Effect of Rosemary Extract on Learning Memory Capacity of Mice

 Abstract: Objective: To investigate the effects of essential oil, water-soluble extract and fat-soluble extract of rosemary on the learning and memory ability of mice modeled with scopolamine-induced learning and memory disorders. Methods A scopolamine-induced learning and memory disorder mouse model was constructed, and the effects of different extracts of rosemary on the learning and memory of mice were investigated by the hopping test, darkness-avoidance test and Y-maze test. The results showed that the essential oil, water-soluble extracts and fat-soluble extracts of rosemary could prolong the latency period of platform jumping and reduce the number of errors (P<0.01), prolong the latency period of darkness avoidance and reduce the number of errors (P<0.01), and increase the number of correct errors in the Y maze (P<0.01) in the mouse model of learning and memory disorders. Conclusion The essential oil, water-soluble extract and fat-soluble extract of rosemary can improve the learning and memory functions of mice with scopolamine-induced learning and memory disorders.

 

Rosemary[1] (Rosmarinus officinalis L.) is a plant of the genus Rosmarinus in the family Labiatae, and the whole herb can be used as a medicine, which is pungent and warm in nature, and has the effect of strengthening the stomach and tranquilizing the mind. As early as 2000 years ago in Europe, rosemary was used to enhance memory and improve speech disorders; drinking rosemary tea can refresh the brain and improve concentration. The present experiment was conducted to investigate the effects of rosemary extract on the learning and memory ability of mice.

 

1 Materials and Methods

1.1 Materials

1.1.1 Laboratory animals   

Ltd. provided 180 Kunming-bred mice, half male and half female, clean grade, with an average body mass of (20±3) g. The mice were used for the study.

1.1.2 Drugs and primary reagents   

The essential oil of rosemary, water-soluble extract of rosemary and fat-soluble extract of rosemary were prepared by steam distillation and solvent extraction [2]; scopolamine hydrobromide injection (Shanghai WoFeng Pharmaceutical Co., Ltd.), with the specification of 1 ml: 0.3 mg; pyrithioxine hydrochloride tablets (Cerebrofloxin, Shanghai Xinya Pharmaceutical Co., Ltd.); saline (Guizhou Tiandi Pharmaceutical Co., Ltd.). (Guizhou Tiandi Pharmaceutical Co., Ltd.).

1.1.3 Instruments  

 DT-200 Jumping Table Automatic Tester (Shanghai Yilianke Teaching Equipment Co., Ltd.), ZH-500x Mouse Darkness Avoidance Instrument (Anhui Zhenghua Biological Instrument Co., Ltd.); BW-MYM103 Y Maze (Shanghai Softlung Technology Development Co., Ltd.).

 

1.2 Methodology

1.2.1 Grouping of animals   

The mice were acclimatized for 1 week and randomly divided into 3 groups of 60 mice each, which were prepared for the platform hopping experiment, the darkness avoidance experiment and the Y-maze experiment, respectively. Each group of 60 mice was randomly divided into blank control group, model group, positive drug group, rosemary essential oil group, water-soluble extract group and fat-soluble extract group (n=10).

1.2.2 Administration   

Each group was given 6d of acclimatization training before the experiment, and the drugs were administered on the 7th day: the blank control group and the model group were given an equal volume of physiological saline, the positive group was given 100mg/kg of cerebral fosfene by gastric gavage, and the test group was given 200mg/kg of essential oil of rosemary, water-soluble extracts and fat-soluble extracts by gastric gavage, for 14d; the rest of the groups were injected intraperitoneal 3mg/kg of scopolamine 20min later, except for the blank group, which was injected with an equal volume of saline one h after the final administration of the drugs. After the last administration, except for the blank control group which was injected with an equal amount of saline intraperitoneally, all the other groups were injected with scopolamine 3mg/kg intraperitoneally[3] , and the experiments were carried out 20min later.

 

1.2.3 Jumping platform experiments   

The mice are acclimatized in a reaction chamber for 3 minutes and then exposed to 36V AC current. The normal response of the animals to a shock is to jump back to the platform to avoid the injurious stimulus. However, most of the animals may jump off the platform to the copper fence again or more times, and then jump back to the platform quickly after being shocked. The number of times the mice received the shock was recorded as the number of errors, and the training period was 5 min, which was used as the learning performance. 24 h later, the test was repeated, and the time when the mice jumped off the platform for the first time was recorded as the latency period and the number of errors during the 5-min period.

 

1.2.4 Darkness avoidance experiments   

The mice are placed in the bright room of the darkness avoidance instrument, the hole between the bright and dark rooms is opened, and the mice are allowed to move freely between the bright and dark rooms to acclimatize to the dark room for 3 min; the dark room is electrified (36V) during the test, the mice are placed into the bright room with their backs to the hole, and then they receive electric shocks when they enter the dark room, and then return to the bright room for a normal response, and the training is repeated for 5 min. The mice are re-tested after 24 h, and the latent period of entry into the dark room and the number of errors made in the 5-min period are recorded.

 

1.2.5 Y Maze Experiment   

The Y maze has 3 arms and a connection zone, each arm can be energized, the unenergized arm is the safe zone, and the other two arms are energized as non-safe zones, and the safe and non-safe zones can be changed randomly. Mice were placed in the starting zone (unenergized) to adapt for 3 min, and then the safe zone was energized to change the position of the safe zone; the position of the safe zone was randomly changed 30 s after the mice arrived at the safe zone, and the bottom of the non-safe zone was energized 5 s after the change to stimulate the soles of the feet of the mice to drive them to the safe arm. If the mouse runs from the non-safe area to the safe area in one go within 10s, it will be judged as correct, otherwise it will be judged as wrong. The number of correct runs out of 10 was used as the learning and memorization score of the mice.

 

1.2.6 Statistical methods   

SPSS 21.0 software was used for statistical analysis, and the experimental data were expressed as x- ±s. Comparison of sample means was analyzed by one-way ANOVA, and P<0.05 was regarded as statistically significant.

 

2 Results

2.1 Effects on the learning and memory ability of mice in platform jumping As shown in Table 1, the learning and memory performance of the model group decreased significantly compared with that of the blank control group, indicating that the memory impairment model was established. Compared with the model group, the positive drug group, the rosemary essential oil group, the water-soluble extract group and the fat-soluble extract group significantly improved the learning performance and memory performance of the mice in platform jumping (P<0.01).

Table 1 Effect of rosemary extract on jumping performance of mice (x- ± s, n=10)

 

3 Discussion          

Learning memory is a high-level thinking activity of the central nervous system, which is a complex neurophysiological activity involving various neurotransmitters such as acetylcholine (Ach), 5-hydroxytryptamine (5-HT), and gamma-ami nobutyric acid (GABA) [3]. Ach, as an important chemical transmitter released from central cholinergic nerve endings, excites the cholinergic system by binding to cholinergic receptors, regulates the transfer process from first-level memory to second-level memory, and is a neurotransmitter that promotes learning and memory. Scopolamine is a cholinergic receptor blocking drug, which affects the acetylcholine-mediated memory function by blocking the binding of Ach to M receptors. In this experiment, scopolamine was used to establish a memory disorder model to study the effect of rosemary extract on the learning and memory ability of mice. The results showed that the scopolamine memory disorder model was successfully constructed, and the essential oil group, the water-soluble extract group, and the fat-soluble extract group could improve the learning and memory ability of the mice in the memory disorder model.

 

 In addition, the essential oil, water-soluble extract, and fat-soluble extract of rosemary contain a variety of components, among which the oil-soluble active ingredient of rosemary extract is mainly rhamnosus acid and the water-soluble active ingredient is mainly rosmarinic acid. Studies have shown that [8, 9], both rosemarinic acid and rosemarinic acid have antioxidant effects, which can inhibit the production of reactive oxygen species (ROS), significantly improve the activity of antioxidant enzymes, reduce the oxidative load of the body, reduce the production of lipid peroxide, and effectively slow down the aging of aging mice. It can be seen that whether the effect of rosemary extract on learning and memory ability is the independent effect of one active ingredient or the combined effect of multiple ingredients, and whether it is a single mechanism or multiple mechanisms, remains to be demonstrated in experimental studies.

 

References:

[1] WU Meng, XU Xiaojun . Recent research progress on chemical composition and pharmacological effects of rosemary [J]. Bio

[2] Zhang Haitao . Research on the new process of extracting and separating the effective components of rosemary [D]. Anhui: Hefei University of Technology, 2011

[3] SHANG Chongzhi, ZHAO Mingliang. Effects of ginsenoside Rg2 on learning memory in scopolamine-induced Alzheimer's disease mice [J]. Chinese Journal of Practical Diagnosis and Therapy, 2017, 31(5):444-447

[4] Gao Li, Peng Xiaoming, Zhang Fuchun, et al. Effects of different doses of scopolamine on learning memory function in mice [J]. Medicine Herald, 2013, 32(5):573-576

[5] Zhang L, Xu J-T, Rong Shuang, et al. Ameliorative effects of Lotus corniculatus proanthocyanidins on scopolamine-induced memory acquisition disorder in mice [J]. Chinese Journal of Neuroimmunology and Neurology, 2009, 16(6):406-410