Application of anthocyanin extract from bayberry fruit in the preparation of drugs for the treatment and prevention of cognitive impairment

Abstract

This invention provides the application of anthocyanin extract from bayberry fruit in the preparation of drugs for treating and preventing cognitive impairment, belonging to the field of biomedical technology. The anthocyanin extract from bayberry fruit is rich in anthocyanins. By regulating the activity of antioxidant enzymes and reducing oxidative stress levels, this extract can prevent cognitive impairment, especially showing a good alleviating effect on cognitive impairment caused by thyrotoxicosis. The anthocyanin extract from bayberry fruit shows promise as a potential natural product for improving cognitive impairment, providing a novel approach for the future development and utilization of bayberry.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of anthocyanin extract from bayberry fruit in the preparation of drugs for the treatment and prevention of cognitive impairment. Background Technology

[0002] The Chinese bayberry (Myrica rubra Sieb.), also known as the waxberry, is an evergreen tree belonging to the Myricaceae family and the Myrica genus. It is prized not only for its delicious flavor but also for its high clinical value. Its fresh, sweet and sour taste and appealing color are widely appreciated. The fruit contains flavonoids considered beneficial to human health, such as myricetin, anthocyanins and proanthocyanidins, anthocyanin-3-glucoside, myricetin alcohol and myricetinone, as well as phenolic compounds. However, whether the Chinese bayberry fruit can prevent cognitive impairment and the specific mechanisms involved are not currently reported in existing research. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing an application of anthocyanin extract from bayberry fruit in the preparation of a drug for treating and preventing cognitive impairment.

[0004] The objective of this invention is achieved through the following technical solution: the application of an anthocyanin extract from bayberry fruit in the preparation of a drug for treating and preventing cognitive impairment.

[0005] Furthermore, the anthocyanin extract from bayberry fruit contains anthocyanins.

[0006] Furthermore, the anthocyanin concentration in the bayberry fruit anthocyanin extract is ≥95%.

[0007] Furthermore, the prevention of cognitive impairment includes the prevention of cognitive impairment caused by thyrotoxicosis.

[0008] Furthermore, the prevention of cognitive impairment caused by thyrotoxicosis includes improving oxidative stress caused by cognitive impairment caused by thyrotoxicosis.

[0009] Furthermore, the improvement of oxidative stress caused by cognitive impairment due to thyrotoxicosis includes reducing MDA levels.

[0010] Furthermore, the anthocyanin extract from the bayberry fruit is prepared through the following steps:

[0011] (1) Extract the fruit of bayberry using formic acid-ethanol solution to obtain an extract. Remove the organic solvent components from the extract to obtain crude extract of bayberry anthocyanins. The mass ratio of formic acid to anhydrous ethanol in the formic acid-ethanol solution is 1:4, and the volume concentration of formic acid is 0.05-0.2 wt%. The mass ratio of the formic acid-ethanol solution to the fruit of bayberry is 5-3:1.

[0012] (2) The crude extract of bayberry anthocyanins obtained by water redissolving is used to obtain a crude extract. The crude extract is then subjected to solid-phase extraction, and the extract is obtained by elution with an eluent. The organic solvent components in the extract are removed to obtain an anthocyanin extract of bayberry fruit. The eluent includes methanol.

[0013] Further, in step (1), the extraction includes ultrasonic extraction; the temperature of the ultrasonic extraction is 15-25°C; and the frequency of the ultrasonic extraction is 20-50kHz.

[0014] Further, in step (2), the solid phase extraction procedure includes: loading the sample, rinsing with pure water, and eluting with methanol.

[0015] Furthermore, the drug includes tablets, pills, capsules, and oral liquids made from anthocyanin extracts from bayberry fruit.

[0016] The beneficial effects of this invention are as follows: The anthocyanin extract from bayberry fruit is rich in anthocyanins (cyanidin-3-O-glucoside). This extract can prevent cognitive impairment by modulating the activity of antioxidant enzymes and reducing oxidative stress levels, especially showing a good alleviating effect on cognitive impairment caused by thyrotoxicosis. The anthocyanin extract from bayberry fruit holds promise as a potential natural product for improving cognitive impairment, providing a novel approach for the future development and utilization of bayberry. Attached Figure Description

[0017] Figure 1 The image shows the UPLC diagram of the anthocyanin extract from bayberry.

[0018] Figure 2 Figure 1 shows the ELISA results of mouse serum T3, T4 and TSH. (A) shows the ELISA results of mouse serum T3, (B) shows the ELISA results of mouse serum T4, and (C) shows the ELISA results of mouse serum TSH.

[0019] Figure 3The figures are OFT statistical analysis results. (A) is the OFT statistical analysis result for the BLANK group, (B) is the OFT statistical analysis result for the CH1 group, (C) is the OFT statistical analysis result for the CH2 group, (D) is the OFT statistical analysis result for the CH3 group, (E) is the OFT statistical analysis result for the SCH1 group, (F) is the OFT statistical analysis result for the SCH2 group, and (G) is the OFT statistical analysis result for the SCH3 group.

[0020] Figure 4 The graph shows the results of the NOR statistical analysis.

[0021] Figure 5 The following are the statistical analysis results of FST, where (A) is the statistical analysis result of mouse swimming time, (B) is the statistical analysis result of mouse climbing time, and (C) is the statistical analysis result of mouse stationary time.

[0022] Figure 6 Figure 1 shows the ELISA analysis results of oxidative stress indicators and antioxidant enzyme activity indicators in mouse brain tissue. Figure 2 shows the statistical analysis results of MDA level in brain tissue, Figure 3 shows the statistical analysis results of GSH level in brain tissue, and Figure 4 shows the statistical analysis results of SOD level in brain tissue.

[0023] Figure 7 The image shows the results of TUNEL analysis of neuronal apoptosis in the mouse hippocampus. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0025] This invention provides the application of anthocyanin extract from bayberry fruit in the preparation of drugs for treating and preventing cognitive impairment. The anthocyanin extract from bayberry fruit can prevent cognitive impairment by regulating the activity of antioxidant enzymes in the body and reducing oxidative stress levels.

[0026] This invention does not have any special requirements for the variety of bayberry. The bayberry variety used in the specific implementation of this invention is Dongkui, which was harvested from Taizhou City, Zhejiang Province.

[0027] In this invention, the preferred raw material for this experiment is a fresh fruit with bright color, plump fruit, no mechanical damage, no pests or diseases, and relatively uniform size and maturity.

[0028] In this invention, the preferred storage temperature for the bayberry fruit is 4°C.

[0029] In this invention, the anthocyanin extract from bayberry fruit is rich in anthocyanins, namely cyanidin-3-O-glucoside. This anthocyanin extract from bayberry fruit can prevent cognitive impairment, especially cognitive impairment caused by thyrotoxicosis, by regulating the activity of antioxidant enzymes and reducing oxidative stress levels.

[0030] In this invention, the anthocyanin mass concentration in the bayberry fruit anthocyanin extract is preferably ≥95%.

[0031] In this invention, the prevention of cognitive impairment includes improving cognitive impairment caused by thyrotoxicosis.

[0032] In this invention, the improvement of oxidative stress caused by cognitive impairment due to thyrotoxicosis is described.

[0033] In this invention, the improvement of oxidative stress caused by cognitive impairment due to thyrotoxicosis includes reducing MDA levels.

[0034] In this invention, the anthocyanin extract from bayberry fruit is prepared through the following steps:

[0035] (1) Extract the fruit of *Myrica rubra* using a formic acid-ethanol solution to obtain an extract. Remove the organic solvent components from the extract to obtain a crude extract of *Myrica rubra* anthocyanins. The mass ratio of formic acid to anhydrous ethanol in the formic acid-ethanol solution is 1:4, and the volume concentration of formic acid is 0.05-0.2 wt%. The mass ratio of the formic acid-ethanol solution to the fruit of *Myrica rubra* is 5-3:1.

[0036] (2) The crude extract of bayberry anthocyanins obtained by water redissolving is used to obtain a crude extract. The crude extract is then subjected to solid-phase extraction, and the extract is obtained by elution with an eluent. The organic solvent components in the extract are removed to obtain an anthocyanin extract of bayberry fruit. The eluent includes methanol.

[0037] This invention first extracts bayberry fruit using a formic acid-ethanol solution to obtain an extract. The organic solvent components in the extract are then removed to obtain a crude extract of bayberry anthocyanins. The volume concentration of formic acid in the formic acid-ethanol solution is 0.05 wt% to 0.2 wt%. In this embodiment, the volume concentration of formic acid in the formic acid-ethanol solution is 0.1%.

[0038] In this invention, the mass ratio of the formic acid-ethanol solution to the bayberry fruit is preferably (5-3:1), and more preferably 4:1.

[0039] In this invention, the preferred method for extracting bayberry fruit using formic acid-ethanol solution includes mixing the formic acid-ethanol solution and bayberry fruit, grinding them, and then extracting. Preferably, the grinding is performed using a high-speed blender; the extraction preferably includes ultrasonic extraction; the ultrasonic extraction temperature is preferably 15-25℃, more preferably 20℃; the ultrasonic extraction frequency is 35kHz; and the ultrasonic extraction time is preferably 1 hour. The advantage of ultrasonic extraction is that it can efficiently extract the active ingredients from the fruit, and the extraction effect is also relatively uniform. Preferably, after extraction, the invention also includes standing for better extraction results; the standing time is preferably 9-16 hours, more preferably 12 hours. After extraction, the preferred method for obtaining the extract is filtration, collecting the filtrate; the filtration preferably includes a first filtration and a second filtration performed sequentially; the first filtration preferably uses four layers of gauze; and the second filtration preferably uses three layers of gauze. In this invention, the preferred method for removing the organic solvent components from the extract is to vacuum rotary evaporate the extract at 30℃ in the dark until the organic solvent components are completely evaporated, leaving the crude extract of bayberry anthocyanins.

[0040] After obtaining the crude extract of bayberry anthocyanins, the present invention re-dissolves the crude extract of bayberry anthocyanins in water to obtain a crude extract, performs solid-phase extraction on the crude extract, elutes with an eluent to obtain an extract, removes the organic solvent components in the extract, and obtains an anthocyanin extract of bayberry fruit.

[0041] In this invention, before performing solid-phase extraction on the crude extract, it is preferable to further filter the crude extract; the filtration preferably uses a 0.22 μm aqueous filter membrane.

[0042] In this invention, the solid-phase extraction column used for the solid-phase extraction preferably includes... A 20cc C18 solid-phase extraction column is used. Before use, the solid-phase extraction column is preferably activated with methanol, and then equilibrated with purified water. The amount of methanol is preferably twice the column volume (bed volume, BV), and the amount of purified water is preferably 2 BV. In this invention, the solid-phase extraction procedure preferably includes: rinsing the sample with purified water, followed by elution with methanol; the amount of sample loaded is preferably 2 / 3 of the solid-phase extraction column's capacity after passing through a water film; the amount of purified water used for rinsing is preferably 20 BV; the purpose of rinsing is to remove impurities; and the amount of methanol used for elution is preferably 2 BV.

[0043] In this invention, the removal of organic solvents from the extract preferably includes: rotary evaporating the extract under vacuum at 30°C in the dark until the organic solvents are completely evaporated. In this invention, the solid-phase extraction serves to remove sugars.

[0044] In this invention, the medicine includes tablets, pills, capsules, and oral liquids made from anthocyanin extracts of bayberry fruit.

[0045] The drugs include tablets, pills, capsules, and oral liquids made from anthocyanin extracts from bayberry fruit.

[0046] Unless otherwise specified, the present invention does not have special requirements on the source of raw materials used, and commercially available products well known to those skilled in the art can be used.

[0047] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0048] Example 1

[0049] 1. Extraction of crude extract of myrica rubra anthocyanins

[0050] The bayberry variety used in this experiment was 'Dongkui'. The fruits were harvested from the planting base (Taizhou, Zhejiang) and immediately sent to the laboratory. Fresh fruits with bright color, plumpness, no mechanical damage, no pests or diseases, and uniform size and maturity were selected. The storage temperature was 4℃ for subsequent anthocyanin extraction. 500g of pitted bayberry fruits were added to 2000mL of formic acid-anhydrous ethanol solution, where the mass ratio of formic acid to anhydrous ethanol was 1:4 and the volume concentration of formic acid was 0.1wt%. The mixture was thoroughly ground using a high-speed blender. Low-temperature ultrasonic extraction was performed for 1 hour (35kHz), followed by standing for 12 hours. The resulting sample was first filtered through four layers of gauze, then through three layers of filter paper. The filtrate was then vacuum-evaporated at 30℃ in the dark until the organic solvent was completely evaporated. The remaining solid was the crude anthocyanin extract from the bayberry.

[0051] 2. Purification of anthocyanins from bayberries

[0052] The remaining solids were dissolved in 100 mL of purified water to obtain a crude extract of myricetin from bayberries. This solution was then filtered through a 0.22 μm aqueous membrane and used for further processing. Sugar removal was performed using a 20cc C18 solid-phase extraction column to obtain relatively pure myricetin. The specific steps are as follows: the column was activated with 2 bed volume (BV) of methanol, and equilibrated with 2 BV of purified water. After loading the sample through a water film, when the solid-phase extraction column reached 2 / 3 capacity, it was rinsed with 20 BV of purified water to remove impurities. Finally, it was eluted with 2 BV of methanol to obtain the fraction rich in myricetin. The collected fraction was then subjected to vacuum rotary evaporation at 30°C in the dark until the organic solvent was completely evaporated.

[0053] 3. Identification of anthocyanins in bayberries

[0054] Weigh 1 mg of powder rich in myricetin of bayberry, use methanol as solvent to prepare a series of solutions of different concentrations, filter the solutions through a 0.22 μm organic filter membrane, and then use ultra-high performance liquid chromatography (UPLC) for detection.

[0055] The UPLC detection conditions were as follows: mobile phase A was an ultrapure aqueous solution containing 1% formic acid, and mobile phase B was acetonitrile / 0.1% formic acid (1:1, v / v). The scanning wavelengths were 280 nm and 520 nm, the flow rate was set to 0.3 mL / min, the column temperature was 28℃, the injection volume was 4 μL, and the column was a Promosil C18 column (4.6 × 250 mm, 5 μm). The gradient elution process was: 0–5 min, 5–15% B; 5–12 min, 15–25% B; 12–20 min, 25–60% B; 20–23 min, 60–100% B; 23–24 min, 100–5% B; 24–28 min, 5% B. Absolute quantitative analysis of the obtained myricetin powder was performed by comparing retention time and peak area with standards.

[0056] The mass spectrometry conditions were as follows: UPLC-Triple-TOF5600+ time-of-flight LC-MS: positive and negative ion scanning mode; scan range: m / z 100-1500; nebulizer gas (GS): 55 psi; curtain gas (CUR): 35 psi; ion source temperature (TEM): 600℃ (positive) 550℃ (negative); ion source voltage (IS): 5500V (positive) - 4500V (negative); scan: declustering voltage (DP): 100V; focusing voltage (CE): 10V; CID energy: 40±20eV. Mass axis calibration was performed before sample injection using a CDS pump to ensure that the mass axis error was less than 2ppm.

[0057] The anthocyanins in the extract of Myrica rubra were analyzed by UPLC-Q-TOF-MS / MS. The UPLC (520 nm) spectrum of the anthocyanin extract of Myrica rubra is shown in the figure. Figure 1 .

[0058] like Figure 1 As shown, the anthocyanin extract of bayberry exhibits a peak at a detection wavelength of 520 nm, indicating that the substance is an anthocyanin compound. Mass spectrometry shows that the molecular weight of the fragment ion of this substance is 449.1088, which is [C]. 21 H 20 O 11 ] + peak.

[0059] 4. Constructing a mouse model of thyrotoxicosis

[0060] Seventy healthy male C57BL / 6 mice, aged 8 weeks, and their feed were purchased from Shanghai Silex Laboratory Animal Co., Ltd. Their body weight (BW) was 25±1.1 g. All mice were housed in a constant temperature and humidity environment (temperature 22±0.5°C, humidity 50±5%) with 12 hours of light and 12 hours of darkness. They had free access to food and water and were acclimatized to the animal facility environment for one week. Our preliminary experimental results verified the feasibility of using thyroxine (200 mg / kg BW and 50 mg / kg BW) to establish CH and SCH mouse models. Parallel gradient experiments determined the lowest effective dose of myricetin powder for treating thyrotoxicosis to be 100 mg / kg BW. Seventy mice were randomly divided into seven groups of ten mice each: a blank control group (BLANK group), a hyperthyroidism group (CH1 group), a CH group treated with 100 mg / kg*BW myricetin solution (CH2 group), a CH group treated with 200 mg / kg*BW myricetin solution (CH3 group), a subclinical hyperthyroidism group (SCH1 group), a SCH group treated with 100 mg / kg*BW myricetin solution (SCH2 group), and a SCH group treated with 200 mg / kg*BW myricetin solution (SCH3 group). Water and myricetin solution were administered orally via gavage using a gavage needle. The myricetin extract prepared in Example 1 was dissolved in water to obtain the myricetin solution, and the volume of liquid administered to each mouse was 0.4 ml. Thyroxine was injected intraperitoneally to establish the hyperthyroidism model. To minimize the impact of human error, all gavage and intraperitoneal injection procedures were performed by the same experienced member of the research group. All mice had free access to the provided standard feed pellets and sterile water. The experiment lasted for 30 days. During the experiment, at 8:00 AM each day, the CH1, CH2, and CH3 groups were intraperitoneally injected with 200 mg / kg*BW of thyroxine, the SCH1, SCH2, and SCH3 groups were intraperitoneally injected with 50 mg / kg*BW of thyroxine, and the BLANK group was intraperitoneally injected with an equal volume of sterile water. Simultaneously, the CH2 and SCH2 groups were gavage with 100 mg / kg*BW of myricetin solution, the CH3 and SCH3 groups were gavage with 200 mg / kg*BW of myricetin solution, and the BLANK, CH1, and SCH1 groups were gavage with an equal volume of sterile water.

[0061] 5. Animal experiment results: Detection results of T3, T4 and TSH in whole blood.

[0062] ELISA was used to quantify T3, T4, and TSH levels in mouse blood. Figure 2 (A) Figure 2 (B) Figure 2 As shown in (C), the final test results indicated that, compared with the BLANK group, T3 levels were significantly increased in the CH1, CH2, and CH3 groups, with statistically significant differences (p < 0.0001). Similar to the changes in T3, T4 levels also increased in the CH1, CH2, and CH3 groups, with p < 0.0001 for CH1 and CH2 groups and p < 0.001 for CH3 group. TSH levels in the three CH groups were significantly lower than in the BLANK group, with the largest decrease in CH1 group (p < 0.0001), followed by CH2 group (p < 0.001) and CH3 group (p < 0.05). The trends in the indicators in the three SCH groups were the same as in the three CH groups, but the changes in T3 levels in the SCH1, SCH2, and SCH3 groups were not statistically different from those in the BLANK group. Compared with the BLANK group, the T4 levels in the SCH1 and SCH2 groups showed statistically significant increases (p<0.01 and p<0.05, respectively). While the T4 level in the SCH3 group was also higher than that in the BLANK group, the difference was not statistically significant. The TSH levels in all three SCH groups were lower than those in the BLANK group to some extent, but the differences were not statistically significant. In conclusion, the animal disease model was successfully established.

[0063] 6. Results of the open field test (OFT)

[0064] First, the total distance traveled by each mouse in each group is recorded. This indicator can suggest whether there are differences in the activity level and performance of the experimental animals. Figure 3 (A) Figure 3 (B) Figure 3 (C) Figure 3 (D) Figure 3 (E) Figure 3 (F) and Figure 3As shown in (G), the experimental results indicate that the total movement distance of groups CH1, CH2, CH3, SCH1, SCH2, or SCH3 was slightly increased compared to the BLANK group, but the difference was not statistically significant. This may be because the increased thyroid hormone levels made the mice more active. Next, the time mice spent in the central region was recorded. In mammals, the length of time spent in the central region is negatively correlated with anxiety level; that is, the more anxious the mouse, the shorter its time spent in the central region, and it prefers to move closer to the wall, i.e., to the periphery. The statistical results show that although the total movement distance of groups CH1, CH2, CH3, SCH1, SCH2, or SCH3 was longer, the time spent in the central region was shorter in all six groups. Furthermore, the CH1 group had a shorter central region time than the SCH1 group, indicating that the CH1 group mice preferred to move to the periphery, suggesting a higher level of anxiety. Mice treated with myricetin spent a longer time in the central region compared to the disease model group. Furthermore, high-concentration myricetin administered via gavage was more effective than low-concentration administration; specifically, the central region residence time in groups CH2 and CH3 was longer than in group CH1, and the central region residence time in groups SCH2 and SCH3 was longer than in group SCH1. The increase in central region residence time in group SCH3 compared to group SCH1 was statistically significant (p < 0.01). Simultaneously, the central region activity time in group CH3 was longer than in group CH2, and the central region activity time in group SCH3 was longer than in group SCH2, but these differences were not statistically significant.

[0065] 7. Results of the Novel Object Recognition (NOR) Experiment

[0066] The time difference between a mouse's exploration of novel and familiar objects can be used to reflect its learning and memory abilities. Under normal circumstances, mice will spend more time exploring novel objects they have never seen before. The Discrimination Ratio (DR) formula is DR = TN - TF / TT, where TN (time of novel object) represents the time spent exploring a novel object, TF (time of familiar object) represents the time spent exploring a familiar object, and TT (total time) represents the total activity time, i.e., 300 seconds. If learning and memory abilities are impaired, mice will forget previously seen objects, which will be reflected in a relatively shorter TN time for exploring novel objects. Figure 4As shown, the experimental results indicate that the DR values ​​in groups CH1, CH2, CH3, SCH1, SCH2, or SCH3 were lower than those in the BLANK group, and the differences were statistically significant. It was also observed that the DR value in group CH1 was lower than that in group SCH1, the DR value in group CH2 was higher than that in group SCH2, and the DR value in group CH1 was lower than that in group SCH1. Further statistical analysis revealed that in groups SCH1, SCH2, and SCH3, mice treated with myricetin (i.e., groups SCH2 and SCH3) had higher DR values ​​than those in group SCH1. Furthermore, the situation in group SCH3 was better than that in group SCH2. Similarly, in groups CH1, CH2, and CH3, mice treated with myricetin (i.e., groups CH2 and CH3) had higher DR values ​​than those in group CH1. The increase in DR value in group CH2 was statistically significant compared to group CH1 (p < 0.05).

[0067] Figure 4 In this study, results are expressed as mean ± standard deviation. * indicates a significant difference compared to the BLANK group, where *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. # indicates a significant difference compared to the corresponding disease model group, where #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001. ns indicates no statistically significant difference.

[0068] 8. Results of the Forced Swimming Test (FST)

[0069] This experiment recorded the swimming time (TS), climbing time (TC), and freezing time (TF) for each mouse in each group.

[0070] like Figure 5 (A) Figure 5 As shown in Figures (B) and (C), the experimental results are as follows: compared to the BLANK group, the TS (transfer time) of the CH1, CH2, CH3, SCH1, SCH2, and SCH3 groups was decreased, and the differences were statistically significant, while the corresponding TC (total calorie concentration) and TF (total tidal volume) were increased. Further analysis revealed that in the CH1, CH2, and CH3 groups, mice that had been given myricetin (i.e., the CH2 and CH3 groups) had higher TS than the CH1 group, while their TC and TF were lower. Mice given high concentrations of myricetin exhibited more swimming behavior than mice given low concentrations of myricetin; that is, the TS of the CH3 and SCH3 groups was longer than that of the CH1 and CH2 groups, and the SCH1 and SCH2 groups, respectively.

[0071] Figure 5 (A) Figure 5 In Figures (B) and (C), results are expressed as mean ± standard deviation. * indicates a significant difference compared to the BLANK group, where *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. # indicates a significant difference compared to the corresponding disease model group, where #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, and ns indicates no statistically significant difference.

[0072] 9. Results of brain tissue oxidative stress indices and antioxidant enzyme activity assays

[0073] This experiment detected oxidative stress indicators and antioxidant enzyme activity in mouse brain tissue. For example... Figure 6 As shown in (A), compared with the BLANK group, the MDA level, representing the degree of lipid peroxidation, was increased in the CH1, CH2, CH3, SCH1, SCH2, and SCH3 groups, with the CH1 group showing the greatest increase. The difference was statistically significant (p < 0.0001). The MDA level in the CH1 group was also higher than that in the SCH1 group. Using myricetin, the MDA levels in the CH2 and CH3 groups were lower than those in the CH1 group, with higher concentrations showing greater relief. However, the decrease in MDA level in the CH3 group was greater than that in the CH2 group. Similarly, the MDA levels in the SCH2 and SCH3 groups were lower than those in the SCH1 group, with higher concentrations showing greater relief. However, the decrease in MDA level in the SCH3 group was greater than that in the SCH2 group.

[0074] GSH is an important component of the non-enzymatic system in the antioxidant system; such as Figure 6 As shown in (B), the GSH levels in groups CH1, CH2, CH3, SCH1, SCH2, and SCH3 were lower than those in the BLANK group, with the largest decrease in GSH level observed in group CH1, and the difference was statistically significant (p < 0.01). The GSH levels in groups CH2 and CH3, which had received the myricetin, recovered to varying degrees compared to group CH1, and this recovery was concentration-dependent, but the recovery rate in group CH3 was greater than that in group CH2. Similarly, the GSH levels in groups SCH2 and SCH3, which had received the myricetin, recovered to varying degrees compared to group SCH1, and this recovery was concentration-dependent, but the recovery rate in group SCH3 was greater than that in group SCH2.

[0075] SOD is an important component of the enzyme system in the antioxidant system; such as Figure 6As shown in (C), the SOD levels in the CH1, CH2, CH3, SCH1, SCH2, and SCH3 groups were lower than those in the BLANK group. The concentrations of the groups that applied myricetin rebounded and showed a concentration-dependent effect.

[0076] Figure 6 (A) Figure 6 (B) Figure 6 In (C), results are expressed as mean ± standard deviation. * indicates a significant difference compared to the BLANK group, where *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. # indicates a significant difference compared to the corresponding disease model group, where #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001, and ns indicates no statistically significant difference.

[0077] 10. Results of TUNEL assay for hippocampal neuronal apoptosis

[0078] This experiment detected neuronal apoptosis in the mouse hippocampus, and the results are as follows: Figure 7 As shown, compared with the BLANK group, the apoptosis rate of neurons in the CH1 group was significantly increased (p<0.0001). The number of apoptotic cells in the low-concentration myricetin-treated group (CH2 group) was also higher than that in the BLANK group (*p<0.05). In contrast, the increase in apoptosis in the high-concentration myricetin-treated group (CH3 group) was reduced, and the difference was not statistically significant compared with the BLANK group. Compared with the CH1 group, apoptosis in both the CH2 and CH3 groups was reduced (##p<0.01, ###p<0.001, respectively). Compared with the BLANK group, the degree of hippocampal neuronal apoptosis in the SCH1 group was also increased (**p<0.01), and apoptosis in the SCH2 group also showed an increasing trend, but the results were not statistically significant. Apoptosis in the SCH3 group showed a decreasing trend compared to the BLANK group, but the results were not statistically significant. However, the decrease in apoptosis in the SCH3 group compared to the SCH1 group was statistically significant (##p<0.01).

[0079] Figure 7 In this study, results are expressed as mean ± standard deviation. * indicates a significant difference compared to the BLANK group, where *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. # indicates a significant difference compared to the corresponding disease model group, where #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001. ns indicates no statistically significant difference.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of an anthocyanin extract from bayberry fruit in the preparation of a medicament for treating and preventing cognitive impairment, wherein the prevention of cognitive impairment is the prevention of cognitive impairment caused by thyrotoxicosis; the anthocyanin extract from bayberry fruit is prepared through the following steps: (1) Extract the fruit of bayberry using formic acid-ethanol solution to obtain an extract, remove the organic solvent components in the extract to obtain crude extract of bayberry anthocyanins; the mass ratio of formic acid to anhydrous ethanol in the formic acid-ethanol solution is 1:4, the volume concentration of formic acid is 0.05~0.2wt%; the mass ratio of formic acid-ethanol solution to bayberry fruit is 5~3:1; (2) The crude extract of bayberry anthocyanins obtained by water redissolving was obtained, and the crude extract was extracted by C18 solid phase extraction. The sample was loaded, washed with pure water, and eluted with methanol to obtain the extract. The organic solvent components in the extract were removed to obtain the bayberry fruit anthocyanin extract.

2. The application according to claim 1, characterized in that, The anthocyanin extract from bayberry fruit contains anthocyanins.

3. The application according to claim 1, characterized in that, In step (1), the extraction is ultrasonic extraction; the temperature of the ultrasonic extraction is 15~25℃; and the frequency of the ultrasonic extraction is 20~50kHz.

4. The application according to claim 1, characterized in that, The drug is a tablet, pill, capsule, or oral liquid made from anthocyanin extract of bayberry fruit.

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