant Ki (yintercept) of galangin (25 ␮M) to AChE was determined from the Hunter–Downs plot, where Ki, app (apparent enzyme-flavonoid inhibition constant at a given substrate concentration) was plot against different substrate concentrations (0.125, 0.25 and 0.5 mM). Data are expressed as means ± SEM, where n = 3, each with triplicate samples.

effect. The structure of galangin was shown in Fig. 1A. The inhibitory activity of galangin was further analyzed, and the results were shown in Fig. 1B and C. The highest inhibitory effect of galangin on AChE activity was 86% at 400 ␮M. The IC50 value for the inhibition of galangin was ∼120 ␮M and the Ki value was ∼74 ␮M, respectively (Fig. 1B and C). Nevertheless, whether galangin can bind to AChE molecules directly or it has the same putative binding site as the substrate ATCh and thereafter compete with ATCh, require further studies. Galangin is a flavonol, a class of flavonoids that contain a 3-hydroxyflavone backbone. It is one of the major flavonoids found in Rhizoma Alpiniae Officinarum, the rhizomes of Alpiniae

248

A.J.Y. Guo et al. / Chemico-Biological Interactions 187 (2010) 246–248

officinarum (Hance.). Results from in vitro and in vivo studies have indicated that galangin has anti-oxidative and free radical scavenging activity. And it has been demonstrated to have biological activities related to some physiological or pathological conditions. It is capable of modulating hypoxia-induced factor [11] and inhibiting the proliferation of human mammary tumor cells by down regulation of cyclins D3, E and A [12]. Moreover, the study has shown that galangin is a potent inhibitor of BChE [9], which was proposed to be beneficial in the treatment of AD. In this study, we provide evidence to show the action of galangin in inhibiting AChE activity. But the action mechanism of this inhibition remains unclear. With the hint from BChE that the inhibitory effect of galangin was attributed to the number of OH groups on its phenyl ring [9], it is possible that this chemical structure of galangin may also contribute to the inhibitory effect on AChE activity. Collectively, it is apparent that the plant-derived AChE inhibitors, such as galangin, may be important for the development of more appropriate drug candidates for the treatment of AD. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements This research was supported by Research Grants Council of Hong Kong SAR (HKUST6419/06M, HKUST662407, N HKUST629/07, HKUST662608, CAS-CF07/08.SC03) to KWKT.

References [1] D.A. Evans, H.H. Funkenstein, M.S. Albert, Prevelance of Alzheimer’s disease in a community population of older persons, JAMA 262 (1989) 2551–2556. [2] A. Nordberg, Pharmacological treatment of cognitive dysfunction in dementia disorders, Acta Neurol. Scand. Suppl. 168 (1996) 87–92. [3] B. Desgranges, J.C. Baron, De La Sayette, The neural substrates of memory systems impairment in Alzheimer’s disease, Brain 121 (1998) 611–631. [4] H. Förstl, F. Hentschel, H. Sattel, Age-associated memory impairment and early Alzheimer’s disease, Drug Res. 45 (1995) 394–397. [5] J. Grafman, H. Weingartner, B. Lawlor, A.M. Mellow, K. ThompsenPutnam, T. Sunderland, Automatic memory processes in patients with dementia—Alzheimer’s type (DAT), Cortex 26 (1990) 361–371. [6] S.J. Birge, The role of estrogen in the treatment and prevention of dementia, Am. J. Med. 103 (1997) 1S–50S. [7] T. Ohkura, K. Isse, K. Akazawa, M. Hamamoto, Y. Yaoi, N. Hagino, Low-dose estrogen replacement therapy for Alzheimer’s disease in women, Menopause: J. N. Am. Menopause Soc. 1 (1994) 125–130. [8] S.E. File, N. Jarrett, E. Fluck, R. Duffy, K. Casey, H. Wiseman, Eating soya improves human memory, Psychopharmacology 157 (2001) 430–436. ˇ ´ G. Rusak, J.D. Barovic, ´ G. Sinko, ´ R. Antolovic, ´ Z. Kovarik, [9] M. Katalinic, D. Jelic, Structural aspects of flavonoids as inhibitors of human butylcholinesterase, Eur. J. Med. Chem. (2009) 1–7. [10] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Feather-Stone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–95. [11] S.S. Park, I. Bae, Y.J. Lee, Flavonoids-induced accumulation of hypoxia-inducible factor (HIF)—1alpha/2alpha is mediated through chelation of iron, J. Cell Biochem. 103 (2008) 1989–1998. [12] T.J. Murray, X. Yang, D.H. Sherr, Growth of a human mammary tumor cell line is blocked by galangin, a naturally occurring bioflavonoid, and is accompanied by down-regulation of cyclins D3, E, and A, Breast Cancer Res. 8 (2006) R17.