Application of Myrica rubra Fruit Anthocyanin Extract in the Preparation of a Drug for Treating Behavioral Disorders

Abstract

The present invention provides an application of a myrica rubra fruit anthocyanin extract in the preparation of a drug for treating behavioral disorders, belonging to the technical field of biomedicine. The myrica rubra fruit extract is rich in anthocyanins, and can treat cognitive disorders and mental disorders related to inflammatory bowel disease by regulating the antioxidant capacity of the brain and changing the permeability of the blood-cerebrospinal fluid barrier in the cerebral ventricle, and has a good alleviating effect on both. The myrica rubra fruit anthocyanin extract is expected to become a potential natural product for treating cognitive disorders and mental disorders such as anxiety and depression complicated with physical diseases, providing a new idea for the future development and utilization of myrica rubra.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of myrica rubra fruit anthocyanin extract in the preparation of drugs for treating behavioral disorders. Background Art

[0002] The pathological changes of inflammatory bowel disease can not only occur in the whole digestive tract, but also be combined with lesions in many extra-intestinal organs, such as the brain, resulting in cognitive disorders, mood disorders, etc. However, there is currently no effective drug treatment plan.

[0003] The brain-gut axis has been widely confirmed, and numerous studies have shown that there is a bidirectional regulatory effect between the intestine and the brain. Previously, many studies have used oral natural antioxidants to treat cognitive disorders, anxiety, depression and other mood disorders associated with somatic diseases, and found that they have a good therapeutic effect on the above-mentioned brain complications. However, the treatment mechanism has not been clarified. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide the application of myrica rubra fruit anthocyanin extract in the preparation of drugs for treating cognitive disorders and mental disorders related to inflammatory bowel disease. The myrica rubra fruit anthocyanin extract can effectively treat cognitive disorders and mental disorders related to inflammatory bowel disease by regulating the antioxidant capacity of the brain and changing the permeability of the blood-cerebrospinal fluid barrier in the choroid plexus.

[0005] The present invention provides the application of myrica rubra fruit anthocyanin extract in the preparation of drugs for treating behavioral disorders.

[0006] The first aspect of the present invention: the application of myrica rubra fruit extract in the preparation of drugs for treating behavioral disorders.

[0007] Specifically, the myrica rubra fruit extract contains anthocyanins, and the mass concentration of anthocyanins in the myrica rubra fruit extract is ≥20%.

[0008] Specifically, the treatment of brain complications includes treating cognitive disorders and mental disorders related to inflammatory bowel disease;

[0009] The treatment of cognitive disorders and mental disorders related to inflammatory bowel disease includes improving the increased level of brain oxidative stress caused by inflammatory bowel disease;

[0010] The increased level of brain oxidative stress caused by inflammatory bowel disease includes increasing the activities of antioxidant enzymes T-SOD and GSH levels, and decreasing the level of lipid peroxidation marker MDA.

[0011] The second aspect of the present invention: the preparation method of the myrica rubra fruit extract includes the following steps:

[0012] 1) An extract is obtained from high-quality and mature bayberry fruits. The organic solvent in the extract is removed to obtain a crude extract of bayberry anthocyanins. The extract used is a formic acid (volume fraction 0.1%) - ethanol solution;

[0013] 2) The crude extract of bayberry anthocyanins obtained in step 1) is re-dissolved with water to obtain a crude extract, which is then subjected to solid-phase extraction. After obtaining an extract using a methanol eluent, the organic solvent therein is removed to obtain an extract of bayberry fruit anthocyanins.

[0014] Specifically, the extraction in step 1) includes ultrasonic extraction at 15 - 25°C, and the ultrasonic extraction is carried out at a frequency of 20 - 50 kHz.

[0015] Specifically, the procedure of the solid-phase extraction in step 2) includes: loading, rinsing with pure water, and eluting with methanol.

[0016] The beneficial effects of the present invention are as follows:

[0017] The present invention provides the application of an extract of bayberry fruit anthocyanins in the preparation of a drug for treating cognitive impairment and mental disorders. The extract of bayberry fruit anthocyanins is rich in anthocyanins. The extract of bayberry fruit anthocyanins can treat cognitive impairment and mental disorders related to inflammatory bowel disease by regulating the antioxidant capacity of the brain and changing the permeability of the blood-cerebrospinal fluid barrier in the cerebral ventricle. Description of the Drawings

[0018] Figure 1 It is the UPLC diagram of the bayberry extract;

[0019] Figure 2 It is the result diagram of the FST, NOR, and OFT experiments after successful modeling; among them, (A) diagram is the immobility time diagram of the control group and the model group in the FST, (B) diagram is the recognition index diagram of the control group and the model group in the NOR, and diagrams C - F are the schematic diagrams of the results of the control group and the model group in the OFT, that is, (C) diagram is the schematic diagram of the total movement distance, (D) diagram is the schematic diagram of the percentage of the movement distance in the central area, (E) diagram is the schematic diagram of the number of entries into the central area, and (F) diagram is the schematic diagram of the percentage of the movement time in the central area;

[0020] Figure 3Schematic diagrams of the results of the FST, NOR, and OFT experiments after the treatment phase; Panels A - E are typical movement trajectory diagrams of the CONTROL, MODEL, Low BAE, High BAE, and SASP groups in the OFT. Panel (F) is a graph of the immobile time of the five groups of mice in the FST. Panel (G) is a graph of the recognition index of the five groups of mice in the NOR. Panels H - K are schematic diagrams of the results of the OFT experiment of the five groups of mice, that is, Panel (H) is a schematic diagram of the total distance, Panel (I) is a schematic diagram of the percentage of movement distance in the central area, Panel (J) is a schematic diagram of the number of entries into the central area, and Panel (K) is a graph of the percentage of movement time in the central area; (* indicates significant difference compared with the control group, # indicates significant difference compared with the model group, where *p < 0.05; **p < 0.01; ***p < 0.001.)

[0021] Figure 4 Schematic diagrams of the basic conditions of mice after drug intervention planning and drug treatment; among them, Panel (A) is a graph of the body weight of the CONTROL, MODEL, Low BAE, High BAE, and SASP groups of mice on the day of sacrifice. Panel (B) is a graph of the body weight changes of the five groups of mice during the whole experiment. Panels C and D are schematic diagrams of the colon length of the five groups of mice on the day of sacrifice; (* indicates significant difference compared with the control group, # indicates significant difference compared with the model group, where *p < 0.05; **p < 0.01; ***p < 0.001)

[0022] Figure 5 Graphs of the effects of BAE on oxidative stress and the results of Pearson correlation analysis; among them, Panels A - C are graphs of the effects of orally administered BAE on the superoxide dismutase (SOD) activity in the mouse brain tissue. Panel (A) is expressed in U / mg protein, the effect on the glutathione (GSH) level, Panel (B) is expressed in μmol / g protein, and the effect on the malondialdehyde (MDA) level. Panel (C) is expressed in nmol / g protein. Panel (D) is a graph of the Pearson correlation analysis coefficients of the data from the behavioral experiments, oxidative stress indicators, and relative BCSFB permeability data; in the upper right triangle of Panel (D), red dots indicate positive correlation between two sets of data, while blue dots indicate negative correlation. In the lower left triangle of Panel (D), the correlation coefficients between each group of data are listed, and * indicates significant difference (p < 0.05);

[0023] Figure 6 Representative images of immunohistochemical staining of Claudin - 2 and Claudin - 11 in the choroid plexus epithelium; containing the quantitative analysis results of the average gray value calculated by ImageJ. (* indicates significant difference compared with the control group, # indicates significant difference compared with the model group, where *p < 0.05; **p < 0.01; ***p < 0.001.)

[0024] Figure 7 Results of immunofluorescence staining of Claudin-3 in choroid plexus epithelium. (A) Representative images of Claudin-3 immunofluorescence in five groups, with blue being DAPI staining. (B) Quantitative analysis results of fluorescence intensity calculated by ImageJ. (C) Blood-cerebrospinal fluid barrier permeability levels of each group relative to the control group, n = 5 / group. (* indicates significant difference compared with the control group, # indicates significant difference compared with the model group, where *p < 0.05; **p < 0.01; ***p < 0.001.)

[0025] Figure 8 Shows the basic conditions of mice after pre-experimental treatment and the results of forced swimming test and open field test. (A) Shows the body weight changes of five groups of mice during the whole experiment; (B) Shows the body weight of five groups of mice on the day of sacrifice; (C) Figure Five Colon length of five groups of mice on the day of sacrifice; (D) Shows the immobility time in FST after treatment with myricetin extract for 7 days; (E-F) Show the results of OFT of five groups of mice after treatment with myricetin extract for 7 days, including the percentage of movement time in the central area in (E) and the total movement distance in (F). Specific implementation mode

[0026] The present invention provides the application of myricetin fruit anthocyanin extract in the preparation of drugs for treating inflammatory bowel disease-related cognitive impairment and mental disorders.

[0027] The present invention has no special requirements for the variety of myricetin, and the variety of myricetin used in the specific implementation process of the present invention is water chestnut, collected from Hangzhou, Zhejiang Province.

[0028] In the present invention, the myricetin fruit is preferably a mature and pest-free myricetin fruit; the myricetin fruit is preferably a myricetin fruit after removing the core. In the present invention, the myricetin fruit is preferably stored at 4°C.

[0029] In the present invention, the myricetin fruit anthocyanin extract is rich in anthocyanins, and the myricetin fruit anthocyanin extract can treat cognitive impairment and mental disorders by regulating the permeability of the blood-cerebrospinal fluid barrier, and has a good relieving effect on cognitive impairment and mental disorders related to inflammatory bowel disease in particular.

[0030] Preferably, the mass concentration of anthocyanins in the myricetin fruit extract ≥ 20%.

[0031] Preferably, the treatment of brain complications includes the treatment of inflammatory bowel disease-related cognitive impairment and mental disorders.

[0032] Preferably, the treatment of inflammatory bowel disease-related cognitive impairment and mental disorders includes improving the increased level of brain oxidative stress caused by inflammatory bowel disease.

[0033] Preferably, the increased brain oxidative stress level caused by inflammatory bowel disease includes increasing the activity of antioxidant enzyme T-SOD and the level of GSH, and decreasing the level of lipid peroxidation marker MDA.

[0034] In the present invention, the preparation method of the myrica rubra fruit anthocyanin extract comprises the following steps:

[0035] 1) Obtain mature and high-quality myrica rubra fruits to get an extract, and remove the organic solvent in the extract to obtain a crude myrica rubra anthocyanin extract. The extract used is a formic acid (volume fraction 0.1%) - ethanol solution.

[0036] 2) After redissolving the crude myrica rubra anthocyanin extract obtained in 1) by the water reconstitution method to obtain a crude extract, perform solid-phase extraction. After obtaining an extract with a methanol eluent, remove the organic solvent therein to obtain the myrica rubra fruit anthocyanin extract.

[0037] In the present invention, first obtain a myrica rubra fruit extract, and remove the organic solvent in the extract to obtain a crude myrica rubra anthocyanin extract; the extract is a formic acid (volume concentration 0.1%) - ethanol solution. In the present invention, the mass ratio of the extract to the myrica rubra fruit is preferably (5 - 3:1), more preferably 4:1.

[0038] In the present invention, the myrica rubra fruit with the extract preferably includes fully grinding the formic acid-ethanol solution and fresh fruits and then performing extraction. In the present invention, the grinding is preferably performed using a wall breaker; the extraction is preferably performed by ultrasonic extraction, the temperature is preferably 15 - 25°C, more preferably 20°C, the frequency is preferably 35 kHz, and the time is preferably 1 h; the advantage of using the ultrasonic extraction is that the extraction efficiency is higher and the extraction effect is more uniform. After the extraction in the present invention, it is preferably further included to fully stand the extract, and the standing time is preferably 9 - 16 h, more preferably 12 h. After the standing in the present invention, the preferred way to obtain the extract is filtration, and collect the filtrate; the filtration preferably includes a first filtration and a second filtration performed in sequence; the first filtration is preferably performed using 4 layers of gauze; the second filtration is preferably performed using 3 layers of gauze. In the present invention, the preferred way to remove the organic solvent in the extract is vacuum rotary evaporation, the temperature is preferably 30°C, the environment is preferably carried out in the dark, and rotary evaporation is performed until the organic solvent completely volatilizes to obtain the crude myrica rubra anthocyanin extract.

[0039] After obtaining the crude extract of bayberry anthocyanins, the present invention uses a water reconstitution method to obtain a crude extract solution from the crude extract. Preferably, after the water reconstitution in the present invention, the crude extract solution is filtered, preferably using a 0.22 μm aqueous filter membrane. After the filtration in the present invention, solid-phase extraction is performed on the crude extract solution, and an extract is obtained after elution. The organic solvent in the extract is removed to obtain an extract of bayberry fruit anthocyanins; the eluent includes methanol.

[0040] In the present invention, the solid-phase extraction column preferably includes a 20 cc C18 solid-phase extraction column; the solid-phase extraction column is preferably activated with methanol before use, and the dosage of the methanol is preferably 2 times the column volume (bed volume, BV); the solid-phase extraction column is preferably equilibrated with pure water before use, and the dosage of the pure water is preferably 4 BV. In the present invention, the procedure of the solid-phase extraction preferably includes: loading and rinsing with pure water, and the loading amount is preferably the sample after passing through the water membrane to 2 / 3 of the loading capacity of the solid-phase extraction column; the dosage of the pure water is preferably 20 BV; the function of the rinsing is to remove impurities; loading and rinsing with pure water and eluting with methanol; the dosage of the methanol is preferably 4 BV.

[0041] In the present invention, removing the organic solvent in the extract preferably includes: vacuum rotary evaporation, the temperature is preferably 30 °C, and the environment is preferably carried out in the dark until the organic solvent completely volatilizes. In the present invention, the function of the solid-phase extraction is to remove impurities such as sugars and acids.

[0042] Unless otherwise specified, the present invention has no special requirements for the sources of the raw materials used, and commercially available products well-known to those skilled in the art can be used.

[0043] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention.

[0044] Example 1

[0045] 1. Preparation of bayberry anthocyanin extract

[0046] The bayberry variety used in this experiment is 'Water Chestnut', which is collected from Lujingtang Ecological Garden, Hangzhou City, Zhejiang Province. After picking, the fresh and mature fruits are immediately transported to the laboratory, frozen with liquid nitrogen and then freeze-dried. The pits are removed to obtain a pulp sample, which is further pulverized and then sealed, packaged and stored in the dark for later use. Weigh 10 g of freeze-dried powder of 'Water Chestnut' bayberry pulp, add 80% ethanol (containing 0.5% formic acid) according to a solid-liquid ratio of 1:20, ultrasonically extract for 30 min, filter through a membrane, and then repeat the extraction 2 times. The supernatant is combined and rotary vacuum concentrated at 37 °C until only the aqueous phase remains. Centrifuge at 10000 rpm for 5 min, and load the supernatant onto the C18 Solid-phase extraction column (Waters 12cc, 2g), rinsed with 4BV of water to remove highly polar impurities, and then eluted with pure methanol (containing 0.5% formic acid) for 4BV. The eluate was evaporated to dryness at 30 °C on a rotary evaporator to obtain the myrica rubra extract powder after passing through the column.

[0047] 2. Identification of Myrica Rubra Anthocyanins

[0048] Waters 2695-2996 DAD detector was used, and the stationary phase was a SunFireTM C18 column (5μm, 4.6×250mm); mobile phase A was 50% acetonitrile (containing 0.1% formic acid); mobile phase B was 0.1% formic acid, and the elution gradient was: 0 - 40 min, 10 - 38% A; 40 - 60 min, 38 - 48% A; 60 - 70 min, 48 - 100% A; 70 - 75 min, 100 - 10% A; 75 - 80 min, 10% A; the scanning wavelength was 200 - 600 nm; the flow rate was 1 mL / min; the injection volume was 10 μL; the column temperature was set at 25 °C. Figure 1 The UPLC chromatogram of the myrica rubra extract at 520 nm. The myrica rubra extract was analyzed by HPLC chromatogram. The first peak indicates that myricetin belongs to anthocyanins and can be detected in the myrica rubra fruit extract we used.

[0049] 3. Construction of Models of Cognitive Impairment and Mental Disorders Associated with Inflammatory Bowel Disease

[0050] The dextran sulfate sodium salt (DSS) used was purchased from MP Biomedicals. Sulfasalazine enteric-coated tablets were purchased from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd. The method for model establishment was: freely drink the DSS (3% w / v) aqueous solution for 7 days. The treatment method was: intragastric administration of the myrica rubra extract or drug at 9 am every day for 7 consecutive days.

[0051] Twenty-five male C57BL / 6J mice (12 weeks old) were purchased from Suzhou Industrial Park Yvette Technology Co., Ltd. All mice were housed in a constant temperature and humidity environment, provided with a 12h light and 12h dark environment, and acclimatized for 1 week. After a 1-week adaptation period, the mice were randomly grouped using a computer, with n = 5 in each group. Group 1, the control group (CONTROL), did not receive DSS modeling and did not receive treatment. Group 2, the IBD-related depression, anxiety, and cognitive impairment group (MODEL), received DSS modeling but no treatment; Groups 3 and 4 received DSS modeling and were treated with different doses of bayberry extract for 7 days. The third group received 50 mg / kg BW of bayberry extract (LowBAE), and Group 4 received 100 mg / kg BW of bayberry extract (High BAE); Group 5 (SASP) received DSS modeling and was treated with sulfasalazine suspension at a dose of 300 mg / kg BW body weight. During the treatment phase, mice in the control group and the model group were given the same amount of clear water by gavage. After the last administration, the forced swim test (FST) and open field test (OFT) were used to examine the depressive-like and anxiety-like behaviors of all animals. The novel object recognition test (NOR) was used to evaluate cognitive function. The preference percentage for the new object was calculated using the following formula: New object preference percentage = (New object exploration time / (New object exploration time + Familiar object exploration time)) * 100%. Figure 2 In A, the difference in the immobility time recorded between the CONTROL group and the MODEL group in the forced swim experiment was statistically significant (p < 0.05), demonstrating the value of this model for depression research. At the same time, in the open field experiment, the number of times the mice in the MODEL group entered the central area was indeed less than that of normal mice ( Figure 2 E), which indicated a tendency towards anxiety. As for cognitive function, the final data showed that the recognition index of the MODEL group was much lower than that of the CONTROL( Figure 2 B), indicating impaired short-term memory.

[0052] After the treatment period, the mice were tested again using the above three experiments to obtain changes in mental disorders and cognitive impairments. As Figure 3 shown, compared with the MODEL group, BAE at both concentrations showed a strong antidepressant effect in a dose-dependent manner, manifested as a significant reduction in the immobility time of the mice (p < 0.05)( Figure 3 F). As Figure 3 J and Figure 3 K shown, BAE also showed anxiolytic activity, and its use increased the number of entries into the central area and the percentage of movement time in the central area. At the same time, there were significant differences in weight loss and colon shortening between the mice receiving DSS and those receiving clear water, and the application of BAE / SASP could alleviate these changes( Figure 4)。This again confirms the conclusion of our previous research that the extract of bayberry anthocyanins is beneficial to gastrointestinal inflammation.

[0053] 4. Immunofluorescence and immunohistochemistry of choroid plexus epithelium in the cerebral ventricles

[0054] The antibodies used were all purchased from Life Technologies. The catalog number of Claudin-2 is 32-5600, the catalog number of Claudin-3 is 34-1700, and the catalog number of Claudin-11 is 36-4500.

[0055] After fixing the mouse brain with formalin for 48 h, dehydrating, paraffin-embedding and sectioning according to the steps, Claudin-3 (1:100) immunofluorescence staining, Claudin-2 and Claudin-11 immunohistochemical staining were performed and photographed. It can be seen under a 100-fold microscope that continuous and strong Claudin-3 immunofluorescence was shown around the choroid plexus epithelial cells of CONTROL mice, while the choroid plexus of MODEL mice showed lighter and discontinuous Claudin-3 immunofluorescence, and the brightness and continuity of the Low BAE, High BAE and SASP groups were between those of the CONTROL and MODEL groups ( Figure 7 B). At the same time, the immunohistochemical results of Claudin-2 and Claudin-11 were similar to the immunofluorescence results of Claudin-3 (such as Figure 6 ), indicating that the tight junction proteins in the choroid plexus of inflammatory bowel disease mice were damaged and the blood-cerebrospinal fluid barrier was impaired, while the bayberry extract could up-regulate the expression of tight junction proteins in the intestine and restore the integrity of the barrier.

[0056] 5. Detection of FITC-dextran permeability

[0057] The FITC-dextran used was purchased from Sigma, catalog number 46944. 75 mg / kg BW 4 kDa FITC-dextran was administered by tail vein injection 1 hour before sacrifice. The obtained cerebrospinal fluid tissue was mixed with sterile phosphate buffer at a ratio of 1:100. After centrifugation at 1000 xg for 5 min, the supernatant was collected. The fluorescence intensity of FITC-dextran in the supernatant was measured at 488 nm / 520 nm using a multifunctional microplate reader (MD iD5). The changes in the permeability of the blood-cerebrospinal fluid barrier of each group of mice were expressed as the relative level of each group relative to the mean of the CONTROL group. The results showed that the permeability of the blood-cerebrospinal fluid barrier in the MODEL group was nearly twice that of the control group, while the permeability of the blood-cerebrospinal fluid barrier decreased significantly after treatment with BAE or SASP. Among them, the effect of BAE at a dose of 100 mg / kg BW was the best, and the permeability could even be restored to the normal level ( Figure 7 C).

[0058] 6. Detection results of oxidative stress level in brain homogenate

[0059] The brain was chopped and mixed in a mixture of PBS (pH 7.4, 100 mM): EDTA (1 mM) at a ratio of 1:10 (% w / v) and centrifuged (12,000 g, 4 °C, 30 min). Thereafter, the supernatant was taken for the detection of T-SOD activity, GSH level and MDA level. By evaluating the degree of lipid peroxidation, we found that the MDA level in the MODEL group was significantly increased compared with the normal group (p < 0.05), indicating a high degree of oxidative stress in the brains of the MODEL group ( Figure 5 C). Treatment with bayberry extract at two concentrations significantly reduced the MDA level. Among them, at a dose of 100 mg / kg BW, its therapeutic effect even exceeded that of the classical inflammatory bowel disease drug SASP. In addition, the T-SOD activity and GSH content in the mouse brain were evaluated to reflect the antioxidant capacity. As Figure 5 shown in A, DSS treatment led to a significant decrease in T-SOD activity and GSH content compared with the normal group (p < 0.05), indicating a weakened antioxidant capacity in the brain. After treatment with bayberry extract or SASP, the levels of these two indicators were partially restored, indicating that bayberry extract and SASP can protect the body from oxidative stress damage. However, bayberry extract or SASP could only partially restore the T-SOD activity and did not reach the level of the CONTROL group. In terms of the GSH level, BAE treatment completely restored the GSH level to the normal level at a higher concentration (100 mg / kg BW) ( Figure 5 B). Combining the above results, it was found that oral administration of bayberry extract could alleviate oxidative stress, reduce the MDA level and increase the body's antioxidant defense ability, and the dose of 100 mg / was superior to 50 mg / kg BW.

[0060] 7. Correlation analysis

[0061] To further explore the relationship between behavioral performance, blood-brain barrier permeability and brain antioxidant capacity, we decided to study the Pearson correlation between all the above data. Through Pearson correlation analysis, we found a correlation between oxidative stress markers and behavioral indicators suggesting anxiety, depression and cognitive impairment. Among them, the parameters most related to depressive symptoms were T-SOD activity and GSH level, and their correlation coefficients both reached +0.65. The correlations of these two indicators with the percentage of time in the central area suggesting the anxiety level were also significant, reaching +0.64 and +0.59 respectively. At the same time, the antioxidant indicators SOD and GSH were positively correlated with the recognition index, while MDA indicating lipid peroxidation was the opposite ( Figure 5D). This indicates that as oxidative stress increases, the time for mice to explore new objects within a certain period decreases, suggesting a decline in short-term memory ability. This confirms the view that there is a link between the antioxidant capacity of the brain and the severity of behavioral disorders. From Figure 5 the results in D, all antioxidant parameters measured in this study were closely related to the relative blood-cerebrospinal fluid barrier permeability. Among them, MDA showed a strong positive correlation with it, while antioxidant enzymes showed a negative correlation. This indicates a close link between blood-cerebrospinal fluid barrier permeability and the level of oxidative stress in the brain parenchyma, confirming our hypothesis about the role of the blood-cerebrospinal fluid barrier in the process of natural antioxidants improving mood and cognitive disorders. Surprisingly, the correlation between blood-cerebrospinal fluid barrier permeability and behavioral blood indicators was also significant. High permeability was associated with depression, anxiety, and cognitive disorders. This indicates that changes in blood-cerebrospinal fluid barrier permeability can affect behavioral performance.

[0062] Example 2

[0063] To verify the feasibility of the protocol and determine the appropriate drug concentration, the extract concentration gradients were set as 50 mg / kg BW, 100 mg / kg BW, and 150 mg / kg BW, and the preliminary experiment was carried out as follows.

[0064] Twenty-five 12-week-old male C57BL / 6J mice were purchased from Suzhou Industrial Park Yvette Technology Co., Ltd. All mice were housed in a constant temperature and humidity environment, provided with a 12 h light and 12 h dark environment, and acclimated for 1 week. The mice were randomly divided into 5 groups (5 mice in each group): control group, model group, treatment group 1, treatment group 2, and treatment group 3. All mice could freely access food. The mice in the model group and the three treatment groups were given 3% DSS aqueous solution for 7 consecutive days. All the modeled mice showed symptoms of weight loss and bloody stools. They showed differences from the control group in behavioral experiments (forced swimming experiment, open field experiment, novel object recognition experiment). During the treatment stage, all mice could freely access food and drinking water, and were administered once by gavage at 9 am every day for 7 consecutive days. The doses for each group were: treatment group 1: 50 mg / kg BW; treatment group 2: 100 mg / kg BW; treatment group 3: 150 mg / kg BW. After the treatment period, the mice were subjected to behavioral tests again. The body weight of the mice was measured regularly every day during the experiment, and the mental state and hair color changes were observed.

[0065] See the figure of the net body weight change of the mice in Figure 8After the modeling period ended, the mice treated with DSS showed symptoms of diarrhea, bloody stools, and weight loss, indicating that the establishment of the acute enteritis mouse model was successful. During the modeling period, the weights of the modeled mice all decreased, with a relatively consistent decrease amplitude and no inter-group differences. Starting from the 9th day, the body weights of the mice in experimental group 1 at 150 mg / kg BW began to increase and did not continue to decrease. Starting from the 10th day, the body weights of the mice at 50 mg / kg BW and 100 mg / kg BW began to increase, while the body weights of the model group still showed a downward trend. Therefore, compared with the model group that recovered naturally, the treatment groups at 50 mg / kg BW, 100 mg / kg BW, and 150 mg / kg BW were all able to resume weight gain. It was found that at the concentrations of 100 mg / kg BW and 150 mg / kg BW, the mice had the least weight loss, and the recovery speed was faster and more stable. There was no significant difference in the performance of the two doses in restoring the body weight of the mice. In terms of restoring the colon length, the performance at the concentration of 100 mg / kg BW was the best.

[0066] After the modeling period and the treatment period, behavioral experiments (forced swimming experiment, open field test) were conducted on the mice to evaluate the levels of anxiety and depression. The evaluation results are shown in Figure 8 。Compared with the model group, the treatment groups at 50 mg / kg BW, 100 mg / kg BW, and 150 mg / kg BW could all reduce the anxiety level to a certain extent. This was manifested in the percentage of movement time in the central area in the open field test of the three groups of mice. All three doses could increase the percentage of movement time in the central area, indicating an increase in the spontaneous behavior of the mice to explore the central area and indicating a decrease in the anxiety level. Among them, the performance at the concentration of 150 mg / kg BW was the best. Similarly, all three concentrations could reduce the depression level to a certain extent, especially the group at 100 mg / kg BW, whose immobility time in the forced swimming experiment was significantly reduced and even restored to the level of the control group. These results indicate that the bayberry extract can not only relieve the intestinal damage of mice with inflammatory bowel disease, but also improve the combined anxiety and depression-like behaviors.

[0067] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of the extract of Chinese bayberry fruit in the preparation of a medicament for treating cognitive disorders and mental disorders related to inflammatory bowel disease. The preparation method of the extract of Chinese bayberry fruit comprises the following steps: 1) An extract is obtained from high-quality ripe Chinese bayberry fruit, and the organic solvent in the extract is removed to obtain a crude extract of anthocyanin of Chinese bayberry. The extract used is a formic acid-ethanol solution, wherein the volume fraction of formic acid is 0.1%; 2) The crude extract of anthocyanin of Chinese bayberry obtained in step 1) is redissolved with water to obtain a crude extract, and then solid-phase extraction is carried out. After an extract is obtained using a methanol eluent and the organic solvent therein is removed, an extract of Chinese bayberry fruit is finally obtained; the extract of Chinese bayberry fruit contains anthocyanin.

2. The application according to claim 1, characterized in that, The mass concentration of anthocyanin in the extract of Chinese bayberry fruit is ≥20%.

3. The application according to claim 1, characterized in that, The treatment of cognitive disorders and mental disorders related to inflammatory bowel disease includes improving the increased level of brain oxidative stress caused by inflammatory bowel disease.

4. The application according to claim 3, characterized in that, The improvement of the increased level of brain oxidative stress caused by inflammatory bowel disease includes increasing the activities of antioxidant enzymes T-SOD and GSH levels and decreasing the level of lipid peroxidation marker MDA.

5. The application according to claim 1, wherein The extraction in step 1) includes ultrasonic extraction at 15-25°C.

6. The application according to claim 5, wherein Ultrasonic extraction is carried out at a frequency of 20-50 kHz.

7. The application according to claim 1, characterized in that The procedure of the solid-phase extraction in step 2) includes: loading, rinsing with pure water, and eluting with methanol.

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