Study on the relationship between the learning and memory ability of the effective component of Zigui Decoction and the dementia model induced by scopolamine

Water labyrinth and platform test related literature: Study on the relationship between the learning and memory ability of the effective component of Zigui Decoction and the dementia model induced by scopolamine

Abstract目的AIM: To evaluate the effect of the effective component of Zigui Decoction on the improvement of learning and memory ability in mice with dementia induced by scopolamine, and to determine the pharmacological effect of the effective component of Zigui Decoction against dementia model. " Relationship and ED50 provide a dose basis for further evaluation of efficacy. METHODS: Kunming mice were used for Morris water maze and platform training. 80 qualified mice, male and female, were randomly divided into normal control group, scopolamine-induced dementia model group, and pharmacodynamic doses 1 to 5 groups. And the positive drug group, the respective treatment drugs were given before the experiment. After 30 days of continuous administration, the scopolamine model was injected intraperitoneally except the normal control group, and the Morris water maze test and the platform test were performed 30 min later to analyze and compare the different dose groups. Learning the difference in memory ability. RESULTS: The mouse Morris water maze test showed that the effective component of Zigui Decoction can significantly improve the learning and memory ability of scopolamine-induced dementia mice, and can significantly shorten the water maze latency of mice. 92 to 43. There was a significant difference in the dose range of 68 g/kg, and it was dose-dependent, with an ED50 of 4. 79g/kg. The experiment of mouse jumping platform showed that the effective component of Zigui Decoction can significantly improve the learning and memory ability of mice with dementia induced by scopolamine, which can prolong the latency of jumping platform and reduce the number of jumping errors. 73 to 43. There was a significant difference in the dose range of 68 g/kg, and it was dose-dependent, with an ED50 of 2. 70g/kg. Conclusion: The effective component of Zigui Decoction can significantly improve the learning and memory ability of mice induced by scopolamine, and the drug effect is more and more obvious with the increase of dose, showing a dose-dependent effect. " Relationship.

Key words Ziguitang effective component; scopolamine dementia mice; Morris water maze experiment; platform experiment; learning and memory ability; "quantity-effect" relationship

Alzheimer disease (AD), also known as senile dementia, is a major disease that seriously threatens the physical and mental health and life safety of the elderly. Modern research has confirmed that Alzheimer's disease is a progressive neurodegenerative disease with cognitive and behavioral disorders, and its pathogenesis is quite complex, involving multiple systems and multiple links. Due to its insidious onset, unclear etiology, long treatment time, and expensive treatment and care, it has become the fourth leading cause of death in the world after heart disease, cancer and stroke. Therefore, the research on aging and anti-dementia drugs has become the focus of the field of geriatric medicine, and has attracted more and more attention. Zigui Decoction is a traditional Chinese medicine compound preparation consisting of Chuanxiong, Angelica and Epimedium, which is composed of Chuanxiong, Angelica and Epimedium. However, due to its complex composition and diverse pharmacological effects, There are few studies on the pharmacodynamics of dementia caused by different pathogenesis, so it is necessary to study the pharmacodynamics of Morris water maze and platform based on the classic scopolamine-induced dementia model. The results are as follows:

1 material

1.1 The test drugs Chuanxiong, Angelica and Epimedium were identified as the dried rhizome of Ligusticum Chuanxiong Hort. by the National Chinese Medicine Creation Project Group according to the 2010 Chinese Pharmacopoeia, batch number 20110312; Umbelliferae Angelica ( Angelia Dry roots of Sinessis (oliv.) Diels), lot number 20110305; dried leaves of Epimedium brevicornu Maxim, batch number 20100328. Both are provided by Anhui World Trade Pharmaceutical Co., Ltd. Zigui Tang is composed of Chuanxiong, Angelica and Epimedium according to 4: 2: 1 (24 g of Chuanxiong, 12 g of Angelica sinensis, 6 g of Epimedium, 70 g of human daily dose is 42 g. Weigh the appropriate amount of Chuanxiong and Angelica, appropriate After pulverization, place in a 3000 ml round bottom flask, soak in 12 times water for 3 hours, heat and condense in a heating jacket to reflux until the volatile oil no longer increases, collect the volatile oil, record the volume (ml). The liquid is filtered in 4 layers of gauze. The aqueous extract was collected and concentrated. The amount of the scum was added to the 3000 ml round bottom flask, and the mixture was added with 10 times, 8 times, 70% ethanol, condensed and refluxed for 2 h and 1.5 h, and filtered through 6 layers of gauze. Combine the two alcohol extracts, recover the ethanol and concentrate. The alcohol extract concentrated extract is mixed with the water extract concentrated extract, and then the volatile oil and the Tween are ground and mixed in a ratio of 1:1, and the mixed concentrate is added to obtain the final Zigui soup. Process-drug extract, yield: 39. 70%, batch number: 2011040101. Combine the amount of the original prescription, the extraction rate of each part of the pharmacy and the pre-experiment data of the drug effect, and calculate the dose of the mouse by the body surface area according to the daily dose. 4, 2, 1, 0.5 times the dose, the dose size is 43.68 g/kg, 21.84 g/kg, 10.92 g/kg, 5.46g / kg, 2.7 g / kg, according to the principle of equal volume and different concentration, the gavage volume of each group was calculated according to 25 ml/kg. Donepezil hydrochloride dispersible tablets , Specifications: Each tablet contains donepezil 5 mg, produced by Shandong Luoxin Pharmaceutical Co., Ltd., batch number: 091243, twice the dose of the body surface area according to the daily dose, the dosage is 1.3mg/ Kg, administered in a volume of 25 ml / kg.

1.2 Animal Kunming mice, SPF pole, body weight 18.0 ~ 22.0g, male and female, SPF grade, provided by the Animal Experimental Center of Shandong University, certificate number: SCXK (Lu) 20090001. SPF-level ordinary mice were fed with feed (provided by Shandong University Animal Experimental Center, experimental animal compound feed (rat), production license number: SCXK (Lu) 20090014).

1.3 Reagent scopolamine hydrobromide, purity ≥ 99.0%, manufactured by Suzhou Industrial Park Yake Chemical Reagent Co., Ltd., batch number: YK2011031201. When used, it should be placed in distilled water to a concentration of 2 mg/kg, and administered intraperitoneally in a volume of 3 ml/kg.

1.4 Instrument XR-XM101 Morris water maze video tracking analysis system, manufactured by Shanghai Xinsoft Information Technology Co., Ltd. The system consists of a water labyrinth instrument: The circular pool has a diameter of 120 cm, a height of 50 cm, a platform height of 20 cm, and a diameter of 8 cm. A display system camera is placed above the maze, and the computer automatically tracks and records the swimming trajectory. XR-3TB mouse jumping test box automatic recording system, manufactured by Shanghai Xinsoft Information Technology Co., Ltd. The system device is a passive anti-reflection box, and the bottom of the box is covered with an electric grid as a stimulating electrode. A circular platform with a height and a diameter of 4.5 cm is placed in the left rear corner of the box to avoid the electric shock.

1.5 Methods Water maze and jumping platform were screened and trained 80 mice, male and female, were randomly divided into normal control group, model control group, Qiguitang effective component dose group 1 to 5 and positive drug group. The effective component doses of Zigui Decoction 1 to 5 and the positive drug group were given the respective therapeutic drugs, and the rats were intragastrically administered in a volume of 25 ml/kg per day. The blank control group was given the same volume of normal saline for 30 days.

1.5.1 Morris water maze experiment newly purchased Kunming mice, after 3 days of stability, Morris water maze screening experiment 2 times. Before the mice were screened for water maze, the mice were placed on the platform for 10 s to allow them to understand the platform, and then the mice were placed into the water from the platform diagonal quadrant head toward the arm to see if the platform could be found within 2 min. Can be found in the formal experiment of the platform, the mouse can not find the platform to guide it to the platform for 10 s, 10 minutes after the second screening experiment, can be found in the formal experiment, can not find the platform of the mouse twice Exclude the experiment. The qualified mice were subjected to water maze training experiments 4 times a day and 2 times in the morning and afternoon. The training experiment was the same as the screening experiment method, so continuous training was carried out 10 to 15 times to ensure that each mouse can be in the prescribed time. Find the platform. During the experiment, the platform was 1 cm below the water surface and the water temperature was 25.0 ± 1.0 °C. The bleach was added to the water and mixed thoroughly to make the water milky white. The labyrinth reference remained unchanged during the experiment. The mice were placed in the water fan from the platform diagonal quadrant head to the barrel arm respectively. The time (latency period) of the mouse to find the platform within 2 min and the number of times of the platform were recorded. As the index of learning and memory ability, the animals were allowed to find the platform. Stay on the platform for 30 s; if the mouse does not find the platform within 120 s, record 120 s. During the test, the position of the spatial reference objects such as lights and articles in the laboratory shall be kept unchanged, and the interference factors shall be excluded.

1.5.2 Jumping test The mice that passed the water maze screening training were placed in different reaction boxes of the jumping platform for 3 min to make them aware of the environment, and then immediately passed the current of 0.2 mA, and the time was 5 min, which was able to be within 5 min. Cognitive platforms and mice that evade electrical stimulation were included in the formal experiment. The mice that could not recognize the platform were manually guided to jump on the platform to avoid electrical stimulation, and again adapted for 3 min, and the second platform experiment was screened. The mice that could not find the platform and evade the electrical stimulation were excluded from the experiment. The screened mice were subjected to a platform training experiment, and the training method was the same as the screening method, so that the training was continuously performed 5 to 10 times. According to Xu Shuyun's classic pharmacological experimental methodology, the platform test was conducted in two days. The first day of study and the second day of memory. During the experiment, the mice were placed in a reaction chamber for 3 min, and then immediately passed through a current of 0.2 mA. Animals are subjected to electric shock. The normal reaction is to jump back to the platform to avoid noxious stimuli. Most animals may jump to the copper grid again or again, and then jump back to the platform after being shocked. At 5 min, the time (latency) of the first jump of the mouse on the platform during the two days (the incubation period) and the number of times the mouse jumped off the platform within 5 minutes (the number of errors) were recorded.

1.5.3 Morris water maze and platform test 30 days after administration 30 min In addition to the normal control group, intraperitoneal injection of scopolamine model, the dose was 2 mg / kg, the injection volume was 3. 0 ml/kg. Morris water maze test was performed 30 min after modeling, and the latency and number of passages of each group of mice within 2 min were recorded. The 32-33 day diving experiment was conducted, and the administration method and time were the same as above. The 32nd day is the platform learning experiment, the 33rd day is the platform memory experiment, the current is controlled at 0.2mA, the timing is 5 min, and the time when the mouse jumps off the platform for the first time (latency period) and the small within 5 minutes are recorded separately. The number of times the mouse jumped off the platform (the number of errors). Comprehensive analysis of the differences in their learning ability.

1.5.4 ED50 calculation of 芎 汤 汤 以 以 以 以 以 以 以 ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED ED Effective ED50. Improvement rate of drug efficacy = incubation period of drug group - model control group latency model control group latency × 100% (effectiveness improvement rate > 25%, effective, recorded as positive)

3 Discussion

Through comparison and analysis of Morris water maze and platform test in each group of mice, the weight of learning and memory decreased gradually from high to low in each dose group of Zigui Decoction, showing a dose-dependent effect. The pharmacological effect of dose group 1 was the strongest. It is suggested that Zigui Decoction has a certain protective effect against the decline of learning and memory ability of scopolamine-induced dementia mice, and its effect size increases with the increase of dose, showing a significant "quantity-effect" relationship. Compared with the model control group, the improvement of learning and memory ability in the Morris water maze test of scopolamine-induced dementia mice was significantly different in the dose range of 10.92 ~ 43.68 g / kg, and it was dose-dependent. The ED50 was 4.79g / kg. The improvement of learning and memory ability of the scopoling mice induced by scopolamine in the dementia mice was significantly different in the dose range of 2.73 ~ 43.68 g / kg, and the dose was LD50. 2.70g/kg. It shows that the effect of Ziguitang in the platform test is better than that in the water maze.

At present, there are different opinions on the pathogenesis of AD. There is no hypothesis that can fully explain the occurrence and development of AD. Among them, the cholinergic damage hypothesis is one of the more widely recognized mainstream theories. It is considered that acetylcholine is an important substance related to learning and memory, and the hippocampus is an important anatomical basis for learning and memory. The choline in the basal forebrain region occurs when AD occurs. The loss of neurons can lead to a decrease in the synthesis, storage, and release of acetylcholine, leading to multiple clinical manifestations based on memory and recognition dysfunction. The form of memory impairment caused by the scopolamine-induced dementia model developed according to the cholinergic hypothesis is very similar to that of senile amnesia, and is therefore widely used in animal experiments to establish cognitive impairment to verify the effects of drugs. Morris water maze experiment and platform test are the most important experimental methods for studying learning and memory ability. Therefore, combined with the scopolamine-induced dementia model, the pharmacodynamic study of drugs using Morris water maze and platform method has become the mainstream of anti-dementia drugs. method. However, in the actual experimental operation, the author found that the Morris water maze experiment and the platform test have their inadequacies. For example, the difference in performance between individuals is huge, the animal's avoidance response is quite different, and it is necessary to detect a large number of animals, etc. Pharmacodynamics laboratories should comprehensively use a variety of behavioral testing methods, comprehensive analysis and pharmacodynamic evaluation to improve the accuracy and persuasiveness of experimental conclusions, and to more effectively and accurately conduct relevant drug activity screening and The evaluation of the efficacy of the drug provides an experimental basis, thereby accelerating the development of the pharmacology of Chinese medicine and improving the level of new drug creation.

Therefore, this experiment used the Morris water maze and the jumping method to conduct a comprehensive evaluation of the pharmacodynamic "quantity-effect" relationship of the effective component of Ziguitang against the scopolamine-induced dementia model, and to analyze the behavior of different dose groups of Ziguitang. The data showed that it had a certain improvement effect on the decline of learning and memory ability of mice with dementia induced by scopolamine, showing a dose-dependent effect. The learning and memory abilities of the different dose groups of Ziguitang in the platform test were better than those in the Morris water maze. The ED50 of the platform was less than the ED50 in the water maze.

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