Effects of Picrasma Quassioides and Its Active Constituents On
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Bioorganic Chemistry 92 (2019) 103258 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg Effects of Picrasma quassioides and its active constituents on Alzheimer's T disease in vitro and in vivo Eryan Guoa, Yunwei Hub, Tao Dua, Huilin Zhua, Lei Chenb, Wei Qua,c, Jie Zhanga, Ning Xied, ⁎ ⁎ Wenyuan Liub, Feng Fenga,c,e, , Jian Xua, a Department of Natural Medicinal Chemistry, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China b Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, China Pharmaceutical University, Nanjing 210009, People’s Republic of China c Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing 211198, People’s Republic of China d State Key Laboratory of Innovative Natural Medicines and TCM Injections, Jiangxi Qingfeng Pharmaceutical Co., Ltd., Ganzhou 341000, Jiangxi, China e Jiangsu Food & Pharmaceutical Science College, Huaian 223003, People’s Republic of China ARTICLE INFO ABSTRACT Keywords: Alzheimer disease (AD), a prevalent neurodegenerative disorder, is one of the leading causes of dementia. Picrasma quassioides However, there is no effective drug for this disease to date. Picrasma quassioides (D.Don) Benn, a Chinese tra- Alkaloids ditional medicine, was used mainly for the treatment of inflammation, fever, microbial infection and dysentery. Alzheimer’s disease In this paper, we reported that the EtOAc extract of Picrasma quassioides stems showed potential neuroprotective activities in L-glutamate-stimulated PC12 and Aβ25-35-stimulated SH-SY5Y cell models, as well as improved memory and cognitive abilities in AD mice induced by amyloid-β peptide. Moreover, it was revealed that the anti-AD mechanism was related to suppressing neuroinflammatory and reducing1-42 Aβ deposition using ELISA assay kits. To clarify the active components of the EtOAc extract of Picrasma quassioides stems, a systematic phytochemistry study led to isolate and identify six β-carboline alkaloids (1–6), seven canthin-6-one alkaloids (7–13), and five quassinoids (14–18). Among them, four β-carbolines (1–3, and 6) and six canthin-6-ones (7–11, and 13) exhibited potential neuroprotective activities in vitro. Based on these date, the structure-activity re- lationships of alkaloids were discussed. Furthermore, molecular docking experiments showed that compounds 2 and 3 have high affinity for both of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYPKIA) and butyrylcholinesterase (BuChE). 1. Introduction reducing neuronal cell death, and suppressing neuronal hyperexcit- ability [8]. Picrasma quassioides (D.Don) Benn, belonging to the family Alzheimer disease (AD), one of the prime leading causes of de- Simaroubaceae, is widely distributed in southern China, Korea, and mentia, is a prevalent neurodegenerative disorder with the character- Japan. This plant was used as anti-inflammation and anti-microbial ization of language disorder, irreversible memory loss, and cognitive infection agent in Chinese traditional medicine [1–3]. Its preparations, impairment [9,10]. The World Alzheimer Report 2015 showed that such as Kumu injection, Kumu mixture, and Xiaoyan Lidan tablets, have over 46 million people have been suffering from AD and this number is been widely used as anti-inflammatory agents. Previous studies re- expected to exceed 131.5 million in 2050 [11]. Currently, there are five vealed that the main chemical constituents of this plant are canthin-6- prescription drugs approved by the U.S. FDA for the symptomatic one alkaloids, β-carboline alkaloids, and quassinoids, and alkaloids are treatment of AD, including acetylcholinesterase inhibitors (donepezil, considered as the major anti-inflammatory active components [4,5]. galantamine, and rivastigmine), and N-methyl-D-aspartate (NMDA) re- Moreover, several alkaloids have been reported to possess neuropro- ceptor (antagonist, and memantine) [12]. Unfortunately, these drugs tective activity [6–8]. For example, picrasidine O exhibited a cerebral only provide symptomatic relief [13]. Hence, it is urgent to develop protective effect through boosting learning and memory performance, more effective and safer drugs and therapeutic strategy forAD. ⁎ Corresponding authors at: Department of Natural Medicinal Chemistry, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China. E-mail addresses: [email protected] (F. Feng), [email protected] (J. Xu). https://doi.org/10.1016/j.bioorg.2019.103258 Received 29 April 2019; Received in revised form 30 July 2019; Accepted 4 September 2019 Available online 05 September 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved. E. Guo, et al. Bioorganic Chemistry 92 (2019) 103258 The etiology of AD remains unclear due to complex and diverse 2.2.4. Cell proliferation assay causes, but several factors, such as neuroinflammation, Aβ deposits, tau EA, NB, and WT were dissolved in DMSO (100 mg/mL) and then hyperphosphorylation, DYRK1A overexpression, oxidative stress, low diluted in medium with 1% FBS to achieve final concentrations. levels of choline (acetylcholine, butyrylcholine), etc., have been greatly Neuroprotection of those extracts were assayed by MTT method on implicated in the AD pathogenesis [12,14]. The neuroinflammation is 10 mM L-glutamate-induced PC12 and 5 μM Aβ25-35 induced SH-SY5Y considered as one of the pathological hallmark of AD [15]. In the pa- cell models. Briefly, the cells (53 ×10 /well) were seeded into 96-well thological state of AD, microglia and astrocytes become activated and plates and incubated overnight. The treatment group were pretreatment release pro-inflammatory cytokines and neurotoxic materials, including with different concentrations of extracts (1, 5, and 10 μg·mL−1, re- tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, nitric spectively) for 2 h. Then, the medium was removed, and the mixtures oxide (NO), and superoxide through activating nuclear factor kappa B with 10 mM L-glutamate (or 5 μM Aβ25-35) and different concentrations (NF-κB) [16,17]. And a series of studies showed that overexpression/ of extracts (1, 5, and 10 μg·mL−1) were added. After incubation for activation of dual-specificity tyrosine phosphorylation-regulated kinase 24 h, 20 μL of MTT solution (5 mg/mL in PBS) was added for each well 1A (DYRK1A) can led to increase Aβ deposits and tau hyperpho- and the cells were incubated in dark for another 4 h. After removal of sphorylation via activating multiple signaling pathways, and DYRK1A the media, 150 μL of DMSO was added. The cell proliferation rate was is considered to be an important target in AD therapy [14]. Besides, the measured, using a microplate reader (Thermo Fisher Scientific Co., BChE is markedly activated in the brains of AD patients, and BuChE Waltham, MA, USA) at 492 nm. inhibitors have become a therapeutic trend for AD [18,19]. However, Cell proliferation rate()( % = A A ) the one-compound-one-target therapeutic strategy for AD has failed, 490 treated 490 model which led to the concern is growing that multi-target therapeutic /A( 490 blankA 490 model) × 100%. strategy could be more promising [20]. Herbal medicines, characterized by multi-component and multi-target, are generally recognized to be effective and safe. Hence, many studies aiming to find a desirable 2.3. Anti-AD tests therapy for AD has focused on traditional medicines. Based on the above considerations, we investigated the neuropro- 2.3.1. Animals and reagents tective effects of different extracts of P. quassioides stems in vitro and in 48 ICR mice (weighing 20 ± 2 g; Qinglong Mountain Animal vivo. Then, we clarified the active constituents of EtOAc extract of Breeding Farm, Nanjing, China) were accommodated in standard an- Picrasma quassioides stems though systematic phytochemical and imal room at temperature 23 ± 2 °C, 60 ± 5% humidity and 12-h pharmacology studies. day/night cycle with free obtain to water and food. The IL-1β, TNF-α, and IL-6 Elisa assay kits were purchased from NeoBioscience Technology Co., Ltd. The Aβ1-42 Elisa assay kit was purchased from 2. Material and methods Nanjing Jin Yibai Biological Technology Co., Ltd. EA was suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) to obtain different 2.1. Plant materials concentration of EA suspensions. Donepezil (Jiangsu Haosen Pharmaceutical Co., Ltd) was used as positive control. The stems of Picrasma quassioides (D.DON) BENN were collected in August 2016 at Ganzhou, Jiangxi Province, People’s Republic of China, 2.3.2. Building of AD animal model and drug treatment and identified by Prof. Feng Feng. A voucher specimen (No. Piqu- After a week of adaptive feeding, all mice were divided randomly 2016GEY-A) was deposited in the Department of Natural Medicinal into 6 groups of 8 each: control group (CT) and model group (Aβ) with Chemistry, China Pharmaceutical University, China. 0.5% CMC-Na, 1.3 mg/kg Donepezil group (Dpz), 25 mg/kg EA group (Low), 50 mg/kg EA group (Medium) and 100 mg/kg EA group (High). 2.2. Active fraction screening After mice were anesthetized fixed on a stereotactic frame (SR-5, Narishige, Tokyo, Japan), 4 μL saline with or without Aβ25–35 (8 μg/ 2.2.1. Sample extraction and fractionation mouse) was infused bilaterally into the left and right hippocampus by a The dry Picrasma quassioides stems (20 kg) was extracted two times glass micropipette with a microinjection pump (Dakumar machinery, with 100 L 80% EtOH by refluxing for 2 h, and the aq. EtOHwas Sweden) at a site 2.0 mm caudal to the bregma, 1.5 mm from the combined and concentrated in vacuum at 60 °C to get the crude extract midline, and 2.0 mm below the dural surface [16]. Then, the heads (290 g). The extract was suspended in water, and then partitioned with were sterilized and stitched. Three days later, mice started to receive ethyl acetate and n-butanol to obtain ethyl acetate extract (64 g, EA), n- 0.5% CMC-Na, Donepezil (1.3 mg/Kg) and EA (25, 50, or 100 mg/kg) butanol extract (78 g, NB) and water extract (130 g, WT), respectively.