Protective effects of bajijiasu in a rat model ofAβ25-35-induced neurotoxicity

Di-Ling Chen a, Peng Zhang b, Li Lin b,n, He-Ming Zhang a, Shao-Dong Deng b,Ze-Qing Wub, Shuai ou b, Song-Hao Liu a, Jin-Yu Wang b

a Southern Institute of Pharmaceutical Research, South China Normal University, Guangzhou, Guangdong 510631, People's Republic of Chinab College of Chinese Materia Medical, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510006, People’s Republic of China

a r t i c l e i n f o

Article history:Received 2 December 2013Received in revised form5 March 2014Accepted 2 April 2014Available online 14 April 2014

Keywords:BajijiasuAlzheimer's diseaseNeurotoxicityMechanismApoptosis

Chemical compounds studied in this article:Beta-amyloid (PubChem CID: 3407255)Norepinephrine bitartrate (PubChem CID:297812)Dopamine hydrochloride (PubChem CID:65340)Serotonin (PubChem CID: 5202)Acetylcholine (PubChem CID: 187)Malondialdehyde (PubChem CID: 10964)

a b s t r a c t

Ethnopharmacological relevence: Neurodegenerative diseases (NDs) caused by neurons and/or myelinloss lead to devastating effects on patients' lives. Although the causes of such complex diseases have notyet been fully elucidated, oxidative stress, mitochondrial and energy metabolism dysfunction, excito-toxicity, inflammation, and apoptosis have been recognized as influential factors. Current therapies thatwere designed to address only a single target are unable to mitigate or prevent disease progression, anddisease-modifying drugs are desperately needed, and Chinese herbs will be a good choice for screeningthe potential drugs. Previous studies have shown that bajijiasu, a dimeric fructose isolated from Morindaofficinalis radix which was used frequently as a tonifying and replenishing natural herb medicine intraditional Chinese medicine clinic practice, can prevent ischemia-induced neuronal damage or death.Materials and methods: In order to investigate whether bajijiasu protects against beta-amyloid (Aβ25-35)-induced neurotoxicity in rats and explore the underlying mechanisms of bajijiasu in vivo, we prepared anAlzheimer's disease (AD) model by injecting Aβ25-35 into the bilateral CA1 region of rat hippocampus andtreated a subset with oral bajijiasu. We observed the effects on learning and memory, antioxidant levels,energy metabolism, neurotransmitter levels, and neuronal apoptosis.Results: Bajijiasu ameliorated Aβ-induced learning and memory dysfunction, enhanced antioxidativeactivity and energy metabolism, and attenuated cholinergic system damage. Our findings suggest thatbajijiasu can enhance antioxidant capacity and prevent free radical damage. It can also enhance energymetabolism and monoamine neurotransmitter levels and inhibit neuronal apoptosis.Conclusion: The results provide a scientific foundation for the use of Morinda officinalis and itsconstituents in the treatment of various AD. Future studies will assess the multi-target activity of thedrug for the treatment of AD.

& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Neurodegenerative diseases (NDs), including Alzheimer's dis-ease (AD), Parkinson's disease (PD), and Huntington's disease (HD)are a major problem worldwide (Hardy and Gwinn-Hardy, 1998;Muqit and Feany, 2002; Palop et al., 2006). According to the WorldHealth Organization, the morbidity due to NDs will soon exceedthat of cancer, becoming the second highest cause of death in2040. As such, it has become a serious social problem and hasattracted considerable attention in the medical profession. NDscan develop for various reasons, including oxidative stress,

mitochondrial dysfunction, excitotoxicity, inflammation, andapoptosis. Over the past several years, a new paradigm hasemerged that simultaneously targets multiple disease pathways.Such “multi-target-designed drugs” have shown great promise asuseful neuroprotective agents in preclinical studies and are able toafford symptomatic relief to decrease the day-to-day burden ofthese illnesses. Currently, there is little that can be done to slowthe progression of NDs. Their multifaceted profiles necessitate achange in the paradigm toward designing effective therapies. Asmore multi-target-designed drugs are tested in clinical trials andclinical applications, support from society is growing, and thefeasibility of this approach is now recognized by many.

Alzheimer’s disease (AD) is the most common form of demen-tia in the elderly and is clinically characterized by the deteriora-tion of learning, memory, and other higher cognitive functions

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Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2014.04.0040378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

n Corresponding author. Tel.: þ86 2039358270.E-mail address: ll76611@126.com (L. Lin).

Journal of Ethnopharmacology 154 (2014) 206–217

(Glenner and Wong, 1984; Grundke-Iqbal et al., 1986). AD is anirreversible and progressive neurodegenerative disease. Senileplaques, neurofibrillary tangles, and extensive neuronal loss arethe main histological hallmarks observed in AD brains (Do Carmoand Cuello, 2013). Beta-amyloid peptide (Aβ), a major proteincomponent of senile plaques, is considered a critical cause ofthe pathogenesis of Alzheimer’s disease (AD). Modulation ofAβ-induced neurotoxicity has emerged as a possible therapeuticapproach to ameliorate AD onset and progression (Selkoe, 2000;Sambamurti et al., 2002).

Morinda officinalis is one of the best-known herbs in China,Korea, and Japan and is especially popular in Southern China. It is acomponent herb that contains many herbal formulae, such ashexasaccharide and heptasaccharide which have been shown toameliorate symptoms in an animal model of depression (Li et al.,2001, 2004, 2008; Deng et al., 2012). Previous studies havedemonstrated that bajijiasu (BJ), a dimeric fructose isolated fromMorinda officinalis (chemical structure is shown in Fig. 1, previousname: Bajisu) is able to reinforce population spikes (PSs) and long-term potentiation (LTP) (Chen et al., 1999), ameliorate cognitivedeficits induced by D-galactose in mice, and protect againstischemia-induced neuronal damage or death (Tan et al., 2000a,2000b). Cell culture experiments have shown that bajijiasu pro-tects against Aβ25-35-induced neurotoxicity in PC12 cells (Chenet al., 2013). The protective effect of bajijiasu might be exertedthrough enhancing antioxidant abilities, stabilizing mitochondrialmembrane potential and intracellular Ca2þ concentration, andreducing neuronal apoptosis (Chen et al., 2000). Collectively, theseresults provide evidence that bajijiasu protects against ischemia-induced neuronal damage or death (Lin et al., 2008). Although theeffects of multi-target bajijiasu have been described in differentmodels of neurotoxicity, there is no direct evidence regarding theprotective function of bajijiasu in the case of Aβ exposure. Thus, theaim of the present study was to investigate whether bajijiasu isprotective against Aβ-induced neurotoxicity in rats and to explorethe underlying mechanisms in vivo.

2. Materials and methods

2.1. Subjects

Adult male Sprague Dawley rats (180–220 g, obtained fromCenter of Laboratory Animal of Guangzhou University of ChineseMedicine, SCXK [Yue] 2008-0020, SYXK [Yue] 2008-0085) werepair-housed in plastic cages in a temperature-controlled (25 1C)colony room on a 12/12-h light/dark cycle. Food and water wereavailable freely. All experiment protocols were approved by theCenter of Laboratory Animals of Guangzhou University of ChineseMedicine. All efforts were made to minimize the number ofanimals used.

2.2. Bajijiasu extraction and acute toxicity

Each 1000 g Morinda officinalis root powder was mixed in3000 mL of distilled water and soaked for 6 h. Then heated refluxextraction 1.0 h in a round-bottom flask twice, merged the filtrate,

concentrated to 1000 mL under reduced pressure in a rotaryevaporator (90 1C). Adjusted the pH value of the concentratedsolution to 6.0 with diluted hydrochloric acid at 45 1C, traversedthe anion exchange resin (D-900, free amine type, purchased fromHebei Cangzhou Baoen adsorption materials technology Co., Ltd.)at the speed of 1.0 Bv/h, repeated 3 times, and then concentratedto 500 mL, when cooled to the room temperature added alcohol tothe concentration of 90% of alcohol, refrigerated at 4 1C for 48 h.The crystallization was extracted by 65% of acetonitrile in aqueoussolution at 80 1C, and the undissolved substance extracted by 65%of acetonitrile in aqueous solution 3 times, the residue crystal-lization was the bajijiasu.

The purity of bajijiasu was analysed by a high performanceliquid chromatography (HPLC) system consisted of a pump(Waters, Smartline system 600, including degasser and autosam-pler system), coupled to an evaporative light scattering detector(ELSD detector Alltech 2000). Briefly, with the hydrophilic chro-matography column of Rezex RCM (Phenomenex, 300 mm�7.8 mm) and column temperature 35 1C, 100% of deionized wateras mobile phase, flow rate at 0.6 mL/min, drift tube temperatureand the nitrogen gas flow rate of ELSD were at 100 1C and 2.0L/min, respectively.

Adult KM mice (18–22 g, half of male, obtained from Center ofLaboratory Animal of Guangdong province, SCXK [Yue] 0058740)were pair-housed in plastic cages in a temperature-controlled(25 1C) colony room on a 12/12-h light/dark cycle. Food and waterwere available freely. All experiment protocols were approved bythe Center of Laboratory Animals of Guangzhou University ofChinese Medicine. All efforts were made to minimize the numberof animals used, and the experimental operation in accordancewith the technical guidelines for acute toxicity studies of Chineseand natural medicines. 20 male and female mice for each group,the test set by the maximum concentration (500 mg/mL) andmaximum volume (0.4 mL/10 g body weight) administered at adose of 20 g/kg body weight last two weeks. And the solventcontrol group was given the same amount of pure water. Theanimal weight, diet, appearance, behavior, secretions, excretionsand animal deaths and so on were observed, especially thereactions of mice after administration 4 h every day, record all ofthe situations, and all the appear, volume, color, texture and otherchanges of the tissues and organs.

2.3. Drugs and treatment procedures

Aβ25-35 (A4559-1 mg), purchased from Sigma-Aldrich (St Louis,MO, USA), was dissolved in stroke-physiological saline solution tothe concentration of 2 g/L and stored at �20 1C. Before being used,it was incubated at 37 1C for 4 days. Other materials includingnorepinephrine bitartrate (NE, 100169-199402), dopamine hydro-chloride (DA, 100070- 200405), 5-hydroxy-indole-3-acetic acid(5-HIAA, 140737-200501) and serotonin hydrochloride (5-HT,111656-200401) were purchased from the China Drugs and Biolo-gical Products Inspection Institute. Acetonitrile (1489730-925)was purchased from Merck & Co. Inc.; Sodium 1-octanesulfonate(B8, AR, 20081229) was obtained from the Tianjin Damao chemicalreagent factory; and phosphoric acid (AR, 20080427), perchloricacid (HPLC, 20070901-1), and sodium dihydrogen phosphate (AR,20080801-1) were purchased from the Guangzhou chemicalreagent factory. Pentobarbital sodium was purchased from MYMBiological (USA). Normal saline and medicinal alcohol were pur-chased from WJ Biotechnology (China). Assay kits for malondial-dehyde (MDA), superoxide dismutase (SOD), catalase (CAT),glutathione reductase (GSH-Px), acetylcholine (ACh), acetylcholi-nesterase (AChE), and Naþ/Kþ-ATPase were purchased fromNanjing JianCheng Bioengineering Institute. (China). All otherreagents and chemicals used in the study were of analytical grade.

Fig. 1. Chemical structure of Bajijiasu.

D.-L. Chen et al. / Journal of Ethnopharmacology 154 (2014) 206–217 207

2.4. Model preparation

The procedures were similar to those described previously(Yamaguchi and Kawashima, 2001). Rats were intraperitoneallyanesthetized with 30 g/L pentobarbital sodium (40 mg/kg, i.p.;Sigma-Aldrich) and placed in a stereotaxic frame (RWD LifeScience Co., Ltd., Shenzhen, China). The hair was shaved, the scalpwas opened, and holes were drilled with an electric dental drill(brushless motor, 30,000 rpm) according to a mouse brain atlas(AP-3.6 mm, ML72.5 mm, DV3.0 mm). Then, 5 μL (10 μg) Aβ25-35,fibrillar state for one hole (Chen et al., 2013), was slowly injectedinto the CA1 region of the hippocampus over 5 min, and theneedle was kept in for 5 min. Then, the wound was sutured, andpenicillin (30 U/kg) was injected intramuscularly to protect againstinfection. Then the rats were isolated in a warm box until theyrecovered consciousness.

After 15 days, the rats were screened with water maze tests toidentify animals that were appropriate models, and they wererandomly divided into seven groups as follows: control group(received oral distilled water), sham-operation group (animalsunderwent surgery but did not received Aβ25-35, oral distilledwater was given), model group (Aβ25-35 and oral distilled waterwere given), positive control group (oral donepezil HCl, 0.125 mg/[kg �d]), low-dose group (oral BJ, 8 mg/[kg �d]), medium-dosegroup (oral BJ, 24 mg/[kg �d]), high-dose group(oral BJ, 48 mg/[kg �d]). Every group had 14 animals, and the experiments lasted25 days.

2.5. Water maze tests

Rat spatial learning and memory abilities were tested in theMorris water maze (MWM, DMS-2, Chinese Academy of MedicalSciences Institute of Medicine) using procedures similar to thosedescribed previously (Novarino et al., 2004; Hamid et al., 2009;Dong, Z.F. et al., 2013). The MWM consisted of a circular fiberglasspool (200-cm diameter) filled with water (2571 1C) that wasmade opaque by non-toxic black paint. The pool was surroundedby light blue curtains, and three distal visual cues were fixed to thecurtains. Four floor light sources of equal power provided uniformillumination to the pool and testing room. A CCD camera sus-pended above the center of the pool recorded animal swim paths,and video output was digitized by an EthoVision tracking system(Noldus, Leesburg, VA). The water maze tests included threeperiods: initial spatial training, spatial reversal training, and theprobe test.

Initial spatial training: Twenty-four hours before spatial train-ing, animals were allowed to adapt to the maze during a 120-s freeswim. Rats were then required to swim to find a hidden platform(15 cm�15 cm, located at NW) submerged 2 cm below the watersurface. Rats were placed into the water facing the pool wall atfour starting positions (N, S, E, and W). Animals were then trainedin the initial spatial learning task for 4 consecutive days. Therewere four trials in every experimental day and between every twotrials were 10-min inter-trial intervals. During each trial, rats wereallowed to swim until they found the hidden platform, where theyremained for 20 s before they were removed to a holding cage.Rats that failed to find the hidden platform in 120 s were guided tothe platform, where they also remained for 20 s.

Spatial reversal training: After the initial spatial learning task, areversal learning protocol was conducted with a subset of rats.During the reversal learning task, the hidden platform was movedto the opposite quadrant (e.g., from NW to SE). The reversallearning task entailed 3 additional days with 4 trials per day,similar to the initial training period. Rats exposed to acute stresswere trained with a modified reversal protocol that could becompleted in a single session. This protocol was used to avoid

confounding the results by exposing the rats to acute stress morethan once.

Probe test: Twenty-four hours after the final reversal trainingtrial, rats were placed in the pool from a new drop point with thehidden platform absent for 120 s, and their swim path wasrecorded.

2.6. Animal parameters

The animals' fur color and appearance and behavior wereobserved and recorded every day. Animal weight was measuredevery 3 days during the drug administration period.

2.7. Enzymatic assays

After the water maze tests, the animals were sacrificed, andtheir hearts were washed twice in 4% paraformaldehyde solution.The entire brain was dissected, and the left brain tissues of half ofthe number of animals in each group were homogenized with9 times the volume of physiological saline in an ice bath andcentrifuged for 10 min at 3500g. The supernatant fluid was storedat �20 1C until being used. The activities of malondialdehyde(MDA), total superoxide dismutase (T-SOD), catalase (CAT), glu-tathione reductase (GSH-Px), and the levels of acetylcholine (ACh),acetylcholinesterase (AChE), and Naþ/Kþ-ATPase were measuredwith detection kits. Protein concentrations were determined usingthe Coomassie Brilliant Blue G250 assay. The kits were allpurchased from Nanjing Jiancheng Bioengineering Institute (Nanj-ing, Jiangsu, PR China). The procedures were performed accordingto the manufacturer’s instructions and following previouslydescribed methods (Wei et al., 2013; Dong, D. et al., 2013). Levelswere normalized to the protein concentration of each sample andexpressed as percentage of non-treated controls.

2.8. Monoamine neurotransmitter assay

The right brain tissue was dissected from eight rats in eachgroup, following previously described methods (Zhou et al., 2011).Briefly, the tissue was homogenized with HClO4 and filtered inultrafiltration centrifuge tubes (3K, volume 1.5 mL, Vivaspin 500)to remove the high-molecular weight proteins. Then, the super-natants were directly injected into the chromatographic system(HPLC-ECD) that contained a chromatographic column (Phenom-enex Luna C18 [150�4.6 mm2, 5 μm]) and a mobile-phase systemthat contained two solvents. Solvent A was prepared by dissolving31.2 g sodium dihydrogen phosphate and 1.0 g sodium octanesulfonate into 1 L deionized water and adjusting the pH value to4.0. Solvent B was 13% acetonitrile (V/V). During the detection, thesolvent speed was 1.0 mL/min, the column temperature was 35 1C,and the detection voltage was set at 350 mV or 700 mV.

2.9. HE staining

After water maze testing, the whole brains were dissected, andsix brains from each group were fixed in 4% paraformaldehydesolution and prepared as paraffin sections that were stained withhematoxylin–eosin (HE) and observed under light microscopy. Thehippocampal histopathological abnormalities were investigatedunder a light microscope. The number of cells in the hippocampalCA1 region of each section was examined by 2 pathologists in ablinded manner, and the average number was taken as the finalresult.

D.-L. Chen et al. / Journal of Ethnopharmacology 154 (2014) 206–217208

2.10. Hippocampal neuron apoptosis assay

Hippocampal neuron apoptosis was measured by flow cyto-metry (FCM) using propidium iodide (PI) staining. The experi-mental procedure was as follows: the bilateral hippocampi wereadded to 2 mL precooled saline, homogenized in an ice bath,filtered with cell strainers, and centrifuged for 10 min at 1200 rpm.The supernatant was removed, and 1.5 mL phosphate-bufferedsaline (PBS) solution was added and dispersed, the sample wasfiltered again, centrifuged 5 min at 1200 rpm, and the supernatantwas discarded. Finally, we added 2 mL 70% ethanol for storage at4 1C for 24 h. Then, the sample was centrifuged, the supernatantwas discarded, 1 mL PBS was added, the sample was centrifugedfor 5 min at 1500 r/min, the supernatant was removed, 200 μL PBSand 200 μL PI dye was added, incubated in the dark for 20 min at4 1C, and then measured in a flow cytometer (Cytomics TM FC 500,Beckman Coulter, USA) at Ex 488 nm and Em 630 nm.

2.11. Statistical analysis

Behavioral assessment data were analyzed using two-wayanalysis of variance (ANOVA) followed by least significant differ-ence (LSD) test. Dunnett's test was used for each test and controlgroup. Significance level was set at po0.05. Statistical Package forthe Social Science software (SPSS 17.0, SPSS Inc.) was used for allstatistical analyses.

3. Results

3.1. Bajijiasu analysis

The HPLC-ELSD test result was show in Fig. 2, there were noother peaks except the bajijiasu.

3.2. Acute toxicity of bajijiasu

In the 14 days of testing, the activity ability, fur and secretionswere all normal, and there were no death. The weight of eachmeasurement time points were no significant difference comparedto the solvent control group (p40.05), as shown in Table 1. Andthe volume, color, texture of the tissues and organs were nochanges.

3.3. Effect of bajijiasu on animal appearance and weight

One animal in the model group died during the treatment; itdid not defecate, and its belly was swollen and angular, suggestingthat this rat was severely constipated. The fur of the treatedanimals was obviously much smoother than that of the modelgroup. Weight records showed that there was no significantdifference (p40.05) in average weight between the treated andmodel groups. They weighed approximately 250 g at the begin-ning and 340 g at the end of the experiment, which implied thatneither the surgery nor the drugs had a serious impact on animalweight.

3.4. Effect of bajijiasu on behavior

The initial spatial training results are shown in Fig. 3. On thefirst day, the latency of the control group (74.25737.01 s) wassignificantly lower than that of model group (103.81716.11 s),and this difference was significant (po0.01). However, the sham-operation group (87.90720.02 s) showed no significant difference(p40.05) compared to the control group. On the fourth day, theincubation period of the control group was (29.3172.66 s), whilefor the model group it was (56.18719.96 s) (compared with thecontrol group, po0.01), and the sham-operation group was(30.4071.29 s) (p40.05 compared with the control group andpo0.01 compared with the model group). These results demon-strate that injecting Aβ25-35 into CA1 could cause memory loss andthat the surgery did not cause any side effect.

Compared with the model group, the incubation period foreach BJ-treated group was significantly shorter. The low-dose BJgroup incubation period was (80.30721.16 s), while for themedium-dose group it was (71.46733.54 s), and for the high-dose group it was (53.47713.93 s), and for the positive controlgroup it was (69.44726.43 s) on the first day. Compared with the

Fig. 2. The HPLC–ELSD fingerprint of bajijiasu extracted from Morinda officinalis.

Table 1Results of the acute toxicity of bajijiasu (mean7SD, g).

Groups n Dose/(g/kg) Measurement time points Death rate/%

D0 D1 D3 D7 D14

Solvent control 40 – 21.471.7 23.472.0 23.472.0 29.971.8 34.072.4 0Bajijiasu 40 20 21.671.8 24.372.1 25.271.7 30.171.9 36.273.1 0

Fig. 3. Effect of bajijiasu on escape latency of Aβ25-35-treated rats in the MWM. Thegraph Control, control group; Sham, sham-operation group; Model, model group;Positive, positive group (Aβ25-35 20 μgþdonepezil HCl, 0.125 mg/[kg �d]); BJ-8 mg,low-dose BJ group (Aβ25-35 20 μgþBJ 8 mg/[kg �d]); BJ-24 mg, medium-dose BJgroup (Aβ25-35 20 μgþBJ 24 mg/[kg �d]); BJ-48 mg, high-dose BJ group (Aβ25-3520 μgþBJ 48 mg/[kg �d]). Values given are the mean7SD n¼14).

D.-L. Chen et al. / Journal of Ethnopharmacology 154 (2014) 206–217 209

model group, the differences were significant (po0.01). On thefourth day, the low-dose BJ group incubation period was(31.3074.69 s), for the medium-dose group it was (27.6473.35 s), for the high-dose group it was (32.0877.20 s), and forthe positive group it was (26.5271.57 s), and the differences weresignificant compared to the model group (po0.01). These resultsshowed that bajijiasu can ameliorate Aβ25–35-induced learning andmemory dysfunction in rats.

The variation in total swimming distance in each group wassimilar to the variation observed for the latency period. The totaldistances of the control group were 81197274, 49477245,39477336, and 32057329 cm from the first to the fourth day,respectively. The corresponding values for the model group were11,3527304, 69367263, 51877347, and 61447356 cm, respec-tively. The differences were significant between the model andcontrol groups (po0.01). The swimming distances of the positivegroup were 75947269, 39467236, 34327331, and 29007327 cm, respectively. Compared to the model group, the differ-ences were significant (po0.01). The swimming distances of thelow-dose BJ group were 87827280, 50757246, 41357338, and34237331 cm; those of the medium-dose group were 78157271,41577238, 29697327, and 30237328 cm; and those of thehigh-dose group were 58477253, 53057249, 36907334, and35087332 cm. Compared to the model group, the differences ofall BJ-treated groups were significant (po0.01). However, thedifferences were not significant compared to the control group(p40.05), and the average swimming speed was similar among allgroups (p40.05).

Probe test results showed that there was no significant differ-ence (p40.05) among the groups with regard to total swimmingdistance or speed. The swimming time of the control group in theNW quadrant (27.3673.38 s) was longer than in the other threequadrants (23.2175.03 s, 25.0074.11 s, 25.4375.27 s), and thedifferences among them were significant (po0.01). The swim-ming time of the model group was 20.7775.63 s, which wassignificantly shorter than the control group (po0.01), suggestingthat the rats remembered the location where the platform wasplaced. The swimming time of the low-, medium- and high-doseBJ groups were 26.0076.95 s, 28.9073.96 s, and 31.9373.39 s,which were significantly shorter than the control group. Comparedto the control group, the differences were significant (po0.01), asshown in Figs. 4 and 5, the swimming trajectory of the BJ-highdose group was obviously denser in the NW quadrant than theothers. These results suggest that bajijiasu could ameliorate Aβ25-35-induced learning and memory dysfunction in the rat model.

3.5. Enzymatic assays

As shown in Fig. 6, values were expressed as percentage ofcontrol group, control group levels of SOD, MDA, CAT and GSH-PXwere 195.91712.71 NU/mg protein, 3.6870.0.44 nmol/pg pro-tein, 8.5370.35 U/mg protein, and 2.9670.20 U/mg protein,respectively. The same measurement values in the model groupwere 177.23712.17, 4.5870.41, 4.5870.12, and 2.6670.36,respectively. The differences for all of these were significant(po0.05), which suggested that Aβ25-35 influenced antioxidantlevels in rat brain tissue, and the same results as in the PC12 cellsexperiment (Chen et al., 2013).

As shown in Fig. 6A, the SOD levels in the low-, medium-, andhigh-dose BJ groups were 192.2279.21, 195.0476.43, and 190.7579.94 NU/mg protein, respectively, compared to 190.3779.27 and177.23712.17, in the positive and model groups, respectively. Thedifferences of low-, medium-, high-dose BJ groups, and the positivecontrol group were significant compared to the model group(po0.05), suggesting that bajijiasu encouraged SOD production.

As shown in Fig. 6B, MDA levels significantly decreased afterbajijiasu treatment. The MDA levels of the low-, medium-, andhigh-dose BJ groups were 3.8170.26, 3.6570.15 and 3.4670.25 nmol/pg protein, respectively, and that of the positive groupwas 3.6870.40, all of which were lower than the model group(4.5870.12). The differences of low-, medium-, high-dose BJgroup, and positive control group were significantly differentcompared to the model group (po0.05), and the MDA level ofthe medium-dose group dropped to the same as the control group,which suggesting that bajijiasu inhibits MDA production.

As shown in Fig. 6C, bajijiasu significantly increased CAT levels.That in the low-, medium and high-dose BJ groups were7.8970.21, 8.2070.17 and 8.3270.21 U/mg protein, respectively,and that of the positive group was 8.6770.35, all of which werehigher than the model group (4.5870.12) and increased in aconcentration-dependent manner. The differences of low-, med-ium-, high-dose BJ group, and positive control group were sig-nificant compared to the model group (po0.05).

As shown in Fig. 6D, bajijiasu treatment significantly increasedGSH-Px levels. The GSH-Px level of the low-, medium- and high-dose groups were 2.5370.27, 2.7770.2 and 3.0370.36 U/mgprotein, and the positive group was 3.2870.48, all of which werehigher than the model group (4.5870.12) and were againconcentration-dependent. The differences of low-, medium-, high-dose BJ groups, and the positive control group were significantcompared to the model group (po0.05).

To evaluate the protective efficacy of bajijiasu on energymetabolism in Aβ25-35-treated rats, we evaluated Naþ/Kþ-ATPaselevels in brain tissue. As shown in Fig. 7, Naþ/Kþ-ATPase wassignificantly lower in the model group (0.9670.52 μmol/pg pro-tein) compared to the control group (1.3170.21) (po0.05). Thelevels of all BJ treated groups were significantly increased, thevalues of the low-, medium-, and high- dose groups and positivegroup were 1.0270.16, 1.4870.45, 1.6270.52 and 1.7770.43,respectively, all of which were higher than the model group(0.9670.52). The differences of low-, medium-, high-dose BJgroup, and positive control group were significantly differentcompared to the model group (po0.05), and they were alsoconcentration-dependent activity, while the specific mechanismneeds further researches.

Cholinergic system damage and abnormal ACh levels areobserved in AD patients. The results are as shown in Fig. 8, thecontrol group levels of ACh and AChE were 119.84718.82 μmol/mgprotein and 4.8670.58 μmol/pg protein, respectively, compared to107.68710.29 and 7.7470.15 in the model group, respectively.Both of these differences were significant (po0.05). After treatmentwith different concentrations (8, 24, 48 mg/[kg �d]) of bajijiasu, the

Fig. 4. The swimming time in the platform quadrant during the spatial probe test.Values given are the mean7SD n¼14, ♯po0.01 vs. control group, npo0.05 vs.model group, nnpo0.01 vs. model group.

D.-L. Chen et al. / Journal of Ethnopharmacology 154 (2014) 206–217210

ACh levels of model rats increased to 118.91719.25, 119.82717.70and 121.07719.32, respectively. Compared with the modelgroup (107.68710.29), the differences were significant (po0.05).

The AchE decreased to 6.7570.19, 6.2470.52 and 5.2870.18,respectively. These differences were significant (po0.05) comparedwith the model group (7.7470.15).

Fig. 5. The swimming trajectory of each group during the probe test in Aβ25-35-induced model rats. CP, control group; SP, sham-operation group; MP, model group; PP,positive group (Aβ25-35 20 μgþdonepezil HCl, 0.125 mg/[kg �d]); BLP, low-dose BJ group (Aβ25-35 20 μgþBJ 8 mg/[kg �d]); BMP, medium-dose BJ group (Aβ25-35 20 μgþBJ24 mg/[kg �d]); BHP, high-dose BJ group (Aβ25-35 20 μgþBJ 48 mg/[kg �d]).

Fig. 6. Effect of bajijiasu on SOD, MDA, CAT, and GSH-Px levels in Aβ25-35-induced model rats. The graphs show levels of (A) SOD, (B) MDA, (C) CAT, (D) GSH-Px. Values givenare the mean7SD n¼8 and expressed as percentage of control group, ♯po0.01 vs. control group, npo0.05 vs. model group, nnpo0.01 vs. model group.

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3.6. Monoamine neurotransmitters levels

As shown in Figs. 9 and 10, levels of NE, DA, 5-HT and 5-HIAA inthe control group were 3.8070.48, 1.8270.16, 2.1870.69, and6.1070.54 μg/g, respectively. In the model group, they were

3.7170.33, 1.8070.08, 1.8770.19, and 5.5170.41 μg/g, respectively.All of these differences were significant (po0.05) except the DAlevel, which suggested that Aβ25-35 influences the levels of mono-amine neurotransmitters in rat brain tissue.

As shown in Fig. 9A, the levels of NE in the low-, medium-, andhigh-dose BJ groups were 3.7270.46, 4.0370.55 and 4.4170.61,and 3.9170.42 in the positive group, except the low- all of whichwere higher than the model group (3.8070.48). The differences ofmedium-, high-dose BJ group, and positive control group weresignificant compared with the control group (po0.05). This resultshowed that bajijiasu promoted NE secretion in a concentration-dependent manner.

As shown in Fig. 9B, DA levels in the low-, medium- and high-dose BJ groups were 1.8370.23, 1.9770.13, and 1.9870.23, andthat in the positive group was 1.8070.06, all of which were higherthan that in the model group (1.8070.08), and the differences ofmedium- and high-dose BJ group were significant compared withthe control group (po0.05), which showed that bajijiasu stimu-lated DA secretion in a concentration-dependent fashion.

As shown in Fig. 10A, the levels of 5-HT in the low-, medium-, andhigh-dose BJ groups were 2.1870.19, 2.0770.38, and 2.8470.84,respectively, and that in the positive control group was 2.2070.36,all of which were higher than the model group (1.8770.19), and thedifferences of low-, medium-dose BJ group, and positive controlgroup were significantly different compared with the control group

Fig. 7. Effect of bajijiasu on Naþ/Kþ-ATPase in Aβ25-35-induced model rats. Valuesgiven are the mean7SD n¼8, ♯po0.01 vs. control group, nnpo0.01 vs. modelgroup.

Fig. 8. Effect of bajijiasu on AchE and Ach in Aβ25-35-induced model rats. Valuesgiven are the mean7SD n¼8, ♯po0.01 vs. control group, npo0.05 vs. modelgroup, nnpo0.01 vs. model group.

Fig. 9. The results of neurotransmitter assays in the brain tissue of Aβ25-35-inducedmodel rats. The graphs show levels of (A) NE and (B) DA. Values given are themean7SD n¼8 and expressed as percentage of control group, npo0.01 vs. controlgroup, npo0.05 vs. model group, nnpo0.01 vs. model group.

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(po0.05), demonstrating that bajijiasu stimulated NE productionand in a concentration-dependent manner.

Fig. 10B illustrates that the levels of 5-HIAA in the low-,medium-, and high-dose groups were 5.8170.90, 6.2670.56,and 6.3571.12, respectively, and that of the positive group was6.0070.65, the medium- and high-dose groups were higher thanthe model group (6.0070.65), and the differences of high-dose BJgroup, and positive control group were significant compared withthe control group (po0.05),which suggested that bajijiasu stimu-lated concentration-dependent 5-HIAA production.

3.7. Histopathologic morphology

HE staining revealed no remarkable neuronal abnormalities inthe hippocampus of rats in the control group. The pyramidal cellsin the CA1 regionwere arranged neatly and tightly, and no cell losswas found. Additionally, for the control group, cells were roundand intact with nuclei stained clear, dark blue (Fig. 11). However,obvious hippocampal histopathological damage was observed inthe model and bajijiasu treated groups. The pyramidal layeredstructure was disintegrated, and neuronal loss was found in theCA1 region. Neurons with pyknotic nuclei and with shrunken orirregular shape were also observed. These abnormalities wereattenuated by bajijiasu treatment. The cells in bajijiasu group hadbetter cell morphology and were more numerous than those in the

untreated and bajijiasu treated groups, but were overall worsethan those in the control group.

Fig. 12A graphs the neuronal counts in each group. The numberof CA1 pyramidal cells in the control group was 3072 (n¼6).However, the number in the model group significantly decreasedto 1573 (po0.01). Compared with the model group, the numberof neurons in the bajijiasu-treated (high-, medium- and low-dose)groups increased to 4474, 3173, and 2573, respectively. Thesevalues were positively correlated to dose, and the differencesbetween all the groups were significant (po0.01). The pyramidalcell number in the positive group was 2773, which was sig-nificantly higher than that in the model group (po0.01) but notwith the medium and low-dose BJ groups (p40.05).

As shown in Fig. 12B, the neuronal number in the CA1 region ofthe hippocampus in the control group was 477752, compared to378760 in the model group (po0.01). The neuronal numbers inthe bajijiasu-treated groups were 491752 (high-dose), 557746(medium-dose), and 503757 (low-dose), all of which weresignificantly different from the model group (po0.01). The num-ber in the positive group was 491752, which was also signifi-cantly different compared with the model group (po0.01).

The results in Fig. 13A demonstrate that the number of neuronsin the cerebral cortex was 474753 in the control group and481757 in the sham-operation group, compared to 378765 inthe model group, which was significantly lower (po0.01). Thisfinding shows that Aβ25-35 had neurotoxic effects. Compared withthe model group, the bajijiasu-treated group numbers increased to489732 (high-dose), 464741 (medium-dose), and 418731(low-dose), and these values were significantly different comparedto the model group (po0.01). The number in the positive groupwas 495743, and this difference was also significant comparedwith the model group (po0.01).

The results as in Fig. 13B, which illustrate that the numbers offorebrain basal ganglia neurons were 643799 in the controlgroup and 648791 in the sham-operation group, comparedto 464740 in the model group, and the difference betweenthem was significant (po0.01). Compared with the model group,the bajijiasu-treated groups increased to 639793 (high-dose),568768 (medium-dose), and 511768 (low-dose), and the differ-ences were significant and dose dependent (po0.01). The numberof neurons in the positive group was 635756, which was alsosignificantly different compared to the model group (po0.01).

3.8. Hippocampal neuron apoptosis assay

As shown in Fig. 14, the apoptotic rate significantly increasedafter rats were treated with Aβ25-35. The apoptotic rate was27.3170.98%. However, after they were treated by bajijiasu con-centrations of 8, 24, 48 mg/(kg d), the apoptotic rate decreasedto 13.2771.29%, 6.3570.89%, and 3.2771.10%, respectively(po0.05 compared with control group).

4. Discussion

Alzheimer’s disease (AD) has been recognized as a senileneurodegenerative disease affecting the life quality of patients. Ithas been characterized as progressive memory capacity damageand cognitive dysfunction in clinic. Pathological evidences supportthe fact that a large number of senile plaques (SP) depositin hippocampus and cerebral cortical neurons of AD patients.A great many evidences have been found to confirm thatthe accumulation of intracellular β-amyloid (Aβ) may be an earlyevent in the development of AD (Pathan et al., 2006; Hampel,2013; Li et al., 2013).

Fig. 10. The results of neurotransmitter assays in the brain tissue of Aβ25-35-induced model rats. The graphs show levels of (A) 5-HT, and (B) 5-HIAA. Valuesgiven are the mean7SD n¼8 and expressed as percentage of control group,♯po0.01 vs. control group, npo0.05 vs. model group, nnpo0.01 vs. model group.

D.-L. Chen et al. / Journal of Ethnopharmacology 154 (2014) 206–217 213

Learning and memory abilities are considered an importantaspect of cognition, and also reflect the advanced integrativefunctions of the brain. Thus, it is useful to assess learning andmemory abilities to diagnose and evaluate the therapeutic effectsof treatments for AD, which is clinically characterized by intellec-tual decline, memory loss, and related behavioral dysfunction. Thewater maze test results showed that the latency induced by Aβ25-35were significantly longer than that of the control group, but thesechanges were ameliorated by bajijiasu, and the time in thequadrant target also increased markedly. These results demon-strated that the model was successfully established and thatbajijiasu improved learning and memory abilities in the dementiamodel rats.

Aβ is a peptide of 36–43 amino acids that is formed by a largetransmembrane glycoprotein expressed on the cell, such as amy-loid precursor protein (APP). Aβ may activate inflammatory andneurotoxic process, including the excessive generation of freeradical and oxidative damage among intracellular proteins andother macromolecules. Oxidative stress is defined as a disturbanceof the balance between the production of reactive oxygen species(ROS) and antioxidant defense systems. Excessive ROS productionis known to cause oxidative damage to major macromolecules incells, including DNA, lipids, and proteins, and disrupts cellularfunctions and integrity (Gardner et al., 1997; Fiers et al., 1999) andcan result in cell death and tissue damage. ROS are implicated inseveral diseases, including cancer, diabetes, cardiovascular dis-eases, and aging. Previous studies (Markesbery, 1997; Gilgun-Sherki et al., 2003; Polidori, 2004) have indicated that oxidativedamage is an early factor in NDs. Therefore, it may be possible todelay AD progression with antioxidants. The results of this studyshowed that SOD, CAT, and GSH-Px levels were lower in the modelgroup compared to the control group, but MDA levels were higherthan the control group. After bajijiasu treatment, the levels of SOD,

CAT and GSH-Px increased, but MDA decreased markedly, whichdemonstrated that bajijiasu alleviated the oxidative damage, andhence delayed the onset of dementia, but further researches willbe encouraged.

Cholinergic deficiencies, particularly in the hippocampus, cor-tex, and septal nuclei of basal forebrain, is another pathologicalphenomenon observed in AD. And studies have showed that theimpaired memory function may also be associated with thecholinergic system synthase such as ChAT and hydrolytic enzymesuch as AChE. The levels of acetylcholine transferase enzyme(ChAT) and AChE are decreased in AD patients, and acetylcholine(Ach) concentration is increased. These changes have been postu-lated to cause delirium and cognitive decline (Volpicelli and Levey,2004; Marcantonio et al., 2006; Hshieh Tammy et al., 2008; Cabezaet al., 2012). Currently, AChE inhibitors (AChEIs) are primarily usedto maintain cognitive functions of AD patients in clinical practice(Cracon et al., 1998; Borroni et al., 2001; De la Fuente et al., 2003;Wolff et al., 2005; Jay, 2005). The results of this study showed thatacetylcholinesterase levels decreased, and those of acetylcholineincreased in the bajijiasu-treated groups.

Modern research has shown that learning and memory abilitiesare closely related to central nervous system function, and mono-amine neurotransmission plays an important role in the regulationof learning and memory, and NE, DA, and 5-HT levels change withage (Coyle et al., 1983; Rehman and Masson, 2001; Bohnen andAlbin, 2011). The results described here demonstrate that thelevels of NE, DA, 5-HT, and 5-HIAA recovered to normal levelsfollowing bajijiasu-treatment.

Previous studies have confirmed that mitochondrial dysfunc-tion and energy metabolism abnormalities are early and commonpathological phenomena (Raichle and Gusnard, 2002; Bouzier-Sore et al., 2003; Wang et al., 2008; Zhao et al., 2010). Thus,ameliorating energy metabolism abnormalities may be effective

Fig. 11. Pathological sections of hippocampus and cerebral cortex from Aβ25-35-induced model rats after stained with hematoxylin–Eosin (HE) (4�200). Rats in the controlgroup did not show histopathological abnormalities. In the model and high-dose BJ groups, the number of cells in the hippocampal CA1 region appeared decreased.Furthermore, the remnants of the pyramidal cells were arranged irregularly and some exhibited shrunken and irregular shape. The cells in the BJ group had better cellmorphology and were more numerous than those in the model and high-dose BJ groups.

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for treating AD. We found that Naþ/Kþ-ATP levels of the modelgroup were lower than those of the control group, but treatmentwith bajijiasu attenuated this decrease, suggesting that bajijiasucan ameliorate energy metabolism abnormalities associated withdementia.

In recent years, apoptosis was found in some specific nerve tissuesof ND models (Chuang et al., 2002; Iqbal et al., 2002; Vassar, 2002;Wang et al., 2006), especially in the hippocampus and cortex ofmutant APP-overexpressing transgenic mice. The evidence indicatesthat apoptosis is involved in AD pathogenesis, and current researchseeks to identify methods or drugs to inhibit apoptosis. We deter-mined that the numbers of neurons in the bajijiasu-treated group,including the CA1 region of the hippocampus, cortex and septalnuclei of basal forebrain, were higher than in the control group, andthe apoptotic rate of hippocampal neurons was lower. It is possiblethat bajijiasu inhibited neuronal apoptosis, but this mechanismshould be confirmed by further study.

One important property of carbohydrate drugs is that most ofthem interact with cell-surface molecules, such as cell recognition,cell differentiation, and cell interaction molecules. These kinds ofinteractions have important effects on human physiology andpathological processes, such as cell growth and differentiation,immune response, bacterial infection, and tumor metastasis(Wang, 2001). Because they interact on the cell surface and do

not enter the intracellular space, they are associated with fewerside effects. Therefore, carbohydrates and their related compoundsare used to both treat disease and promote health; they are evenbeing developed as functional foods. These novel treatments have

Fig. 12. Effect of bajijiasu on pyramidal cells and neurons in hippocampus of Aβ25–35-induced model rats, cerebral cortex and forebrain basal ganglia. The graph(A) pyramidal cells number in CA1 region of hippocampus, (B) neurons number inCA1 region of hippocampus. Values given are the mean7SD n¼6, ♯po0.01 vs.control group, nnpo0.01 vs. model group.

Fig. 13. Effect of bajijiasu on neurons in hippocampus of Aβ25-35-induced modelrats, cerebral cortex and forebrain basal ganglia. The graph (A) neurons number incerebral cortex and (B) neurons number in forebrain basal ganglia. Values given arethe mean7SD n¼6, ♯po0.01 vs. control group, npo0.05 vs. model group, nnpo0.01vs. model group.

Fig. 14. Effect of bajijiasu on hippocampal neuron apoptosis in Aβ25-35-inducedmodel rats. Values given are the mean7SD n¼6, ♯po0.01 vs. control group,nnpo0.01 vs. model group.

D.-L. Chen et al. / Journal of Ethnopharmacology 154 (2014) 206–217 215

great potential as anti-dementia drugs. Bajijiasu, a dimeric fruc-tose, may be one choice for AD, similar to the marine sulfatedoligosaccharide HSH-971.

In the present study, our results demonstrate that bajijiasuprotected against Aβ25-35-induced neurotoxicity in rats. The pro-tective effect was mediated through inhibition of oxidative stressand neuronal apoptosis, restoration of normal energy metabolism,and amelioration of cholinergic system defects. Also in our pre-vious vitro study have demonstrated that treatment with bajijiasusignificantly increased the cell viability and mitochondrial mem-brane potential, while decreased oxidative stress, and alteration ofP21, CDK4, E2F1, Bax, NF-κB p65, Caspase-3 protein and mRNAwasinvolved in neuroprotective action of bajijiasu against Aβ-inducedcellular toxicity (Chen et al., 2013). These observations laid thescientific foundation for the use of Morinda officinalis in thetreatment of various AD. In addition bajijiasu is considered apromising natural chemical constituent for treating AD, and themulti-target activity of this drug will be further studied fortreating AD.

Acknowledgments

This work was supported by the financial support from theChina National Ministry of Science and Technology Plan Projects(2011BAI01B02), and Guangdong Science and Technology PlanProjects (2012A030100005), and Guangdong Natural ScienceFoundation Grant (S2013040016159), and Science and TechnologyAgency HaiNan province Grant 090603.

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