Bioassay-Guided Isolation of Neuroprotective Compounds from Uncaria rhynchophylla against Beta-Amyloid-Induced Neurotoxicity

Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 802625, 8 pages doi:10.1155/2012/802625 Research Article Bioassay-Guided Isolation of Neuroprotective Compounds from Uncaria rhynchophylla against Beta-Amyloid-Induced Neurotoxicity Yan-Fang Xian,1 Zhi-Xiu Lin,1 Qing-Qiu Mao,1 Zhen Hu,1 Ming Zhao,2 Chun-Tao Che,2 and Siu-Po Ip1 1 School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong 2 Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago IL 60607, USA Correspondence should be addressed to Zhi-Xiu Lin, and Siu-Po Ip, Received 27 March 2012; Accepted 12 May 2012 Academic Editor: Karl Wah-Keung Tsim Copyright © 2012 Yan-Fang Xian et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Uncaria rhynchophylla is a component herb of many Chinese herbal formulae for the treatment of neurodegenerative diseases. Previous study in our laboratory has demonstrated that an ethanol extract of Uncaria rhynchophylla ameliorated cognitive deficits in a mouse model of Alzheimer’s disease induced by D-galactose. However, the active ingredients of Uncaria rhynchophylla responsible for the anti-Alzheimer’s disease activity have not been identified. This study aims to identify the active ingredients of Uncaria rhynchophylla by a bioassay-guided fractionation approach and explore the acting mechanism of these active ingredients by using a well-established cellular model of Alzheimer’s disease, beta-amyloid- (Aβ-) induced neurotoxicity in PC12 cells. The results showed that six alkaloids, namely, corynoxine, corynoxine B, corynoxeine, isorhynchophylline, isocorynoxeine, and rhynchophylline were isolated from the extract of Uncaria rhynchophylla. Among them, rhynchophylline and isorhynchophylline significantly decreased Aβ-induced cell death, intracellular calcium overloading, and tau protein hyperphosphorylation in PC12 cells. These results suggest that rhynchophylline and isorhynchophylline are the major active ingredients responsible for the protective action of Uncaria rhynchophylla against Aβ-induced neuronal toxicity, and their neuroprotective effect may be mediated, at least in part, by inhibiting intracellular calcium overloading and tau protein hyperphosphorylation. 1. Introduction Alzheimer’s disease (AD), a neurodegenerative disorder characterized by a progressive loss of learning, memory, and other cognitive functions, is the most common form of dementia in the elderly. The pathological hallmarks of AD are extracellular senile plaques and intracellular neurofibrillary tangles [1]. It is well known that deposition of β-amyloid (Aβ) is a pivotal event in initiating the neuronal degeneration of AD [2]. Aβ aggregates into amyloid fibrils, which have been reported to be neurotoxic in vitro [3] and in vivo [4]. In this connection, the toxic effect of Aβ on a cultured neuronal cells can be used as a screening tool for identifying potential therapeutic agents for AD. Current clinical treatments of AD patients use acetylcholinesterase inhibitors (AChEIs) and antagonists of Nmethyl-D-aspartate receptors (NMDA) to slow down the progress of the deterioration of AD. However, effective approaches for delaying the progression of AD are yet to be found to date. Thus, searching for safer, better-tolerated, and effective drugs for the treatment of AD remains an important area of drug discovery. Traditional Chinese herbal medicine has been practiced in China for thousands of years, and vast experience has been accumulated for using medicinal herbs for clinical treatment of diseases. Thus, Chinese herbal medicine may be a promising source of effective drugs for treating AD. Uncaria rhynchophylla has been extensively used in Chinese 2 herbal medicine to relieve headache, dizziness, tremors, and hypertension-induced convulsion [5–7]. In recent years, Uncaria rhynchophylla has been shown to be effective for inhibiting Aβ fibril formation, disassembling performed Aβ fibrils [8] and antiacetylcholinesterase [9]. Previous study in our laboratory demonstrated that an ethanol extract of Uncaria rhynchophylla significantly reversed cognitive deficits induced by D-galactose, a mouse model of AD [10]. Phytochemical study has shown that alkaloids, terpenoids, and flavonoids are the major chemical ingredients of Uncaria rhynchophylla [7]. However, the bioactive principles responsible for the protective action of Uncaria rhynchophylla have not been identified. The present study aims to identify the active constituents of Uncaria rhynchophylla using bioassayguided fractionation. Furthermore, the acting mechanism of these active ingredients is explored by using a wellestablished cellular model of Alzheimer’s disease, betaamyloid- (Aβ-) induced neurotoxicity in PC12 cells. 2. Materials and Methods 2.1. Chemicals and Reagents. Nerve growth factor (NGF), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and the fragment of β-amyloid peptide (Aβ25−35 ) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fura 2-AM, Dulbecco’s modified Eagle’s medium (DMEM), horse serum, fetal bovine serum, and a penicillin/streptomycin mixture were purchased from Gibco-Invitrogen (Grand Island, NY, USA). All other solvents and chemicals used in the study were of analytical grade. 2.2. Plant Material. The dried stem with hooks of Uncaria rhynchophylla was purchased from Zhixin Pharmaceutical Co., a GMP-certified supplier of Chinese medicinal herbal materials (Guangzhou, China). It was authenticated to be the dried rhizome of Uncaria rhynchophylla (Miq.) Miq. ex Havil. by Ms. Y. Y. Zong, School of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, where a voucher specimen (no. 091220) has been deposited. 2.3. Preparation of Aggregated Aβ25−35 . The aggregated Aβ25−35 was prepared according to a method described previously [11]. Briefly, Aβ25−35 was dissolved in sterile distilled water at a concentration of 1 mM and incubated at 37◦ C for 4 days to form the aggregation. It was stored at −20◦ C until use. 2.4. Extraction, Fractionation, Isolation, and Identification. Uncaria rhynchophylla (1 kg) was macerated in 6 L of 70% aqueous ethanol for 24 h at room temperature and then refluxed for 30 min. The extraction was repeated twice. The pooled fractions were concentrated using a rotary evaporator at reduced pressure at 40◦ C to yield 140 g of extract (UR-E). The extract was resuspended in water and then transferred into a separatory funnel. The solution was partitioned with ethyl acetate and 1-butanol successively to obtain the ethyl acetate-soluble fraction (UR-E-EA, 43 g), the 1-butanolsoluble fraction (UR-E-B, 28 g), and the water-soluble Evidence-Based Complementary and Alternative Medicine (...truncated)