Use of dendrobium polysaccharide in preparation of medicine for preventing and / or treating alcohol-induced neuroinflammation and a dendrobium polysaccharide liquor

Abstract

The present application provides the application of dendrobium polysaccharide in the preparation of medicine for preventing and / or treating alcohol-induced neuroinflammation and a dendrobium polysaccharide liqueur, belonging to the field of biological medicine. The present application prepares dendrobium polysaccharide into a liqueur, and applies it to a mouse model exposed to alcohol for a long time, and finds that the dendrobium polysaccharide liqueur can improve the cognitive dysfunction, the decrease of motor coordination ability and the anxiety-like behavior and other neurofunctional abnormalities caused by long-term drinking; can intervene in the neuroinflammation of long-term drinking mice. It is shown that the dendrobium polysaccharide has a significant intervention effect on the neurodegenerative diseases caused by excessive drinking of alcohol, including Alzheimer's disease, Parkinson's disease and the like. It provides a new way for the development and utilization of dendrobium resources, and helps to promote the modernization of traditional Chinese medicine and the development of the health industry.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and particularly relates to the application of Dendrobium polysaccharide in the preparation of drugs for the prevention and / or treatment of alcohol-induced neuroinflammation, and a Dendrobium polysaccharide liqueur. Background Technology

[0002] Chronic alcohol abuse is a global health problem. Alcohol has a variety of damaging effects on the nervous system, among which neuroinflammation is one of the important pathological mechanisms. Alcohol-induced neuroinflammation can lead to a series of neurological diseases, such as alcoholic encephalopathy and neurodegenerative diseases, including neuronal damage, neurotransmitter imbalance, and cognitive impairment.

[0003] Liqueurs are alcoholic beverages made by blending fermented wine, distilled spirits, or edible alcohol as a base with edible or medicinal ingredients, possessing unique flavors and certain health benefits. Dendrobium is a traditional and precious Chinese medicinal herb, rich in various bioactive components, among which Dendrobium polysaccharides exhibit a variety of pharmacological activities, including antioxidant, antitumor, and immunomodulatory effects. However, there are no reports on the intervention effect of Dendrobium polysaccharide liqueurs on neuroinflammation caused by long-term alcohol exposure.

[0004] Currently, there are limited treatment options available for alcoholic neuritis in clinical practice, and these options often have significant side effects. Therefore, developing safe and effective interventions is of great importance. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of Dendrobium polysaccharides in the preparation of drugs for the prevention and / or treatment of alcohol-induced neuroinflammation, and a Dendrobium polysaccharide liqueur. This invention, by preparing Dendrobium polysaccharides into a liqueur and applying it to a mouse model of long-term alcohol exposure, found that the Dendrobium polysaccharide liqueur has a significant intervention effect on neuroinflammation. Furthermore, the Dendrobium polysaccharide liqueur has advantages such as convenient administration, good taste, and high safety, making it easily acceptable to consumers and possessing broad market application prospects.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the use of Dendrobium polysaccharide in the preparation of medicaments for the prevention and / or treatment of alcohol-induced neuroinflammation, wherein the alcohol-induced neuroinflammation is an alcoholic neurological disease caused by excessive drinking.

[0008] Preferably, the alcohol-induced neurological diseases caused by excessive drinking include alcoholic encephalopathy and neurodegenerative diseases caused by excessive drinking.

[0009] Preferably, the drug is in the form of tablets, powders, granules, capsules, pills, liquids, tinctures, or syrups.

[0010] This invention provides a Dendrobium polysaccharide liqueur for the prevention and / or treatment of alcohol-induced neuroinflammation, comprising a base liquor with an alcohol concentration of 30-50% and Dendrobium polysaccharides; wherein the mass-to-volume ratio of Dendrobium polysaccharides to base liquor is 1-10 g: 1 L.

[0011] As a preferred option, 1L of base spirit also includes 50-200g of flavoring agent.

[0012] Preferably, the flavoring agent is honey.

[0013] The present invention also provides a method for preparing the Dendrobium polysaccharide liqueur, wherein Dendrobium polysaccharide is dissolved in base liquor, homogenized, and aged to obtain Dendrobium polysaccharide liqueur.

[0014] Preferably, the homogenization pressure is 20-30 MPa, the homogenization is performed 2-3 times, and the homogenization time is 3-5 min / time.

[0015] Preferably, the aging temperature is 15~25℃, the relative humidity is 60%~75%, and the aging time is 0.5~1.5 months.

[0016] Preferably, before homogenization, a flavoring step is added, and after aging, a filtration operation is also included.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention is the first to discover that Dendrobium polysaccharides and Dendrobium polysaccharide liqueur have a significant intervention effect on neuroinflammation caused by long-term alcohol exposure. Specifically, Dendrobium polysaccharide liqueur can improve neurological dysfunctions such as cognitive impairment, decreased motor coordination, and anxiety-like behavior caused by long-term alcohol consumption; it can intervene in neuroinflammation in mice with long-term alcohol consumption by altering its neurobehavioral, inflammatory factors, neurotransmitters, hippocampal structure, and oxidative stress physiological and biochemical indicators. This indicates that Dendrobium polysaccharides have a significant intervention effect on neurodegenerative diseases caused by excessive alcohol consumption, including Alzheimer's disease and Parkinson's disease. Compared with traditional drug treatments, Dendrobium polysaccharide liqueur has the advantages of convenient administration, good taste, and high safety, making it easily accepted by consumers and having broad market application prospects.

[0019] (2) This invention provides a new way for the development and utilization of Dendrobium resources, which helps to promote the modernization of traditional Chinese medicine and the development of the big health industry. Attached Figure Description

[0020] Figure 1Figure 1 shows the behavioral effects of DOP on mice with long-term alcohol consumption; where a represents the Y-maze test results; b represents the mine test results; c represents the fatigue rotundus test results; BLK group is the blank control group; E_ALC group is the model group; LU group is the positive control group; and DOP is the Dendrobium polysaccharide liqueur group.

[0021] Figure 2 The figure shows the effects of DOP on inflammatory factors in the brain tissue of mice with long-term alcohol consumption; where a represents the effect on IL-6 levels; b represents the effect on IL-1β levels; and c represents the effect on TNF-α levels.

[0022] Figure 3 The figure shows the effects of DOP on neurotransmitters in the hippocampus of mice with long-term alcohol consumption; where a represents the effect on serotonin (5-HT) levels; b represents the effect on dopamine (DA) levels; c represents the effect on γ-aminobutyric acid (GABA) levels; and d represents the effect on glutamate (Glu) levels.

[0023] Figure 4 The figure shows the effect of DOP on the CA3 structure in the hippocampus of mice with long-term alcohol consumption; where a is the BLK group; b is the E_ALC group; c is the LU group; and d is the DOP group.

[0024] Figure 5 The figure shows the effects of DOP on oxidative stress indicators in the brain tissue of mice with long-term alcohol consumption; where a represents the effect on the content of reduced glutathione (GSH); b represents the effect on the content of malondialdehyde (MDA); and c represents the effect on the content of superoxide dismutase (T-SOD). Detailed Implementation

[0025] This invention provides the use of Dendrobium polysaccharide in the preparation of medicaments for the prevention and / or treatment of alcohol-induced neuroinflammation, wherein the alcohol-induced neuroinflammation is an alcoholic neurological disease caused by excessive drinking.

[0026] In this invention, the alcohol-induced neurological diseases caused by excessive drinking include alcoholic encephalopathy and neurodegenerative diseases caused by excessive drinking, including Alzheimer's disease and Parkinson's disease.

[0027] In this invention, the drug is a tablet, powder, granule, capsule, pill, liquid, medicated wine, or syrup.

[0028] This invention provides a Dendrobium polysaccharide liqueur for the prevention and / or treatment of alcohol-induced neuroinflammation, comprising a base liquor with an alcohol concentration of 30-50% and Dendrobium polysaccharides; wherein the mass-to-volume ratio of Dendrobium polysaccharides to base liquor is 1-10 g: 1 L.

[0029] In this invention, 1L of base liquor also includes 50-200g of flavoring agent; the base liquor can be any commercially available white liquor, and the flavoring agent is honey.

[0030] The present invention also provides a method for preparing the Dendrobium polysaccharide liqueur, wherein Dendrobium polysaccharide is dissolved in base liquor, homogenized, and aged to obtain Dendrobium polysaccharide liqueur.

[0031] In this invention, Dendrobium polysaccharides are dissolved in base liquor. After the Dendrobium polysaccharides are completely dissolved, flavoring agents are added and stirred until completely dissolved to obtain a solution. The solution is homogenized 2-3 times using a homogenizer. The homogenized solution is then placed in a sealed container and aged in a cool, dry place for 0.5-1.5 months. After aging, the solution is filtered to obtain Dendrobium polysaccharide liqueur. The homogenization pressure is 20-30 MPa, and the homogenization time is 3-5 min / time. The aging temperature is 15-25℃, the relative humidity is 60%-75%, and the aging time is preferably 1 month. The filtration method involves double filtration using gauze and filter paper. The gauze consists of 4 layers, and the filter paper is preferably coffee filter paper with a pore size of 20-30 μm.

[0032] 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.

[0033] Example: Preparation of Dendrobium polysaccharide liqueur (DOP)

[0034] 1. Raw materials

[0035] Dendrobium polysaccharide (BR, 60%, Shanghai Yuanye Biotechnology Co., Ltd., product number: S27808), Baijiu (52% vol Fupingchun Baijiu, Henan Fupingchun Winery Co., Ltd.), honey.

[0036] 2. Preparation process

[0037] The base liquor was prepared by adjusting the alcohol content of the baijiu (Chinese white liquor) to 38° (i.e., 38% vol). One gram of Dendrobium polysaccharide powder and 50 grams of honey were added to every liter of base liquor. The Dendrobium polysaccharide powder was slowly added to the base liquor while stirring continuously until fully dissolved. Honey was then added as a flavoring agent to improve the flavor and taste of the liquor, and stirred until completely dissolved to obtain a solution. The solution was homogenized three times at 20 MPa for 5 minutes to ensure a finer and more uniform consistency. The homogenized solution was then placed in a sealed container and aged for one month in a cool place (temperature: 15–25°C, relative humidity: 60%–75%) to allow the components to fully blend and result in a richer, more mellow flavor. After aging, the product is filtered twice using four layers of gauze and coffee filter paper (purchased from the PAKCHOICE flagship store, 100 pieces of fan-shaped V-shaped filter paper and 100 pieces of cone-shaped 02 filter paper for hand-drip coffee makers) to remove any possible impurities, thus obtaining the finished Dendrobium polysaccharide liqueur.

[0038] Experimental Example

[0039] 1. Grouping setup and construction of a liquor-induced model

[0040] Six-week-old c57bl / 6j mice, purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China), were randomly divided into four groups (n=8 per group) according to body weight after one week of acclimatization in the animal facility: a blank control group (BLK group), a model group (E_ALC group), a positive control group (LU group; Chinese liquor, purchased from Hubei Jinpai Health Wine Co., Ltd.), and a Dendrobium polysaccharide liqueur group (DOP group). Except for the blank control group, all other groups of mice were administered alcohol by gavage (3.6 g ethanol / kg / day) for 4 weeks to establish a long-term alcohol exposure animal model (baijiu-induced model). Subsequently, the mice in each group underwent intervention treatment for 4 weeks according to the following gavage dosage. The Chinese Dietary Guidelines recommend that adult males consume 25g of ethanol daily, with an alcohol content of 38%. Based on the dose-multiplication ratio between experimental animals and humans (the dose for mice is typically 8.65 times the human dose), the gavage doses for each group of mice were calculated as follows: blank control group (3.6g saline / kg / day), model group (3.6g ethanol / kg / day), positive control group (3.6g ethanol / kg / day), and Dendrobium polysaccharide liqueur group (3.6g Dendrobium polysaccharide liqueur obtained in Example 1 / kg / day). All baijiu and jinjiu used in the above experiments were diluted with purified water to 38% alcohol before being administered by gavage once daily according to the mouse weight and gavage dose standards. After 4 weeks of intervention, neurobehavioral tests were performed, followed by dissection and collection of mouse brain tissue, hippocampus, and other experimental samples for subsequent detection of neuroinflammatory-related indicators.

[0041] 2. Neurobehavioral science

[0042] 2.1 Y-maze Test

[0043] The Y-maze test is a behavioral test commonly used to assess spatial memory and cognitive function in animals. This test uses frosted, non-transparent organic plastic, shaped like a "Y," divided into three arms (A, B, and C, 35cm long, 10cm wide, and 20cm high), with an angle of 120° between each arm. At the start of the experiment, mice are placed at the junction of the three arms in the same direction and allowed to move freely within the apparatus. The order in which the mice enter each arm is recorded within 5 minutes. A mouse entering two-thirds or more of an arm is considered a successful entry; otherwise, it is not considered an entry. To eliminate animal odors and ensure the hygiene of the experimental equipment, it is wiped with 75% alcohol before and after the experiment. The alcohol is allowed to evaporate completely before the next round of the experiment, as odors and residual alcohol can affect the behavioral results of the experimental animals. After the experiment, the order of entry into each arm is analyzed, and a correct transition is recorded as "ABC," "ACB," "BCA," "BAC," "CAB," and "CBA." The spontaneous alternation rate in the Y-maze test can directly reflect the degree of influence of neuroinflammation on the cognitive function of mice, especially spatial memory. The results are shown in [Figure 1]. Figure 1 .

[0044] Depend on Figure 1 As shown in 'a', the spontaneous alternation rate of mice in the E_ALC group in the Y maze was significantly lower than that in the BLK and DOP groups. P The value was <0.05), and there was no significant difference between the DOP group and the BLK group, indicating that DOP intervention can improve the symptoms of decreased spatial memory in mice with long-term alcohol consumption.

[0045] 2.2 Open Field Experiment (OPT)

[0046] The open field test can be used to evaluate changes in cognitive function and anxiety in mice after long-term alcohol exposure. Mice were individually placed in a 50cm*50cm*50cm opaque rectangular medical ABS test chamber. Tracking software was used to record and analyze the mice's spontaneous activities, such as movement trajectory, distance traveled, movement time, and dwell time. The animals' activities within the chamber were automatically recorded. The test duration was typically 5 minutes. To avoid interference from previous experiments, the test apparatus was cleaned with 75% ethanol after each test. The central area dwell time index in the open field test reflects changes in anxiety-like and exploratory behaviors in mice. Results are shown in [Figure number missing]. Figure 1 .

[0047] Depend on Figure 1 As shown in b, the E_ALC group mice spent significantly less time in the central region than the BLK group ( P <0.05, after DOP intervention, the time mice spent in the central region increased significantly ( PThe levels of DOP (<0.05) returned to normal, indicating that DOP can improve anxiety and exploratory behavior in mice treated with baijiu (Chinese liquor).

[0048] 2.3 Rotard test

[0049] The fatigue rotundus test can be used to assess the motor coordination and balance of mice with long-term alcohol exposure. In the experiment, a group of mice are placed in a channel (if mice are difficult to handle, an empty channel is left between two mice; only three mice are measured per run). The mice on the bar are encouraged to walk forward while maintaining balance. The bar initially rotates at a constant speed of 4 rpm to allow all mice to be positioned in their respective channels. Once all mice are "ready" (i.e., checked to see if they can walk forward at 4 rpm for a few seconds), the start button is pressed, and the bar accelerates from 4 rpm to 40 rpm over 300 seconds. Throughout the experiment, the rotundus automatically records the latency, fall speed, and distance traveled by each mouse from the bar, and records the reason for the end of the test (fall, jump, passive rotation). After one set of experiments, the equipment is cleaned with water, dried with 75% ethanol, and the above steps are repeated to test the remaining mice. Neuroinflammatory responses interfere with the transmission of nerve signals, affecting muscle contraction and relaxation, thus leading to decreased exercise endurance in animals. In the fatigue rotundus test, this manifests as a shortened distance traveled; the results are shown in [Figure number missing]. Figure 1 .

[0050] like Figure 1 As shown in c, the movement distance of mice in the E_ALC group was significantly lower than that in the BLK group ( P <0.05), after DOP intervention, the movement distance of mice increased significantly ( P The levels of DOP (<0.05) returned to normal, indicating that DOP can restore the motor function of mice that have been drinking alcohol for a long time.

[0051] In summary, the results indicate that Dendrobium polysaccharide liqueur can improve cognitive dysfunction, decreased motor coordination, and anxiety-like behavior in animals caused by long-term alcohol consumption, suggesting that it has a comprehensive protective effect against alcohol-induced neurological damage.

[0052] 3. Detection of physiological and biochemical indicators

[0053] 3.1 Measurement of inflammatory factor markers in brain tissue

[0054] Inflammatory factors can affect the blood-brain barrier and neurodegeneration. This invention uses an ELISA kit (Merck Biotech Ltd.) to detect the levels of neuroinflammatory factors such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in mouse brain tissue. The experimental procedures were strictly performed according to the instructions provided by the ELISA kit manufacturer. Results are shown below. Figure 2 .

[0055] like Figure 2 As shown, in mice in the E_ALC group who were exposed to baijiu (Chinese liquor) for a long period of time, the levels of inflammatory factors such as IL-6, IL-1β, and TNF-α in their brains were significantly higher than those in the BLK group. P <0.05%, after DOP intervention, the levels of IL-1β and TNF-α in the mouse brain were significantly decreased ( P <0.05), returning to the BLK group level, indicating that DOP inhibits the expression of inflammatory factors in the brains of mice with long-term alcohol consumption.

[0056] 3.2. Measurement of neurotransmitters in the hippocampus

[0057] This invention employs ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS) to determine the levels of neurotransmitters such as 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), glutamate (Glu), and dopamine (DA) in the hippocampus. The results are shown in the figure. Figure 3 .

[0058] Pretreatment of mouse hippocampal samples: Mice were euthanized by decapitation, and their brains were immediately removed from an ice plate and rinsed in cold physiological saline. The saline was blotted dry with filter paper, and the hippocampus was quickly dissected from the brain on an ice plate and weighed. Cold 0.1% formic acid was added to the hippocampus at a ratio of 1:10 (mg / mL) for homogenization. The sample was centrifuged at 12000 rpm for 20 min at 4°C, and the supernatant was collected and filtered through a 0.22 μm microporous membrane for analysis.

[0059] Preparation of standard solutions: Weigh approximately 5.0 mg of 5-HT, GABA, Glu, and DA standards respectively, dissolve each in a solvent (0.2% formic acid-methanol, 8:2, V / V), and dilute to 5 mL to obtain standard stock solutions. Then prepare mixed standards.

[0060] Liquid chromatography-mass spectrometry conditions:

[0061] ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm);

[0062] Mobile phase A: 0.1% formic acid aqueous solution;

[0063] Mobile phase B: Acetonitrile;

[0064] Gradient elution program: 0-2.5 min, 5% B; 2.5-4 min, 5%-20% B; 4-7 min, 20%-60% B; 7-7.5 min, 60%-5% B; 7.5-10 min, 5% B. Flow rate: 0.2 mL / min. Column temperature: 30℃. Injection volume: 3 μL.

[0065] MS conditions: Electrospray ionization source (ESI): positive ion mode; multiple reaction monitoring mode (MRM); curtain gas: 40.0 psi; collision gas: N2; spray voltage: 5500 V; ion source temperature: 500 °C; GS1: 50 psi, GS2: 50 psi; gas flow rate: 12 L / min.

[0066] Depend on Figure 3 It can be seen that, compared with the blank control group, long-term exposure to alcohol treatment significantly reduced ( P <0.05) The levels of 5-HT, DA, and GABA in the hippocampus were significantly increased ( P <0.05) The level of Glu in the hippocampus significantly reversed the alcohol-induced decrease in DA content in the hippocampus after DOP intervention treatment. P <0.05) and the increase in Glu content ( P Although the levels of 5-HT and GABA were not significantly reduced (<0.05), there was no statistically significant difference compared to the BLK group. The results indicate that DOP can regulate the concentrations of neurotransmitters such as 5-HT, DA, GABA and Glu in the hippocampus of mice that have been drinking alcohol for a long time.

[0067] 3.3. HE staining of mouse hippocampal tissue

[0068] The hippocampus was preserved intact, and the brain tissue was excised along the coronal plane, fixed with 10% formaldehyde solution, embedded, dehydrated, cleared with xylene, stained with hematoxylin and eosin (HE), and automatically mounted with neutral resin using an automated mounting machine. The slides were then observed under an optical microscope. Results are shown in [Figure number missing]. Figure 4 .

[0069] The effect of DOP on the structure of the CA3 region of the hippocampus in alcohol-exposed mice was detected using HE staining. Figure 4As shown, the number of neurons in the CA3 region of the hippocampus of BLK group mice was 4-5 layers. The cells had normal morphology, intact cell bodies, prominent nucleoli, and were arranged neatly and tightly. The number of pyramidal cells in the CA3 region of E_ALC group mice was significantly reduced, and the pyramidal cells exhibited various abnormal morphologies such as cell body swelling, cytoplasmic vacuolation, disappearance or marginalization of Nissl bodies, and nuclear pyknosis. After DOP intervention, the number of neurons in the CA3 region of mice increased, the morphology of most pyramidal cells returned to normal, the intercellular spaces narrowed, and the cells returned to a neat and tightly arranged state, indicating that DOP can alleviate the inflammatory damage to nerve cells caused by long-term alcohol consumption and restore the damaged hippocampal structure.

[0070] 3.4 Measurement of oxidative indicators in the brain

[0071] Brain samples were removed from a -80°C freezer and homogenized with physiological saline to form a 10% homogenate. The homogenates were then centrifuged at 4°C and 3500 rpm for 10 min to obtain the liver and brain supernatants. The levels of AST and ALT in the liver, and the levels of reduced glutathione (GSH) (to assess the degree of nerve cell damage), malondialdehyde (MDA) (to reflect the degree of lipid peroxidation), and superoxide dismutase (T-SOD) activity (to measure antioxidant capacity) in the liver and brain tissue homogenates were measured according to the kit instructions. The results are shown in the table below. Figure 5 .

[0072] DOP can alleviate the oxidative imbalance in the brains of mice that have been drinking alcohol for a long time. Figure 5 As shown, compared with the BLK group, the levels of oxidative stress indicators such as GSH and T-SOD in the brains of mice in the E_ALC group were significantly reduced. P <0.05%, MDA content increased significantly ( P <0.05%, after DOP intervention, the levels of GSH and T-SOD in the mouse brain were significantly increased ( P <0.05%, MDA content decreased significantly ( P <0.05) There was no significant difference between the group and the BLK group.

[0073] In summary, DOP can intervene in neuroinflammation in mice with long-term alcohol consumption by altering physiological and biochemical indicators such as neurobehavioral, inflammatory factors, neurotransmitters, hippocampal structure, and oxidative stress.

[0074] 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. The use of Dendrobium polysaccharide in the preparation of a medicine for preventing and / or treating alcohol-induced neuroinflammation, characterized in that, The alcohol-induced neuroinflammation is alcoholic encephalopathy.

2. Use according to claim 1, characterized in that, The medicament is a tablet, a powder, a granule, a capsule, a pill, an aqueous preparation, a liquor or a syrup.

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