Application of plateau safflower flavonoids in preparation of products for improving senile cognitive dysfunction

Abstract

This invention relates to the field of biomedical technology, and in particular to the application of plateau cattail pollen flavonoids in the preparation of products that improve age-related cognitive dysfunction. This invention provides plateau cattail pollen flavonoids derived from the Lalu Wetland National Nature Reserve in Tibet. These plateau cattail pollen flavonoids can achieve safe and efficient relief of age-related cognitive dysfunction through multi-target synergistic effects, including "regulating gut microbiota to reshape microecological balance, repairing the intestinal barrier to inhibit peripheral inflammation, and crossing the blood-brain barrier to directly inhibit acetylcholinesterase activity." This overcomes the shortcomings of existing technologies, such as the large side effects and poor long-term tolerance of synthetic drugs, and the single target and difficulty in effectively regulating the "microbe-gut-brain axis" of ordinary natural products.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of flavonoids from the high-altitude cattail pollen in the preparation of products that improve age-related cognitive impairment. Background Technology

[0002] Age-related cognitive dysfunction (ARCD) is a clinical phenomenon characterized by a gradual decline in cognitive abilities with age. It primarily manifests as impairment in one or more cognitive functions, such as learning, memory, attention, and executive function, significantly impacting the quality of life of older adults. With the increasing aging of the global population, ARCD has become a major challenge in the field of public health.

[0003] The pathogenesis of ARCD is not yet fully understood, but it is generally believed to be closely related to multiple factors such as oxidative stress, neuroinflammation, synaptic aging, and gut microbiota imbalance. Among these, the "microbe-gut-brain axis" theory provides a new perspective for understanding the pathogenesis of ARCD and developing intervention strategies. Gut microbiota dysbiosis can lead to impaired intestinal barrier function, allowing bacterial endotoxins (such as lipopolysaccharides) to enter the bloodstream, inducing systemic chronic inflammation and neuroinflammation, thereby damaging the central nervous system and exacerbating cognitive decline.

[0004] Clinically, the main drugs used to treat ARCD are acetylcholinesterase inhibitors (such as donepezil and galantamine) and N-methyl-D-aspartate receptor antagonists (such as memantine). While these drugs can improve symptoms to some extent, they have significant limitations: firstly, they cannot reverse or stop the progression of the disease; secondly, long-term use is often accompanied by side effects such as nausea, diarrhea, dizziness, and insomnia. Therefore, developing naturally derived drugs that can intervene in the pathological process of ARCD at multiple targets and have high safety is of significant clinical importance and market demand.

[0005] Flavonoids are a class of secondary metabolites widely found in plants, attracting significant attention due to their excellent antioxidant, anti-inflammatory, and neuroprotective activities. The unique geographical and climatic environment of the Qinghai-Tibet Plateau (such as low pressure, low oxygen, and strong ultraviolet radiation) has fostered plateau plant resources with special adaptation mechanisms. Typha pollen, the dried pollen of Typha orientalis or related species, is abundant in the Lalu Wetland National Nature Reserve in Tibet. The flavonoid components in its pollen (plateau Typha pollen flavonoids) may possess unique chemical compositions and stronger biological activities due to the stresses of the plateau environment. However, there are currently no research reports or applications regarding whether plateau Typha pollen flavonoids can improve ARCD through a dual pathway of regulating the "microbe-gut-brain axis" and directly inhibiting acetylcholinesterase in the brain. Summary of the Invention

[0006] The purpose of this invention is to provide the application of flavonoids from Typha orientalis in the preparation of products that improve age-related cognitive impairment.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of flavonoids from Typha orientalis in the preparation of products that improve age-related cognitive impairment.

[0008] Preferably, the plateau cattail pollen flavonoids are obtained by extraction, separation and purification of cattail pollen.

[0009] Preferably, the plateau cattail flavonoids include rutin, typhain, narcissin, apigenin, naringin chalcone, quercetin, isoquercitrin, and quercetin-3-O-rutin-(1→2)-O-rhamnoside.

[0010] Preferably, the improvement of age-related cognitive impairment includes one or more of the following effects: (1) Improve learning and memory impairments; (2) Reduce damage to hippocampal neurons; (3) Enhances the antioxidant capacity of brain and colon tissues; (4) Inhibits the expression of pro-inflammatory factors in brain and colon tissues; (5) Repairing intestinal barrier function; (6) Regulate the structure of intestinal flora, increase the abundance of beneficial bacteria, and reduce the abundance of harmful bacteria; (7) Promotes the production of beneficial metabolites; (8) Inhibits the activity of acetylcholinesterase in the brain and increases the level of acetylcholine.

[0011] The present invention also provides a formulation for improving age-related cognitive impairment, the formulation comprising flavonoids from Typha orientalis and a pharmaceutically acceptable carrier.

[0012] Preferably, the dosage form of the preparation is tablets, capsules, granules, oral liquids or powders.

[0013] This invention also provides the application of flavonoid monomers from Typha orientalis in the preparation of products that protect cognitive function, wherein the flavonoid monomers from Typha orientalis are rutin.

[0014] Preferably, the plateau cattail flavescens flavonoid monomer protects cognitive function by maintaining acetylcholine homeostasis in the brain.

[0015] The present invention also provides a formulation for protecting cognitive function, the formulation comprising rutin and a pharmaceutically acceptable carrier.

[0016] Preferably, the dosage form of the preparation is tablets, capsules, granules, oral liquids or powders.

[0017] Beneficial effects: This invention, based on the unique environment of the plateau, provides a plateau cattail pollen flavonoid sourced from the Lalu Wetland National Nature Reserve in Tibet. This plateau cattail pollen flavonoid can achieve safe and efficient relief of age-related cognitive impairment through multi-target synergistic effects, including "regulating the intestinal flora to reshape the microecological balance, repairing the intestinal barrier to inhibit peripheral inflammation, and crossing the blood-brain barrier to directly inhibit acetylcholinesterase activity." It overcomes the shortcomings of existing technologies, such as the large side effects and poor long-term tolerance of synthetic drugs, and the single target of ordinary natural products, which are difficult to effectively regulate the "microbe-gut-brain axis." Moreover, it is of great significance for the development and utilization of plateau-specific plant resources and the research and development of cognitive function protection products with independent intellectual property rights, and has significant scientific value and application prospects.

[0018] The plateau cattail pollen flavonoids described in this invention possess active monomers (such as rutin) capable of crossing the blood-brain barrier, directly binding to and inhibiting the activity of acetylcholinesterase in the brain, thereby maintaining acetylcholine homeostasis and exerting a direct protective effect on cognitive function. Furthermore, as a prebiotic, plateau cattail pollen flavonoids can regulate the intestinal flora structure through the "microbe-gut-brain axis," promoting the production of beneficial metabolites such as short-chain fatty acids and indole derivatives, reducing peripheral and central oxidative stress and inflammatory responses, and repairing intestinal barrier function. This synergistic effect of the "gut-brain dual pathway" enables it to comprehensively improve ARCD through multiple targets and pathways. Attached Figure Description

[0019] Figure 1 Fourier transform infrared spectrum of flavonoids from Typha orientalis; Figure 2 The results show the in vitro antioxidant activity of flavonoids from Typha orientalis. Figure 3 The results are as follows: A is a representative swimming trajectory of mice in the water maze test; B is a curve showing the change in escape latency of mice in each group during the localization and navigation test; C is the escape latency of mice in each group during the spatial exploration test; D is the number of times mice in each group crossed the original platform during the spatial exploration test; and E is the time mice in each group stayed in the third quadrant during the spatial exploration test. Figure 4 Image showing Nissl staining results of mouse brain tissue; Figure 5 The figure shows the effects of oxidative stress and inflammatory factor levels on mouse brain tissue. In the figure, A is the measurement of brain SOD activity, B is the measurement of brain GSH content, C is the measurement of brain MDA content, D is the measurement of brain IL-1β content, E is the measurement of brain IL-6 content, and F is the measurement of brain TNF-α content. Figure 6 Image showing the results of immunofluorescence staining of mouse colon tissue; Figure 7 The figure shows the effect of flavonoids from Typha orientalis on improving gut microbiota dysbiosis in D-galactose-induced aging mice. A represents the α-diversity results, B represents the β-diversity results, and C represents the changes in the relative abundance of key bacterial genera. Figure 8 A graph showing the results of short-chain fatty acid content in mouse feces; Figure 9 These are key intestinal microbial metabolites in mouse feces, of which A consists of deoxycholic acid and tauroursodeoxycholic acid in the bile acid pathway, B consists of choline and trimethylamine in the gut-liver axis, and C consists of tryptophan, indole-3-lactic acid, indole-3-acetic acid and indole in the indole pathway. Figure 10 The effect of flavonoids from Typha orientalis on the levels of neurotransmitters acetylcholine and glutamate and the activity of acetylcholinesterase in the brains of D-galactose-induced aging mice is shown in the figure. Figure 11 The diagram shows the results of the molecular docking between rutin and acetylcholinesterase, where A is the active site of acetylcholinesterase and B is the result of the molecular docking between rutin and acetylcholinesterase. Figure 12 The results of surface plasmon resonance detection of the binding affinity between rutin and acetylcholinesterase are shown in Figure 1. In Figure 2, A is the binding curve of rutin molecules of different concentrations with AChE, and B is the fitting curve of rutin molecules of different concentrations with AChE. Detailed Implementation

[0020] This invention provides the application of flavonoids from Typha orientalis in the preparation of products that improve age-related cognitive impairment.

[0021] In this invention, the plateau cattail pollen flavonoids are obtained by extraction, separation and purification of cattail pollen; The specific preparation process of flavonoids from Typha orientalis: Mature male cattail flowers are collected, dried naturally in the sun, and the pollen is collected. The pollen is then sieved through a 120-mesh sieve to obtain coarse cattail pollen powder, which is stored at -80℃ for later use. Weigh 25 g of dried crude cattail pollen powder and add 56% ethanol solution at a material-to-liquid ratio of 1:60. Under ultrasonic-assisted extraction conditions of 600 W and 42.6℃ for 35.5 min, collect the extract. Centrifuge the extract at 4000 rpm for 30 min and collect the supernatant. Purify the supernatant using macroporous adsorption resin D-101, then elute sequentially with distilled water and 55% ethanol, collecting the eluent. Dry the eluent in a freeze dryer with the following program: -42℃ for 4 h, -35℃ for 3 h, -25℃ for 2 h, -15℃ for 1 h, 0℃ for 2 h, 10℃ for 3 h, 20℃ for 4 h, 25℃ for 12 h, with vacuum stage 2 and constant temperature stage 8. After drying, plateau cattail pollen flavonoids (…) are obtained. Pollen typhae flavonoids (PTF).

[0022] In this invention, the plateau cattail flavonoids include rutin, typhain, narcissin, apigenin, naringin chalcone, quercetin, isoquercitrin, and quercetin-3-O-rutin-(1→2)-O-rhamnoside.

[0023] In this invention, the improvement of age-related cognitive dysfunction includes one or more of the following effects: (1) Improve learning and memory impairments; (2) Reduce damage to hippocampal neurons; (3) Enhances the antioxidant capacity of brain and colon tissues; (4) Inhibits the expression of pro-inflammatory factors in brain and colon tissues; (5) Repairing intestinal barrier function; (6) Regulate the structure of intestinal flora, increase the abundance of beneficial bacteria, and reduce the abundance of potentially harmful bacteria; The regulation of gut microbiota structure includes increasing Bifidobacterium, Lactobacillus, Adlercreutzia,Ligilactobacillus The relative abundance of fungal genera, and / or decrease. Bacteroides, Colidextribacter,unidentified_Lachnospiraceae Relative abundance of fungal genera; (7) Promotes the production of beneficial metabolites; The beneficial metabolites include short-chain fatty acids, deoxycholic acid, tauroursodeoxycholic acid, and indole and its derivatives. (8) Inhibits the activity of acetylcholinesterase in the brain and increases the level of acetylcholine.

[0024] The present invention also provides a formulation for improving age-related cognitive impairment, the formulation comprising flavonoids from Typha orientalis and a pharmaceutically acceptable carrier.

[0025] In this invention, the dosage form of the preparation is tablets, capsules, granules, oral liquids, or powders.

[0026] This invention also provides the application of flavonoid monomers from Typha orientalis in the preparation of products that protect cognitive function, wherein the flavonoid monomers from Typha orientalis are rutin.

[0027] In this invention, the plateau cattail flavescens flavonoid monomer protects cognitive function by maintaining acetylcholine homeostasis in the brain.

[0028] The present invention also provides a formulation for protecting cognitive function, the formulation comprising rutin and a pharmaceutically acceptable carrier.

[0029] In this invention, the dosage form of the preparation is tablets, capsules, granules, oral liquids, or powders.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] In the embodiments of the present invention, human normal colonic epithelial cells NCM460 were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; SPF-grade C57BL / 6J male mice were purchased from the Medical Animal Experiment Center of Lanzhou University.

[0032] Example 1: Preparation, molecular structure, and component analysis of flavonoids from Typha orientalis.

[0033] Sample collection: Mature male cattail flowers were collected from July 2 to 5, 2025, in the Lalu Wetland National Nature Reserve (altitude approximately 3650 meters) in Lhasa, Tibet Autonomous Region. The flowers were naturally dried in the sun, and the pollen was collected. The pollen was sieved through a 120-mesh sieve to obtain coarse cattail pollen powder, which was stored at -80℃ for later use.

[0034] Preparation of plateau cattail pollen flavonoids: 25 g of dried crude cattail pollen powder was weighed and added to a 56% ethanol solution at a material-to-liquid ratio of 1:60. The mixture was ultrasonically extracted for 35.5 min at an ultrasonic power of 600 W and an ultrasonic temperature of 42.6℃. The extract was collected. The extract was centrifuged at 4000 rpm for 30 min, and the supernatant was collected. The supernatant was purified using macroporous adsorption resin D-101, followed by elution with distilled water and 55% ethanol, and the eluent was collected. The eluent was then dried in a freeze dryer with the following program: -42℃ for 4 h, -35℃ for 3 h, -25℃ for 2 h, -15℃ for 1 h, 0℃ for 2 h, 10℃ for 3 h, 20℃ for 4 h, and 25℃ for 12 h. The vacuum stage was set to 2, and the isothermal stage to 8. After drying, plateau PTF freeze-dried powder was obtained.

[0035] The functional group composition and molecular structure of flavonoids from *Typha orientalis* were analyzed using Fourier transform infrared spectroscopy (FT-IR). The results are as follows: Figure 1 As shown.

[0036] The results showed that 354.1 cm -1 The broad, intense peak at 2925.7 cm⁻¹ is the absorption peak of the stretching vibration of the phenolic hydroxyl group. -1 and 2855.1 cm -1 This absorption peak is caused by the asymmetric stretching vibration of CH in the methylene group; 1658.2 cm⁻¹ -1 The characteristic absorption peak at 1605.2 cm⁻¹ represents the carbonyl stretching vibration of the flavonoid skeleton. -1 1513.5 cm -1 1453.9 cm -1 This is a characteristic absorption peak generated by the vibration of the benzene ring in the flavonoid skeleton; 1205.5 cm⁻¹ -1 and 1068.6 cm -1 The absorption peaks are caused by the asymmetric stretching vibration of the ether bond on the epoxide ring in the C6-C3-C6 flavonoid skeleton; the above characteristic absorption peaks indicate that the plateau cattail pollen flavonoids prepared in this invention have a complete basic flavonoid skeleton.

[0037] The flavonoid components in the flavonoids of Typha orientalis were qualitatively and quantitatively determined by liquid chromatography-mass spectrometry (LC-MS), and the results are shown in Table 1. Table 1 Flavonoid components in Typha pollen from the plateau

[0038] Note: All data are mean ± standard deviation (n=3).

[0039] As shown in Table 1, a total of 8 flavonoid components were detected, with rutin having the highest content (94.41 μg / g), followed by typhain (55.89 μg / g) and narcissin (32.43 μg / g).

[0040] Example 2 Evaluation of the in vitro antioxidant activity of Typhae pollen flavonoids from the plateau

[0041] The groups were set up as follows: control group (Con group), H2O2 group (H2O2 group), and PTF+H2O2 group (PTF group), with at least three replicate controls in each group; PTF was dissolved in PBS to prepare a sample solution of 4 mg / mL.

[0042] 1 mL of NCM460 cell suspension was seeded into each well of a 24-well plate and incubated at 37°C with 5% CO2 for 12 h. 143 μL of PTF sample solution was added to the wells corresponding to the PTF group to achieve a working concentration of 0.5 mg / mL, and the plates were incubated again for another 12 h. After incubation, 5.1 μL of 30% H2O2 solution was added to the wells corresponding to the H2O2 and PTF groups to achieve a working concentration of 50 mM, inducing oxidative stress in NCM460 cells. After 10 min of treatment, 1 mL of 2',7'-dichlorofluorescein diacetate (DCFH-DA) solution was added to all wells according to the reactive oxygen species detection kit instructions, and the plates were incubated for 20 min. After washing the cells three times with PBS, the plates were observed under a fluorescence microscope. The exported images were analyzed and calculated using FIJI / ImageJ. The results are shown below. Figure 2 As shown.

[0043] The results showed that, compared with the control group, the intensity of green fluorescence in the cells of the H2O2 group was significantly enhanced, indicating a sharp increase in ROS levels, while the fluorescence intensity of the PTF group was significantly lower than that of the H2O2 group, indicating that high-altitude PTF can effectively prevent H2O2-induced cellular oxidative stress damage.

[0044] Example 3: Determination of the effect of flavonoids from Typhae pollen on age-related cognitive impairment in high-altitude areas

[0045] SPF-grade C57BL / 6J male mice were randomly divided into 5 groups (n=12): normal control group (CON), model group (D-gal), low-dose PTF group (LPTF, 100 mg / kg), high-dose PTF group (HPTF, 200 mg / kg) and positive control group (VC, 40 mg / kg). Except for the CON group, the other groups were injected intraperitoneally with D-galactose (500 mg / kg) daily to establish an aging mouse model. The CON group was injected with the same dose of physiological saline. At the same time, each drug administration group was administered the corresponding dose of the drug by gavage daily. The CON group and the D-gal group were administered the same volume of PBS by gavage for 12 consecutive weeks.

[0046] Behavioral testing: The Morris water maze (MWM) test was performed after the last administration of medication. The MWM test is commonly used to assess the learning and memory abilities of mice. The main experimental apparatus consists of a circular water tank with a diameter of 100 cm and a height of 50 cm, divided into four quadrants. A transparent cylindrical platform with a diameter of 9 cm is located in the center of the third quadrant. Before the experiment, the entire tank was placed in a dimly lit area. Water was poured into the water maze until it submerged the transparent cylindrical platform in the third quadrant by 0.5 cm. Non-toxic titanium dioxide was added to make the water opaque. Different colored and patterned pictures were affixed to the four quadrants to help the mice remember the platform's location. The water temperature was maintained at 22±1℃ during the experiment. The experiment consisted of two phases: (1) Positioning and navigation experiment: The experiment lasted for 4 days. Each mouse was trained 4 times a day. During the training, the mice were placed in water facing the wall from four different quadrants. The time it took for the mice to find the hidden platform within 120 seconds was recorded as the escape latency. If the mice failed to find the platform within 120 seconds, they were guided to the platform with a transparent glass rod and allowed to stay there for 15 seconds to help the mice remember the location of the platform. (2) Spatial exploration experiment: On the second day after the positioning and navigation experiment, i.e., the fifth day of the experiment, the transparent cylindrical platform in the third quadrant was removed from the pool. Then, the mice were gently placed into the pool from the first quadrant facing the wall. The time it took for the mouse to first reach the original location of the transparent cylindrical platform, the number of times it crossed the original location of the transparent cylindrical platform, and the time it spent in the third quadrant were recorded. The test time for each mouse was 90 s. The results are as follows: Figure 3 As shown.

[0047] The results showed that, compared with the CON group, the escape latency of mice in the D-gal group was significantly prolonged ( p < 0.05, the number of times the original platform was crossed and the time spent in the target quadrant were significantly reduced ( p < 0.05 indicates impaired learning and memory abilities. Compared to the D-gal group, the HPTF group mice showed a significantly shorter escape latency, a significantly increased number of platform crossings, and a significantly increased time spent in the target quadrant. p The effect was < 0.05%, which was comparable to that of the VC group, indicating that high-altitude PTF can significantly improve cognitive dysfunction in aging mice.

[0048] Histopathological examination: Mouse brain tissue was collected after the last administration and stained with Nissl. The results are as follows: Figure 4 As shown.

[0049] The results showed that neurons in the CA1, CA3, and DG regions of the hippocampus in the CON group were neatly arranged with no significant morphological changes; neurons in the CA3 and DG regions of the D-gal group were disordered, with significant cell loss and nuclear condensation; neuronal damage was significantly reduced and cell morphology was restored in the PTF intervention group.

[0050] Oxidative stress and inflammatory factor detection: Oxidative stress levels in mouse brain tissue homogenates were detected using SOD, GSH, and MDA kits, and inflammatory factor levels were detected using IL-1β, IL-6, and TNF-α kits. Results are as follows: Figure 5 As shown.

[0051] The results showed that, compared with the CON group, the SOD activity and GSH content in the brain tissue of the D-gal group were significantly reduced. p <0.05), MDA, IL-6, and TNF-α levels were significantly increased ( p < 0.05); Compared with the D-gal group, the HPTF group showed significantly increased SOD activity and GSH content, and significantly decreased MDA, IL-6, and TNF-α content. p The result was < 0.05, indicating that high-altitude PTF can effectively reduce oxidative stress and neuroinflammation in the brain tissue of aging mice.

[0052] Example 4: The regulatory effect of flavonoids from Typha orientalis on the gut-brain axis

[0053] Intestinal barrier function assessment: In Example 3, after the last administration, mouse colon tissue was taken for immunofluorescence staining to detect tight junction proteins. The results are as follows: Figure 6 As shown.

[0054] The results showed that the fluorescence intensity of Occludin and Claudin-1 in the colon tissue of D-gal group mice was weaker than that in CON group, indicating that the intestinal barrier was damaged. After PTF intervention, the expression of these two proteins was enhanced, indicating that plateau PTF can effectively restore the integrity of the intestinal barrier and thus maintain its normal function.

[0055] Gut microbiota 16S rRNA sequencing: In Example 3, 16S rRNA was sequenced from mouse feces after the last administration. Results Figure 7 As shown.

[0056] The results showed that the gut microbiota α diversity (Shannon and Simpson indices) of D-gal group mice was significantly reduced. p < 0.05), β-diversity analysis showed significant changes in the bacterial community structure. At the genus level, beneficial bacteria in the D-gal group, such as Bifidobacterium , Lactobacillus , Adlercreutzia and Ligilactobacillus The relative abundance of these bacteria decreased significantly, while potentially harmful bacteria such as Bacteroides , Colidextribacter and unidentified_Lachnospiraceae The abundance of was significantly increased ( p < 0.05). These changes were all significantly reversed after PTF intervention ( p < 0.05).

[0057] Detection of short-chain fatty acids (SCFAs): In Example 3, the content of SCFAs in feces was detected by gas chromatography (GC) after the last administration. The results are as follows: Figure 8 As shown.

[0058] The results showed that, compared with the CON group, the contents of SCFAs such as acetic acid, propionic acid, and butyric acid in the feces of mice in the D-gal group were significantly reduced. p < 0.05), after PTF intervention, the content of SCFAs significantly rebounded ( p < 0.05).

[0059] Detection of intestinal microbial metabolites: In Example 3, after the last administration, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was used to detect intestinal microbial metabolites in feces without targeting. The results are as follows: Figure 9 As shown; The results showed that, compared with the CON group, the levels of beneficial metabolites tauroursodeoxycholic acid, choline, tryptamine, indole-3-lactic acid, indole-3-acetic acid, and indole in the feces of mice in the D-gal group were significantly reduced. p < 0.05), after PTF intervention, the levels of these beneficial metabolites significantly rebounded ( p < 0.05).

[0060] Detection of neurotransmitters and acetylcholinesterase (AChE) activity in the brain: In Example 3, after the last administration, the neurotransmitter content in the mouse brain was detected by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), and the AChE activity was detected using an AChE kit. The results are as follows: Figure 10 As shown.

[0061] The results showed that, compared with the CON group, the D-gal group had a significantly lower level of ACh in the brain ( p < 0.05), AChE activity was significantly increased ( p < 0.05), after PTF intervention, ACh levels significantly rebounded, and AChE activity was significantly inhibited ( p <0.05).

[0062] Example 5: Verification of the molecular interaction between the active monomer of Typhae pollen flavonoids and acetylcholinesterase.

[0063] The ability of PTF active monomers to cross the blood-brain barrier (BBB): Six male C57BL / 6J mice, aged 8-10 weeks and weighing approximately 22±2 g, were selected. Mice were acclimatized in an SPF-grade laboratory for one week, given standard maintenance feed and pure water. The laboratory environment was maintained at 22℃, with a circadian rhythm of 12 h light + 12 h dark. After gavage with 200 mg / kg PTF and cardiac perfusion, brain tissue samples were obtained through dissection. The samples were weighed, added to physiological saline at a 1:9 ratio, homogenized, and centrifuged in a refrigerated centrifuge (3500 rpm, 4℃) for 10 min to obtain the brain tissue supernatant. LC-MS was used to detect the high-altitude PTF active monomers entering the mouse brain. The results are shown in Table 2.

[0064] Table 2. Content of plateau PTF active monomers in mouse brain

[0065] The results showed that rutin, typhain, and narcissin in high-altitude PTF could cross the BBB and enter the brain.

[0066] Molecular docking: Rutin was searched for in the Traditional Chinese Medicine Systems Pharmacology Database and analysis platform, and its corresponding chemical structure file was downloaded. AChE was searched in the Structural Bioinformatics Research Collaboration Protein Database, and AChE containing ligands with similar structures (PDB code: 6O4W) was screened against the chemical structure of rutin, and the protein file was downloaded. The Pymol molecular graphics system was used to process the AChE and rutin small molecules, removing water molecules from AChE, separating the protein and ligand, and converting them to PDBQT format. The Autodock vina-1.2.3 software was used to locate the active pocket of AChE. After identifying the active pocket, molecular docking of rutin small molecules with AChE was performed, and the binding result file was exported after docking. Data processing and visualization were performed using the Pymol molecular graphics system. Results are as follows: Figure 11 As shown.

[0067] The results showed that the binding energy of rutin to AChE was -7.3 kcal / mol, and the binding site was located in the active domains of AChE (CAS and PAS sites), which highly overlapped with the binding site of the positive drug donepezil.

[0068] Surface plasmon resonance (SPR): The affinity between rutin and AChE was detected using a dual-channel SPR instrument at 25°C. The concentrations of the rutin small molecule sample solutions were 15.625, 31.25, 62.5, 125, and 250 μmol / L. AChE was diluted with HEPES buffer. The protein-ligand binding time and spontaneous dissociation time were both 60 s. Data analysis and processing were performed using Trace Drawer™ software to plot the binding curves between AChE and rutin, and the affinity constant KD(M) was calculated. The results are shown below. Figure 12 As shown.

[0069] The results showed that the binding response value increased in a dose-dependent manner with increasing rutin concentration. The equilibrium dissociation constant KD of rutin and AChE was calculated to be 6.71 × 10⁻⁶. -4 M indicates that the two have a good affinity.

[0070] As can be seen from the above embodiments, the present invention provides the application of high-altitude cattail pollen flavonoids in the preparation of products that improve age-related cognitive dysfunction. High-altitude cattail pollen flavonoids can achieve safe and efficient relief of age-related cognitive dysfunction through multi-target synergistic effects such as "regulating intestinal flora to reshape microecological balance, repairing intestinal barrier to inhibit peripheral inflammation, and crossing blood-brain barrier to directly inhibit acetylcholinesterase activity". This overcomes the shortcomings of existing technologies, such as large side effects of synthetic drugs, poor long-term tolerance, and single target of ordinary natural products, which are difficult to effectively regulate the "microbe-gut-brain axis".

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of flavonoids from Typha orientalis in the preparation of products that improve age-related cognitive impairment.

2. The application according to claim 1, characterized in that, The plateau cattail pollen flavonoids are obtained by extraction, separation and purification of cattail pollen.

3. The application according to claim 1, characterized in that, The plateau cattail flavonoids include rutin, typhain, narcissin, apigenin, naringin chalcone, quercetin, isoquercitrin, and quercetin-3-O-rutin-(1→2)-O-rhamnoside.

4. The application according to claim 1, characterized in that, The improvement in age-related cognitive impairment includes one or more of the following effects: (1) Improve learning and memory impairments; (2) Reduce damage to hippocampal neurons; (3) Enhances the antioxidant capacity of brain and colon tissues; (4) Inhibits the expression of pro-inflammatory factors in brain and colon tissues; (5) Repairing intestinal barrier function; (6) Regulate the structure of intestinal flora, increase the abundance of beneficial bacteria, and reduce the abundance of potentially harmful bacteria; (7) Promotes the production of beneficial metabolites; (8) Inhibits the activity of acetylcholinesterase in the brain and increases the level of acetylcholine.

5. A formulation for improving age-related cognitive impairment, characterized in that, The formulation comprises flavonoids from Typha orientalis and a pharmaceutically acceptable carrier.

6. The formulation according to claim 5, characterized in that, The dosage form of the preparation is tablets, capsules, granules, oral liquids, or powders.

7. Application of flavonoid monomers from Typha orientalis in the preparation of products that protect cognitive function, wherein the flavonoid monomers from Typha orientalis are rutin.

8. The application according to claim 7, characterized in that, The flavonoid monomers from Typha orientalis protect cognitive function by maintaining acetylcholine homeostasis in the brain.

9. A formulation that protects cognitive function, characterized in that, The formulation includes rutin and a pharmaceutically acceptable carrier.

10. The formulation according to claim 9, characterized in that, The dosage form of the preparation is tablets, capsules, granules, oral liquids, or powders.

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