Effect of Shenyi Anshen Tablet on Improving Learning and Memory in Mice and Its Mechanism
Shenyi Anshen Tablet is a prescription based on the theory of traditional Chinese medicine in the literature of Shenmao Wuzizi Tablet. It consists of four kinds of Chinese herbal medicines: ginseng, schisandra, jujube kernel and puzzle. Ginseng and Yizhiren are commonly used medicines for meditation and soothing. Suanzaoren has the effect of nourishing the heart and soothing the nerves. It is not only a medicine for treating insomnia, but also nourishing strong and strong, long-term service can raise the heart and brain; Schisandra, there are sour and contagious, qi and qi and other effects, good governance of heart and kidney, the loss or discomfort caused by uneasy, cold and insomnia. However, whether the compound preparation has obvious effects of improving memory, sedative and hypnosis has not been reported. This article mainly studies the pharmacodynamics of the compound preparation Shenyi Anshen Tablet to improve memory and explores its mechanism of action.
1 Materials and methods
1. 1 experimental drug and reagent compound preparation Shenyi Anshen tablets, homemade. 293 tablets containing raw materials (the required medicinal materials were purchased from Changchun Hongda Pharmacy); Zaodong Anshen granules (Beijing Youyou Special Pharmaceutical Co., Ltd.) batch number: 130501; the required drugs for the experiment were prepared in distilled water. . Acid n-butanol, n-heptane, phthalic acid heptachloric acid solution, iodine test solution, hemi-histidine solution, pH 7.2 phosphate buffer solution (PBS), ethylenediaminetetraacetic acid (EDTA) test solution.
1. 2 Animal Health Kunming Mouse (KM), 18-22g, male and female, purchased from Jitai Small Animal Products Distribution Department: the test mice were introduced and placed in the animal laboratory of Changchun University of Traditional Chinese Medicine. Temperature: 20 ± 3 degrees Celsius, relative humidity: 50% - 70%.
1. 3 experimental instruments: AL 204 million balance (METTLER MAN MOLOLOL Instruments (Shanghai) Co., Ltd.); CaryEc; lip, molecular fluorescence photometer (Zhengzhou Zhongpu Instrument Equipment Co., Ltd.) 8205 constant temperature water bath ( Shanghai Shensheng Technology Co., Ltd.); T18 basic: tissue homogenizer (Guangzhou Yike Laboratory Technology Co., Ltd.); KQ3200DB CNC ultrasonic cleaner (Guangzhou Yike Laboratory Technology Co., Ltd.); XR-3TB type jumping platform experimental system ( ); XR-XB110 mouse darkening experimental system (Shanghai Xinsoft Information Technology Co., Ltd.); Morris water maze experimental system (Shanghai Xinsoft Information Technology Co., Ltd.).
1. 4 Methods 50 mice were randomly divided into blank control group, positive control group, high dose group (1. 606 5 g / kg), medium dose group (0.535 5 g / kg), low dose group (0.267 8 g/kg), the three dose groups were administered with Shenyi Anshen tablets by gavage, the blank control group was given the same amount of normal saline, and the positive control group was given Zaodong Anshen Granules. After 15 days of infusion, the mice in each group were started. Train and measure.
1. 4. 1 Platform test: Place the mouse on the insulation platform (time starts). The latency of the second jump off the platform, the number of errors in the jump platform within 3 minutes, and the number of animals jumping off the platform in each group were recorded. Start the test after 3 days of training.
1.4.2 Avoiding dark experiment: Place the mouse in the electric shock chamber, back to the hole, first give conditional stimulation (lighting) for 15 s, and start energizing for 5 s after 10 s (lighting intensity is 40 V, 50 Hz). The latency of each mouse entering the darkroom, the number of errors entering the darkroom within 5, min, and the number of animals entering the darkroom in each group were recorded. And the percentage of the dark room (error reaction) within 5 minutes was trained, and the test was started after 5 days of training.
1.4.3 Water maze test Starting from the first day of training, one mouse per batch of each experiment was administered, in parallel, and the second batch of mice was injected 5 minutes later, and so on. Train 4 times a day, randomly select an entry point each time, observe and record the route map and time required (latency period) of the mouse to find and climb the platform. The 4 training mice were fed into water from 4 different water inlet points. If the mouse did not find the platform within 120 s, it should be led to the platform. At this time, the incubation period is 120 s, each training interval
For 60 s, the average time and distance of 4 times was used as the daily training score, and the measurement was started after 5 days of continuous training.
1.4.4 The effect of Fuwan preparation Shenyi Anshen Tablet on the monoamine neurotransmitters norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in the rat brain.
1.4.4.1 Determination of NE content in brain tissue 50 mice in the above experiment were decapitated, brain tissue was washed with ice physiological saline, and the water was absorbed. The balance of the mouse brain was weighed and the mouse brain was cut along the midline of the brain. , take left half of the brain tissue and add 2 ml of acid
After the mixture is homogenized with butanol, and then centrifuged at 2 500 r/min for 5 min, respectively, and the supernatant is taken as 0. 4 ml, plus n-heptane 0. 8 ml, plus 0.1 mol/L of HCl 0. 2 ml of ultrasound The bottom aqueous phase was taken at 1 min, 2 500 r/min for 5 min. 50 ul of aqueous phase, add 150 ul of PBS, add 40 ul of EDTA, add 20 ul of freshly prepared iodine solution, mix and let stand for 3 min, add alkaline sodium phosphate 40, mix separately, let stand for 3 min, add acetic acid 40 respectively. Ul, mix, 80 ° C water bath for 10 min, cooling is available. Pipette 200 ul of each sample and place it in a 96-well plate for measurement. The excitation wavelength is 382/4880.
1.4.4.2 Determination of DA content in brain tissue 50 mice of the above experimental mice were decapitated, brain tissue was washed with ice physiological saline, and water was absorbed. The balance of the brain was weighed and the mouse brain was along the midline of the brain. The HCl 0. 2 mol / L of HCl 0. 2 ml, plus 0. 4 ml, plus 0. 1 mol / L of HCl 0. 2 The supernatant was taken for 1 min and centrifuged at 2 500 r/min for 5 min to obtain the bottom aqueous phase. 50 ul of aqueous phase, plus PBS 150 }, 1 plus EDTA
40 flash, add freshly prepared iodine solution 20, mix, let stand for 3 min, add alkaline sodium phosphate 40 ul, mix, let stand for 3 min, add 5 mol / L acetic acid 40 ul, mix, 100 ° C water bath for 15 min, cooling is available. Precision extraction of 200 ul of each sample, placed in a 96-well plate, content determination, excitation wavelength of 325 / 385. Record the results.
1.4.4.3 Determination of 5-HT content in brain tissue 50 mice of the above experimental mice were decapitated, brain tissue was washed with ice physiological saline, and water was absorbed. The balance of the mouse brain was weighed and the mouse brain was The midline of the brain is cut and the left hemisphere is added.
After 2 ml of acidic n-butanol, the mixture was homogenized, and the supernatant was taken as 0. 4 ml, plus n-heptane 0. 8 ml, plus 0.1 mol/L of HCl 0. 2 ml ultrasonic for 1 min, 2 500 r/ The bottom aqueous phase was taken by centrifugation for 5 min. 05%åŠè‚¤æ°¨ã€‚ Take the water phase 0. 4 ml, add 0. 25% half skin ammonia
Acid (new) 0. 1 ml, then add 0. 002% OPTO NHCI 3 mlo mix, boil water bath for 10 min, cool it. Precision extraction of each sample 200 flash 96-well plate for content determination, excitation wavelength is 368 /480 0
2 results
2. 1 compound preparation Shenyi Anshen tablets platform test results positive control group, middle dose group latency and error times were significantly different from the blank control group (P <0.05 or P <0. O1), high dose group latency and error times and There was a significant difference in the blank control group (P<0. O1).
2. 2 Fuwan preparations Shenyi Anshen tablets dark test results in the number of errors in the number of groups were significantly different from the blank control group (P <0.05), high-dose group, positive control group error times compared with the blank control group There was a significant difference (P < 0. O1), and the latency of the other 4 groups increased compared with the blank control group.
2. 3 compound preparation Shenyi Anshen tablets water maze test results in the middle dose group, high dose group experiment completion time [(69. 84士23. 37) e, X65. 45士22.98) e] and the blank control group [(114 82 士7.96) e) The difference is significant (P<0. O5). The positive control group was (101. 72 ± 26.32) e, and the low dose group was (80. 50 ± 27.65) eo
2. 4 The monoamines in the brain of the mice were measured by the sputum of the sputum. The levels of 5-HT, DA and NE in the high and middle dose groups were significantly increased compared with the blank control group (P<0. OS). The low-dose group was higher than the blank control group, but there was no significant difference (P>0. OS).
3 discussion
Shenyi Anshen tablets selected four commonly used traditional Chinese medicines for compatibility. It can be seen through the darkness test, the platform test, and the water maze test that both the wrong number and the latent period of the drug-administered group improved, and the drug efficacy was compared with the positive control drug. The efficacy is quite the same. Therefore, from the perspective of pharmacodynamics, the improvement of learning and memory of Shenyi Anshen Tablet can be fully affirmed and has a good educational effect.
Monoamine neurotransmitters include catechins and guanamines. The former include DA, NE and epinephrine, the latter is 5-HT, and there are a large number of DA and 5-HT nerves in the cerebral cortex and striatum. At the end, they play an important role in high-level activities such as feelings, learning, and memory. Decreased DA content is one of the manifestations of brain aging. Learning and memory enhancement is often accompanied by an increase in NE and DA, but the report on 5-HT is not particularly clear. Shenyi Anshen tablets can improve the learning and memory ability of mice and may be related to the mutual regulation of 5-HT, DA and NE. Related literature reports [7], DA metabolism in the nerve center will affect the metabolism of NE, NE may regulate synaptic afferent activity in a wide range of brain regions, increase the meaningful information in the environment, and inhibit other afferent stimuli. Interference, thereby increasing attention and enhancing learning ability.
In this experiment, the content of neurotransmitters was determined by fluorescence method to study the mechanism of improving memory. Fluorescence method was compared with liquid chromatography. Fluorescence method can improve the efficiency of experiments, and the application of pre-oral kits is increasing. Extensive, can
Optimization is carried out in subsequent mechanism research trials.
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