ARTICLE

Cognitive enhancement of volatile oil from the stems ofSchisandra chinensis Baill. in Alzheimer’s disease ratsBingyou Yang, Bo Liu, Yan Liu, Hua Han, and Haixue Kuang

Abstract: The volatile oil (VO), extracted from the stems of Schisandra chinensis Baill. (SCS), was separated and identified bygas chromatography – mass spectrometry. The study was devised to investigate the effects of VO on oxidative stress andcognitive deficits induced by amyloid-� (A�(1-42)). Alzheimer’s disease (AD) models were established by injecting A�(1-42)into the rat hippocampus and the effects of learning and memory were observed by a Morris water maze test, immuno-histological alterations, and correlative indicators covering nerve growth (brain-derived neurotrophic factor, glial-cell-derived trophic factor, and nerve growth factor), interleukin 1�, tumor necrosis factor, superoxide dismutase (SOD),glutathione peroxidase (GSH-Px), malondialdehyde (MDA), glial fibrillary acidic protein (GFAP), and microglial CD11b in ADrats. And activities of SOD, MDA, and GSH-Px were ameliorated by VO. The neurotrophic factors GFAP and microglial CD11bwere noticeably improved in histopathologic changes. These data suggested that VO from SCS had potential activities forthe prevention and treatment of AD.

Key words: A�, oxidative stress, memory impairment, anti-oxidation, anti-inflammatory.

Résumé : Nous avons séparé de l’huile volatile (HV) extraite de tiges de Schisandra chinensis Baill. (SCS), et nous l’avonsanalysée à l’aide de la chromatographie gazeuse et de la spectrométrie de masse. Nous avons conçu ces travaux pour étudierles effets de l’HV sur le stress oxydatif et les déficits cognitifs provoqués par la bêta-amyloïde (A�(1-42)). Nous avons mis enplace des modèles de maladie d’Alzheimer (MA) en injectant de l’A�(1-42) dans l’hippocampe de rats et mesuré les effets del’apprentissage et de la mémoire à l’aide du test de la piscine de Morris, les modifications immunohistologiques ainsi quele taux des indicateurs de corrélation couvrant la croissance des nerfs (facteur neurotrophique dérivé du cerveau, facteur neurotro-phique dérivé des cellules gliales et facteur de croissance nerveuse), d’interleukine 1�, de facteur de nécrose tumorale, de superoxydedismutase (SOD), de glutathion peroxydase (GSH-Px), de malondialdéhyde (MDA), de protéine acide fibrillaire gliale (GFAP) et de CD11bmicroglial chez des rats atteints de MA. Aussi l’HV permettait d’améliorer l’activité de la SOD, du MDA et de la GSH-Px. Les tauxde facteurs neurotrophiques de GFAP et de CD11b microglial s’amélioraient de façon notable en cas de variations histo-pathologiques. Ces données laissaient entendre que l’HV de SCS aurait eu une activité éventuelle dans la prévention et letraitement de la MA. [Traduit par la Rédaction]

Mots-clés : A�, stress oxydatif, trouble mnésique, anti-oxydation, anti-inflammatoire.

IntroductionAlzheimer’s disease (AD) is a common neurodegenerative disor-

der with cognitive function impairment in the elderly (Pasqualettiet al. 2015). Research showed that the pathogenesis of AD wasabnormal metabolism of the amyloid precursor protein anddeposition of the extracellular amyloid-� (A�) protein (Cioancaet al. 2013). The A�(1-42) deposition triggered a variety of patho-genic events such as oxidative injury, neurofibrillary tangles,proinflammatory apoptosis, and neurotransmitter deficits thatultimately led to neurodegeneration (Hardy and Selkoe 2002).

Schisandra chinensis Baill. (SC) has been long used as a traditionaldrug in China (Zhou et al. 2011), with the effects of nootropic, salu-brity, and prolonging life as well as the activities of anti-oxidation,anti-inflammation, anti-ager, and anti-tumor (Song et al. 2011). Oncurrent clinic, it was used to improve cognitive function morecommonly (Jeong et al. 2013). Modern medicine suggested that SCcould decrease the activity of �-secretase 1, inhibit JKN/p38 of theMAPKs inflammatory signaling pathways, and protect againstneurodegeneration and prevent A�-induced neuronal dysfunc-

tion (Giridharan et al. 2015; Zhao et al. 2016), and neurodegenera-tion could cause a series of reactions of cells protection,inflammation, and oxidative stress (Zhu et al. 2014). Furthermore,plentiful volatile oil (VO) from SC was reported to have variouspharmacological activities, especially anti-oxidation and anti-inflammation (Hancke et al. 1999; Teng and Lee 2014; Jeong et al.2015). According to the research, the chemical constituents of VObetween the stems of SC (SCS) and SC were exactly the same(Hancke et al. 1999; Ma et al. 2012; Li et al. 2013). As a consequence,we reasonably surmised and speculated that SCS might have sim-ilar or better activities than SC. The good news was that VO of SCSwas discovered to have obvious effects against AD in our previousstudy. The more important point was, as we know, the millionsof tons of SCS were wasted seriously in the process of pruningeach year at the places of origin. Therefore, based on furtherinvestigation of pharmacological activities and rationally uti-lizing resources, the effects against AD of the VO from SCS weredetermined including the Morris water maze test, immunohis-tological alterations, and correlative indicators in AD rats in-duced by A�(1-42).

Received 18 April 2016. Accepted 28 May 2016.

B. Yang, B. Liu, Y. Liu, H. Han, and H. Kuang. Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of ChineseMedicine, Ministry of Education, Harbin 150040, China.Corresponding author: Haixue Kuang (email:hxkuang@hotmail.com).Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.

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Can. J. Physiol. Pharmacol. 00: 1–6 (0000) dx.doi.org/10.1139/cjpp-2016-0194 Published at www.nrcresearchpress.com/cjpp on xx xxx xxxx.

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Materials and methods

Plant materialThe SCS was collected in August 2010 from Shuangyashan City

of Hei Longjiang Province, China. The plant materials (HerbariumNo. 20130985) were identified by Lianjie Su (Department of Chi-nese Medicine Resources, Heilongjiang University of ChineseMedicine). The VO from SCS was obtained by hydrodistillation for3 h and dried with anhydrous sodium sulfate and kept at −4 °Cuntil analysis.

Animals and drugsAdult SD rats (half male and half female, certificate SCXK2008-

0001), weighing 350–400 g, were obtained from the LaboratoryAnimal Center of Heilongjiang University of Chinese Medicineand allowed access to food and water ad libitum and maintainedunder a constant temperature (22 ± 1 °C) with a 12 h light–dark

cycle. All procedures performed in studies involving animalswere in accordance with the ethical standards of the institutionand Canadian Council on Animal Care, and efforts were madeto minimize animal affliction and to reduce the number ofanimals used.

A�(1-42) peptide (≥95% purified by HPLC, catalog No. bs-0107P; Boaosen Shengwujishu Co., Ltd., Beijing, China) wasdissolved in physiological saline. The dissolved A�(1-42) wasincubated at 37 °C for 3 days to obtain the fibrillated form ofA�(1-42) to induce AD in the rats. The rats were divided into fivegroups (eight animals per group): (1) control group (CG), (2)A�(1-42)-treated group (ATG), (3) A�(1-42)-treated with Aricept (ATA)(0.02 g·kg–1·day–1; Weicai Pharmacy Co., Ltd., Suzhou, China), (4)A�(1-42)-treated with VO of SCS (ATVH) (0.2 g·kg–1·day–1), and (5)A�(1-42)-treated with VO of SCS (ATVL) (0.067 g·kg–1·day–1).

Table 1. Compounds from volatile oil identified by GC−MS.

No.Retention time(min) Compound(s) CAS No.

Molecularformula

Content(%)

1 12.334 3-Thujene 002867-05-2 C10H16 0.6932 12.679 1 R-�-pinene 007785-70-8 C10H16 3.6033 13.549 Camphene 000079-92-5 C10H16 1.0664 15.246 �-Terpinene 000099-84-3 C10H16 19.4975 15.645 �-Phellandrene 000555-10-2 C10H16 1.2286 16.231 �-Phellandrene 000099-83-2 C10H16 0.7257 16.866 Bicyclo[4.1.0]hept-2-ene, 3,7,7-trimethyl- 000554-61-0 C10H16 1.1718 17.307 �-Limonene 005989-27-5 C10H16 1.1379 18.153 Benzene, 1-methyl-2-(1-methylethyl)- 000527-84-4 C10H14 4.62810 18.424 1,3,6-Octatriene, 3,7-dimethyl- 013877-91-3 C10H16 0.09411 18.835 1,4-Cyclohexadiene, 1-methyl-4-(1-methylethyl)- 000099-85-4 C10H16 1.97812 20.116 Terpinolene 000586-62-9 C10H16 0.6113 23.204 2-Nonanone 000821-55-6 C9H18O 0.32414 23.681 Linalool 000078-70-6 C10H18O 1.88515 26.074 6-Octenal, 3,7-dimethyl- 000106-23-0 C10H18O 0.82816 26.726 DL-Camphor 021368-68-3 C10H16O 0.07717 27.276 Terpinen-4-ol 000562-74-3 C10H18O 4.91718 27.983 Borneol 010385-78-1 C10H18O 0.31819 28.75 Benzene, 2-methoxy-4-methyl-1-(1-methylethyl)- 001076-56-8 C11H16O 4.56620 30.297 6-Octen-1-ol, 3,7-dimethyl-, (R)- 001117-61-9 C10H20O 0.24321 31.62 Bicyclo[2.2.1]heptan-2-ol, 1,7,7-trimethyl-, acetate, (1S-endo)- 005655-61-8 C12H20O2 2.19222 31.765 �-Cubebene 017699-14-8 C15H24 0.36423 32.569 2-Undecanone 000112-12-9 C11H22O 2.5124 33.137 Copaene 003856-25-5 C15H24 1.49525 33.977 �-Elemene 000515-13-9 C15H24 0.09226 34.206 Terpinyl acetate 000080-26-2 C12H20O2 1.46227 34.508 Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)- 110823-68-2 C15H24 2.29728 35.934 1,5,5-Trimethyl-6-methylene-cyclohexene 000514-95-4 C10H16 0.3629 36.714 Germacrene D 023986-74-5 C15H24 0.98630 38.139 Spiro[5.5]undec-2-ene, 3,7,7-trimethyl-11-methylene-, (−)- 018431-82-8 C15H24 0.84231 38.768 (+)-Valencene 004630-07-3 C15H24 1.44132 39.009 1 H-cyclopropa[a]naphthalene, decahydro-1,1,3a-trimethyl-7-methylene-,

[1aS-(1a.alpha, 3a.alpha, 7a.beta, 7b.alpha)]-020071-49-2 C15H24 1.767

33 39.197 Spiro[5.5]undeca-1,8-diene, 1,5,5,9-tetramethyl-, (R)- 019912-83-5 C15H24 0.5434 39.614 Naphthalene, 1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)- 000483-75-0 C15H24 0.94335 39.819 �-Cadinene 000483-76-1 C15H24 2.97136 40.23 Benzene,1-methyl-4-(1,2,2-trimethylcyclopentyl)-, (R)- 016982-00-6 C15H22 0.77537 40.387 Naphthalene, 1,2,3,4,4a,7-hexahydro-1,6-dimethyl-4-(1-methylethyl)- 016728-99-7 C15H24 0.20738 40.538 (+)-�-Muurolene 017627-24-6 C15H24 0.42539 40.768 l-Calamenene 000483-77-2 C15H22 0.20640 41.034 2-Tridecanone 000593-08-8 C13H26O 1.33341 43.033 Benzene, 1,2-dimethoxy-4-(1-propenyl)- 000093-16-3 C11H14O2 1.13542 44.151 Nerolidol 007212-44-4 C15H26O 6.70543 47.154 (+)-�-Cedrene 000546-28-1 C15H24 0.84844 47.408 1-Isopropyl-7-methyl-4-methylene-1,2,3,4,4a,5,6,8a-octahydron-aphthalene 030021-74-0 C15H24 0.67845 48.381 (−)-g-Cadinene 039029-41-9 C15H24 4.31846 48.81 �-Cedrene 000469-61-4 C15H24 0.65247 50.048 2-Pentadecanone 002345-28-0 C15H30O 0.171

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Gas chromatography – mass spectrometry (GC–MS)GC analysis was carried out using an Agilent Technologies 7890-

5975C gas chromatograph. Samples were analyzed on a DB-1701column (60 m × 250 �m inside diameter and 0.25 �m filmthickness). Helium was used as the gas carrier with a flow rate of1.2 mL/min. The oven temperature program was initially set at50 °C and heated to 160 °C at a rate of 3 °C/min, then at 2 °C/min to170 °C and held for 2 min, and finally at 3 °C/min to 200 °C andheld for 2 min. The total run time was 56 min.

Animal model establishmentSD rats were anesthetized with an intraperitoneal injection of

3.6 mL/kg 10% chloral hydrate and fixed with a stereotaxic appa-ratus (DW-2000D Chengdu Taimeng Co., Ltd., Shenzhen, China).Then, according to a rat brain atlas, rats were injected with theaggregated A� (1-42) (1 mg/mL 0.01 mg/rat) into the right and leftlateral ventricles. The aggregated A�(1-42) was implementedwithin 5 min (1 �L/min), and the syringe was left for 5 min afterinjection and retained the needle for 5 min to dispersion and thenthe incision was sutured. From the next day, rats of each groupwere administered the corresponding drug daily for 14 consecu-tive days by intragastric infusion.

Morris water maze testThe Morris water maze test was started 10 days after A�(1–42)

peptide injection. The water maze constituted a metal pool (diam-eter 150 cm, height 60 cm) and filled with 22 ± 1 °C water. The poolwas crossed at the center by two imaginary lines and divided intofour quadrants. A black cylindrical platform (8 cm in diameter)was centered in one of the quadrants of the pool and submerged1.0 cm under the water surface.

In training trials, rats were lightly released into the water at onesame starting position and allowed to swim for a maximum of60 s. When a rat could not find the platform within 60 s, it wasallowed to stay on the platform for 15 s. Such training trials wererepeated for 5 days. On the last training trial, the rats were al-lowed to swim for 60 s to search for the platform. The behaviorwas recorded by video.

Serum and brain tissue acquisitionAfter a probing trial of the Morris water maze test, all rats were

intraperitoneally anesthetized with 10% chloral hydrate. Bloodsamples were collected from a hepatic portal vein and the bloodserums were collected by centrifuging (4 °C, 3500g for 20 min) andstored at −80 °C until biochemical assay. Then, the hearts of anes-thetized rats were perfused with 0.9% saline solution followed by4% paraformaldehyde fixing solution containing 0.1 mol/L phos-phate buffer solution (pH 7.2). Brains were immediately removed.Parts of brains were dissected out and quickly frozen in liquidnitrogen and then stored at −80 °C and other parts of brains werefixed in 10% formalin at 4 °C and then embedded in paraffin forhistopathological analysis.

Determinations of nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF), glial-cell-derived neurotrophicfactor (GDNF), tumor necrosis factor (TNF-�), andinterleukin 1� (IL-1�) in serums

The serums were collected to measure concentrations of neu-rotrophic, nerve growth (BDNF, GDNF, and NGF), and inflamma-tory factors (TNF-� and IL-1�) by means of ELISA kits (NanjingJiancheng Bioengineering Institute, Nanjing, China). Absorbancevalues were measured at 450 nm and the averages of the signalswere considered to represent the BDNF, GDNF, NGF, TNF-�, andIL-1� concentrations for the samples.

Oxidative stress analysisAnti-oxidative enzyme levels of supernatants from brains were

measured including superoxide dismutase (SOD), glutathioneperoxidase (GSH-Px), and malondialdehyde (MDA) by means of

assay kits (Nanjing Jiancheng Bioengineering Institute, Nan-jing, China).

Histopathological examinationThe brains of rats were fixed in ice-cold 10% formalin and em-

bedded in paraffin and cut into 4 �m thick sections. The sectionswere processed for hematoxylin and eosin. The brain slices weredeparaffinized and rehydrated. Then, the brain tissues wererinsed in phosphate buffer solution and incubated in 0.5% H2O2for 15 min to block peroxidase activity in the tissue. Sections wereblocked for 1 h with 3% goat serum and incubated in polyclonalantibodies against rat glial fibrillary acidic protein (GFAP) (catalogNo. bs-0199R, 1:500; Boaosen Shengwujishu Co., Ltd., Beijing,China) and microglial CD11b (catalog No. bs-11127R, 1:500; Boaosen

Fig. 1. Effects of volatile oil (VO) from the stems of Schisandrachinensis (SCS) on escape latency in Alzheimer’s disease rats ofamyloid-� (A�)(1-42)-treated control group (CG), A�(1-42)-treatedgroup (ATG), A�(1-42)-treated with Aricept (ATA) (20 mg·kg–1·day–1),A�(1-42)-treated with VO of SCS (ATVH) (0.2 mg·kg–1·day–1), andA�(1-42)-treated with VO of SCS (ATVL) (0.067 mg·kg–1·day–1).

Table 2. Escape latency of volatile oil on the performance of amyloid-�(1-42)-treated rats in the water maze test.

Escape latency (s)

Group 1 day 2 days 3 days 4 days 5 days

CG 49.85±6.05 43.03±8.46 49.91±5.82 38.42±4.97 29.79±8.56**ATG 59.88±0.10 59.94±0.08 47.72±15.48 44.56±10.86 48.28±7.66ATA 59.99±0.08 44.04±13.99 24.97±7.92 40.68±14.21 30.22±5.06**ATVH 56.16±17.35 54.49±8.09 49.67±11.28 45.51±17.18 39.29±5.11*ATVL 57.96±2.48 57.10±5.81 49.03±15.74 46.54±16.47 41.64±6.10

Note: CG, amyloid-� (A�)(1-42)-treated control group; ATG, amyloid-� (A�)(1-42)-treated group; ATA, A�(1-42)-treated with Aricept (20 mg·kg−1·day−1); ATVH,A�(1-42)-treated with VO of SCS (0.2 mg·kg−1·day−1); ATVL, A�(1-42)-treated withVO of SCS (0.067 mg·kg−1·day−1). Values are mean ± SD and were analyzed byone-way ANOVA. **P < 0.01 and *P < 0.05 compared with ATG.

Table 3. Expression of volatile oil on the levels of glial-cell-derivedtrophic factor (GDNF), brain-derived neurotrophic factor (BDNF),nerve growth factor (NGF), tumor necrosis factor (TNF-�), and inter-leukin 1� (IL-1�) in the serums.

Group(ng/L protein) GDNF BDNF NGF TNF-� IL-1�

CG 7.73±1.89* 22.26±4.54* 35.59±5.27** 32.26±4.43** 2.43±0.35*ATG 3.88±0.68 15.3±3.71 16.34±3.26 54.47±7.03 3.86±0.42ATA 7.23±1.08* 22.13±2.06 33.28±4.72* 33.66±4.57** 2.42±0.22*ATVH 6.91±1.25 20.67±2.16* 31.32±4.28* 36.32±3.42** 2.64±0.50ATVL 6.65±1.37 17.43±4.57 30.65±3.92* 42.48±4.72 3.33±0.43

Note: CG, amyloid-� (A�)(1-42)-treated control group; ATG, amyloid-� (A�)(1-42)-treated group; ATA, A�(1-42)-treated with Aricept (20 mg·kg−1·day−1); ATVH,A�(1-42)-treated with VO of SCS (0.2 mg·kg−1·day−1); ATVL, A�(1-42)-treated withVO of SCS (0.067 mg·kg−1·day−1). Values are mean ± SD and were analyzed byone-way ANOVA. **P < 0.01 and *P < 0.05 compared with ATG.

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Shengwujishu Co., Ltd., Beijing, China) at 4 °C overnight. Later,the secondary antibody (1:100) was incubated at room tempera-ture for 2 h and the sections were rinsed with wash buffer threetimes for 5 min each. The sections of stain were dehydrated andmounted and photomicrographs (400×) of representative CA1 areaswere taken.

Statistical analysisData of all of the experimental results were analyzed using SPSS

19.0 software (SPSS Inc., Chicago, Illinois) and displayed as themean ± SD. The differences among the groups were assessed usingANOVA followed by Student–Newman–Keuls multiple range tests.Immunoreactivities of GFAP and CD11b were analyzed in the CPU.A value of P < 0.05 was deemed to be statistically significant.

Results

GC–MSThe components of VO were identified by the GC – MS – flame

ionizarion detection (Table 1) (Wang et al. 2008; Ma et al. 2011;Chen et al. 2012). The results revealed that monoterpenes were thehighest content of approximate 55%, sesquiterpenes were the sec-ond highest (25%), and aliphatics were the third highest (close to10%).

Morris water mazeIn the 14 days of testing (Fig. 1), the escape latencies of all groups

decreased with a gradual increase in the number of days (Table 2).Compared with CG, the escape latency of ATG was obviously in-creased with an extremely significant difference (P < 0.01). Thelatencies of ATVH and ATVL were notably lower than that of ATG.Moreover, the latency of ATVH (P < 0.05) was less to a greaterdegree than that ofATVL, and the treatment of ATVH was moreeffective. The escape latency of ATA was remarkably reduced com-pared with ATG.

Effects on the expressions of GDNF, BDNF, NGF, TNF-�, andIL-1� in the serums of VO

To evaluate the effects of VO on anti-dementia, the expressionsof GDNF, BDNF, and NGF and the levels of TNF-� and IL-1� wereevaluated (Table 3). The expressions of GDNF, BDNF, and NGF inserums were dramatically reduced in ATG compared with CG(P < 0.05, P < 0.05, and P < 0.01, respectively). Compared with ATG,ATVH, ATVL, and ATA of SCS produced an augment in GDNF (ATAP < 0.05), BDNF (ATVH P < 0.05), and NGF (ATVH, ATVL, and ATAP < 0.05) activities, while comparing CG and ATA with ATG, theTNF-� (P < 0.01 and P < 0.01) and IL-1� (P < 0.05 and P < 0.05)activities were relatively lower. ATVH (P < 0.01) and ATVL of SCSalleviated the TNF-� and IL-1� levels when compared with ATG.

Effects on the levels of SOD, GSH-Px, and MDA in thehippocampus of VO

The effects of oxidative stress on the AD induced by A� (1-42)were discovered in our present research. The levels of major anti-oxidation factors including SOD, MDA, and GSH-Px were found tobe obviously changed (Fig. 2).

The expressions of GFAP and CD11b in the hippocampusand hematoxylin and eosin staining

Hematoxylin and eosin staining (Fig. 3) was performed after theMorris water test. Compared with ATG, the neurons of CG andATA in the hippocampus were tight, aligned of nuclei, and evenlystained. Clearly, it was discovered that there was no significantdifference between CG and ATVH of SCS. The microglial and as-trocyte activations were observed by immunolabeling of CD11b(Fig. 4) and GFAP (Fig. 5). In CG, the neurons in the CA1 region weredisordered and rarefied. However, ATVH of SCS was not signifi-cantly different from CG.

DiscussionAD, a neurodegenerative disorder, was characterized by the

sustained higher nervous disorders of the activities and functionsof the brain (Su et al. 2014). On the basis of recent clinical news,oxidative stress, inflammation, neurotrophic factors, and NGFs weremainly elements to induce learning and memory deficits (Al-Rejaieet al. 2015) and could interrupt the neurotransmission as well astrigger the neurological disorders in rats. These pro-inflammatorycytokines of TNF-� and IL-1� could enhance neuronal injurythrough apoptotic pathways during neuro-inflammatory pro-cesses (Zeng et al. 2012). Neurotrophic factors (GDNF and BDNF)and NGF affected hippocampal neurogenesis and thereby couldcause stress-induced cellular and behavioral deficits (Lang andBorgwardt 2013). MDA was one of the final products of lipid per-oxidation, as an indicator of free radical mediated injury (Slater1984). SOD and GSH-Px were important antioxidant enzymes andprotected cells from injury caused by oxygen-derived free radicalsby way of hindering the lipid peroxidation chain reaction andremoving harmful peroxide metabolites (Hu et al 2012). Notably,the microglia and astrocytes played prominent roles in immunesurveillance and neuro-inflammatory processes of the brain. Acti-vated microglia and astrocytes possessed inflammatory and immuneproperties in many neuronal injury and neurodegeneration, partic-ularly AD (Lian and Zheng 2016).

In the text, the Morris water maze test was used to intuitivelyexplain the spatial memory formation and assess spatial learningability. In our study, ATVH restored the activities of SOD andGSH-Px, inhibited the additions of lipid peroxidation, and de-creased the damage of oxygen-derived free radicals. The expres-sions of GDNF, BDNF, and NGF were improved by treatment of VO,and VO of SCS extraordinarily improved the pathological feature

Fig. 2. Effects of volatile oil (VO) from the stems of Schisandra chinensis (SCS) on the oxidative stress in the hippocampus of Alzheimer’s diseaserats (mean ± SD). (A) Superoxide dismutase (SOD) level; (B) malondialdehyde (MDA) activity; (C) glutathione peroxidase (GSH-Px) activity. CG,amyloid-� (A�)(1-42)-treated control group; ATG, amyloid-� (A�)(1-42)-treated group; ATA, A�(1-42)-treated with Aricept (20 mg·kg–1·day–1);ATVH, A�(1-42)-treated with VO of SCS (0.2 mg·kg–1·day–1); ATVL, A�(1-42)-treated with VO of SCS (0.067 mg·kg–1·day–1). Data are presented asmean ± SD. **P < 0.01 as compared with ATG.

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of A�(1-42)-induced rats. In immunohistochemical experiments,neurons were restored in the hippocampus of ATVH. To betterilluminate the variations of neurons, activities of GFAP and CD11bwere examined in the hippocampus of AD rats. Our resultsshowed that the neurons were significantly rarefied and the VO ofSCS could protect against neurotoxicity and potentially alter cog-nitive behaviors in AD rats. The protective effects were realizedthrough inhibition of neuronal apoptosis and oxidative stress.

With the population aging, the problem of AD affects approxi-mately 35 million people worldwide (Konietzko 2015). In clinicalapplications, the actual drugs of AD treatment had significant sideeffects and the dependence on the drug deeply troubled patientswith a long-term course of treatment. However, traditional Chi-nese medicine had the advantages of good effects, safety, andabundant resources, attracting extensive attention. SCS had obvi-ous preponderances of low toxicity, bargain, and prolificacy as ahomology of medicine and food. Furthermore, the results of theexperiments showed that the VO of SCS could improve cognitiveand behavioral deficits of AD rats. These made contributed to drugdevelopment and provided a possibility for the treatment of AD inupcoming further studies.

Conflict of interestThe authors declare that there is no conflict of interest associ-

ated with this work.

AcknowledgementThis work was financed by the National Natural Science Foun-

dation of China (No. 81473325).

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