[논문]사이프러스 에센셜 오일의 흡입이 전임상 실험동물의 손상된 학습능력과 기억력에 미치는 영향

이길용; 이찬; 백정인; 배근영; 박찬익; 장정희

doi:10.6116/kjh.2020.35.5.33.

사이프러스 에센셜 오일의 흡입이 전임상 실험동물의 손상된 학습능력과 기억력에 미치는 영향
Cypress Essential Oil Improves Scopolamine-induced Learning and Memory Deficit in C57BL/6 mice 원문보기

大韓本草學會誌 = The Korea journal of herbology, v.35 no.5, 2020년, pp.33 - 39

이길용 (대구한의대학교 향산업전공) , 이찬 (계명대학교 의과대학 약리학교실) , 백정인 (대구한의대학교 향산업전공) , 배근영 (펜실베니아 대학교, 생명과학과 경영 복수 학위과정) , 박찬익 (대구한의대학교 향산업전공) , 장정희 (계명대학교 의과대학 약리학교실)

Abstract ▼ AI-Helper

Objectives : Increasing evidence supports the biological and pharmacological activities of essential oils on the central nervous system such as pain, anxiety, attention, arousal, relaxation, sedation and learning and memory. The purpose of present work is to investigate the protective effect and molecular mechanism of cypress essential oil (CEO) against scopolamine (SCO)-induced cognitive impairments in C57BL/6 mice. Methods : A series of behavior tests such as Morris water maze, passive avoidance, and fear conditioning tests were conducted to monitor learning and memory functions. Immunoblotting and RT-PCR were also performed in the hippocampal tissue to determine the underlying mechanism of CEO. Results : SCO induced cognitive impairments as assessed by decreased step-through latency in passive avoidance test, relatively low freezing time in fear conditioning test, and increased time spent to find the hidden platform in Morris water maze test. Conversely, CEO inhalation significantly reversed the SCO-induced cognitive impairments in C57BL/6 mice comparable to control levels. To elucidate the molecular mechanisms of memory enhancing effect of CEO we have examined the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. CEO effectively elevated the protein as well as mRNA expression of BDNF via activation of cAMP response element binding protein (CREB). Conclusions : Our findings suggest that CEO inhalation effectively restored the SCO-impaired cognitive functions in C56BL/6 mice. This learning and memory enhancing effect of CEO was partly mediated by up-regulation of BDNF via activation of CREB.

주제어

표/그림 (5)

표 Table 1. Chemical Composition of Cypress Essential Oil (CEO)
그림 Fig. 1. Effect of CEO against SCO-induced memory impairment in the acquisition and retention trials of passive avoidance test. CEO was exposed for 1 h/day or 3 h/day and scopolamine (1 ㎎/㎏) was intraperitonically injected 30 min before acquisition trial. Retention trial was conducted 24 h after acquisition trial. Data represent mean ± S.E.M (n=7). **p < 0.01 compared with the sham control group. ^#p < 0.05, ^##p < 0.01 compared with the scopolamine-injected model group.
그림 Fig. 2. Effect of CEO on SCO-induced learning and memory deficits as assessed by Morris water maze test. (A) Mean escape latency to find the hidden platform over 5 consecutive days of the training trials. (B) Swimming time spent in the target quadrant during probe trials without platform. Data are shown as ± S.E.M (n=6-7). **p < 0.01 vs. sham control group. ^#p < 0.05, ^##p < 0.01 vs. scopolamine-induced model group. (C) Representative pathways of Morris water maze test on days 1, 3, and 5 from each group.
그림 Fig. 3. Effect of CEO against SCO-induced cognitive dysfunction in the retention session of contextual fear conditioning test. Mice were exposed to CEO for 1 h/day or 3 h/day and intraperitonically injected with scopolamine (1 ㎎/㎏) 30 min before starting the retention trial. Values are expressed as means ± S.E.M. (n=7). **p < 0.01 compared with the sham control group. ^#p < 0.05, ^##p < 0.01 compared with the scopolamine-treated model group.
그림 Fig. 4. Effects of CEO on the expression of BDNF and activation of CREB in the hippocampus. The protein (A) and mRNA (B) expression of BDNF was determined by immunoblotting and RT-PCR using specific antibody and primers for BDNF. Actin and GAPDH levels were measured for the confirmation of equal amount of protein and mRNA loading, respectively. (C) The activation of CREB was determined by Western blot analysis using anti-phospho-CREB (upper panel) and anti-CREB (lower panel) antibodies.

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문제 정의

This study was supported by a grant of the Traditional Korean Medicine (TKM) Project, Ministry of Health & Welfare, Republic of Korea to commercialize TKM products.

제안 방법

Based on these ideas, the purpose of the present work is to investigate the memory enhancing effect of CEO against the SCO-induced amnesia in C57BL/6 mice by conducting diverse behavior tests. Furthermore, to elucidate an underlying molecular mechanism, we have examined the activation of cAMP response element binding (CREB) protein and subsequent expression of brain-derived neurotrophic factor (BDNF) in the hippocampus.
The location and swimming parameter of each experimental mouse, from the start point to the platform, was recorded and analyzed by a video tracking system (Ethovision system, Noldus, Wageningen, Netherlands). During five successive days of training session, the mice were given three trials per day. Once the mouse found and climbed onto the platform, the escape latency was recorded, and it was allowed to remain on it for additional 10 sec.
In the next experiment, Morris water maze test was conducted to examine the effect of CEO on the hippocampus-dependent spatial recognition and long-term memory. Spatial memory is hippocampus-mediated function that appears to be compromised in the elderly subjects and in patients suffering from AD.
The experimental mice were divided into six groups (6-7 animals per group) as follows, Group 1 : sham control group, Group 2 : SCO-induced amnesia group, Croup 3 and 4 : SCO + CEO treatment groups, Group 5 and 6 : CEO alone-exposed groups (1 and 3 hours per day, respectively). The inhalation apparatus (Hyochang Science, Daegu, South Korea) was made of acrylic fiber capped with an air filter paper, which allowed minimum respiration air to pass.
The maze was divided into four quadrants containing visual external cues and an escape platform (10 ㎝ in diameter and 30 ㎝ in height) was located in one quadrant submerged 2 ㎝ below the water surface. The location and swimming parameter of each experimental mouse, from the start point to the platform, was recorded and analyzed by a video tracking system (Ethovision system, Noldus, Wageningen, Netherlands). During five successive days of training session, the mice were given three trials per day.
Membranes were blocked with 5% non-fat dried milk in PBST (phosphate-buffered saline containing Tween-20) for 1 h at room temperature (RT) and then incubated with primary antibodies at 4℃ for overnight, followed by reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. The signals were developed using the enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham Biosciences, Piscataway, NJ, USA) and the relative density of the bands were analyzed using the ImageQuant LAS 4000 Multi Gauge software (Fujifilm, Tokyo, Japan).
To analyze the composition of CEO, GC/MS was performed in a 7890A-5975C GC/MSD System (Agilent Technologies, California, USA) using a split ratio of 1:50. An HP-5 capillary column (30 m × 0.

대상 데이터

Cypress essential oil (CEO) was supplied by CPL Aromas (Bishop's Stortfod, UK).
Scopolamine (SCO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cypress essential oil (CEO) was supplied by CPL Aromas (Bishop's Stortfod, UK).
The Morris water maze test was conducted in a circular pool (120 ㎝ in diameter and 45 cm in height) filled with water (21 ± 2℃), which was made to be opaquely white with non-fat milk powder. The maze was divided into four quadrants containing visual external cues and an escape platform (10 ㎝ in diameter and 30 ㎝ in height) was located in one quadrant submerged 2 ㎝ below the water surface. The location and swimming parameter of each experimental mouse, from the start point to the platform, was recorded and analyzed by a video tracking system (Ethovision system, Noldus, Wageningen, Netherlands).

데이터처리

For statistical analysis, one-way ANOVA followed by the Tukey’s test was conducted using SPSS software (SPSS 12.0 KO for windows) as multiple comparisons.

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