A traditional Chinese medicine composition for improving senile cognitive impairment and a preparation method thereof

Abstract

The application discloses a traditional Chinese medicine composition for improving senile cognitive impairment and a preparation method thereof, and belongs to the technical field of traditional Chinese medicine. The composition is composed of the following components in parts by weight: 2-60 parts of blueberry freeze-dried powder, 5-60 parts of rhizoma polygonati, 10-70 parts of radix astragali, 5-50 parts of medlar and 3-40 parts of ganoderma lucidum spore powder. The method comprises the following steps: soaking the rhizoma polygonati, radix astragali and medlar; decocting the raw materials for 1-3 times; combining the medicinal liquid obtained after decoction, and concentrating to obtain concentrated medicinal liquid; preparing the concentrated medicinal liquid into freeze-dried powder; and adding the blueberry freeze-dried powder and the ganoderma lucidum spore powder into the freeze-dried powder and uniformly stirring. The anti-aging pharmacodynamic study results of the application are as follows: (1) the tested medicine can significantly prolong the longest life span of natural aging nematodes by 33.79%; (2) the tested medicine can improve senile behavior; (3) the tested medicine can improve antioxidant capacity; (4) the tested medicine can reduce senile markers; (5) the tested medicine can up-regulate SIRT; (6) the tested medicine can improve nematode cognitive impairment, improve mechanical stimulation withdrawal response rate, temperature-seeking learning ability and olfactory chemotaxis learning ability, and reduce 5-HT sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a traditional Chinese medicine composition for improving age-related cognitive impairment and its preparation method. Background Technology

[0002] Age-related cognitive impairment (ACI) is a critical transitional stage from aging to dementia, representing a golden window for intervention. Missing this window leads to irreversible dementia. Currently, clinically used drugs for ACI include donepezil, memantine, rivastigmine, galantamine, and huperzine. While these drugs have some efficacy, they commonly cause adverse reactions such as nausea, vomiting, dizziness, headache, gastrointestinal discomfort, and thrombocytopenia. Furthermore, they cannot cure or reverse the disease progression, have poor long-term efficacy, and their long-term safety remains controversial. Therefore, there is an urgent clinical need for safe, effective, and long-term-use innovative drugs to open up new avenues for treating ACI.

[0003] Traditional Chinese medicine (TCM) boasts a thousand-year history of human use and demonstrates unique advantages in combating age-related diseases. TCM herbs, often considered both food and medicine, possess the dual attributes of "anti-cognitive impairment efficacy" and "food-like safety for long-term consumption," aligning with consumer demand for natural, safe, and effective anti-aging health products. However, currently, there are no approved TCM-food homologous formulas for improving age-related cognitive impairment. Summary of the Invention

[0004] To address the problem of how to develop a traditional Chinese medicine product that is effective and safe in improving age-related cognitive impairment, this invention provides a traditional Chinese medicine composition for improving age-related cognitive impairment and its preparation method.

[0005] This invention comprises the following traditional Chinese medicine components: blueberry, polygonatum, astragalus, wolfberry, and ganoderma spore powder. This combination of traditional Chinese medicine is based on the core theory of "preventive treatment" in traditional Chinese medicine. Following the principle of tonifying the kidneys and replenishing marrow, it scientifically combines selected medicinal and edible herbs, clearly quantifies the proportions, and employs optimized processes. In this formula, blueberry is the principal ingredient, tonifying the kidneys and securing essence; polygonatum is the assistant ingredient, tonifying the kidneys and replenishing essence; astragalus is the adjuvant ingredient, tonifying qi and raising yang; and wolfberry and ganoderma spore powder are the guiding ingredients, nourishing the liver and kidneys, tonifying qi and calming the mind. This embodies the classic anti-aging theory of "tonifying the kidneys and replenishing marrow," extending lifespan by activating longevity factors and significantly improving age-related cognitive impairment. It is superior in effect, lower in cost, and safer than current Western medicines for delaying aging, such as nicotinamide mononucleotide (NMN), making it suitable for widespread application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A traditional Chinese medicine composition for improving age-related cognitive impairment, the composition comprising, by weight, the following components: 2-60 parts of freeze-dried blueberry powder (equivalent to 20-600 parts of fresh blueberries), 5-60 parts of Polygonatum sibiricum, 10-70 parts of Astragalus membranaceus, 5-50 parts of Lycium barbarum, and 3-40 parts of Ganoderma lucidum spore powder.

[0008] A method for preparing the above-mentioned traditional Chinese medicine composition for improving age-related cognitive impairment, the method comprising the following steps:

[0009] Step 1: Soak all ingredients except for the freeze-dried blueberry powder and Ganoderma lucidum spore powder;

[0010] Step 2: Boil the ingredients 1-3 times;

[0011] Step 3: Combine the decoctions obtained from several decoctions and concentrate them to obtain a concentrated decoction;

[0012] Step 4: Prepare the concentrated drug solution into lyophilized powder;

[0013] Step 5: Add the freeze-dried blueberry powder and Ganoderma lucidum spore powder to the freeze-dried powder and stir well. For clinical use, Ganoderma lucidum spore powder is best taken by dissolving in water or dissolving in water; when used in compound prescriptions, it must be added last and never decocted together throughout the entire process. This is because its active ingredient, Ganoderma lucidum triterpenoids, has poor stability and is more easily degraded at higher temperatures.

[0014] Furthermore, in step one, the soaking time is 30-60 minutes.

[0015] Furthermore, in step two, the mass ratio of the soaking liquid to the raw materials is 12.5:1 for each decoction.

[0016] Furthermore, in step two, first bring the raw materials and all the soaking liquid to a boil over high heat, then reduce the heat to low and continue simmering for 30-60 minutes. After simmering, separate the liquid and dregs, add water to the dregs for the next simmering, and each simmering time is 30-60 minutes.

[0017] The beneficial effects of this invention compared to the prior art are as follows: The pharmacodynamic study results of this invention in improving age-related cognitive impairment are as follows: (1) The test drug can significantly prolong the longest lifespan of naturally aging nematodes by 33.79%, which is superior to the Western medicine NMN; (2) Improve aging behavior: enhance motor function, feeding ability and nervous system function; (3) Enhance antioxidant capacity: reduce the fluorescence intensity of lipofuscin, increase the survival rate of nematodes after H2O2 stress stimulation, and increase the activity of catalase (CAT) and superoxide dismutase (SOD); (4) Reduce aging markers -Galactosidase activity, upregulating the expression of the longevity factor SIRT; (5) improving reproductive capacity; (6) improving cognitive impairment in nematodes: by improving the mechanostimulation habituation, thermotactic learning ability, aversion-type olfactory chemotactic learning ability, and reducing sensitivity to 5-HT in aging nematodes, age-related cognitive impairment is improved. In summary, this invention activates the activity of longevity factors, prolongs the lifespan of aging nematodes, improves the behavioral changes of aging, resists oxidative stress, improves age-related cognitive impairment, is of medicinal and edible origin, has high safety, and is suitable for widespread application. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0019] Traditional Chinese medicine classifies cognitive impairment as "forgetfulness" or "morbid forgetfulness." The text states that "the kidneys store essence, which generates marrow, and the brain is the sea of ​​marrow." The Suwen states, "If the kidneys do not generate essence, the marrow cannot be full." The Yibian emphasizes, "The kidneys are below and the brain is above; deficiency in either leads to deficiency in both." Therefore, kidney deficiency and marrow depletion, as well as an empty sea of ​​marrow, are the fundamental pathogenesis of cognitive impairment, and tonifying the kidneys and nourishing the marrow is the core treatment method.

[0020] This invention is based on the core theories of traditional Chinese medicine, namely "prevention of disease" and "medicine and food sharing the same origin, strengthening the body's resistance and consolidating its foundation." Following the principles of tonifying the kidneys and marrow, and nourishing the five internal organs simultaneously, it scientifically selects medicinal herbs that are both food and medicine, clearly quantifies the proportions, and employs optimized processes. Integrating modern cutting-edge technology, it has developed a safe, effective, and daily-use anti-aging compound product made from medicinal herbs and food, primarily comprising the following Chinese herbal components: Polygonatum sibiricum, Astragalus membranaceus, Lycium barbarum, Ganoderma lucidum spore powder, and freeze-dried blueberry powder.

[0021] Blueberry, the principal ingredient in the formula, is a typical functional food, used in the largest quantity. It enters the liver, kidney, and spleen meridians, and has the functions of tonifying the kidneys and strengthening essence, strengthening muscles and improving eyesight, and regulating qi and relieving bloating. Clinical studies have confirmed that blueberries improve age-related mild cognitive impairment by enhancing episodic memory, delayed recall, and vocabulary recognition abilities in older adults. Its mechanism of action may involve protecting cognitive function through anti-oxidative stress, inhibiting inflammation, reducing neurotoxicity, and promoting nerve repair.

[0022] The principal herb, Polygonatum sibiricum, is a "key herb for replenishing both Qi and Yin." It enters the lung, spleen, and kidney meridians and has the effects of replenishing Qi and nourishing Yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys and replenishing essence. It exerts its neuroprotective effects through anti-inflammatory, antioxidant, brain-gut axis regulation, and improvement of synaptic plasticity pathways, thereby improving age-related cognitive impairment and assisting the principal herb in enhancing efficacy.

[0023] Astragalus, an adjuvant herb, is considered a "leader among tonics," entering the lung and spleen meridians. It possesses the functions of tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing edema, generating fluids and nourishing blood, promoting circulation and relieving pain, promoting pus drainage and detoxification, and astringing sores and promoting tissue regeneration. Based on the principle of preventing disease before it occurs, strengthening the spleen can tonify the kidneys, thus enhancing the efficacy of the principal herb. Astragalus is a frequently used herb for treating cognitive impairment, improving age-related cognitive impairment by enhancing learning and memory abilities and inhibiting nerve cell damage and neuroinflammation. Astragalus assists the principal and adjuvant herbs in strengthening their therapeutic effects.

[0024] The guiding herb, wolfberry (also known as "longevity fruit"), enters the liver and kidney meridians; it nourishes the liver and kidneys, benefits essence and improves eyesight, and also has anti-aging and neuroprotective effects. The guiding herb, Ganoderma lucidum spore powder, consists of the reproductive cells of Ganoderma lucidum. Ganoderma lucidum enters the heart, liver, lung, and kidney meridians and is a medicinal and edible anti-aging herb, possessing the effects of tonifying qi and calming the mind, and strengthening the body's resistance. Wolfberry, as a guiding herb, can guide the medicine to the kidneys to replenish essence, thus fulfilling the guiding role of the guiding herbs.

[0025] The various medicinal and edible herbs in this formula work synergistically to tonify the kidneys and replenish the marrow, providing a safe, effective, and long-term edible product for age-related cognitive impairment.

[0026] Example 1: A traditional Chinese medicine composition for improving age-related cognitive impairment, comprising the following components: 2-5 parts of freeze-dried blueberry powder (equivalent to 20-50 parts of fresh blueberries), 5-7 parts of Polygonatum sibiricum, 10-11 parts of Astragalus membranaceus, 5-6 parts of Lycium barbarum, and 3-5 parts of Ganoderma lucidum spore powder.

[0027] Example 2: A traditional Chinese medicine composition for improving age-related cognitive impairment, comprising the following components: 6-9 parts of freeze-dried blueberry powder (equivalent to 60-90 parts of fresh blueberries), 8-12 parts of Polygonatum sibiricum, 12-14 parts of Astragalus membranaceus, 7-9 parts of Lycium barbarum, and 6-9 parts of Ganoderma lucidum spore powder.

[0028] Example 3: A traditional Chinese medicine composition for improving age-related cognitive impairment, comprising the following components: 10-20 parts of freeze-dried blueberry powder (equivalent to 100-200 parts of fresh blueberries), 13-20 parts of Polygonatum sibiricum, 15-25 parts of Astragalus membranaceus, 10-19 parts of Lycium barbarum, and 10-17 parts of Ganoderma lucidum spore powder.

[0029] Example 4: A traditional Chinese medicine composition for improving age-related cognitive impairment, comprising the following components: 21-40 parts of freeze-dried blueberry powder (equivalent to 210-400 parts of fresh blueberries), 21-35 parts of Polygonatum sibiricum, 26-40 parts of Astragalus membranaceus, 20-35 parts of Lycium barbarum, and 18-24 parts of Ganoderma lucidum spore powder.

[0030] Example 5: A traditional Chinese medicine composition for improving age-related cognitive impairment, comprising the following components: 41-60 parts of freeze-dried blueberry powder (equivalent to 410-600 parts of fresh blueberries), 36-60 parts of Polygonatum sibiricum, 41-70 parts of Astragalus membranaceus, 36-50 parts of Lycium barbarum, and 25-40 parts of Ganoderma lucidum spore powder.

[0031] The above composition was prepared by the following method:

[0032] Step 1: Soak all ingredients except blueberry freeze-dried powder and Ganoderma lucidum spore powder for 40-50 minutes;

[0033] Step 2: Boil the raw materials twice; each time, the mass ratio of the soaking liquid to the raw materials is 12.5:1; first, bring the raw materials and all the soaking liquid to a boil over high heat, then turn the high heat to low heat and continue to boil for 40-50 minutes. After boiling, separate the liquid and the dregs, add water to the dregs and boil again. Each boiling time is 40-50 minutes.

[0034] Step 3: Combine the decoctions obtained from several decoctions and concentrate them to obtain a concentrated decoction;

[0035] Step 4: Prepare the concentrated drug solution into lyophilized powder;

[0036] Step 5: Add the freeze-dried blueberry powder and Ganoderma lucidum spore powder to the freeze-dried powder and stir well. For clinical use, Ganoderma lucidum spore powder is best taken by dissolving in water or melting in water; when used in compound prescriptions, it must be added last and never decocted together throughout the entire process. This is because its active ingredient, Ganoderma lucidum triterpenoids, has poor stability and is more easily degraded at higher temperatures.

[0037] The traditional Chinese medicine composition obtained in Example 3 was used as the low-dose group of the test drug, the traditional Chinese medicine composition obtained in Example 4 was used as the medium-dose group of the test drug, and the traditional Chinese medicine composition obtained in Example 5 was used as the high-dose group of the test drug. The following experiments were conducted:

[0038] I. Experimental Methods

[0039] Nematode lifespan experiments and behavioral observations

[0040] Nematodes were transferred to culture dishes containing three dose groups of the test drug (0.5 mg / mL, 1 mg / mL, and 2 mg / mL, denoted as LL, LM, and LH), M9 buffer (blank control group), three dose groups of blueberry lyophilized powder (0.5 mg / mL, 1 mg / mL, and 2 mg / mL, denoted as BL, BM, and BH, denoted as positive control group), and 1 μg / mL NMN (positive control group for Western medicine) (150 nematodes per group; n=150), and cultured in a 20°C biological incubator. During the reproduction period, nematodes were transferred to new plates every other day at room temperature to prevent interference from hatching embryos. The number of surviving nematodes in each dish was recorded daily until all nematodes died. Nematodes were considered dead when they did not respond to repeated poking and showed no pharyngeal pumping. On days 0, 3, 6, 9, 12, and 15 of culture, 15 nematodes were randomly selected from each group, and the frequency of pharyngeal pumping, head shaking, and body bending was observed for 20 seconds at room temperature.

[0041] Nematode Reproduction Experiment

[0042] L4 stage nematodes were transferred to culture dishes containing three dose groups of the test drug (0.5 mg / mL, 1 mg / mL, and 2 mg / mL, denoted as LL, LW, and LH), M9 buffer (blank control group), three dose groups of blueberry lyophilized powder (0.5 mg / mL, 1 mg / mL, and 2 mg / mL, denoted as BL, BM, and BH, denoted as positive control group), and 1 μg / mL NMN (positive control group) (one nematode per group; n=5). The dishes were then incubated in a 20°C biological incubator. Every 24 hours, the nematodes were transferred to a new food plate. The number of eggs on the culture dishes from the previous day was counted daily until the nematodes stopped laying eggs or died. The total number of eggs laid each day was the sum of the daily egg counts.

[0043] L4 stage nematodes were transferred to culture dishes containing three dose groups of the test drug (0.5 mg / mL, 1 mg / mL, and 2 mg / mL, denoted as LL, LW, and LH), M9 buffer (blank control group), three dose groups of blueberry lyophilized powder (0.5 mg / mL, 1 mg / mL, and 2 mg / mL, denoted as BL, BM, and BH, denoted as positive control group), and 1 μg / mL NMN (positive control group) (one nematode per group; n=6). The dishes were then incubated in a 20°C biological incubator. After approximately 24 hours, the nematodes were transferred to a new food plate, and the time was recorded for 3 hours. The number of eggs laid by the nematodes within 3 hours was counted, and the oviposition rate of the nematodes was calculated.

[0044] Detection of lipofuscin in nematodes

[0045] After incubating the test drug, lyophilized blueberry powder, and NMN in nematodes (20 nematodes per dish) for 5 days, the nematodes were placed on 2% agarose plates and anesthetized with 10 mM levamisole. Images were captured using a fluorescence microscope, and fluorescence intensity was calculated using ImageJ software.

[0046] Detection of ROS fluorescence in nematodes

[0047] After incubating the test drug, lyophilized blueberry powder, and NMN in nematodes (20 nematodes per dish) for 5 days, the mixture was transferred to 100 μL of LDCFH-DA and incubated at room temperature in the dark for 2 hours. ROS levels were detected by fluorescence microscopy, and fluorescence intensity was calculated using ImageJ software.

[0048] H2O2-induced oxidative stress experiment in nematodes

[0049] After incubating the test drug, lyophilized blueberry powder, and NMN in nematodes for 5 days (20 nematodes per plate), the mixture was transferred to an NGM plate containing 50 μL of 3% H2O2. After 5 h, the survival rate of the nematodes was measured to assess the antioxidant stress resistance of the test drug.

[0050] nematodes -Galactosidase activity assay

[0051] After incubating nematodes with the test drug and NMN for 14 days, nematode samples were collected and washed with M9 buffer. The samples were fixed in fixative for 15 minutes, then the fixative was removed. Subsequently, the samples were stained at 37°C in a carbon dioxide-free environment for 20 hours. Afterwards, the staining was removed with buffer. -Galactosidase ( Wash three times with gal) staining solution. Bright-field images were captured using a fluorescence microscope, and the fluorescence area was calculated using ImageJ software.

[0052] ELISA

[0053] After incubating nematodes with the test drug, lyophilized blueberry powder, and NMN for 15 days (at least 500 nematodes per dish), the nematodes were washed with M9 buffer at 5000 rpm for 1 min. This process was repeated 2-3 times. The nematode pellet was then ground, sonicated, centrifuged at 12000 rpm, and the supernatant was collected. The supernatant was then detected using an ELISA kit for nematode longevity protein (SIRT1), catalase (CAT), and superoxide dismutase (SOD).

[0054] Nematode mechanical stimulation habituation experiment

[0055] This study was used to detect the neural reflex adaptation and synaptic plasticity of nematodes. The experimental groups were: a young control group (adults aged 1 day), an aging model group (adults aged 5 days), and a test drug group (test drug incubated for 5 days from the age of 1 adult) (5 nematodes / group, 3 groups). The nematodes were cleaned with M9 granules, and one nematode was placed on a 35mm food-free NGM plate each time. Testing began after each nematode had adapted to the environment for 1 minute. The nematode's posterior pharyngeal wall was gently touched, and observation was performed for 1 second immediately after the touch. A positive reaction was defined as the nematode retreating after stimulation; no reaction was defined as no change in the nematode's movement. A positive reaction was recorded as +, and no reaction as -. A total of 10 touches were performed, with 10-second intervals between each. The withdrawal reaction rate was calculated using the formula: Withdrawal Reaction Rate (%) = Number of Reactions / Total Number of Stimulations × 100%.

[0056] nematode 5-hydroxytryptamine (5-HT) sensitivity test

[0057] This study reflects synaptic function, neural regulation, and cognitive status. The experimental groups were: a young control group (adults aged 1 day), an aging model group (adults aged 5 days), and a test drug group (test drug incubated for 5 days from adult day 1) (20 nematodes per plate, n=3). The nematodes were washed with M9 solution. Twenty nematodes from each group were placed in a 35 mm culture dish on a food-free plate. Immediately afterward, 50 μL of 5 mg / mL 5-HT solution was added to the center of the plate. After 5 min of treatment, the number of paralyzed nematodes was immediately counted. The paralysis rate (%) = (number of paralyzed nematodes / total number of paralyzed nematodes) × 100%.

[0058] Nematode temperature chemotaxis experiment

[0059] The memory and tactic ability of nematodes to a culture temperature of 20℃ were investigated. The experimental groups were: a juvenile control group (adults aged 1 day), an aging model group (adults aged 5 days), and a test drug group (test drug incubated for 5 days from adult day 1) (200 nematodes / group, n=3). The incubation temperature was 20℃. The nematodes were cleaned with M9 nematodes, placed in the center of a culture dish, and kept unfed with a temperature gradient of 14-20℃. The dish was divided into eight zones (1-8) along this temperature gradient. After allowing the nematodes to move freely for 1 hour, they were treated with chloroform. The number of nematodes in each of the eight zones along the temperature gradient was counted under a stereomicroscope, and the thermotactic index was calculated. The thermotactic index = (number of nematodes in zone 7 + number of nematodes in zone 8) / total number of nematodes.

[0060] Nematode aversion olfactory chemotaxis experiment

[0061] The study investigated the odor-food association learning and memory retention abilities of nematodes. Experimental groups were: a young control group (adults aged 1 day), an aging model group (adults aged 5 days), and a test drug group (test drug incubated for 5 days from adult day 1) (each group was further divided into a Naïve group (untrained) (50 nematodes) and a Trained group (trained) (50 nematodes), n=3). Nematodes were washed with M9 and placed on food-free plates for 60 min of starvation. The Naïve group was washed with M9 before the experiment. The Trained group was placed upside down on food-free medium, with 100 μL of a 1:10000 diacetyl solution diluted in anhydrous ethanol added to the plate lid, and trained for 60 min; then washed with M9 before the experiment. Fifty nematodes were placed at the starting point on a 90 mm food-free NGM medium, allowing them to choose between a spot containing 1 μL of a 1:10000 diacetyl solution diluted in anhydrous ethanol and a equidistant control group containing 1 μL of ethanol. Both spots contained 1 μL of 1 M levamisole. One hour later, observe the distribution of nematodes on the culture medium and calculate the CI (chemotaxis index) and LI (learning index) values ​​using the following formulas: CI = (number of diacetyl spotted nematodes - anhydrous ethanol) / (number of main nematodes - number of starting point nematodes). LI = average CI of the Naïve group - CI value of the Trained group.

[0062] Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism 10.1.2 software, employing t-tests or one-way ANOVA. p < 0.05 was considered statistically significant.

[0063] II. Experimental Results

[0064] 1. The tested drug can prolong the lifespan of naturally aging nematodes.

[0065] Mean survival time (MSG) results showed that, compared with the blank control group, the MSG, medium-, and high-dose groups of the test drug had MSG extended by 8.06%, 12.65%, and 19.15%, respectively; the low-, medium-, and high-dose groups of blueberry freeze-dried powder had MSG extended by 6.07%, 10.40%, and 15.34%, respectively; and the NMN group had MSG extended by 14.82%. Median survival time (MSG) results showed that, compared with the blank control group, the MSG, medium-, and high-dose groups of the test drug had MSG extended by 2.83%, 11.40%, and 14.22%, respectively; the low-, medium-, and high-dose groups of blueberry freeze-dried powder had MSG extended by 2.83%, 2.83%, and 5.66%, respectively; and the NMN group had MSG extended by 8.57%. The maximum survival time results showed that the longest survival time of nematodes in the blank control group was 22.67 days. Compared with the blank control group, the lifespan of the low, medium and high dose groups of the test drug was extended by 19.10%, 22.06% and 33.79% respectively. The lifespan of the low, medium and high dose groups of blueberry freeze-dried powder was extended by 16.14%, 20.56% and 27.92% respectively, and that of NMN was extended by 32.33%.

[0066] Table 1. Effects of the test drugs on the lifespan of naturally aging nematodes (mean ± standard deviation)

[0067]

[0068] Note: Compared with Group C, p < 0.05; p < 0.01; p < 0.001; compared with the NMN group, ## p < 0.01.

[0069] 2. Behavioral manifestations of the investigational drug in improving aging

[0070] Behavioral results of nematodes showed that on day 15, naturally aging nematodes exhibited 2.60 twisting movements, 10.73 head shaking movements, and 40.93 pharyngeal pumping movements. After intervention with low, medium, and high doses of the test drug, the number of body twisting movements increased by 15.38%, 33.46%, and 71.92%, respectively; the number of head shaking movements increased by 16.22%, 25.54%, and 34.86%, respectively; and the pharyngeal pumping frequency increased by 24.43%, 33.40%, and 40.90%, respectively. In the blueberry freeze-dried powder group, the number of body twisting movements in the low, medium, and high dose groups were 3.00, 3.80, and 4.00 times, respectively; the number of head shaking movements were 12.20, 13.00, and 13.87 times, respectively; and the pharyngeal pump frequency was 49.40, 52.33, and 56.00 times, respectively. The number of body twisting movements increased by 15.38%, 46.15%, and 53.85%, respectively; the number of head shaking movements increased by 13.70%, 21.16%, and 29.26%, respectively; and the pharyngeal pump frequency increased by 20.69%, 27.85%, and 36.82%, respectively. After NMN intervention, the number of body twisting movements increased by 69.23%, the number of head shaking movements increased by 39.14%, and the pharyngeal pump frequency increased by 36.65%.

[0071] Table 2. Effects of the test drugs on the aging behavior of nematodes (mean ± standard deviation)

[0072]

[0073] Note: Compared with Group C, p < 0.05; p < 0.01; p < 0.001.

[0074] 3. The test drug enhances the reproductive capacity of nematodes.

[0075] The average egg production of naturally aging nematodes was 204.60. After intervention with low, medium, and high doses of the tested drugs, the average egg production was 241.40, 259.80, and 285.00, respectively, representing increases of 17.99%, 26.98%, and 39.30%. After intervention with low, medium, and high doses of blueberry freeze-dried powder, the average egg production was 239.40, 261.80, and 270.80, respectively, representing increases of 17.01%, 27.96%, and 32.36%. The average egg production in the NMN group (1 μg / mL) was 292.60, an increase of 43.01%.

[0076] Intervention with low, medium, and high doses of the test drug increased the oviposition rate of nematodes by 35.00%, 45.00%, and 63.25%, respectively; intervention with low, medium, and high doses of blueberry freeze-dried powder increased the oviposition rate of nematodes by 23.25%, 36.75%, and 46.75%, respectively; and the NMN group (1 μg / mL) increased the oviposition rate by 55.00%.

[0077] Table 3. Effects of the test drugs on the reproductive capacity of nematodes (mean ± standard deviation)

[0078]

[0079] Note: Compared with Group C, p < 0.05; p < 0.01; p < 0.001.

[0080] 4. The test drug enhances the antioxidant capacity of nematodes.

[0081] After low, medium, and high doses of the test drug, the fluorescence intensity of lipofuscin in naturally aging nematodes decreased by 23.00%, 26.00%, and 30.00%, respectively; the fluorescence intensity of lipofuscin in the low, medium, and high dose groups of blueberry freeze-dried powder decreased by 24.00%, 25.00%, and 26.00%, respectively; and NMN intervention reduced it by 21.00%.

[0082] After intervention with low, medium, and high doses of the test drug, the fluorescence intensity of reactive oxygen species (ROS) in naturally aging nematodes decreased by 19.00%, 27.00%, and 35.00%, respectively; the low, medium, and high doses of blueberry freeze-dried powder decreased by 14.00%, 23.00%, and 28.00%, respectively; and the NMN intervention decreased by 19.00%.

[0083] After intervention with low, medium, and high doses of the test drug and NMN, the survival rate of nematodes after H2O2 stress stimulation increased by 83.40%, 166.60%, and 266.60%, respectively; after intervention with low, medium, and high doses of blueberry freeze-dried powder, the survival rate of nematodes after H2O2 stress stimulation increased by 50.00%, 116.60%, and 150.00%, respectively; and after NMN intervention, the survival rate of nematodes after H2O2 stress stimulation increased by 200.00%.

[0084] Table 4 Antioxidant effects of the test drugs (mean ± standard deviation)

[0085]

[0086] Note: Compared with Group C, p < 0.05; p < 0.01; p < 0.001.

[0087] After intervention with low, medium, and high doses of the test drug and NMN, the activities of catalase (CAT) in nematodes were increased by 17.00%, 19.00%, 35.00%, and 22.00%, respectively; and the activities of superoxide dismutase (SOD) in nematodes were increased by 19.00%, 35.00%, 63.00%, and 49.00%, respectively.

[0088] Table 5. Effects of test drugs on antioxidant enzyme activity (mean ± standard deviation)

[0089]

[0090] Note: Compared with Group C, p < 0.05; p < 0.01; p < 0.001.

[0091] 5. Effects of the test drug on nematodes Effect of galactosidase activity

[0092] After intervention with low, medium, and high doses of the test drug, naturally aging nematodes -Galactosidase (SA- The fluorescence area of ​​-gal decreased by 53.00%, 73.00%, and 93.00%, respectively; after NMN intervention, it decreased by 93.00%.

[0093] Table 6. Effects of test drugs on nematodes - Effect of galactosidase activity (mean ± standard deviation)

[0094]

[0095] Note: Compared with Group C, p < 0.001.

[0096] 6. The test drug enhanced the activity of SIRT1, a longevity factor, in naturally aging nematodes.

[0097] Compared with the control group, the activity of SIRT1, a longevity protein in nematodes, increased by 13.00%, 29.00%, and 50.00% respectively after intervention in the low, medium, and high dose groups of the test drug; the activity of SIRT1, a longevity protein in nematodes, increased by 8.00%, 19.00%, and 41.00% respectively after intervention in the low, medium, and high dose groups of blueberry freeze-dried powder; and the activity of SIRT1 increased by 89.00% in the NMN group.

[0098] Table 7. Effects of the test drug on the activity of SIRT1, a longevity protein in naturally aging nematodes (mean ± standard deviation)

[0099]

[0100] Note: Compared with Group C, p < 0.05; p < 0.01; p < 0.001.

[0101] 7. The test drug increased the rate of mechanical stimulation withdrawal response in naturally aging nematodes.

[0102] The results of the mechanical stimulation habituation experiment of nematodes showed that the withdrawal response rate of naturally aging nematodes was 30.00%. After intervention with low, medium and high doses of the test drug, the withdrawal response rates were 59.33%, 66.67% and 76.67%, respectively, which were 97.77%, 122.23% and 155.57% higher than those of naturally aging nematodes. The withdrawal response rate of nematodes in the young control group was 84.67%, which was 182.23% higher than that of the young control group. There was no difference between the test drug group and the young control group, suggesting that the test drug can significantly improve the mechanical stimulation withdrawal response rate of aging nematodes.

[0103] Table 8 Effects of the test drugs on mechanical stimulation of naturally aging nematodes (mean ± standard deviation)

[0104]

[0105] Note: Compared with Group C, p < 0.001; compared with group M, ### p < 0.001.

[0106] 8. The tested drug reduced the sensitivity of naturally aging nematodes to 5-HT.

[0107] Using 5-HT to detect synaptic function and neural regulation ability in nematodes, the experimental results showed that the paralysis rate of naturally aging nematodes was 83.33%. After intervention with the test drug, the paralysis rates of low, medium, and high doses were 68.33%, 63.33%, and 55.00%, respectively, which were reduced by 18.00%, 24.00%, and 34.00% compared with naturally aging nematodes. The paralysis rate of nematodes in the young control group was 46.67%, a reduction of 43.99%, indicating that aging nematodes are more sensitive to 5-HT. There was no difference between the test drug group and the young control group, suggesting that the test drug can significantly reduce the sensitivity of naturally aging nematodes to 5-HT.

[0108] Table 9. Effects of test drugs on the 5-HT sensitivity of naturally aging nematodes (mean ± standard deviation)

[0109]

[0110] Note: Compared with Group C, p < 0.001; compared with group M, ## p < 0.01; ### p < 0.001.

[0111] 9. The test drug enhances the thermotaxis learning ability of naturally aging nematodes.

[0112] A temperature chemotaxis experiment was conducted on nematodes. The results showed that the temperature chemotaxis learning index of naturally aged nematodes was 0.32. After intervention with the test drug, the temperature chemotaxis learning indices of low, medium, and high doses were 0.36, 0.39, and 0.41, respectively, which were 12.50%, 21.88%, and 28.13% higher than those of naturally aged nematodes. The temperature chemotaxis learning index of the young control group was 0.48, an increase of 50.00%. This suggests that the test drug can significantly improve the temperature chemotaxis learning ability of aged nematodes and has an ameliorative effect on neurological function decline.

[0113] Table 10 Effects of the test drugs on the thermotropic learning ability of naturally aging nematodes (mean ± standard deviation)

[0114]

[0115] Note: Compared with Group C, p < 0.001; compared with group M, # p < 0.05; ## p < 0.01.

[0116] 10. The test drug enhances the aversion-type olfactory chemotaxis learning ability of naturally aging nematodes.

[0117] An aversion-type olfactory chemotaxis experiment was conducted on nematodes. The results showed that the learning index of naturally aged nematodes was 0.10. After intervention with the test drug, the learning indices of low, medium, and high doses were 0.21, 0.25, and 0.56, respectively, which were 110.00%, 150.00%, and 460.00% higher than those of naturally aged nematodes. The learning index of the young control group was 0.62, which was 520.00% higher. This suggests that the test drug can significantly improve the olfactory chemotaxis training ability of aged nematodes and has an improving effect on neurological function and cognitive decline.

[0118] Table 11 Effects of the test drugs on the aversion-type olfactory chemotaxis learning ability of naturally aging nematodes (mean ± standard deviation)

[0119]

[0120] Note: Compared with Group C, p < 0.001; compared with group M, ## p < 0.01.

Claims

1. A traditional Chinese medicine composition for improving age-related cognitive impairment, characterized in that: The composition comprises, by weight, the following components: 2-60 parts of freeze-dried blueberry powder, 5-60 parts of Polygonatum sibiricum, 10-70 parts of Astragalus membranaceus, 5-50 parts of Lycium barbarum, and 3-40 parts of Ganoderma lucidum spore powder.

2. A method for preparing the traditional Chinese medicine composition for improving age-related cognitive impairment as described in claim 1, characterized in that: The method includes the following steps: Step 1: Soak all ingredients except for the freeze-dried blueberry powder and Ganoderma lucidum spore powder; Step 2: Boil the ingredients 1-3 times; Step 3: Combine the decoctions obtained from several decoctions and concentrate them to obtain a concentrated decoction; Step 4: Prepare the concentrated drug solution into lyophilized powder; Step 5: Add the freeze-dried powder to the blueberry freeze-dried powder and Ganoderma lucidum spore powder, and stir well.

3. The preparation method according to claim 2, characterized in that: In step one, the soaking time is 30-60 minutes.

4. The preparation method according to claim 2, characterized in that: In step two, the mass ratio of the soaking liquid to the raw materials is 12.5:1 for each decoction.

5. The preparation method according to claim 2, characterized in that: In step two, first bring the raw materials and all the soaking liquid to a boil over high heat, then reduce the heat to low and continue simmering for 30-60 minutes. After simmering, separate the liquid and dregs, add water to the dregs and simmer for the next time. Each simmering time is 30-60 minutes.

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