Application of low-temperature ripening black yam oligosaccharides in preparation of drugs for preventing or treating cognitive impairment

Abstract

The application belongs to the field of medicine and relates to a new use of low-temperature ripening black shilajit oligosaccharide, in particular to application of the low-temperature ripening black shilajit oligosaccharide in preparation of a medicine for preventing or treating cognitive impairment. The low-temperature ripening black shilajit oligosaccharide can significantly improve the defects of the recognition and spatial memory ability of mice, and also reduce the expression of acetylcholinesterase and the like in brain tissues to improve the cholinergic system. The low-temperature ripening black shilajit oligosaccharide can cause significant up-regulation of SOD, GSH, GPx and Keap-1 in brain tissues, and significant down-regulation of MDA and Nrf2, to improve the oxidative stress system. It is also observed that the low-temperature ripening black shilajit oligosaccharide intervention increases probiotics in the intestinal tract, such as Barnesiella, Staphylococcus, Clostridium_XlVb, and reduces opportunistic pathogens, such as Eisenbergiella and Intestinimonas. It can be seen that the low-temperature ripening black shilajit oligosaccharide has the advantages of significant curative effect and small toxic and side effects.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to a new use of low-temperature matured black ginseng oligosaccharide (BGOS), specifically the application of low-temperature matured black ginseng oligosaccharide (BGOS) in the preparation of drugs for the prevention or treatment of cognitive impairment. Background Technology

[0002] Cognitive impairment is a progressive decline in cognitive function caused by disease or brain injury, and the rate of decline is much higher than the normal aging process. It is mainly manifested as a decrease in cognitive and learning functions, memory loss or impairment, and decline in social skills while the individual is conscious. It can further develop into dementia (see Chen Shengdi). Deng Yulei “Current Status of Domestic Research on Cognitive Impairment”, Chinese Journal of Practical Internal Medicine, 2010, 10, 30(10): 875-878. Cognitive impairment is more common in the elderly. With my country’s rapid development towards an aging society, the incidence of cognitive impairment is showing a rapid upward trend, which seriously affects the quality of life of patients and imposes a burden on families and society.

[0003] Currently, clinically used drugs for treating cognitive impairment mainly include acetylcholinesterase inhibitors (donepezil (DON), istigmine, galantamine, etc.), NMDA receptor antagonists (memantine, etc.), and nimodipine. Most of these drugs are synthetic, have low bioavailability, and exhibit varying degrees of toxic side effects. Therefore, developing safe, effective, and economical drugs for treating cognitive impairment is imperative. Natural medicines are an important source of substances for the prevention and treatment of diseases, and the diversity of the structure and biological activity of natural products provides inspiration for new drug development.

[0004] Ginseng (Panax genseng CAMey) is the dried root of the ginseng plant (Panax ginseng), a member of the Araliaceae family. It is a renowned tonic medicine throughout history. It is neutral in nature, sweet, slightly bitter, and slightly warm in taste. It is used to greatly replenish vital energy, promote the production of body fluids and quench thirst, and calm the mind and improve intelligence.

[0005] Black ginseng (BG) is a processed form of ginseng. One traditional method of its production is based on the principle of "nine steaming and nine drying," involving soaking, washing, sorting, steaming, sun-drying, and baking. Black ginseng can also be produced through a low-temperature maturation process, where fresh ginseng is matured at 70℃ for 15 days. Another method involves fermentation, where fresh ginseng is repeatedly steamed and dried, then fermented with brewer's yeast. Alternatively, white ginseng can be soaked in garlic juice, then sterilized under high temperature and pressure, and subsequently dried in an oven to obtain a flavorful black ginseng. Current research has confirmed that processing techniques significantly improve the chemical composition and pharmacological activity of black ginseng, and compared to white and red ginseng products, black ginseng exhibits stronger pharmacological activities, such as inhibiting cancer cell growth, anti-tumor effects, antioxidant properties, and lowering blood sugar and cholesterol.

[0006] Low-temperature matured black ginseng oligosaccharide (BGOS) is a water-soluble low-temperature matured black ginseng oligosaccharide with a molecular weight of <3000 Da extracted from black ginseng. To date, there have been no reports on the use of low-temperature matured black ginseng oligosaccharide (BGOS) in the preparation of drugs for the prevention or treatment of cognitive impairment. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies, realize the development and utilization of natural products using modern drug research methods, and combine extensive pharmacodynamic experiments to provide the application of low-temperature matured black ginseng oligosaccharides (BGOS) in the preparation of drugs for the prevention or treatment of cognitive impairment.

[0008] To achieve the objectives of this invention, the following technical solution is adopted:

[0009] Application of low-temperature aged black ginseng oligosaccharides (BGOS) in the preparation of drugs for the prevention or treatment of cognitive impairment.

[0010] The drug comprises low-temperature aged black ginseng oligosaccharide (BGOS) and pharmaceutically acceptable excipients, wherein the low-temperature aged black ginseng oligosaccharide (BGOS) is the active ingredient in the cognitive impairment drug.

[0011] The drug improves the recognition and spatial memory abilities of patients with cognitive impairment, reduces the expression of acetylcholinesterase (AChE) in brain tissue, and thus improves the cholinesterase system.

[0012] The drug upregulates SOD (superoxide dismutase), GSH (glutathione), GPx (glutathione peroxidase), and Keap-1 in brain tissue, and downregulates MDA (malondialdehyde) and Nrf2, thereby improving the oxidative stress system.

[0013] The drug increases beneficial bacteria in the gut, such as Barnesiella, Staphylococcus, and Clostridium XlVb, and reduces opportunistic pathogens, such as Eisenbergiella and Intestinimonas.

[0014] The drug is in the form of an oral dosage form.

[0015] The oral dosage form is a capsule, tablet, granule, or oral liquid.

[0016] The pharmaceutically acceptable carrier refers to a conventional drug carrier in the field of pharmaceutical formulations, selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0017] The fillers of this invention include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binders include, but are not limited to, water, ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone, etc.; the disintegrants include, but are not limited to, starch effervescent mixtures, i.e., sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agents include, but are not limited to, polysaccharides such as farnesian gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan, etc.; and the solvents include, but are not limited to, water, ethanol, salt solutions, etc.

[0018] The low-temperature cooked black ginseng oligosaccharide (BGOS) is prepared by the following method: black ginseng obtained after low-temperature cooking is pulverized, the powder is extracted with warm water, ultrafiltered, graded and freeze-dried to obtain water-soluble low-temperature cooked black ginseng oligosaccharide with a molecular weight of <3000 Da.

[0019] Preferably, the low-temperature matured black ginseng oligosaccharide (BGOS) is prepared by the following method: dried black ginseng root that has been processed by low-temperature maturation is crushed, soaked in distilled water overnight at room temperature, extracted with warm water, filtered, and the residue is repeatedly extracted. The extracts are combined, centrifuged, and the supernatant is evaporated and freeze-dried to obtain low-temperature matured black ginseng crude oligosaccharide. Then, it is mixed with water and ultrafiltration fractionated using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The filtrate is concentrated and freeze-dried to obtain purified low-temperature matured black ginseng oligosaccharide (BGOS) with a molecular weight of <3000 Da.

[0020] Most preferably, the low-temperature matured black ginseng oligosaccharide (BGOS) is prepared by the following method: Dry black ginseng root obtained through low-temperature matured processing is pulverized, soaked overnight in distilled water at a ratio of 1g:10mL at room temperature, extracted at 70℃ for 3h, filtered through four layers of gauze, and the residue is extracted twice more. The three extracts are combined, centrifuged at 3500rpm for 15min, the precipitate is discarded, and the supernatant is then rotary evaporated and freeze-dried to obtain low-temperature matured black ginseng crude oligosaccharide. The low-temperature matured black ginseng crude oligosaccharide is then mixed with water at a mass ratio of 7:1000, and ultrafiltration fractionated using an ultrafiltration membrane with a molecular weight cutoff of 3000Da. The filtrate is monitored in real time using the phenol-sulfuric acid method until no color is detected. The filtrate is collected, concentrated by rotary evaporation, and freeze-dried to obtain purified low-temperature matured black ginseng oligosaccharide (BGOS) with a molecular weight <3000Da.

[0021] The present invention also provides a low-temperature matured black ginseng oligosaccharide for the prevention or treatment of cognitive impairment, wherein the low-temperature matured black ginseng oligosaccharide has a molecular weight of <3000 Da and the monosaccharide residues are mannose, rhamnose, glucose, glucuronic acid, galactose, arabinose and fructose.

[0022] In the pharmaceutical applications described above, the timing, frequency, and duration of administration of low-temperature cooked black ginseng oligosaccharides (BGOS) need to be determined based on the specific diagnostic results of the condition, which is within the technical scope of those skilled in the art.

[0023] The black ginseng used in this invention is prepared by low-temperature cooking (see Simeng Shao, et al. Distinctive carbohydrate profiles of black ginseng revealed by IM-MS combined with PMP labeling and multivariate data analysis. Current Research in Food Science. 2022(5):2243–2250).

[0024] To better understand the essence of the present invention, the following detailed embodiments section uses pharmacodynamic experiments and their results to further illustrate the new uses of low-temperature matured black ginseng oligosaccharides (BGOS) in the pharmaceutical field.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] This invention, leveraging my country's strengths in natural product research, is the first to verify the efficacy of low-temperature ripened black ginseng oligosaccharides (BGOS) in improving cognitive impairment. Low-temperature ripened black ginseng oligosaccharides (BGOS) significantly improved the deficits in recognition and spatial memory in mice, while also reducing the expression of acetylcholinesterase and other enzymes in brain tissue, thereby improving the cholinesterase system. Low-temperature ripened black ginseng oligosaccharides (BGOS) significantly upregulated SOD, GSH, GPx, and Keap-1 in brain tissue, and significantly downregulated MDA and Nrf2, thereby improving the oxidative stress system. Furthermore, intervention with low-temperature ripened black ginseng oligosaccharides (BGOS) increased beneficial bacteria in the gut, such as Barnesiella, Staphylococcus, and Clostridium XLVb, and reduced opportunistic pathogens, such as Eisenbergiella and Intestinimonas. Therefore, low-temperature ripened black ginseng oligosaccharides (BGOS) have the advantages of significant therapeutic effects and minimal toxic side effects. Attached Figure Description

[0027] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0028] Figure 1 To analyze the total ion chromatograms of (A) seven monosaccharide standards, (B) monosaccharide residues in low-temperature cooked black ginseng oligosaccharides, (C) eleven sugar standards, and (D) low-temperature cooked black ginseng oligosaccharides in negative ion mode using PMP derivatization HPLC, fructose, glucose, sucrose, maltose, isomaltulose, fructotriose, maltotriose, fructotetraose, isomalttriose, maltotetraose, and isomalttetraose were designated as peaks 1–11.

[0029] Figure 2 The effects of low-temperature-cooked black ginseng oligosaccharides on scopolamine (SCO)-induced memory and learning impairments were investigated. (A) Escape latency over 5 consecutive days; (B) Target quadrant time; (C) Number of platform entry attempts in the spatial exploration test; (D) Total swimming distance; (E) NORDI values ​​were statistically analyzed using ANOVA and Tukey's post-hoc test. (Compared with the control group, #p<0.05, ##p<0.01, ###p<0.001; compared with the SCO group, *p<0.05, **p<0.01, ***p<0.001; ns not significant).

[0030] Figure 3To investigate the effects of low-temperature matured black ginseng oligosaccharides on scopolamine-induced cholinergic nervous system. (A) Hippocampal ACh (acetylcholine) and AChE (acetylcholinesterase) activities; (B) Hippocampal ChAT and AChE mRNA levels; (C) Cortical ACh and AChE levels; (D) Cortical ChAT and AChE mRNA levels; (E) Comparison of ACh and AChE levels in the hippocampus and cortex of rats in low-dose groups of low-temperature matured black ginseng oligosaccharides; (F) Hippocampal and cortical cholinergic nervous system mRNA levels in the low-dose group of low-temperature matured black ginseng oligosaccharides; (G) Comparison of ACh and AChE levels in the hippocampus and cortex of rats in high-dose groups of low-temperature matured black ginseng oligosaccharides; (H) Hippocampal and cortical cholinergic nervous system mRNA levels in the high-dose group of low-temperature matured black ginseng oligosaccharides are expressed as mean ± SEM (n = 6). Statistical analysis included Tukey retrospective analysis and one-way ANOVA for A, B, C, and D, and Student's t-test for E and F. Using the control group as a reference, p < 0.01, p < 0.001, and p < 0.0001 were considered statistically significant. Compared with the control group, p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 were considered statistically significant. Any insignificant differences were expressed as ns. (H-BGOSL: hippocampal-low-temperature-ripened black ginseng oligosaccharide low-dose group; C-BGOSL: cortical-low-temperature-ripened black ginseng oligosaccharide low-dose group; H-BGOSH: hippocampal-low-temperature-ripened black ginseng oligosaccharide high-dose group; C-BGOSH: cortical-low-temperature-ripened black ginseng oligosaccharide high-dose group.)

[0031] Figure 4 To investigate the effects of low-dose, low-temperature-aged black ginseng oligosaccharides on oxidative stress. (A) Detection of SOD, MDA, and GSH activities in the hippocampus; (B) Detection of SOD mRNA and GPx mRNA levels in the hippocampus of rats in each group; (C) Measurement of cortical SOD, MDA, and GSH levels; (D) Detection of SOD mRNA and GPx mRNA expression levels in the cortex of rats in each group; (E) Comparison of antioxidant factors SOD, MDA, and GSH levels in the hippocampus and cortex of rats in the low-dose low-temperature-aged black ginseng oligosaccharide group; (F) Detection of SOD and GPx mRNA levels in the hippocampus and cortex of rats in the low-dose low-temperature-aged black ginseng oligosaccharide group. All histograms represent mean ± SEM (n = 6). Similar studies were conducted. Figure 3Labeling. Low-dose, low-temperature-cooked black ginseng oligosaccharides activate the Keap-1 / Nrf2 pathway to counteract scopolamine-induced oxidative stress; (G) Representative immunoblots of Keap-1 and Nrf2. Control, SCO, and low-temperature-cooked black ginseng oligosaccharide samples were run on the same gel. (H) Keap-1 / Gapdh and Nrf2 / Gapdh (n=3) ratios. Bar charts are presented as ±SEM; significance was analyzed by Tukey's test using ANOVA; *P<0.05, **P<0.01.

[0032] Figure 5 To assess the bacterial taxa identified in the CON, SCO, and low-temperature cooked black ginseng oligosaccharide groups using multiple diversity indices, the Alpha diversity index was used to evaluate bacterial richness and intragroup diversity. (a) Sparse curves; (B) Species, Chao1, ACE, Shannon, and Simpson indices showing significant intergroup differences under five test conditions (p<0.05); (C) PLS-DA score plots showing the bacterial communities.

[0033] Figure 6 Enrichment and predictive metabolism of bacterial taxa in fecal samples. (A) Venn plot shows α-diversity at the OTU level; (B) Relative abundance of bacterial taxa was calculated using the LEfSe tool, with each bar representing the log 10 effect size (LDAscore) of a specific taxa. Higher LDA scores indicate more significant effect sizes, represented by longer bars; (C) A clade plot was generated by LEfSe analysis, displaying taxa rank in a circular radial pattern. The outermost circle represents phylum, while the innermost circle represents species, each colored based on its most abundant taxa. Red represents the control group, green represents the SCO group, and dark blue represents the black ginseng oligosaccharide group. The node size on the clade plot reflects the relative abundance of species. Furthermore, the letters 'p, c, o, f, g, s' represent phylum, class, order, family, genus, and species, respectively; (D) A heatmap shows enriched KEGG pathways, with the top 30 pathways representing intergroup abundance differences. The figure shows a color gradient from light to dark to represent relative abundance from low to high.

[0034] Figure 7 (A) shows the mean relative abundance of bacterial communities at the phylum level (n=6). Statistical analysis was performed using one-way ANOVA and Tukey's posthoc analysis. Significance levels were expressed as *p<0.05 and **p<0.01, respectively. (B) shows the analysis of fecal microbiota at the genus level for the three population groups through bacterial taxonomy. A heatmap analysis was performed, with the color gradient from blue to red representing the relative abundance of species; blue represents lower abundance, and red represents higher abundance. Detailed Implementation

[0035] The embodiments of the present invention are described in detail below. The embodiments are provided to better illustrate the content of the present invention and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0036] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0037] Example 1: Preparation of low-temperature aged black ginseng oligosaccharides (BGOS)

[0038] The dried black ginseng root, processed by low-temperature aging, was pulverized and soaked overnight in distilled water at a ratio of 1g:10mL at room temperature. It was then extracted at 70℃ for 3 hours, filtered through four layers of gauze, and the residue was extracted twice more. The three extracts were combined, centrifuged at 3500rpm for 15 minutes, and the precipitate was discarded. The supernatant was then rotary evaporated and freeze-dried to obtain low-temperature aged black ginseng crude oligosaccharide. The low-temperature aged black ginseng crude oligosaccharide was then mixed with water at a mass ratio of 7:1000 and ultrafiltration fractionated using an ultrafiltration membrane with a molecular weight cutoff of 3000Da. The filtrate was monitored in real time using the phenol-sulfuric acid method until it was colorless. The filtrate was collected, concentrated by rotary evaporation, and freeze-dried to obtain purified low-temperature aged black ginseng oligosaccharide (BGOS) with a molecular weight <3000Da.

[0039] The black ginseng used was prepared by low-temperature cooking (see Simeng Shao, et al. Distinctive carbohydrate profiles of black ginseng revealed by IM-MS combined with PMP labeling and multivariate data analysis. Current Research in Food Science. 2022(5):2243–2250).

[0040] Example 2: Characterization of low-temperature aged black ginseng oligosaccharides (BGOS)

[0041] 2.1 HPLC analysis of monosaccharide composition

[0042] 2.1.1 Sample Derivatization: To prepare samples for HPLC analysis, seven monosaccharide standards and the low-temperature matured black ginseng oligosaccharide (BGOS) sample prepared in Example 1 were dissolved in NH3·H2O, and 0.5 MPa PMP methanol solution was added. The mixture was reacted at 70°C for 1 h. After the reaction, the mixture was centrifuged at 10,000 rpm for 10 min to remove the precipitate. The supernatant was then dried under a nitrogen stream to remove excess ammonia. The residue was dissolved in ultrapure water and extracted five times with chloroform to remove excess PMP. Before HPLC analysis, the aqueous layer was filtered through a 0.45 μm filter membrane.

[0043] 2.1.2 HPLC chromatographic conditions: A Dikma Di-amonsil Plus C18 column (4.6 mm) was used.

[0044] PMP-labeled monosaccharides were analyzed using a Shimadzu LC-15C HPLC system (Shimadzu Corporation, Tokyo, Japan) with an ID size of 250 mm and a particle size of 5 μm. The mobile phase consisted of 82% 0.05 mM phosphate buffer (pH 6.86, A) and 82% acetonitrile (B). Elution was performed at a flow rate of 1.0 mL / min at room temperature, and UV detection was performed at 250 nm.

[0045] 2.2. UPLC-MS Conditions

[0046] UPLC analysis was performed using an ExionLCAD liquid chromatography system (AB SCIEX, USA), which includes a binary pump, autosampler, solvent degasser, and column oven. Analytes were separated using a BEHAmide column (150 mm × 2.1 mm i.d., 1.7 μm, Waters, Ireland) at a flow rate of 0.25 mL / min and a column temperature of 40 °C. A gradient solvent system was used with solvent A (water containing 0.02% NH3) and solvent B (acetonitrile). Initially, 85% B was used at injection, then linearly increased to 75% B over 5 minutes, then linearly increased to 65% B over 5 minutes, and finally gradient to 70% B over 5 minutes.

[0047] Using AB SCIEX The 6500 mass spectrometer, with an electrospray ionization (ESI) source, was used in multiple reaction monitoring (MRM) mode. ESI source parameters—source temperature (350°C), curtain gas (30 psi), gas 1 (50 psi), and gas 2 (50 psi)—were all pre-optimized and preset. Two unique MRM transitions for each analyte were monitored using Analyst 1.6.2 software for accurate identification. In negative ion mode, the ion spray voltage was set to -4,500 V, and the mass spectrometer parameters were optimized to maximize sensitivity for all analytes.

[0048] 2.3 Experimental Results

[0049] The low-temperature aged black ginseng oligosaccharides (BGOS) prepared in Example 1 were analyzed by HPLC and UPLC-MS / MS. The retention time of BGOS in the test sample was compared with that of the reference standard (e.g., ...). Figure 1 A and Figure 1 Based on the retention time shown in B), the monosaccharide residues in the low-temperature cooked black ginseng oligosaccharides (BGOS) were identified as mannose, rhamnose, glucose, glucuronic acid, galactose, arabinose, and fructose. Direct comparison... Figure 1 C and Figure 1 As can be seen from Table 1, UPLC-MS / MS in MRM mode detected eight carbohydrates in low-temperature cooked black ginseng oligosaccharides (BGOS), including glucose, fructose, sucrose, maltose, isomaltulose, maltotriose, nitrosose, and maltotetraose. As shown in Table 1, low-temperature cooked black ginseng oligosaccharides (BGOS) had higher contents of fructose, maltose, and glucose, while the contents of other components were lower.

[0050] Table 1: Parameters and Quantitative Results of BGOS under MRM Mode

[0051]

[0052] Example 3: Pharmacodynamic study of low-temperature cooked black ginseng oligosaccharides (BGOS) in the prevention or treatment of cognitive impairment

[0053] 3.1 Study on the effects of low-temperature aged black ginseng oligosaccharides (BGOS) on scopolamine (SCO)-induced memory and learning impairments

[0054] 3.1.1 Animals

[0055] Thirty wild-type C57BL / 6N mice, half male and half female, weighing 20-22g and 6-7 weeks old, were provided by Liaoning Changsheng Biotechnology Co., Ltd. (Shenyang, China). The animals were housed at the Experimental Animal Center of Changchun University of Traditional Chinese Medicine, with strict control over temperature (25±2℃), relative humidity (55-60%), and a 12-hour light-dark cycle. Prior to the experiment, the animals were acclimatized for one week in a local animal facility. All experimental procedures were approved by the Experimental Animal Management Committee of Changchun University of Traditional Chinese Medicine.

[0056] 3.1.2 Drugs and Experimental Protocol

[0057] To investigate the effects of low-temperature ripened black ginseng oligosaccharide (BGOS) on learning and memory in mice, 30 mice were randomly divided into 5 treatment groups (n=6), as shown in Table 2. Different pretreatments with 40 mg / kg and 80 mg / kg of low-temperature ripened black ginseng oligosaccharide (BGOS) were performed for 5 consecutive days. Starting from day 6, the first treatment was the same as the pretreatment. To establish the SCO-induced mouse model, 15 minutes after the first treatment, mice were intraperitoneally injected with 3 mg / kg of SCO for 7 consecutive days. Behavioral tests were performed 30 minutes after the second injection. DON and low-temperature ripened black ginseng oligosaccharide (BGOS) were administered orally, followed by intraperitoneal injection of SCO.

[0058] Table 2: Experimental Groups and Drug Treatments

[0059]

[0060] 3.1.3 Behavioral Testing

[0061] The behavioral experiments were conducted from 8:00 AM to 11:00 AM.

[0062] 3.1.3.1 Morris Water Maze

[0063] Mice underwent the Morris water maze test to assess spatial learning and working memory. The maze consisted of a 120 cm diameter plastic pool divided into four quadrants, with a circular platform (10 cm in diameter) 1 cm below the water surface. The water temperature was maintained at 22 ± 2 °C, and four types of visual-spatial cues (circle, square, triangle, and pentagram) were placed on the pool walls. During the acquisition phase, mice were trained four times a day for five consecutive days, with the platform fixed in one location. Each 90-second test ended only when the mouse found the platform; unsuccessful subjects were guided to the platform by the experimenter and allowed to stay there for 10 seconds. After the final training test, mice underwent a 60-second exploration test without the platform. Swimming activity was recorded using a camera-based computer monitoring system, and all parameters were analyzed using Etho Vision XT software.

[0064] 3.1.3.2 New Object Recognition Experiment

[0065] Mice underwent a 3-day novel object recognition test. On day 1, mice were allowed to explore their environment for 10 minutes during the adaptation period. On day 2, two identical toys (4cm x 4cm) were placed in a box, and the exploration time for each object was recorded. On day 3, a novel object replaced one of the toys, and the exploration duration for each object was measured. The Discriminant Index (DI) was used to assess recognition memory, calculated as (novel object - familiar object) / (novel object + familiar object) × 100%, where DI represents the number of discriminations.

[0066] 3.1.3.3 Experimental Results

[0067] We performed the Morris water maze and Novel Object Recognition (NOR) tests to assess whether low-temperature cured black ginseng oligosaccharide (BGOS) treatment could prevent scopolamine-induced cognitive impairment in mice. Scopolamine administration resulted in prolonged latency in all behavioral tests, indicating significant impairment in learning and memory. During the 5-day training period, escape latency (swimming time for mice to find the platform) gradually decreased (e.g., Figure 2 (As shown in A). In the space exploration experiment, the platform was removed, and the mice were placed in the contralateral quadrant and allowed to swim freely in the contralateral quadrant for 90 seconds. Mice treated with scopolamine showed impaired memory function, manifested as quadrant time and target quadrant crossing (e.g., Figure 2 B and Figure 2 The time in the target quadrant was significantly reduced (as shown in C). Mice treated with low-temperature cooked black ginseng oligosaccharides (40, 80 mg / kg) or DON (1 mg / kg) were significantly shorter than those in the SCO-treated group (as shown in C). Figure 2 As shown in Figure B, p < 0.01) the length. However, compared with the SCO treatment group, the low-temperature cooked black ginseng oligosaccharide-(40, 80 mg / kg) treatment significantly increased (e.g.) Figure 2 As shown in C (p < 0.05), the total swimming distance was significantly reduced in the DON and low- and high-dose low-temperature matured black ginseng oligosaccharide treatment groups compared to the model group (e.g., as shown in C). Figure 2 As shown in Figure D), this indicates that both DON and low-temperature ripened black ginseng oligosaccharide (BGOS) can improve spatial memory. SCO (p<0.01) significantly reduced the object recognition index, but both doses of low-temperature ripened black ginseng oligosaccharide (BGOS) increased the RI, suggesting their potential to reduce the forgetting effect of SCO (e.g., as shown in Figure D). Figure 2 As shown in E.

[0068] Low-temperature cooked black ginseng oligosaccharide low-dose group and high-dose group (e.g.) Figure 2 B~ Figure 2 The differences (as shown in E) were not statistically significant. These results indicate that low-temperature cooked black ginseng oligosaccharides (BGOS) can alleviate SCO-induced memory impairment, but did not show a dose-dependent effect.

[0069] 3.2 Study on the effects of low-temperature aged black ginseng oligosaccharides (BGOS) on scopolamine-induced cholinergic nervous system

[0070] 3.2.1 Brain tissue preparation

[0071] After the aforementioned behavioral tests were completed, the mice were euthanized, and their brains were quickly removed by decapitation. Hippocampal and cortical tissues were harvested from both cerebral hemispheres, rapidly frozen, and stored at -80°C for later analysis.

[0072] 3.2.2 Enzyme-linked immunosorbent assay (ELISA)

[0073] For biochemical analysis, tissue samples were homogenized with ice-cold physiological saline (0.9%, v / w) at a ratio of 1:9 (w / v). The homogenate was centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant was collected. The supernatant was diluted with an appropriate buffer before determining relevant biochemical parameters. ACh, AChE, SOD, GSH, and MDA levels were determined using an ELISA kit (Hubei, China, mouse-specific) provided by Huangshi Yansheng Biotechnology Co., Ltd., according to the manufacturer's instructions.

[0074] 3.2.3 qRT-PCR analysis

[0075] RNA was isolated from the hippocampus and cerebral cortex using Trizol reagent (Tiangen Biotech, Beijing, China) and quantified using a One 5225 UV-Vis nanodroplet spectrophotometer (Termo Fisher Scientific Inc., Walsam, MA, USA). cDNA was synthesized using a commercial reverse transcription kit (Tiangen Biotech, Beijing, China). qRT-PCR analysis was performed using the SYBR Green system from Bio-Rad Laboratories, USA. qRT-PCR primers were purchased from Sangon Biotech (Shanghai, China). Primer sequences are as follows:

[0076] ChAT: (F:5'-AGGGGTGATCTGTTCASCTCAG-3'

[0077] R:5'-TCTTGTGGCCTGTCAATCATA-3');

[0078] AChE(F:5'-AGAAAATATTGCAGCCTTTG-3'

[0079] R:5'-CTGCAGGTCTTGAAAATCTC-3');

[0080] SOD(F:5'-AACCAGTTGTGTTGTCAGGAC-3'

[0081] R:5'-CCACCATGTTTCTTAGAGTGAGG-3');

[0082] GPx(F:5'-CCACCGTGTATGCCTTCTCC-3'

[0083] R:5'-AGAGAGACGCGACATTCTCAAT-3');

[0084] GAPDH(F:5'-GCCAAGGT CATCCATGACAAC-3'

[0085] R:5'-AGTGTAGCCCAGGATGCCC-3').

[0086] GAPDH is a housekeeping gene. The qRT-PCR program consists of 40 cycles of denaturation at 95°C for 15 minutes, 95°C for 10 seconds, and 60°C for 32 seconds.

[0087] 3.2.4 Western blot analysis

[0088] To detect the expression levels of Keap-1 and Nrf2 proteins in the cortex, cortical tissue was homogenized in RIPA lysis buffer (1:5, w / v) containing PMSF and protease inhibitor cocktail (100:1:1, v / v / v) at ice temperature. The mixture was then centrifuged at 12,000 rpm for 15 minutes at 4°C. Protein concentrations were determined using the Bradford method with BSA as a standard. 10% SDS-PAGE gels were prepared using an SDS-PAGE gel kit, with 25 μg sample loaded per well for subsequent electrophoresis and transfer. Primary antibody Keap-1 (1:2000), Nrf2 (1:1000), and internal control GAPDH (1:1000) were diluted with primary antibody dilution buffer. The transferred samples were blocked in TBST with 5% (w / v) skim milk at room temperature for 1 hour, then incubated with primary antibody overnight at 4°C. After washing the membrane five times with TBST, horseradish peroxidase-conjugated secondary antibody was added and incubated at room temperature for 1 hour. Protein bands were treated with ECL solution, and each protein band was exposed using a ChemiDoc MP imaging system (BIO-RAD, USA). Quantification was performed using ImageJ software.

[0089] 3.2.5 Experimental Results

[0090] We examined the levels of various biochemical factors associated with the cholinergic system to better understand the potential mechanism by which low-temperature-ripened black ginseng oligosaccharides (BGOS) improve SCO-induced memory impairment. We examined ACh levels in the hippocampus and cortex and found that compared with the control group ( Figure 3 A, Figure 3 Compared to the left side panel (B), SCO significantly reduced ACh levels in both tissues. Pre-administration of DON (1 mg / kg) and low-temperature-ripened black ginseng oligosaccharides (40 and 80 mg / kg) significantly increased ACh levels in the hippocampus and cortex. There was no significant difference between the high-dose and low-dose groups of low-temperature-ripened black ginseng oligosaccharides. We compared the ACh content in the two tissues at the same dosage and found that the ACh content in the hippocampus was significantly higher than that in the cortex (e.g., left side panel). Figure 3 C Figure 3 (As shown in the left figure). Figure 3 A and Figure 3 Figure B (right) shows that AChE expression was significantly increased in the scopolamine-treated group compared to the control group. However, in both tissues, AChE levels were significantly decreased in the low-temperature-ripened black ginseng oligosaccharide-treated group compared to the scopolamine-treated group. Figure 3 C and Figure 3 As shown in Figure D (right), there were no significant differences in AChE levels between the high-dose and low-dose low-temperature cooked black ginseng oligosaccharide groups, as well as between the two tissues.

[0091] To validate the ELISA results, we used qRT-PCR to detect the mRNA expression levels of ChAT and AChE in the cortex and hippocampus. Figure 3 (Left figure) shows that ChAT activity was significantly decreased in the scopolamine-treated group, while ChAT activity in the hippocampus was significantly increased in the low-dose group of low-temperature-ripened black ginseng oligosaccharides; other comparative results showed no significant differences. AChE mRNA (e.g.) Figure 3 E, Figure 3 The results of the assay (shown in the right-hand figure) were consistent with the ELISA results described above. Interestingly, at any dose, the hippocampal tissue ( Figure 3 G and Figure 3 In the right-hand panel (H), both ChAT activity and AChE expression were high. In summary, we found that low-temperature-ripened black ginseng oligosaccharides (BGOS) have a neuroprotective effect on scopolamine-induced cognitive impairment in mice, and this effect is tissue-dependent and not dose-dependent.

[0092] 3.3 Study on the antioxidant effect of low-dose, low-temperature matured black ginseng oligosaccharides (BGOS) in brain tissue of scopolamine-induced cognitive impairment mice.

[0093] Studies have reported that SCO induces oxidative stress through multiple mechanisms, including reducing T-AOC, SOD, GSH-Px, and CAT levels, and increasing MDA levels. Furthermore, MDA and SOD are closely associated with AD, therefore this study selected MDA and SOD. Figure 4 A and Figure 4 As shown in Figure B, compared with the control group, the levels of SOD and GSH in the hippocampus and cortex of the SCO group were significantly reduced. Compared with mice treated with SCO alone, administration of low-dose, low-temperature-ripened black ginseng oligosaccharide led to increased levels of SOD and GSH in the hippocampus and cortex. Furthermore, compared with the SCO group, low-dose, low-temperature-ripened black ginseng oligosaccharide significantly reduced MDA content in both brain regions. Figure 4 As shown in Figure C, there was no statistically significant difference in the levels of SOD, MDA, and GSH in the hippocampus and cortical tissues of rats in the low-dose group of low-temperature cooked black ginseng oligosaccharides compared with the control group.

[0094] To investigate whether low-temperature-ripened black ginseng oligosaccharides (BGOS) alleviate scopolamine-induced damage through their antioxidant effects, we used qRT-PCR to assess the transcriptional levels of antioxidant enzymes, such as SOD and GPx, in the hippocampus and cortex. Figure 4 D and Figure 4 As shown in E, scopolamine treatment not only downregulated SOD levels but also GPx levels. As expected, DON and the low-temperature matured black ginseng oligosaccharide group (such as...) Figure 4 D and Figure 4 The expression levels of SOD and GPx were significantly increased in the hippocampus and cortex (as shown in E). Similarly, in the low-dose, low-temperature-ripened black ginseng oligosaccharide group, there was no difference in the expression levels of SOD and GPx between the hippocampus and cortex (as shown in E). Figure 4 (As shown in F). Since no tissue-dependent differences were observed as described above, we next explored whether low-temperature-cooked black ginseng oligosaccharides (BGOS) could activate the Keap-1 / Nrf2 pathway to exert antioxidant effects in cortical tissue. According to... Figure 4 G and Figure 4 H showed that Nrf2 levels were significantly higher in the SCO group than in the control group, but this increase was reversed by administration of low-temperature matured black ginseng oligosaccharide (BGOS). Compared with the control group, Keap-1 protein expression was significantly decreased in the SCO group, but Keap-1 levels were increased by administration of low-temperature matured black ginseng oligosaccharide (BGOS) compared to the SCO group. Our collected data support our hypothesis that low-temperature matured black ginseng oligosaccharide (BGOS) exerts its antioxidant activity by activating the Keap-1 / Nrf2 pathway.

[0095] 3.4 Study on changes in gut microbiota in mice with scopolamine-induced memory impairment treated with low-dose, low-temperature-aged black ginseng oligosaccharides (BGOS).

[0096] 3.4.1 Microbial Community Analysis

[0097] Fecal samples collected on the last day of the aforementioned behavioral tests were cryopreserved at -80°C. A total of 30 samples were subjected to 16S rRNA gene amplicon sequencing analysis, with 6 samples collected from each group. DNA was extracted from mouse feces using the Top Taq DNA Polymerase Kit (Transgenic, China). The V3-V4 variable region of the bacterial 16S rDNA gene was amplified using primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-GACTACHVGGGTATCTAATCC-3'), and then sequenced on an Illumina Mi Seq clean bench sequencer (Illumina, USA). The samples were sequenced using the Illumina Hi Seq platform, and operational taxonomic units (OTUs) were identified with 97% similarity. Taxonomic classification of the typical sequences of each OTU was performed using the Ribosome Database Project (RDP) classifier. Taxonomic differences among dominant species were analyzed using R software, and all indices in the samples were calculated using QIIME software (version 2.0).

[0098] 3.4.2 Experimental Results

[0099] As previously mentioned, the gut-brain axis plays a crucial role in regulating brain activities such as memory and learning. We evaluated the effects of administration of low-temperature cooked black ginseng oligosaccharides (BGOS) on the gut microbiota of mice using 16S rRNA gene sequencing for fecal microbiota analysis. We assessed the composition of the gut microbiome from abundance, α-diversity (abundance and richness of OTUs within each participant), and β-diversity (similarity or difference in microbiome composition among participants). When sufficient sequencing data were available, Figure 5 The dilution curves plotted in Figure A reached a relatively stable state, indicating that the diversity index increased slowly as the sequencing platform approached saturation. Alpha diversity, based on the Chao1 index, Shannon index, observed index, and ACE index, showed significant differences among groups (e.g., ...). Figure 5 (As shown in B). β-diversity analysis using the PLS-DA method showed that the gut microbiota composition of the three groups of mice exhibited significant clustering (e.g., ...). Figure 5 (as shown in C).

[0100] exist Figure 6In group A, we observed a total of 362 OTUs across three groups, with the low-temperature cooked black ginseng oligosaccharide group containing an additional 588 unique OTUs. To identify gut microbiota biomarkers across different groups, we used linear discriminant analysis (LDA) effect size (LEfSe) to analyze the data. Significant biomarkers were identified through histograms of LDA scores (based on LDA scores > 4) and clamor analysis, such as… Figure 6 B and Figure 6 As shown in C, the two OUT values ​​of g-Barnesiella.s-uncultured-Bacteroidales-bacterium and g-Bameslella showed significant differences in the low-temperature matured black ginseng oligosaccharide group (e.g., ...). Figure 6 (As shown in B). Important microbial groups in the oligosaccharide group of low-temperature cooked black ginseng are represented by blue nodes (e.g., Figure 6 (As shown in C). Using the PICRUSt2 method, we analyzed the potential functional changes in the mouse gut microbiome by exploring the KEGG database. We... Figure 6 Analysis of D revealed a significant enrichment of metabolic pathways and functions in the gut microbiome from the SCO and low-temperature matured black ginseng oligosaccharide groups, such as meiosis-yeast, toluene degradation, geraniol degradation, lysine biosynthesis, proteasome, lipopolysaccharide biosynthesis, pentose phosphate pathway, and folic acid biosynthesis.

[0101] We analyzed the relative abundance of taxa at the phylum and genus levels. The results showed that, compared to the control group, the abundance of Bacteroidetes was decreased and the abundance of Firmicutes was increased in the SCO group, leading to an increased Firmicutes / Bacteroidetes ratio (e.g., ...). Figure 7 As shown in A). As expected, the low-temperature cooked black ginseng oligosaccharide group had lower Firmicutes abundance and higher Bacteroidetes abundance, resulting in an increased Bacteroidetes / Firmite ratio (e.g., Figure 7 As shown in Figure A). Diversity of gut microbiota at the genus level can be observed on the community heatmap, such as... Figure 7As shown in B, the genus-level taxonomic composition showed that the dominant genera were Barnesiella, Staphylococcus, Clostridium XlVb, Eisenbergiella, Corynebacterium, Anaerofustis, Enterococcus, Parabacteroides, Vampirovibrio, Anaerorotruncus, Ruminococcus, Aerococcus, Intestinimonas, and Alloprevotella. Compared with the SCO group (P<0.05), the relative abundance of Eisenbergiella and Intestinimonas was significantly decreased in the low-temperature matured black ginseng oligosaccharide treatment group, while the relative abundance of Barnesiella, Staphylococcus, Clostridium XlVb, Anaerofustis, Corynebacterium, Parabacteroides, Anaerorotruncus, and Alloprevotella was significantly increased.

[0102] 3.5 Data Analysis

[0103] Statistical results are expressed as mean ± SEM and visualized using box plots and whisker plots. Statistical analysis was performed using one-way ANOVA, with unpaired Student's t-tests or Tukey's multiple comparison tests used where necessary. Each experiment was performed at least three times, and analysis was conducted using GraphPadPrism 9.1 software (Graphpad, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant. Tukey's HSD test was used to compare the relative abundance of OTUs between the two groups.

[0104] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. The application of low-temperature cured black ginseng oligosaccharides in the preparation of drugs for the prevention or treatment of cognitive impairment, characterized in that, The monosaccharide residues of the low-temperature cooked black ginseng oligosaccharide are mannose, rhamnose, glucose, glucuronic acid, galactose, arabinose, and fructose. The low-temperature cooked black ginseng oligosaccharide also contains glucose, fructose, sucrose, maltose, isomaltulose, maltotriose, nisose, and maltotetraose. The low-temperature matured black ginseng oligosaccharide is prepared by the following method: Dry black ginseng root obtained through low-temperature matured processing is pulverized, soaked overnight in distilled water at a ratio of 1 g: 10 mL at room temperature, extracted at 70°C for 3 h, filtered through four layers of gauze, and the residue is extracted twice more. The three extracts are combined, centrifuged at 3500 rpm for 15 min, and the precipitate is discarded. The supernatant is then rotary evaporated and freeze-dried to obtain low-temperature matured black ginseng crude oligosaccharide. The low-temperature matured black ginseng crude oligosaccharide is then mixed with water at a mass ratio of 7:1000, and ultrafiltration fractionated using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. The filtrate is monitored in real time using the phenol-sulfuric acid method until it is colorless. The filtrate is collected, concentrated by rotary evaporation, and freeze-dried to obtain purified low-temperature matured black ginseng oligosaccharide with a molecular weight <3000 Da. The low-temperature matured processing refers to maturing fresh ginseng at 70°C for 15 days.

2. The application according to claim 1, characterized in that, The drug comprises low-temperature matured black ginseng oligosaccharides and pharmaceutically acceptable excipients, wherein the low-temperature matured black ginseng oligosaccharides are the active ingredient in the cognitive impairment drug.

3. The application according to claim 2, characterized in that, The drug improves the recognition and spatial memory abilities of patients with cognitive impairment, reduces the expression of acetylcholinesterase in brain tissue, and thus improves the cholinesterase system.

4. The application according to claim 2, characterized in that, The drug upregulates SOD, GSH, GPx and Keap-1 in brain tissue, and downregulates MDA and Nrf2, thereby improving the oxidative stress system.

5. The application according to claim 2, characterized in that, The drug increases beneficial bacteria in the gut. Barnesiella , Staphylococcus , Clostridium _XlVb, and reduced opportunistic pathogens. Eisenbergiella and Intestinimonas .

6. The application according to claim 2, characterized in that, The drug is in oral dosage form.

7. The application according to claim 6, characterized in that, The oral dosage form is a capsule, tablet, granule, or oral liquid.

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