Study On The Intelligence-promoting Effect Of The Combination Of Total Glycosides Of Cistanche Deserticola And Lycium Barbarum Polysaccharides On C57BL/6J Mice At Different Ages

Summary

Objective: To investigate the effects of total glycosides of cistanche deserticola combined with Lycium barbarum polysaccharides on the learning and memory abilities, the synthesis and degradation of cholinergic neurotransmitters, and the level of oxidative stress in the brain of C57BL/6J mice at different months of age. Methods: 48 C57BL/6J mice aged 3, 6, and 9 months each were randomly divided into a control group, total glycosides of cistanche deserticola 100 mg/kg group, Lycium barbarum polysaccharide 100 mg/kg group, and combined (total glycosides of cistanche deserticola 100 mg/kg+lycium barbarum polysaccharide 100 mg/kg) group. After 30 days of intragastric administration, the learning and memory abilities of mice at different months of age were evaluated by the Morris water maze test, dark avoidance test, and platform jumping test; The levels of AChE, ChAT, T-SOD, GSH-Px, and MDA in the cerebral cortex of mice at different months of age were measured by colorimetry. Results: Compared with the control group, the learning and memory abilities of mice in each month-old combination group were improved, the activities of ChAT and T-SOD in the cortex of mice in the 3 and 6-month-old combination group were significantly increased, the activities of ChAT and GSH-Px in the cortex of mice in the 9-month-old combination group were significantly increased, and the content of MDA in the cerebral cortex of mice in the 6 and 9-month-old combination group was significantly decreased (P<0.05) 05 or P<0 01)。 Conclusion: The combined administration of Cistanche glycosides and Lycium barbarum polysaccharides can improve the learning and memory abilities of C57BL/6J mice aged 3, 6, and 9 months, and its mechanism may be related to regulating the synthesis and degradation of cholinergic neurotransmitters in the brain and inhibiting the level of oxidative stress in the brain.

Keywords 

Total glycosides of Cistanche deserticola; Lycium barbarum polysaccharides; Acetylcholine; oxidative stress

Learning and memory are advanced intelligent activities of the central nervous system, and they are closely related. At the end of the last century, the World Health Organization formulated and conducted a "Brain Science Research Plan", and countries such as the United States and Japan actively carried out research plans to solve the mysteries of the human brain, solve the mystery of memory, and seek effective methods and drugs to improve human learning and memory functions and prevent and treat human learning and memory disorders. There is no doubt that improving learning and memory functions and preventing and treating related diseases will be major tasks in the field of life science.

Cistanche deserticola and Lycium barbarum are two traditional tonifying herbs commonly used in clinical practice. The total glycosides of Cistanche deserticola are ethanol extracts from the dried, scaly, fleshy stems of the Orobaceae plant Cistanche deserticola or Cistanche tubulosa. Studies have found that the total glycosides of Cistanche deserticola have significant effects on A β 1-42 induced PC12 cell damage have a significant protective effect [2] and can improve A β 1-42 induced learning and cognitive dysfunction and oxidative stress levels in Alzheimer's disease rats. Lycium barbarum polysaccharide is one of the main effective components extracted from the fruit of Lycium barbarum, which has good anti-fatigue, anti-aging, antioxidant, and neuroprotective effects. In ancient prescription research, it was found that Cistanche deserticola often plays an anti-aging role in combination with Chinese wolfberry. However, there are no relevant literature reports on the use of the combination of the two in promoting intelligence. In this experiment, C57BL/6J mice aged 3, 6, and 9 months were used to investigate the effects of total glycosides of cistanche deserticola combined with Lycium barbarum polysaccharides on the learning and memory abilities of C57BL/6J mice aged different months and the related mechanisms through behavioral experiments, determination of cholinergic and oxidative stress-related indicators in the brain, so as to provide a certain research basis for its clinical application in promoting intelligence.

Chinese medicines such as ginseng, Cistanche deserticola, and astragalus

1 Material and Instruments

1.1 Experimental animals

There are 48 SPF grade C57BL/6J mice aged 3, 6, and 9 months each, half male and a half female purchased from Nanjing Junke Biotechnology Co., Ltd., with the laboratory animal certificate number 20180006018886. During the experiment, free intake of water and 12 hours of alternating light and dark illumination were conducted. All experiments were conducted in accordance with the experimental regulations, and the operation and management were in accordance with relevant laboratory animal management regulations.

Chinese medicinal materials such as cistanche

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1.2 Drugs and reagents

Total glycosides of cistanche deserticola (mass fraction ≥ 80%, batch number: JZ20092513), Lycium barbarum polysaccharides (mass fraction ≥ 90%, batch number: JZ21042710), Nanjing Jingzhu Biotechnology Co., Ltd; BCA Kit, Acetylcholinesterase (AChE) Kit, Acetylcholine transferase (ChAT) Kit, Malondialdehyde (MDA) Kit, Total Superoxide Dismutase (T-SOD) Kit, Glutathione Catalase (GSH-Px) Kit, Nanjing Jiancheng Biological Engineering Research Institute.

1.3 Main instruments

DMS-2 Morris Water Maze Experimental Device, DTT-2 Mouse Platform Jumping Device, SBA-2 Mouse Darkness Avoidance Test Device, Institute of Medicine, Chinese Academy of Medical Sciences; 4503R low-temperature high-speed centrifuge, Eppendorf, Germany; VARIOSKAN FLASH ELISA, Thermo Corporation, USA.

2 Methods

2.1 Animal grouping and administration

48 C57BL/6J mice aged 3, 6, and 9 months each were randomly divided into 4 groups, with 12 mice in each group, half male and half female, including the blank control group, the total glycosides of Cistanche deserticola group (100 mg/kg), the Lycium barbarum polysaccharide group (100 mg/kg), and the combined group (total glycosides of Cistanche deserticola+lycium barbarum polysaccharide 100 mg/kg). They were administered daily by gavage for 1 month.

2.2 Morris water maze experiment

The device consists of a black circular pool, a transparent circular platform, a camera, and an automatic image acquisition and processing system. The outer edge of the pool is equidistantly divided into four water entry points: east, west, south, and north. A circular transparent platform with a diameter of 15 cm is placed inside the pool. During the experiment, maintain a constant temperature, sound insulation, and no direct light to prevent external factors from interfering with the mice. The experiment was conducted for a total of six days: from the first day to the fifth day, a positioning navigation experiment was conducted, recording the time when the mouse found the platform within 120 seconds, which is the escape latency. If the mouse did not find the platform within 120 seconds, its escape latency was 120 seconds. After training, each mouse, regardless of whether it found the platform, was placed on the platform to learn for 15 seconds, and entered the water from the adjacent relative quadrant of the platform to learn twice a day; On the sixth day, a space exploration experiment was conducted, and the platform was removed. The first time it crossed the platform after entering the water from the adjacent relative quadrant and the number of times it crossed the platform within 120 seconds were measured.

2.3 Darkness avoidance experiment

The device is divided into two chambers, open and dark. The two chambers are connected by a small hole for mice to enter and exit. A 40 V current is applied to the bottom of the dark chamber, while no electricity is applied to the bottom of the bright chamber. The experiment is divided into two stages: The first day is the learning stage. First, put the mouse back in the hole and put it into the bright room. After allowing it to freely move in two rooms for 2 to 3 minutes to adapt to the environment, put the mouse back in the hole and put it into the bright room. The mouse's habit of tending to darkness and avoiding light and liking to drill holes will cause it to enter the dark room and receive an electric shock. If the mouse does not enter, it will be driven into the dark room to generate memory; The second day is the testing stage. Put the mouse back into the hole in the light chamber, and record the incubation period of the mouse entering the dark chamber from the light chamber and the number of times it enters the dark chamber within 3 minutes as the test result.

2.4 Platform diving experiment

The diving platform device is a 20 cm × 20 cm × A 30 cm rectangular body with an insulated rubber platform on the bottom for mice to stand on and a 36 V current applied. The experiment was conducted for two consecutive days, and the first day was a learning and training phase. First, the mice were placed in a jumping platform test box and allowed to freely move for 3 minutes to adapt to the environment. Then, the mice were placed on an insulating platform, with the bottom copper grid energized for continuous stimulation for 180 seconds. After receiving an electric shock, the mice jumped onto the platform to avoid the electric shock. If the mice did not jump onto the platform, they were manually guided to the insulating platform by the experimental operator; The second day is the testing phase. Place the mouse on an insulated platform, and then energize the bottom copper grid. Record the time when the mouse first jumped off the platform as a latency period. The number of times the mouse jumped off the platform within 180 seconds is recorded as an error number. If the mouse does not jump off the platform within 180 seconds, the latency period is recorded as 180 seconds.

2.5 Index detection

At the end of the behavioral experiment, the mouse was decapitated, the cortex was quickly separated at low temperatures, and a certain amount of cortical tissue was accurately weighed. Nine times the volume of normal saline was added. The homogenate was lysed to 10% tissue homogenate, and centrifuged at 4 ℃ at 3000 r/min for 10 minutes. After the supernatant was taken and the protein concentration was measured with a BCA kit, the experiment was conducted in strict accordance with the steps in the instructions of the AChE, ChAT, MDA, T SOD, and GSH-Px kits.

2.6 Statistical processing

Use SPSS 19 for all data 0 software for statistical analysis. In the Morris water maze experiment, the positioning navigation experiment data were analyzed using two-way ANOVA, while other experimental data were analyzed using one-way ANOVA, combined with the LSD test, to compare the differences between the groups. The results were expressed as (􀭰 x ± s), with P<0 05 as a statistically significant difference, using Origin 2021 for mapping.

3 Results

Cistanche deserticola ma

3.1 Effect of the compatibility of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides on Morris water maze test in C57BL/6J mice of different months of age

As shown in Figure 1, compared with the control group, there was no significant change in the escape latency of the 3 and 6-month-old mice in each treatment group, while the escape latency of the 9-month-old mice in each treatment group on the 4th day was significantly shortened (P<0. 05) 05)。 In the space exploration experiment, compared with the control group, the first time crossing platform time of the 6 and 9-month-old mouse combination group was significantly shortened, the number of times crossing platform in each month-old mouse combination group was significantly increased, the target quadrant residence time was significantly prolonged, and the target quadrant residence time of the 3 and 9-month-old mice in the total glycosides of cistanche deserticola group was significantly prolonged (P<0.05) 05 or P<0 01)。 The effect of the combination of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides on the dark avoidance experiment in C57BL/6J mice at different months of age is shown in Figure 2. Compared with the control group, the incubation period of mice in the 3-month combined group was significantly prolonged, the number of errors in each month combined group was significantly reduced, and the number of errors in the 9-month Lycium barbarum polysaccharide group was significantly reduced (P<0. 05) 05 or P<0 .01)。

3.2 Effect of the compatibility of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides on platform jumping test in C57BL/6J mice of different months of age

As shown in Figure 3, there was no statistically significant difference in latency and the number of errors in platform jumping experiments among mice in each group (P>0. 05) 05)。

3.3 Effects of the compatibility of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides on the synthesis and degradation of ACh in the cerebral cortex of C57BL/6J mice at different months of age

Desert ginseng

As shown in Figure 4, compared with the control group, the activity of ChAT in the cerebral cortex of mice in the combined group at each month of age significantly increased (P<0. 05) 05 or P<0 01)。 

3.4 Effects of the compatibility of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides on the synthesis and degradation of ACh in the cerebral cortex of C57BL/6J mice at different months of age

As shown in Figure 4, compared with the control group, the activity of ChAT in the cerebral cortex of mice in the combined group at each month of age significantly increased (P<0. 05) 05 or P<0 01)。

3.5 Effects of the compatibility of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides on the levels of MDA, T-SOD, GSH-Px in the cerebral cortex of C57BL/6J mice at different months of age

As shown in Figure 5, compared with the control group, the MDA level in the cerebral cortex of mice in the 6-month-old and 9-month-old combination groups and the 6-month-old total glycosides group of cistanche deserticola significantly decreased, the T-SOD level in the cerebral cortex of mice in the 3-month-old and 6-month-old combination groups significantly increased, and the GSH-Px level in the 9-month-old Lycium barbarum polysaccharide group and the combination group significantly increased (P<0. 05) 05 or P<0 01)。

Figure 1   Effect of total glycosides of cistanche deserticola combined with lycium barbarum polysaccharides on Morris water maze test in C57BL/6J mice of different months of age

A. Control group B. Cistanche glycosides group C. Lycium barbarum polysaccharide group D. Combination group

Note: Compared with the control group, ∗ P<0 05, ∗∗ P<0. 01

Figure 2   Effect of total glycosides of cistanche deserticola combined with lycium barbarum polysaccharides on dark avoidance experiment in C57BL/6J mice at different months of age

A. Control group B. Cistanche glycosides group C. Lycium barbarum polysaccharide group D. Combination group

Note: Compared with the control group, ∗ P<0 05, ∗∗ P<0. 01

Figure 3   Effect of the compatibility of total glycosides of cistanche deserticola and lycium barbarum polysaccharides on platform jumping test in C57BL/6J mice of different months of age

A. Control group B. Cistanche glycosides group C. Lycium barbarum polysaccharide group D. Combination group

Note: Compared with the control group, ∗ P<0 05, ∗∗ P<0. 01

Figure 4   Effect of the combination of total glycosides of cistanche deserticola and lycium barbarum polysaccharides on the synthesis and degradation of ACh in the cerebral cortex of C57BL/6J mice at different months of age

A. Control group B. Cistanche glycosides group C. Lycium barbarum polysaccharide group D. Combination group

Note: Compared with the control group, ∗ P<0 05, ∗∗ P<0. 01

Figure 5   Effect of total glycosides of cistanche deserticola combined with lycium barbarum polysaccharides on the levels of MDA, T-SOD, and GSH-Px in the cerebral cortex of C57BL/6J mice at different months of age

A. Control group B. Cistanche glycosides group C. Lycium barbarum polysaccharide group D. Combination group

Note: Compared with the control group, ∗ P<0 05, ∗∗ P<0. 01

4 Discussion

Learning and memory are one of the important advanced neurophysiological activities of the brain. Learning refers to the process of receiving information from the external environment and influencing one's own behavior. Memory is the process of storing, processing and extracting acquired information or experiences in the brain. Both are important functions for humans to understand and adapt to the world. Therefore, developing drugs to improve human learning and memory functions and prevent and treat related diseases will be a major challenge in the field of life science. Currently, there are various behavioral testing methods for evaluating learning and memory functions, such as the Morris water maze experiment, the Y-word maze experiment, the new object recognition experiment, the platform diving experiment, and the dark avoidance experiment. The Morris water maze experiment is a classic experiment aimed at detecting the spatial learning and memory abilities of rodents, while the dark avoidance experiment and the platform jumping experiment are two commonly used behavioral experiments to evaluate the short-term learning and memory abilities of mice. In this experiment, the above three behavioral experiments were used to evaluate the effect of total glycosides of cistanche deserticola combined with Lycium barbarum polysaccharides on the learning and memory abilities of C57BL/6J mice aged 3, 6, and 9 months. The results showed that in the Morris water maze experiment, the combination of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides significantly shortened the first crossing platform time of C57BL/6J mice aged 6 and 9 months, increased the number of times C57BL/6J mice crossed the platform and the target quadrant residence time, significantly prolonged the incubation period of C57BL/6J mice aged 3 months in the dark avoidance experiment, and decreased the number of errors in C57BL/6J mice aged 3, 6 and 9 months. The latency increased most and the number of errors decreased most in the group of mice administered with total glycosides of cistanche deserticola and Lycium barbarum polysaccharides at the age of each month in the platform jumping experiment. The above behavioral results suggest that the combination of total glycosides of cistanche and Lycium barbarum polysaccharides can better improve the learning and memory abilities of C57BL/6J mice aged 3, 6, and 9 months compared to the use of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides alone.

Superman herbs cistanche

In the central nervous system, cholinergic neurons dominate most brain regions. ACh is a neurotransmitter released from the terminals of cholinergic neurons, and is one of the most important neurotransmitters in the central nervous system, participating in most physiological activities of the brain. AChE and ChAT are two important key enzymes in the central cholinergic system, which regulate the degradation and synthesis of ACh in the central cholinergic system, respectively. Their activities indirectly affect the content of ACh. Therefore, the content of the neurotransmitter ACh and the activity of its marker enzymes have a direct or indirect impact on the learning and memory signal pathway [10,11]. The results of this experiment showed that the AChE activity in the cerebral cortex of mice in each month-old group treated with the combination of total glycosides of cistanche deserticola ma and Lycium barbarum polysaccharides decreased the most, and the ChAT activity in the cerebral cortex of C57BL/6J mice in different months old group was also significantly increased. Compared with the use of total glycosides of cistanche and Lycium barbarum polysaccharides alone, the combination of total glycosides of cistanche and Lycium barbarum polysaccharides significantly increased the synthesis rate of ACh and decreased its hydrolysis, increasing the content of ACh in the brain, To improve the learning and memory abilities of C57BL/6J mice at different months of age.

Due to the particularity of brain tissue structure, it is more susceptible to attack by oxygen free radicals, causing damage to the macromolecular structure in the brain, inducing neuronal apoptosis in the central nervous system, and leading to learning and memory impairment. MDA is a marker of oxidative stress, and its increased content indicates the enhancement of oxidative stress. SOD and GSH-Px are important antioxidant enzymes in the body. Research has found that their activity decreases in patients with learning and memory impairment, which can cause damage to brain tissue structure and function. The results of this experiment showed that the combined use of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides could significantly reduce the content of MDA in the cerebral cortex of C57BL/6J mice at the age of 6 and 9 months, increase the activity of T-SOD in the cerebral cortex of C57BL/6J mice at the age of 3 and 6 months, and increase the activity of GSH-Px in the cerebral cortex of C57BL/6J mice at the age of 9 months, indicating that the combined use of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides could better improve the level of oxidative stress in the brain of C57BL/6J mice at the age of 3, 6, and 9 months, compared with the.

Benefits of cistanche tubulosa

In summary, compared to the use of total glycosides of cistanche deserticola and Lycium barbarum polysaccharides alone, the combination of total glycosides of cistanche and Lycium barbarum polysaccharides can better improve the learning and memory abilities of C57BL/6J mice aged 3, 6, and 9 months, which may be closely related to regulating the central cholinergic nervous system function and improving the level of oxidative stress in the brain.

reference

[1] Wu Tao Study on the Extraction, Purification, Fingerprint and Memory Improvement of Total Glucosides from Cistanche Tubulata [D] Urumqi: Xinjiang University, 2010

[2] Ju Bowei, Yang Jianhua, Yan Yao, et al Total glycosides of phenylethanol from Cistanche deserticola β Protective effect of 1-42 induced PC12 cell injury [J] Journal of Xinjiang Medical University, 2017,40 (5): 634-637

[3] Wang Lu, Bai Yumeng, Li Xiaoyu, et al Effects of total glycosides from cistanche deserticola on learning, cognitive function, and oxidative stress in Alzheimer's disease model rats [J] Journal of Anatomy, 2020,43 (3): 194-199

[4] Wei Fenfen, Wang Wenjuan, Zhang Bo Study on relieving physical fatigue in mice with lycium barbarum polysaccharides [J] Food Research and Development, 2020,41 (6): 48-52

[5] Yi R,Liu X,Dong Q. A study of Lycium barbarum polysaccharides extraction technology and its antiaging effect[J]. AJTCAM,2013,10(4):171-174. 

[6] Zhang Chunli, Lu Li, Fang Huijun, etc Determination of lycium barbarum polysaccharide content and evaluation of its antioxidant capacity in different regions of northwest China [J] Northwest Journal of National Defense Medicine, 2020,41 (7): 414-417

[7] Cao S,Du J,Hei Q. Lycium barbarum polysaccharide protects against neurotoxicity via the Nrf2-HO-1 pathway[ J]. Experimental and Therapeutic Medicine, 2017, 14 ( 5 ): 4919-4927. [8] Zhang Min Study on the Medication Law of Ancient Anti aging Prescriptions [D] Beijing: Chinese Academy of Traditional Chinese Medicine, 2018

[9] Haam J,Yakel JL. Cholinergic modulation of the hippocampal region and memory function[J]. Journal of neurochemistry,2017,142(2):111-121. 

[10] Eric T,Eugenia T. Oxidative stress,synaptic dysfunction, and Alzheimer′s disease[ J]. Journal of Alzheimer′s Disease,2017,57(4):1105-1121. 

[11] Skaper SD,Facci L, Zusso M, et al. Synaptic plasticity, dementia and alzheimer disease[ J]. CNS & neurological Disorders Drug Targets,2017,16(3):220-233. 

[12] Noreen S, Imran I, Iqra Z,et al. Ameliorative effect of lithium chloride against d-galactose induced behavioral and memory impairment,oxidative stress and alteration in serotonin function in rats [ J]. Pharmacological Reports, 2019,71(5):909-916. 

[13] Chen H, Lv K,Dai Z, et al. Intramuscular injection of mechano growth factor E domain peptide regulated expression of memory-related sod,miR-134 and miR-125b-3p in rat hippocampus under simulated weightlessness[ J]. Biotechnology letters,2016,38(12):2071-2080.