Prunella vulgaris L., an Edible and Medicinal Plant, Attenuates

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Prunella vulgaris L., an edible and medicinal plant, attenuates scopolamine induced memory impairment in rats Zhuo Qu, Jingze Zhang, Honggai Yang, Jing Gao, Hong Chen, Changxiao Liu, and Wenyuan Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04597 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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Journal of Agricultural and Food Chemistry

Prunella vulgaris L., an edible and medicinal plant, attenuates scopolamine induced memory impairment in rats Zhuo Qu†, Jingze Zhang‡, Honggai Yang†, Jing Gao†, Hong Chen‡, Changxiao Liu#, Wenyuan Gao*, †

†

Tianjin Key Laboratory for Modern Drug Delivery and High-Efﬁciency, School of Pharmaceutical

Science and Technology, Tianjin University, Tianjin 300072, China ‡

Department of Pharmacy, Tianjin Key Laboratory of Cardiovascular Remodeling and Target Organ

Injury, Logistics University of Chinese People’s Armed Police Forces, Tianjin 300162, China #

The State Key Laboratories of Pharmacodynamics and Pharmacokinetics, Tianjin 300193, China

* Corresponding author: Wenyuan Gao. Tianjin Key Laboratory for Modern Drug Delivery and High-Efﬁciency, School of Pharmaceutical Science and Technology, Tianjin University, Weijin Road, Tianjin, 300072, China. E-mail address: pharmgao@tju,edu.cn. Tel: +86-22-87401895; fax: +86-22-87401895.

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1

ABSTRACT

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Prunella vulgaris L. is as a major plant in Chinese traditional functional beverage Guangdong Herbal

3

Tea for the treatment of fevers, diarrhea, and sore mouth. In this study, ethyl acetate parts of aqueous

4

extracts from Prunella vulgaris L. (EtOAc-APV) were found to demonstrate potent

5

acetylcholinesterase (AChE) inhibition in vitro. Therefore, this study was designed to further

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investigate the effects of EtOAc-APV on scopolamine (SCOP)-induced aging rats. Male Wistar rats

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were randomly divided into 4 groups (n = 12) and given orally by gavage EtOAc-APV (100 mg/kg)

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for 3 weeks. SCOP (1 mg/kg, i.p.) was administrated to rats 30 min before starting behavioral tests

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consecutively for 3 days. EtOAc-APV could attenuate SCOP-induced brain senescence in rats by

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improving behavioral performance and decreasing brain cell damage, which was associated with a

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notable reduction in AChE activity and MDA level, as well as an increase in SOD and GPx activities.

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Additionally, EtOAc-APV administration could reduce the expression of NF-κB and GFAP, which

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played an anti-neuroinflammatory effect on the SCOP-treated rat. Overall, the current study

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highlights Prunella vulgaris L. as an anti-dementia dietary supplement.

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KEYWORDS: Prunella vulgaris L., ethyl acetate parts of aqueous extracts from Prunella vulgaris

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L., acetylcholinesterase inhibitor, neuroprotection, chemical components


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INTRODUCTION

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Acetylcholinesterase (AChE) is a major enzyme associated with the hydrolysis of acetylcholine,

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which plays a principal role in memory and cognition. AChE inhibitors are the standard therapeutic

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medications for the treatment of Alzheimer’s disease. It is documented that AChE inhibitors can

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postpone disease progress and alleviate symptom. However, lack or loss of the therapeutic benefits

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or safety/tolerability concerns has limited the duration of AChE inhibitor therapy 1. It has been

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recently reported that natural substances and dietary components exhibit inhibitory activity against

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AChE in different tissues 2. Several synthetic and natural origin AChE inhibitors are available in the

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drug market. However, side effects and relatively low bioavailability limit their uses in medicine.

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Therefore, there is still a great demand to discover new AChE inhibitors 3.

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Prunella vulgaris L., commonly known as self-heal, is a perennial herb widely distributed in

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Korea, Japan, China, and Europe. In Europe, Prunella vulgaris L. is a popular therapy since the 17th

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century for the treatment of mild fever, sore throat, and wound healing 4. In Asian countries, it has

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been commonly used as food or tea for a long time

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spikes were used in typical dose of 9-15 g per day for different ailments 7. It also has been

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extensively used as a health-promoting food or tea in China 8. For example, Prunella vulgaris L. is as

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a major plant in Guangdong Herbal Tea. Guangdong Herbal Tea, well known as

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Wanglaoji-Liang-Cha, has been consumed as a healthy beverage in Southern China for a long time 9.

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In addition, the aerial parts of Prunella vulgaris L. are often used as pig feed and its leaves are used

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as raw or cooked in salads and soups in the cuisine of Southern China 10, 11.

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5, 6

. In China, the aqueous extract from its fruit

Phytochemical studies on Prunella vulgaris L. have revealed the presence of a large amount of compounds (rosmarinic acid (RA), caffeic acid (CA), triterpenoids, and tannins)


12

. Plenty of cell


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culture studies have demonstrated that dietary phenolics like RA and CA, which are all found in

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extracts of Prunella vulgaris L., have antioxidant, anti-inflammatory, anti-mutagenic, anti-viral, and

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neuroprotective activities

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vulgaris L. colud attenuated scopolamine (SCOP)-induced memory deficits in mice

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study documented that nutritional antioxidants-rich dietary could improve cognitive function

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Although a variety of pharmacological activities of Prunella vulgaris L. and its main active

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ingredients, such as CA and RA, have been documented, few studies have assessed their

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neuroprotective effects and potential mechanisms. Thus, we examined the neuroprotective effects

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and potential mechanisms of ethyl acetate parts of aqueous extracts from Prunella vulgaris L.

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(EtOAc-APV) both in vitro and in vivo. In vitro AChE inhibitory activity and in vivo SCOP-induced

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aging rat model were used in the present study.

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EXPERIMENTAL PROCEDURES

13, 14

. Recently it has been reported that the ethanol extract of Prunella 15

. Previous 16

.

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Materials and Reagents. SCOP, CA, RA, and donepezil (DON) were obtained from National

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Institute for Control of Pharmaceutical and Biological Products (Beijing, China). The purity of CA

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and RA were over 99.0%. Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione

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(GSH), nitric oxide (NO), glutathione peroxidase (GPx), AChE, and acetylcholine (ACh) assay kits

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were obtained from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China).

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Anti-nuclear factor (NF)-κB p65, anti-Caspase-3, and anti-glial fibrillary acidic protein (GFAP)

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antibodies were purchased from Boster Biological Engineering Co., Ltd. (Wuhan, China).

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Biotinylated goat anti-rabbit secondary antibody and 3, 3’-diaminobenzidine tetrahydrochloride were

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purchased from ZSGB-BIO (Beijing, China). AChE was produced by Sigma–Aldrich Co. (St. Louis,

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MO, USA).


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Prunella vulgaris L. was obtained from Tianjin Tasly Pharmaceutical Co., Ltd. (Tianjin, China).

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The crude herb was authenticated by Prof. Wenyuan Gao (Tianjin University, China). Prunella

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vulgaris L. powder (500 g) was extracted with 5 L distilled water for 2 h. The filtrate was collected

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and evaporated. The yield of the aqueous extract of Prunella vulgaris L. (APV) was 3.49% (w/w).

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Next, APV (10 g) was suspended in 500 mL of distilled water and extracted with 500 mL of the

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following solvents in a stepwise manner: hexane, ethyl acetate, and butanol. Each fraction was

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collected and evaporated under reduced pressure and then lyophilized with a vacuum and freeze

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dryer (FD-10-50, Beijing Boyikang Experimental Instrument Co., Ltd, Beijing, China). Finally, the

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extraction of hexane (Hexane-APV), ethyl acetate (EtOAc-APV), butanol (Butanol-APV), and water

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(Water-APV) were obtained. The dry residues were stored at -20°C.

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Determination of Total Phenolics Content. The total phenolics amount in APV and its fractions

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were measured using the Folin-Ciocalteu method described previously 17. Briefly, APV, Hexane-APV,

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EtOAc-APV, Butanol-APV, or Water-APV 100 µL was transferred to separate tubes containing 120

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µL distilled water and 40 µL Folin-Ciocalteu reagent. After incubation at room temperature for 5 min,

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the solution was mixed with 40 µL of 20% sodium carbonate. An absorbance was detected at 680 nm

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using UV visible spectrophotometer (UV-754, Tianjin Precise Instrument Co., Ltd, Tianjin, China).

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The concentration of total phenolics was calculated from a calibration curve of catechin. The data

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were expressed as milligrams catechin equivalent (CE)/g dry weight of extract.

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Determination of Total Flavonoids Content. The total flavonoids amount in APV and its

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fractions were determined using the previous method with some modifications. APV, Hexane-APV,

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EtOAc-APV, Butanol-APV, or Water-APV 2.0 mL was mixed with 0.4 mL 5% NaNO2 solution in a

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10 mL flask. The mixture was allowed to stand for 6 min. Then 0.3 mL 5% NaNO2 solution was



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added to the mixture. After 6 min, 4 mL 4% NaOH solution and 3.2 mL ethanol were added. After

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thoroughly mixing, the absorbance was measured at 510 nm using UV visible spectrophotometer

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(UV-754, Tianjin Precise Instrument Co., Ltd, Tianjin, China). The concentration of total flavonoids

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was calculated from a calibration curve of rutin. The data were expressed as milligrams rutin

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equivalent (RE)/g dry weight of extract.

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Quantitative Analysis of CA and RA. Contents of CA and RA were determined in APV and

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different fractions by HPLC-PAD. CA 3.29 mg and RA 4.90 mg were accurately weighed and

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dissolved with 5 mL of methanol in a volumetric flask as standard solution, respectively. APV 10.33

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mg, Hexane-APV 10.04 mg, EtOAc-APV 10.77 mg, Butanol-APV 10.40 mg, and Water-APV 10.62

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mg were accurately weighed and dissolved with 5 mL of solvent in a volumetric flask. The

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supernatant was filtered through a 0.45 µm syringe filter before HPLC analysis. Contents of CA and

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RA were determined using the standard curve. The HPLC system (Waters Co., USA) was used for

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sample analysis. The HPLC system equipped with a Waters 2998 photodiode array detector, a Waters

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1525 binary HPLC pump, a waters column heater module and Empower Pro data station.

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Chromatographic separation was performed at 30oC on Phenomenex C18 column (4.6 mm × 250

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mm I.D., 5 µm). Flow rate of mobile phase, consists of 0.1% aqueous formic acid (A) and methanol

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(B), was 1 mL/min. The gradient profile was optimized as follows: 0 ~ 30 min: 30% ~ 60% B. The

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samples were detected at 330 nm and injection volume was 20 µL.

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UHPLC-Q-TOF-MS/MS Analysis Conditions. EtOAc-APV (30.12 mg) was accurately weighed

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and dissolved with 25 mL of methanol in a volumetric flask. A 0.45 µm syringe filter was used to

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filter before UHPLC analysis. The UHPLC-Q-TOF-MS instrument and MS conditions were

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described as our previous study

18

. Chromatographic separation was performed at 30oC on an


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reversed phase C18 column (150 mm × 2.1 mm I.D., 5 µm, ZORBAX SB-C18). Flow rate of mobile

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phase, consists of 1% acetic acid in water (A) and acetonitrile (B), was 0.4 mL/min. The gradient

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profile was optimized as follows: 5% B to 10% B during first 2 min, followed by 10% B to 45% B

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during 2 ~ 5 min, 45% B to 75% B during 5 ~ 8 min, 75% B to 100% B during 8 ~ 18 min. The

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samples were detected at 330 nm and injection volume was 5 µL.

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In Vitro Microplate Assay for AChE Inhibitory Activity. AChE inhibitory activity was 2, 19

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determined using the method described by previous research

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and sample solution 400 µL (7.0, 14.0, 28.0, 56.0, 112.0, or 224.0 µg/mL) were added to 860 µL

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phosphate buffer saline, and the mixture was incubated at 37oC for 30 min. Then the reaction was

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started by adding 5,5'-dithiobis-(2-nitrobenzoic acid) (125 µL, 3 mM in phosphate buffer saline) and

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acetylthiocholine iodide (25 µL, 15 mM in phosphate buffer saline). The reaction mixture was

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incubated at 37oC for 30 min and was terminated by adding physostigmine (20 µL, 0.1 mM in

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methanol). The absorbance of the mixture was detected at 412 nm using a microplate reader

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(DNM-9602, Beijing Pulangxin Technology Co., Ltd, Beijing, China). The inhibition ratio was

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calculated using the equation.

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. Briefly, 25 µL 0.22 U/mL AChE

I (%) = 100 – (Asample/Acontrol) × 100

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Animals and Drug Administration Protocols. Male Wistar rats, weighing about 280-330 g, were

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purchased from Experimental Animal Center, Chinese Academy of Medical Sciences, Peking, SCXK

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(Jing) -2014-0013. Animal feeding conditions and animal ethics were described by our previous

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study 18. In the pharmacodynamics study, the rats (n = 48) that were able to find the hidden platform

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within 90 s were randomly divided into 4 groups (n = 12): control group (Control), SCOP alone

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(SCOP), SCOP + DON 1 mg/kg (DON), and SCOP + EtOAc-APV 100 mg/kg (EtOAc-APV).



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EtOAc-APV and DON were suspended in carboxymethylcellulose sodium aqueous solution.

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EtOAc-APV or DON was administered orally once daily for 3 weeks. SCOP (1 mg/kg, i.p.) was

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administrated to rats 30 min before starting behavioral tests consecutively for 3 days. Control rats

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were administered with an equal volume saline. The dosages of SCOP, DON, and EtOAc-APV were

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established by previous work 12, 20. Figure 1 exhibited the experimental protocol.

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Morris Water Maze Test. After the 3 weeks treatment, Morris water maze test was carried out to

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assess spatial memory of the rats (n = 12 each group). The test was conducted as described

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previously with minor modifications

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diameter, 40 cm in height) filled with a non-toxic black colored water (23 ± 1oC). A circular escape

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platform (10 cm in diameter, 20 cm in height) was submerged 1 cm below the water surface and

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located in the center of the target quadrant. Each rat received 4 trials per day for 6 consecutive days.

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Escape latency (the time required to find the submerged escape platform) were measured. On the 7th

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day, a probe test was conducted to assess memory consolidation. During the probe test, the platform

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was removed and the rats swam in the tank for 90 s. For the probe trials, target quadrant occupancy

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(the time that rat spent in the target quadrant which previously contained the platform) and crossing

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times (the times that the rats crossed the position of the platform during learning session) were

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measured. All of the tests were recorded using a camera installed on the roof and analyzed using a

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behavioral analysis system (Anhui Zhenghua Biological Equipment Co., Ltd, Anhui, China).

21

. Briefly, the maze was a black circular pool (100 cm in

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Biochemical Estimation in Brain. For the biochemical determination, the levels or activities of

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ACh, AChE, GPx, GSH, MDA, NO, and SOD in the hippocampus were determined using

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commercially available assay kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing,

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China) according to the manufacturer’s protocol.


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Immunohistochemical Analysis. For immunohistochemical studies, brains were fixed with 4%

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paraformaldehyde solution after transcardially perfused with normal saline. After dehydrating, the

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sections (4 µm thickness) were rinsed in phosphate buffer saline for 3 times and then incubated with

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3% H2O2 and 3% goat serum in order to block the endogenous peroxidase activity. The brain sections

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were immunostained overnight at 4°C with rabbit anti-Caspase-3, anti-GFAP, and anti-NF-κB p65

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antibodies diluted 1:100, then with biotinylated goat anti-rabbit secondary antibody diluted 1:500 in

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phosphate buffer saline at 37°C for 1 h. The slides were subsequently exposed to an

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avidin–biotin–horseradish peroxidase complex for 15 min at 37oC. The reaction subsequently was

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visualized with 3, 3’-diaminobenzidine tetrahydrochloride. Images were captured with a microscope

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and digital sight camera (ECLIPSE TS 100, Nikon). Measure of the immunoreactivity of NF-κB,

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Caspase-3, and GFAP proteins were quantified using Image Pro Plus software (IPP 6.0, Media

160

Cybernetics).

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Histopathological Analysis. For the tissue analysis (n = 6 rats per group), brain tissues were fixed

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with 4% paraformaldehyde solution for 24 h. After dehydrating, the brain was embedded in paraffin

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blocks. Then, coronal sections of 4 µm thicknesses were analyzed using Nissl staining. The images

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were captured by a digital camera (ECLIPSE TS 100, Nikon) and analyzed by IPP 6.0.

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Statistical Analysis. The statistical analyses were carried out using SPSS 20.0 system (Chicago,

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IL). Data were analyzed by One-way ANOVA for comparison of mean values across the groups.

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Multiple comparisons were made by LSD test. Pearson correlation coefficients were used to

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represent the correlation. Differences at P < 0.05 was regarded as being statistically significant.

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RESULTS

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Total Phenolics and Total flavonoids Contents in APV and the Different Fractions. Total



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phenolics contents in APV, Hexane-APV, EtOAc-APV, Butanol-APV, and Water-APV tested in this

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study are presented in Table 1. In general, the highest total phenolics content was observed in

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EtOAc-APV fraction, followed by Butanol-APV, APV, and Hexane-APV. Water-APV exhibited

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significantly lower total phenolics content than the other fractions. In addition, the contents of total

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flavonoids in different fractions of Prunella vulgaris L. were obvious lower than the total phenolics

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(Table 1).

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Contents of CA and RA in APV and the Different Fractions. Amounts of CA and RA in APV,

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Hexane-APV, EtOAc-APV, Butanol-APV, and Water-APV were finally quantified using

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corresponding calibration curves y = 53032 x + 39125 (r2 = 0.999) and y = 57333 x + 11207 (r2 =

180

0.999), respectively. The representative HPLC chromatograms were shown in Figure 2. The contents

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of CA and RA were summarized in Table 1.

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Analysis of Chemical Composition of EtOAc-APV by UHPLC-Q-TOF-MS. Based on the

183

obtained UV spectra, retention time, and mass spectra, totally 8 compounds were identified in

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EtOAc-APV, including protocatechuate, benzaldehyde, salviaflaside, CA, RA, and 3 unknown

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compounds (Table 2, Figure 3). These 8 identified compounds represented the major components of

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EtOAc-APV.

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In Vitro AChE Inhibitory Activities of APV and the Different Fractions. As shown in Figure 4,

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all of the 5 fractions exhibited AChE inhibitory activities. The IC50 values of AChE inhibitory

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activities of APV, Hexane-APV, EtOAc-APV, Butanol-APV, and Water-APV were 146.8 ± 9.4, 156.0

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± 11.6, 101.8 ± 6.9, 217.9 ± 17.43, and 296.3 ± 15.7 µg/mL, respectively. AChE inhibitory activity of

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EtOAc-APV was more powerful than others.

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In Vivo Neuroprotective Effect of EtOAc-APV on SCOP-induced Memory Injury Rats.


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EtOAc-APV showed more powerful AChE inhibitory activity in vitro than others. Therefore, we

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only investigated the in vivo neuroprotective effect of EtOAc-APV in the following study.

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EtOAc-APV Restored Memory Impairment Caused by SCOP Administration. Morris water

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maze test is a most commonly used laboratory tools for investigating spatial learning and memory.

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During the training trials, all rats improved their performance as their escape latency decreased

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across successive days (Figure 5A). The present results showed that the platform crossing times and

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the time in the target quadrant were significantly lower in SCOP-treated rats than those in control

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rats in probe trials. These results indicated that the memory ability was impaired in SCOP-treated

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rats. However, EtOAc-APV treatment reversed the extension of escape latency caused by SCOP on

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the last day of the training session (Figure 5A). Moreover, the reduced platform crossing times

203

(Figure 5B) and the shortened time in the target quadrant (Figure 5C) triggered by SCOP were

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significantly increased by EtOAc-APV treatment. Figure 5D represented the traces on the last day of

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hidden platform training.

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EtOAc-APV Attenuated Oxidative Damage Caused by SCOP Administration. To examine

207

whether the anti-aging effect of EtOAc-APV was involved in attenuating oxidative damage, SOD

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and GPx activities, as well as MDA and GSH levels in the hippocampus were measured. As shown in

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Table 3, the levels of MDA and NO in SCOP group were significantly increased compared to control

210

group. However, the rise of MDA and NO levels were attenuated by treatment of EtOAc-APV. We

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also found that SCOP administration caused a remarkable decline in GSH level, GPx level, and SOD

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activity, which were significantly reversed by EtOAc-APV. In addition, relationships between

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learning ability and oxidative markers or antioxidant enzymes were analyzed by Pearson correlation.

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Results revealed that the escape latency was positively correlated with MDA level (r = 0.931,



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Supporting Information Figure SA) and NO level (r = 0.896, Supporting Information Figure SB); but

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negatively correlated with SOD activity (r = - 0.668, Supporting Information Figure SC), GSH level

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(r = - 0.785, Supporting Information Figure SD), and GPx activity (r = - 0.403, Supporting

218

Information Figure SE).

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EtOAc-APV Inhibited AChE Activity and Enhanced ACh Level in the Hippocampus of

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SCOP-treated Rats. In the present study, cholinergic markers like ACh level and AChE activity in

221

the hippocampus were investigated. Compared with the control group, ACh level decreased

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significantly and AChE activity increased remarkably in SCOP administration group (Figure 6).

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Meanwhile, EtOAc-APV rescued the reduction of ACh level significantly, and partially rescued the

224

increase of AChE activity (Figure 6).

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EtOAc-APV Attenuated the Activation of NF-κB in the Hippocampus of SCOP-treated Rats.

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To examine whether EtOAc-APV could inhibit the inflammation caused by SCOP, we performed

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immunohistochemistry analysis to measure the expression of NF-κB. SCOP administration

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dramatically elevated the expression of NF-κB in the hippocampus compared with that of control

229

group (Figure 7). Interestingly, oral administration of EtOAc-APV notablely suppressed the

230

expression of NF-κB as demonstrated by the immunohistochemistry staining (Figure 7).

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EtOAc-APV Suppressed the Activation of Astrocytes in the Cortex of SCOP-treated Rats. To

232

further investigate whether EtOAc-APV suppressed the activation of astrocytes in SCOP treated rat

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brain to exert its neuroprotection, we measured the GFAP expression in rat brain. GFAP

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immunohistochemistry demonstrated that the pathological changes of astrocytes were serious in the

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cortex of rats injected with SCOP. However, treatment with EtOAc-APV markedly inhibited

236

astrocytes activation compared to the SCOP group (Figure 7), showing the reduced number of


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GFAP-positive cells in the cortex. EtOAc-APV Prevented Apoptosis and Neurodegeneration in SCOP-treated Rats. To evaluate

239

the

effect of EtOAc-APV on SCOP caused apoptosis,

we measured Caspase-3 by

240

immunohistochemistry analysis. Results are dislayed in Figure 7, revealing that SCOP led to marked

241

augment in the expression of Caspase-3 in the hippocampus of SCOP-treated rats. As expected,

242

EtOAc-APV remarkably reduced Caspase-3 level. In SCOP group, the number of the

243

Caspase-3-positive staining was markedly rised compared with that in control group. In EtOAc-APV

244

group, Caspase-3-positive staining was moderately detected in the hippocampus, and the mean

245

number of the Caspase-3-positive staining was markedly declined compared with that in SCOP

246

group.

247

Nissl staining results revealed that the neuron loss and nucleus shrinkage/disappearance appeared

248

in the CA1 region of SCOP rats. Moreover, the numbers of Nissl bodies remarkably declined in

249

SCOP rats compared to control rats. EtOAc-APV treatment for 3 weeks remarkably reduced the

250

neuropathological changes and elevated the density of healthy neurons in the CA1 region in SCOP

251

rats (Figure 8).

252

DISCUSSION

253

The present study showed that EtOAc-APV exhibited the AChE inhibitory activity in vitro and could

254

prevent SCOP-induced rat memory injury in vivo. The neuroprotective effect of EtOAc-APV could

255

be related to the antioxidant system, since it reduced the elevation of MDA and NO, as well as the

256

reduction of SOD, GSH, and GPx triggered by SCOP. In addition, EtOAc-APV improved the

257

function of cholinergic system, as it reversed the abnormality of AChE activity and ACh level in

258

SCOP rat brain. What’s more, EtOAc-APV could also exert neuroprotective activity by inhibiting the



259

neuroinflammation, like as attenuating the activation of astrocyte and the expression of NF-κB. All

260

above observation could be strengthened by the histopathological analysis, showing the

261

morphological changes of neurons. The mechanisms and pathways responsible for the

262

neuroprotective effect of EtOAc-APV are depicted in Figure 9.

263

Previous chemical components studies have shown that Prunella vulgaris L. contains multiple

264

ingredients and is rich in phenolics, flavonoids, triterpenes, polysaccharides, and anthraquinones.

265

These active components are known to significantly affect human health 17. In the present study, the

266

contents of total phenolics, flavonoids, CA, and RA in APV and the different solvent extractions of

267

APV were determined. Results showed that the different solvent extractions of APV contained much

268

more total phenolics than flavonoids. Also, CA and RA in EtOAc-APV were much higher than those

269

in other solvent extractions.

270

Next, we further investigated the bioactivity of APV, Hexane-APV, EtOAc-APV, Butanol-APV,

271

and Water-APV. It has been documented that the diversity ingredients of Prunella vulgaris L.

272

contributes to a wide range of pharmacological activities, including anti-oxidant, anti-allergic,

273

anti-microbial, etc. 22. In addition, it has been reported that the ethanol extract and active ingredients

274

derived from Prunella vulgaris L. showed neuroprotective acitivities. Therefore, we further

275

investigated the AChE inhibitory activities of APV, Hexane-APV, EtOAc-APV, Butanol-APV, and

276

Water-APV. AChE inhibitors may also be insecticides and exert neurotoxicity. Neuroprotective and

277

neurotoxic activities of AChE inhibitors may be related to the chemical structure. Therefore, both

278

sides of the AChE inhibitors’ effects should be considered. For example, AChE inhibitors used for

279

the treatment of Alzheimer’s disease are mostly piperidine derivatives and alkaloid. However, the

280

toxic AChE inhibitors are carbamate and organophosphorus compounds 23. The main components in


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281

Prunella vulgaris L. are phenolics. According to the present results, EtOAc-APV showed potent

282

AChE inhibitory activity in vitro. Besides, it has been documented that CA and RA, the active

283

ingredients in EtOAc-APV, exert neuroprotective effect. Therefore, we further assessed the

284

neuroprotective effect of EtOAc-APV on SCOP-induced aging rats in vivo.

285

Morris water maze test is a most commonly used laboratory tools for investigating spatial learning

286

and memory 24. SCOP, a muscarinic cholinergic antagonist, is regard as an agent for the induction of

287

cognitive impairment in healthy individuals. A chronic administration with a high dose of SCOP

288

induces changes that resemble natural aging in animals, such as cognitive dysfunction, oxidative

289

stress, cholinergic dysfunction, biochemical and pathological alterations of astrocytes and apoptosis

290

25

291

function

292

salivation and smooth muscle function have not been directly assessed. In the present study, we

293

observed that EtOAc-APV could reverse the memory deficits caused by SCOP as evident from the

294

Morris water maze test. To further study the molecular mechanisms involved in the recovery ability

295

of aged brain by treatment with EtOAc-APV after SCOP-induced impairment, we used

296

immunohistochemistry and biochemical assay to measure the expression of a representative protein

297

or enzyme-associated aging in rat brain.

. Besides, SCOP has non-behavioral effects on pupil diameter, salivation and smooth muscle 26

. To the best of our knowledge, effects of Prunella vulgaris L. on pupil diameter,

298

Age-related disruptions of cholinergic function have been demonstrated 27. It is well known that

299

the central cholinergic system is associated with learning and memory process regulated by ACh 28.

300

AChE is a key cholinergic enzyme that regulates the level of ACh in the brain 27. AChE inhibitors

301

possess positive effects on cognition in Alzheimer’s disease patients. In this study, we found that

302

chronic injection of SCOP significantly elevated AChE activity; this effect could be reversed by



303

EtOAc-APV treatment. Therefore, our results suggested that the potentiality of EtOAc-APV in

304

attenuating the reduction of memory impairment induced by SCOP might be via the inhibition of

305

AChE activity in rat brain.

306

Oxidative stress of reactive oxygen species may be involved in aging. SOD and GPx are key

307

enzymes to scavenge reactive oxygen species. MDA is a marker of lipid peroxidation; while GSH is

308

the substrate of GPx and its consumption decreases with age 29. Therefore, SOD and GPx activities,

309

as well as MDA and GSH levels in the hippocampus were determined to examine whether the

310

anti-aging effect of EtOAc-APV was involved in attenuating oxidative damage in the present study.

311

Earlier research reported that the RA-rich extract from Prunella vulgaris L. could inhibit

312

lipoperoxidation and scavenge superoxide radical 30. The present results indicated that EtOAc-APV

313

treatment reduced SCOP-induced oxidative damage by reducing MDA and increasing SOD and GSH.

314

However, it has been reported that phenolics are rapidly conjugated by phase II enzymes and

315

therefore do have no or limited antioxidant activity in vivo. Nevertheless, phenolics may have

316

indirect antioxidant effects via activating HO-1 by the ARE/Nrf2 pathway to prevent neurons

317

oxidative damage

318

contribute to potent antioxidant activity of phenolics-rich EtOAc-APV.

31

. Therefore, we suppose that the activation of the ARE/Nrf2 pathway may

319

Astrocytes are the brain phagocytic cells and their activation is considered to be a part of the

320

brain’s inflammatory reaction in response to neurodegeneration. Astrocyte activation is generally

321

evidenced by the over-expression of GFAP 32. Therefore, we evaluated the expression of GFAP by

322

immunohistochemisty in this study. Results displayed that rat administration of EtOAc-APV could

323

reverse the elevation of GFAP induced by SCOP, suggesting that the neuroprotective property of

324

EtOAc-APV was partly through its anti-inflammatory activity. In addition, neuronal loss is an


Page 16 of 38

Page 17 of 38


325

important characteristic alteration in Alzheimer’s disease. Neurodegenerative changes of Alzheimer’s

326

disease were related with inflammatory cytokines and apoptogenic signaling. Accordingly, activity of

327

Caspase-3 suggested the apoptosis and the neuronal loss triggered by SCOP 33. In the present study,

328

we found that EtOAc-APV intake 100 mg/kg/day could substantially down-regulate Caspase-3

329

expression, suggesting that EtOAc-APV is neuroprotective.

330

Analysis of chemical composition of EtOAc-APV showed that phenolics were the main

331

constituents, especially CA and RA. CA and RA could be detected in rat plasma after oral

332

administration. RA is immediately metabolized, and one of its metabolites is CA 34. Accumulating

333

data from experimental studies indicated that CA and RA had neuroprotective effects, and CA had

334

central analgesic activity, possibly by crossing the blood-brain barrier. Therefore, we suspected that

335

the outstanding neuroprotective activity of EtOAc-APV might be associated with the high amounts

336

of CA and RA in EtOAc-APV.

337

In summary, the present study demonstrated that EtOAc-APV could attenuate SCOP-induced

338

brain senescence in rats by improving behavioral performance and decreasing brain cell damage,

339

which was associated with a notable reduction in AChE activity and MDA level, as well as an

340

increase in SOD and GPx activities. Additionally, EtOAc-APV administration could reduce the

341

expression of NF-κB and GFAP, which played an anti-neuroinflammatory effect on the SCOP-treated

342

rat. In general, these data will help reveal more about Prunella vulgaris L. as an anti-dementia

343

dietary supplement.

344 345

ACKNOWLEDGMENTS

346

The work was supported by Special Financial Grant from the China Postdoctoral Science Foundation



347

(No. 2015T81140), PhD research startup foundation of Logistics University of Chinese People's

348

Armed Police Forces (No. WHB201509) and Science and Technology Support Program Foundation

349

of Tianjin China (No. 15CZDSY01020).

350

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351

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Impairment in Rats. Prev. Nutr. & Food Sci. 2015, 20, 126.

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(30) Škottová, N.; Kazdová, L.; Oliyarnyk, O.; Večeřa, R.; Sobolová, L.; Ulrichová, J., Phenolics-rich extracts from

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Silybum marianum and Prunella vulgaris reduce a high-sucrose diet induced oxidative stress in hereditary

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hypertriglyceridemic rats. Pharmacol. Res. 2004, 50, 123-130.

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(31) Scapagnini, G.; Sonya, V.; Nader, A. G.; Calogero, C.; Zella, D.; Fabio, G., Modulation of Nrf2/ARE pathway

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Neurobiol. 2011, 44, 192-201.

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nanoparticles for oral delivery of estradiol to rat brain in a model of Alzheimer's pathology. J. Control. Release.

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Neurosci. 2012, 220, 191-200.

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440

FIGURE CAPTION

441

Figure 1. The sequence of in vivo study procedures. After 1 week acclimatization, all rats were

442

screened by the Morris water maze test. DON and EtOAc-APV were administered by oral gavage for

443

2 weeks prior to the training for Morris water maze test, and continued when the rats were undergone

444

the test. During Morris water maze test, DON and EtOAc-APV were administered 1 h before the trial,

445

and SCOP was intraperitoneally (i.p.) injected 30 min thereafter consecutively for 3 days. Morris

446

water maze test were performed at 3rd week. After behavioral tests, rats were sacrificed and brain

447

tissues were collected for biochemical analysis or histological examination.

448

Figure 2. Representive HPLC chromatograms of (A) APV, (B) Hexane-APV, (C) EtOAc-APV, (D)

449

Butanol-APV, and (E) Water-APV.

450

Figure 3. The base peak chromatogram (BPC) of EtOAc-APV by UHPLC-Q-TOF-MS in negative

451

ion mode.

452

Figure 4. AChE inhibitory activities of (A) APV, Hexane-APV, EtOAc-APV, Butanol-APV,

453

Water-APV, and (B) the positive control DON.

454

Figure 5. EtOAc-APV improved the memory ability on SCOP-induced rat memory impairment.

455

EtOAc-APV was administered (100 mg/kg/day) by oral gavage 2 weeks prior to the training for

456

Morris water maze test, and continued when the rats were undergone the test. Memory impairment

457

was induced by SCOP (1 mg/kg, i.p). (A) Escape latency of rats in hidden platform tests for 6

458

consecutive days; (B) Platform crossing times in probe trials; (C) The time in the target quadrant in

459

probe trials; (D) Swimming tracks on the last day of hidden platform training. The results are

460

presented as mean ± standard errors (n = 6). Different letters represent significantly different values

461

as assessed by One-way ANOVA and LSD tests with P < 0.05.

462

Figure 6. EtOAc-APV treatment attenuated the increase of AChE and decrease of ACh induced by



463

SCOP on rats. The results are presented as mean ± standard errors (n = 6). Different letters represent

464

significantly different values as assessed by One-way ANOVA and LSD tests with P < 0.05.

465

Figure 7. EtOAc-APV reduced the expression of Caspase-3 and NF-κB in the CA1 regions of the

466

hippocampus of SCOP-treated rats and GFAP in the cortex of SCOP-treated rats. The results are

467

presented as mean ± standard errors (n = 6). Different letters represent significantly different values

468

as assessed by One-way ANOVA and LSD tests with P < 0.05.

469

Figure 8. Representative photomicrograph of Nissl staining in the CA1 regions of the hippocampus

470

of SCOP-treated rats. Neuronal cell death increased after injection of SCOP in the hippocampus of

471

rat brain. EtOAc-APV treatment significantly decreased neuronal cell death in SCOP-treated rats.

472

The results are presented as mean ± standard errors (n = 6). Different letters represent significantly

473

different values as assessed by One-way ANOVA and LSD tests with P < 0.05.

474

Figure 9. Proposed mechanism of the neuroprotective effect of EtOAc-APV on SCOP-induced rat

475

memory injury. EtOAc-APV produces neuroprotective effects via inhibition of the cholinergic

476

deficiency, oxidative damage, neurons apoptosis, and neuroinflammation.


Page 24 of 38

Page 25 of 38


Table 1 Contents of total phenolics, total flavonoids, CA, and RA in various solvent fractions obtained from the water extract of Prunella vulgaris L. Fractions

Total flavonoids ( RE µg/mg)

Total phenolics (µg CE/mg)

CA (µg/mg ext.)

RA (µg/mg ext.)

APV

3.16 ± 0.27

102.69 ± 17.56

6.14 ± 0.11

29.36 ± 0.35

Hexane-APV

2.97 ± 0.51

79.47 ± 4.44

2.99 ± 0.04

34.59 ± 0.33

EtOAc-APV

11.95 ± 0.54

316.75 ± 17.72

54.89 ± 0.51

233.09 ± 1.92

BuOH-APV

6.14 ± 1.05

120.17 ± 10.02

2.62 ± 0.02

38.63 ± 0.63

Water-APV

7.51 ± 0.64

40.18 ± 2.49

ND

2.68 ± 0.06

Total phenolics contents expressed in milligrams of catechin equivalent (CE)/g dry weight. Total flavonoids contents expressed in milligrams of rutin equivalent (RE)/g dry weight. Values are mean ± standard errors (n = 3). ND: not detection.



Page 26 of 38

Table 2 Chromatographic and mass spectral data of the 8 compounds of EtOAc-APV analyzed by UHPLC–Q-TOF-MS. Peak No. Retention time

Identified compound

Chemical structure

Formula

Molecular weigh (Da)

Negative ion (m/z)

198

197[M-H]-, 295[2M-H]-

1

2.681

Unknown

2

2.962

Protocatechuate

C7H6O4

154

153[M-H]-

3

3.538

Benzaldehyde

C7H6O3

138

137[M-H]-

4

3.867

Caffeic acid

C9H8O4

180

179[M-H]-

5

4.501

Salviaflaside

C24H26O13

522

521[M-H]-


Page 27 of 38


360

359[M-H]-

Unknown

344

343[M-H]-, 687[2M-H]-

Unknown

388

387[M-H]-, 775[2M-H]-

6

5.358

Rosmarinic acid

7

6.310

8

8.917

C18H16O8



Page 28 of 38

Table 3 Effect of EtOAc-APV on oxidative and anti-oxidant parameters in SCOP-induced aging rats Groups

MDA (nM/mg protein)

NO (nM/mg protein)

GSH (mg/g protein)

GPx (mU/mg protein)

SOD (U/mg protein)

Control

91.10 ± 3.97 a

158.09 ± 9.53 a

36.90 ± 4.07 b

22.07 ± 2.53 c

0.75 ± 0.04 b

SCOP

130.58 ± 17.41 c

198.20 ± 9.19 d

24.13 ± 2.55 a

16.54 ± 3.82 a

0.65 ± 0.05 a

DON

102.74 ± 14.92 b

167.25 ± 8.96 b

35.00 ± 4.53 b

18.51 ± 3.34 b

0.73 ± 0.04 b

EtOAc-APV

109.90 ± 16.93 b

176.75 ± 6.93 c

32.89 ± 3.72 b

19.22 ± 2.60 b

0.72 ± 0.04 b

The results are presented as mean ± standard errors (n = 6). Different letters represent significantly different values as assessed by One-way ANOVA and LSD tests with P < 0.05.


Page 29 of 38


Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6


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Figure 7



Figure 8


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Figure 9



Table of contents


Page 38 of 38

Prunella vulgaris L., an Edible and Medicinal Plant, Attenuates

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