**1. Introduction**

Black chokeberry (*Aronia melanocarpa*) is used as an ornamental plant and as a food and colorant. It is rich in the secondary metabolites such as anthocyanins and flavonoids which play vital roles in protecting against oxidative stress and biotic stress [1]. The main anthocyanins in the black chokeberry are cyanidin 3-*O*-galactoside, cyanidin 3-*O*arabinoside, cyanidin 3-*O*-glucoside, and cyanidin 3-*O*-xyloside. These compounds exhibit many bioactivities such as antioxidant, antiproliferative, antimicrobial, anti-inflammation, and modulate hepatic lipid metabolism activities [1–4]. Meanwhile, anthocyanins have been shown to prevent and remedy diseases such as cardiovascular disease, liver failure, obesity, and diabetes [5]. Moreover, anthocyanins can cross the blood-brain barrier (BBB) and delay aging-related degenerative diseases [6–8]. However, the stability of anthocyanins is influenced by many factors such as structure, the presence of solvents, pH, temperature,

**Citation:** Wen, H.; Cui, H.; Tian, H.; Zhang, X.; Ma, L.; Ramassamy, C.; Li, J. Isolation of Neuroprotective Anthocyanins from Black Chokeberry (*Aronia melanocarpa*) against Amyloid-β-Induced Cognitive Impairment. *Foods* **2021**, *10*, 63. https://doi.org/10.3390/ foods10010063

Received: 16 November 2020 Accepted: 24 December 2020 Published: 29 December 2020

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oxygen, and enzymes and other concomitant substances; as such it is still impossible to isolate and purify monomeric anthocyanin from complex natural compounds [9].

Neurodegenerative disorders are becoming more and more prevalent, leading to living and economic burdens on the family members of affected individuals. Alzheimer's disease (AD), one of the most common causes of dementia, is associated with many risk factors including alcohol use, smoking, hypertension, exposure to metals, and oxidative stress [10]. A hallmark of AD is the accumulation of insoluble forms of amyloid-β (Aβ) in the plaques in extracellular spaces and in the walls of blood vessels [10]. Although the pathogenesis of AD is not fully understood, a great deal of research has supported the hypothesis that reactive oxygen species (ROS) impair antioxidant defense systems and induce neuron apoptosis [11]. Many secondary metabolites, like phenolic compounds from Ginkgo biloba, green tea, curcumin, grape, and blueberry, have been reported to protect neuronal cells against oxidative stress in in vivo and in vitro models [12–16].

Simulated moving bed (SMB) chromatography is a continuous countercurrent process which has been used in the separation stage for the large-scale production of compounds such as glucose and fructose [17] and chiral drugs. SMB chromatography is characterized by the separation of a few grams of thermally unstable compounds [18] and chiral drugs, which differ little in terms of their physicochemical properties [19]. Compared to semipreparative liquid chromatography, SMB requires less adsorbent and solvent, and target compounds may be derived with low loss [17,20]. Although SMB is suitable for the isolation of unstable secondary metabolites like phenolic compounds, further studies on its use are necessary. To the best of our knowledge, this study is the first to isolate and purify anthocyanins by SMB chromatography and study both their protective effects against oxidative stress and their neuroprotective ability against Aβ-induced damage in rats.

The specific aims of this study are to develop a new, highly-efficient method to isolate and purify the anthocyanins from black chokeberry, and to investigate the antioxidant activity of the SMB purified anthocyanin extract. An Aβ-induced damage animal model was used to test the effects of SMB-purified anthocyanins on spatial learning and memory ability, as well as nerve cell viability in vivo.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Cyanidin 3-*O*-glucoside standard (99%) was purchased from Sichuan Weikeqi Biological Technology CO., LTD (Chengdu, Sichuan, China) and Cyanidin 3-*O*-galactoside (Cyn-3-gal, 95%) was obtained from HaoChen Ecological Agriculture Development CO., LTD (Shanghai, China). The Aβ1–40 peptides were purchased from Beijing Biosynthesis Biotechnology CO., LTD (Beijing, China). 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from TCI (Shanghai) Development CO., LTD (Shanghai, China). All other organic solvents were of analytical grade.

#### *2.2. Plants Material*

Black chokeberry fruits were harvested at the full maturity stage in September 2017 from black chokeberry demonstration planting base in Wafangdian City (39◦49 21" N, 121◦54 32" E, Dalian, Liaoning, China). Fruits were transported at 4 ◦C and stored at −40 ◦C for a maximum of 6 months.

#### *2.3. Extraction of Anthocyanins*

The black chokeberry fruits were crushed and homogenized using a stainless steel blender, and then extracted twice with 65% ethanol in a 1:8 (*w*/*v*) ratio with 1% acetic acid in an ultrasonic wave bath for 10 min. Then, the supernatants were collected after centrifugation at 6485× *g* (10,000 rpm in an Anke GL-20G-II centrifuge, Shanghai Anke company, Ltd., Shanghai, China) at 25 ◦C for 15 min. The supernatants were evaporated until ap-

proximately a 90% volume was achieved, and then loaded onto a column (2.6 × 60 cm) containing 100 g of the Amberlite® XAD-7HP macroporous resin (Sigma Aldrich Co., St. Louis, MO, USA). The column was washed with deionized water, and then eluted with 1% acetic acid in 35% ethanol at a flow rate of 1 mL/min. Then, the crude extract solutions were evaporated, lyophilized, and stored at −40 ◦C.

#### *2.4. Purification of Anthocyanins by SMB*

Simulated moving bed chromatography (HYSMB6-500) was provided by Beijing Xiang Yue Huang Yu Technology Development Co., Ltd. (Beijing, China). The mathematical principle model is based on a rigorous mathematical first-principles model and the accurate dynamic models of multicolumn continuous chromatographic processes [18]. The anthocyanin extract solutions were homogenized by ultrasound and dissolved in 25% ethanol with 1% acetic acid at a concentration of 50 g/L through a 0.45 μm membrane to form the raw material solution used as the first SMB feed. In this SMB system (Figure 1), a four-zone working model was set up including an eluting zone (zone I), a refining (zone II) and an adsorbing zone (zone III), and a washing zone (zone IV) with two columns per zone. The SMB was equipped with eight columns (10 mm × 150 mm) with C18 (30 μm) filler configured as 2/2/2/2. The fluid was 25% ethanol with 1% acetic acid. Pump F and D pumped into the feeding and desorbent for eluting, and pump E and R pumped out the extract and raffinate. QE, QR, QD, and QF are the flow rates of extract, raffinate, desorbent and feed, respectively. The SMB purified anthocyanin extract (SMB ACN) solutions were dried under vacuum in a rotary evaporator and lyophilized into powder, and then stored at −40 ◦C. HPLC-PDA was used to monitor of the extract and raffinate during the SMB process, and the purity was calculated by the area of the corresponding anthocyanin peak to the total peak area at 278 nm.

**Figure 1.** Schematic illustration of the isolation and purification of anthocyanins from black chokeberry fruits. The anthocyanins were isolated by an ultrasonic extraction method and the crude extract was prepared by XAD-7HP macroporous resins as the feed of simulated moving bed chromatography (SMB). The separation zones of SMB were zones II and III, and the solid phase was regenerated in zones I and IV with two columns per zone. The liquid phase (black) consisted of feed and desorbent for inlets, and extract and raffinate for outlets by pump E and pump R. The liquid phase was continuously fed the crude extract by pump F and eluted with ethanol by pump D. Then, SMB ACN was collected from the extract. The columns switched counter-clockwise (gray) at the switching time intervals.
