Polygonatum sibiricum fibrous root fermentation liquor as well as preparation method and application thereof
By inoculating Lactobacillus plantarum bacteria solution into the fibrosus root of Polygonatum for fermentation, a fermentation liquid of Polygonatum root with intestinal protective effect was prepared, which solved the problem that existing traditional Chinese medicine processing and treatment methods were difficult to fully utilize the advantages of traditional Chinese medicine, and achieved effective treatment for alcoholic liver damage, significantly improving liver function and blood lipid metabolism.
Patent Information
- Application Number
- CN202510297139.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-13
- Publication Date
- 2025-06-13
AI Technical Summary
The existing traditional Chinese medicine processing methods are difficult to fully utilize the advantages of traditional Chinese medicine, and the effect of treating alcoholic liver damage is not ideal.
By inoculating the Lactobacillus plantarum bacteria solution into the sterilized Polygonatum fibrous root fermentation liquid, a Polygonatum fibrous root fermentation liquid is prepared. This liquid has an intestinal protective effect, can reduce the LPS content in the serum, and improve the permeability of the intestinal barrier in mice caused by alcohol.
Polygonatum fibrostridium fermentation broth can significantly reduce the content of ALT, AST, TC and TG in the serum, improve blood lipid metabolism disorders and liver abnormalities, reduce oxidative stress damage, and protect the liver.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biomedicine, and particularly relates to a fermented liquid of polygonatum odoratum rootlets, a preparation method thereof and an application thereof. Background Art
[0002] Alcoholic liver injury is a common liver disease caused by long-term heavy drinking. In the early stage, it is usually manifested as alcoholic fatty liver. If not effectively treated, it may develop into alcoholic hepatitis, liver fibrosis, cirrhosis or even hepatocellular carcinoma. The pathogenesis of alcoholic liver injury is very complex, and multiple pathways are involved. Oxidative stress has been considered to play an important role in the occurrence and development of alcoholic liver disease. When liver function is impaired or a large amount of alcohol is consumed, exceeding the body's metabolic capacity, the rate of ethanol oxidation and decomposition slows down, and liver injury is likely to occur. Among them, alcohol can inhibit the synthesis of antioxidant enzymes in liver tissue, and at the same time cause a large amount of free radicals to be generated, leading to lipid peroxidation, generating oxidative stress response, and resulting in hepatocyte injury. At the same time, alcohol-induced oxidative stress causes mucosal barrier dysfunction and disruption of epithelial tight junctions, resulting in leakage of luminal lipopolysaccharide in the colonic mucosa and exacerbating liver injury.
[0003] In recent years, traditional Chinese medicines for treating alcoholic liver injury include licorice, bupleurum and isatis root. However, existing traditional Chinese medicine processing methods often cannot fully exert the advantages of traditional Chinese medicine.
[0004] Therefore, it is crucial to develop a drug that can exert the advantages of traditional Chinese medicine and treat alcoholic liver injury. Summary of the Invention
[0005] In order to exert the advantages of traditional Chinese medicine, the present invention provides a fermented liquid of polygonatum odoratum rootlets, a preparation method thereof and an application thereof. The fermented liquid of polygonatum odoratum rootlets provided by the present invention has an intestinal protection effect, can reduce the content of lipopolysaccharide in serum, and improve the increased intestinal barrier permeability in mice caused by alcohol, providing a basis for the research and development of the fermented liquid of polygonatum odoratum rootlets to improve alcoholic liver injury.
[0006] An application of a fermented liquid of polygonatum odoratum rootlets in the preparation of a drug for treating alcoholic liver injury, wherein the drug takes the fermented liquid of polygonatum odoratum rootlets as the only active ingredient, and the fermented liquid of polygonatum odoratum rootlets is obtained by inoculating lactic acid bacteria into a homogenate of polygonatum odoratum rootlets for fermentation.
[0007] The fermented liquid of polygonatum odoratum rootlets provided by the present invention is obtained by inoculating a plant lactobacillus bacterial solution into sterilized polygonatum odoratum rootlets for fermentation. The fermented liquid of polygonatum odoratum rootlets provided by the present invention has an intestinal protection effect, can reduce the LPS content in serum, and improve the increased intestinal barrier permeability in mice caused by alcohol, providing a basis for the research and development of the fermented liquid of polygonatum odoratum rootlets to improve alcoholic liver injury.
[0008] Furthermore, the drug is used to improve liver tissue structure damage.
[0009] Furthermore, the drug also contains pharmaceutically acceptable excipients.
[0010] Furthermore, the excipients are sodium bicarbonate, magnesium stearate or carboxymethyl cellulose.
[0011] The present invention provides a method for preparing the fermented liquid of polygonatum odoratum rhizome fibrous roots, comprising the following steps:
[0012] Wash fresh polygonatum odoratum, trim the rhizome fibrous roots, take the rhizome fibrous roots and homogenize them at a rotation speed of 8000 r / min to 10000 r / min for 3 min to 7 min to obtain a homogenized liquid of polygonatum odoratum rhizome fibrous roots;
[0013] Ferment lactic acid bacteria into a lactic acid bacteria liquid with a bacterial concentration of 1×10 7 CFU / mL to 1×10 9 CFU / mL, add the lactic acid bacteria liquid to the fresh homogenized liquid of polygonatum odoratum rhizome fibrous roots according to an inoculation amount of 3% to 7%, and culture at 34°C to 40°C for 3 d to 5 d to obtain the fermented liquid of polygonatum odoratum rhizome fibrous roots.
[0014] Furthermore, the lactic acid bacteria are Lactobacillus plantarum.
[0015] Furthermore, the culture conditions of the Lactobacillus plantarum suspension are to culture at 34°C to 40°C for 18 h to 36 h in a sterile liquid MRS medium.
[0016] The present invention provides a fermented liquid of polygonatum odoratum rhizome fibrous roots.
[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0018] 1. The present invention inoculates a Lactobacillus plantarum liquid into sterilized polygonatum odoratum rhizome fibrous roots for fermentation to obtain a fermented liquid of polygonatum odoratum rhizome fibrous roots; the fermented liquid of polygonatum odoratum rhizome fibrous roots provided by the present invention has an intestinal protection effect, can reduce the LPS content in the serum, and improve the increase in the intestinal barrier permeability of mice caused by alcohol, providing a basis for the research and development of the fermented liquid of polygonatum odoratum rhizome fibrous roots to improve alcoholic liver injury.
[0019] 2. The present invention uses the non-medicinal part of polygonatum odoratum, namely polygonatum odoratum rhizome fibrous roots, for fermenting and preparing a fermented liquid, which can improve the utilization rate of the non-medicinal part of polygonatum odoratum, increase the high added value of the non-medicinal part of polygonatum odoratum, and realize the maximum utilization of polygonatum odoratum resources.
[0020] 3. By constructing an animal model and setting up a blank control group, a model group, a low-dose polygonatum odoratum root fermented liquid group, and a high-dose polygonatum odoratum root fermented liquid group, the present invention found that the polygonatum odoratum root fermented liquid group could alleviate the weight loss of mice caused by alcohol, significantly reduce the contents of ALT, AST, TC, and TG in the serum, improve the disorder of blood lipid metabolism and abnormal liver function, and at the same time reduce the content of MDA in the serum, increase the activities or regeneration of SOD, CAT, and GSH-Px, so as to improve the ability of the body to scavenge free radicals and reduce oxidative stress damage, and thus protect the liver. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.
[0022] Figure 1 Shows the changes in the body weights of mice in different animal model groups; Note: * indicates P < 0.05, ** indicates P < 0.005, *** indicates P < 0.001.
[0023] Figure 2 Shows the HE staining results of liver tissue sections of mice in different animal model groups, and the scale bar is 100 μm.
[0024] Figure 3 Shows the contents of ALT and AST in the serum of mice in different animal model groups; In the figure, A shows the content of ALT in the serum of mice in different animal model groups; B shows the content of AST in the serum of mice in different animal model groups.
[0025] Figure 4 Shows the contents of TC and TG in the serum of mice in different animal model groups; In the figure, A shows the content of TC in the serum of mice in different animal model groups; B shows the content of TG in the serum of mice in different animal model groups.
[0026] Figure 5 Shows the activities of SOD, GSH-Px, and CAT in the serum of mice in different animal model groups; In the figure, A shows the activity of SOD in the serum of mice in different animal model groups; B shows the activity of GSH-Px in the serum of mice in different animal model groups; C shows the activity of CAT in the serum of mice in different animal model groups.
[0027] Figure 6 Shows the content of MDA in the serum of mice in different animal model groups.
[0028] Figure 7The staining results of mouse ileum tissue sections in different animal model groups, with the scale bar being 150 μm; the first row is HE staining, and the second row is AB-PAS staining.
[0029] Figure 8 It is the content of LPS in the sera of mice in different animal model groups.
[0030] Figure 9 It is the influence of different animal model groups on the mRNA expression levels of tight junction proteins in mouse intestinal tissues; in the figure, A is the relative expression level of the tight junction protein Claudin-1 in mouse intestinal tissues in different animal model groups; B is the relative expression level of the tight junction protein Occludin in mouse intestinal tissues in different animal model groups; C is the relative expression level of the tight junction protein ZO-1 in mouse intestinal tissues in different animal model groups. Specific embodiments
[0031] The specific embodiments of the present invention will be described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.
[0032] Experimental materials: Polygonatum sibiricum root, provided by Chongqing Institute of Traditional Chinese Medicine; Lactobacillus plantarum ATCC14917, Lactobacillus plantarum subsp. plantarum, purchased from Shanghai Fuxiang Biotechnology Co., Ltd. (Shanghai, China); C57BL / 6 male mice (body weight 20±2g, 8 weeks old, male, animal license number: SCXK(Xiang)2019-0004), Hunan Slake Jingda Experimental Animal Co., Ltd.; MRS culture medium, Guangdong Huankai Microbiological Technology Co., Ltd.; Lieber-DeCarli liquid diet feed, Research Diets, USA; catalase (CAT) detection kit, Wuhan Saiweier Biotechnology Co., Ltd.; glutathione peroxidase (GSH-Px) determination kit, Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.; malondialdehyde (MDA) determination kit, Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.; superoxide dismutase (SOD) test kit, Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.; alanine aminotransferase (ALT) test kit, Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. ALT assay kit was purchased from Shenzhen Raydu Life Science Co., Ltd.; aspartate aminotransferase (AST) assay kit was purchased from Shenzhen Raydu Life Science Co., Ltd.; triglyceride (TG) assay kit was purchased from Shenzhen Raydu Life Science Co., Ltd.; total cholesterol (TC) assay kit was purchased from Shenzhen Raydu Life Science Co., Ltd.; LPS ELISA kit was purchased from Jiangsu Enzyme Immunity Industry Co., Ltd.; hematoxylin-eosin (HE) high-definition constant staining kit was purchased from Wuhan Sevier Biotechnology Co., Ltd.; Alcian blue-periodic acid Schiff (AB-PAS) stain solution was purchased from Wuhan Sevier Biotechnology Co., Ltd.; other organic solvents and chemical reagents (all of analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd.
[0033] Instruments and equipment: YC-330 drug refrigerator, Aucma; HH-S electric constant temperature water bath, Great Wall; pipette, Eppendorf, Germany; 5430R desktop refrigerated high-speed centrifuge, Eppendorf, Germany; K5600C spectrophotometer, Kayo Technology; VORTEX-6 vortex mixer, Chilin Bell; ELISA detector, Meigu Molecular Co., Ltd.; fully automatic biochemical analyzer, Shenzhen Redu Life Science Co., Ltd.
[0034] Example 1: A fermented liquor of Polygonatum sibiricum root, and a preparation method and application thereof.
[0035] Preparation of Polygonatum odoratum rootlet homogenate: Wash fresh Polygonatum odoratum with distilled water, then trim the rootlets. Place 1 kg of rootlets in a tissue homogenizer and homogenize at 10,000 r / min for 5 min to obtain the Polygonatum odoratum rootlet homogenate.
[0036] Preparation of Polygonatum odoratum rootlet fermentation broth: Culture Lactobacillus plantarum in sterile liquid MRS medium at 37 °C for 24 h, then centrifuge at 500 r / min for 5 min to collect the bacterial cells. Dilute the bacterial cells with sterile normal saline to obtain a bacterial suspension with a concentration of 1×10 8 CFU / mL. Then add the bacterial suspension to the fresh Polygonatum odoratum rootlet homogenate (after ultra-high temperature instantaneous sterilization) at an inoculation amount of 5%, and culture at 37 °C for 4 d. When the pH of the fermentation product is <2.5, the fermentation ends and it is centrifuged at 8,000 r / min for 10 min. The supernatant is sterilized at 95 °C for 30 min, and the pH of the supernatant is adjusted to 3.5 with food-grade sodium bicarbonate to obtain the Polygonatum odoratum rootlet fermentation broth.
[0037] MRS medium. The composition of each liter of MRS medium is as follows: 10.0 g of peptone, 4.0 g of yeast extract powder, 1.0 g of Tween 80, 2.0 g of dipotassium hydrogen phosphate heptahydrate, 5.0 g of sodium acetate trihydrate, 2.0 g of ammonium citrate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate tetrahydrate, 10.0 g of maltose, 15.0 g of agar, 0.01 g of vancomycin, and 0.0048 g of bromocresol purple. Make up to 1 L with distilled water.
[0038] Establishment of an animal model: Place 24 male C57BL / 6 mice in a humidity of 50±5%, temperature of 23±1 °C, and a 12 h / 12 h light-dark cycle. Allow them to feed and drink freely. After one week of adaptive culture, randomly divide them into 4 groups (n = 6): blank control group, model group, low-dose Polygonatum odoratum rootlet fermentation broth group, and high-dose Polygonatum odoratum rootlet fermentation broth group. The specific diet is shown in Table 1.
[0039] Table 1 Diet types of different animal models
[0040]
[0041] From day 1 to day 5, all mice were given Lieber-DeCarli control diet to adapt to the liquid diet. From day 6 to day 16, the blank control group continued to be fed Lieber-DeCarli control diet, while the model group, the low-dose polygonatum root fermented liquid group, and the high-dose polygonatum root fermented liquid group were fed Lieber-DeCarli diet containing 5% ethanol. 9 hours before the end of the experiment on day 16, the mice in the blank control group were intragastrically administered maltodextrin solution (5 g / kg), and the mice in the model group, the low-dose polygonatum root fermented liquid group, and the high-dose polygonatum root fermented liquid group were intragastrically administered ethanol solution (5 g / kg) respectively. During the experiment, the mice in the low-dose polygonatum root fermented liquid group and the high-dose polygonatum root fermented liquid group were intragastrically administered 0.05 mL / 10 g and 0.1 mL / 10 g of polygonatum root fermented liquid respectively at a fixed time every day. At the same time, the mice in the blank control group and the model group were intragastrically administered an equal amount of normal saline.
[0042] After the experiment, the mice were anesthetized with 1% sodium pentobarbital, blood was collected from the orbital venous plexus. After blood collection, the mice were sacrificed by cervical dislocation. The abdominal cavity was opened to take out the liver and intestinal tissues, and the tissues were quickly separated on an ice table for subsequent analysis and detection.
[0043] Example 2: A polygonatum root fermented liquid, its preparation method and application.
[0044] The experimental steps of Example 2 are the same as those of Example 1, and the main differences are as follows:
[0045] Preparation of polygonatum root homogenate: Fresh polygonatum was washed clean with distilled water, and then the fibrous roots were trimmed. 1 kg of fibrous roots were placed in a tissue homogenizer and homogenized at 10000 r / min for 5 min to obtain polygonatum root homogenate.
[0046] Preparation of polygonatum root fermented liquid: Lactobacillus plantarum was cultured in sterile liquid MRS medium at 37 °C for 24 h, and then centrifuged at 500 r / min for 5 min to collect the bacteria. The bacteria were diluted with sterile normal saline to obtain a bacterial solution with a concentration of 1×10 7 CFU / mL. Then, the bacterial solution was added to the fresh polygonatum root homogenate according to an inoculation amount of 3%, and cultured at 34 °C for 3 d. When the pH of the fermentation product was <2.5, the fermentation was completed and it was centrifuged at 8000 r / min for 10 min. The supernatant was sterilized at 95 °C for 30 min, and the pH of the supernatant was adjusted to 3 with food-grade Na 2 CO 3 to obtain polygonatum root fermented liquid.
[0047] Example 3: A polygonatum root fermented liquid, its preparation method and application.
[0048] The experimental steps of Example 3 are the same as those of Example 1, and the main differences are as follows:
[0049] Preparation of fermented liquid from the fibrous roots of Polygonatum sibiricum: Wash fresh Polygonatum sibiricum with distilled water, then trim the fibrous roots. Place 1 kg of fibrous roots in a tissue homogenizer and homogenize at a speed of 10,000 r / min for 5 min to obtain the fermented liquid from the fibrous roots of Polygonatum sibiricum.
[0050] Preparation of fermented liquid from the fibrous roots of Polygonatum sibiricum: Culture Lactobacillus plantarum in a sterile liquid MRS medium at 37 °C for 24 h, and then centrifuge at 500 r / min for 5 min to collect the bacterial cells. Dilute the bacterial cells with sterile normal saline to obtain a bacterial suspension with a concentration of 1×10 9 CFU / mL. Then add the bacterial suspension to the fresh fermented liquid from the fibrous roots of Polygonatum sibiricum according to an inoculation amount of 7%, and culture at 40 °C for 5 d. When the pH of the fermentation product is < 2.5, the fermentation ends and it is centrifuged at 8,000 r / min for 10 min. The supernatant is sterilized at 95 °C for 30 min, and the pH of the supernatant is adjusted to 4 with food-grade Na 2 CO 3 to obtain the fermented liquid from the fibrous roots of Polygonatum sibiricum.
[0051] Using the fermented liquid from the fibrous roots of Polygonatum sibiricum prepared in Examples 1 to 3, study its protective effect on mice with alcoholic liver injury. The effects of Examples 1 to 3 are similar. For the convenience of subsequent discussion and reference, the results of Example 1 are used as the standard.
[0052] I. Effect of the fermented liquid from the fibrous roots of Polygonatum sibiricum on the body weight of mice
[0053] 1. Experimental method
[0054] During the experiment, observe the survival of each group of mice, and weigh and record them with an electronic scale at the beginning and end of the experiment.
[0055] 2. Experimental results
[0056] The changes in the body weight of each group of mice are as Figure 1 shown. At the beginning of the experiment, the body weights of each group of mice were close and there were no significant differences. At the end of the experiment, the body weight of the mice in the model group decreased significantly (P < 0.001) under the action of the Lieber-DeCarli alcohol diet, and was lower than that of the mice in the low-dose and high-dose groups of the fermented liquid from the fibrous roots of Polygonatum sibiricum, with significant differences (P < 0.05, P < 0.005), indicating that the fermented liquid from the fibrous roots of Polygonatum sibiricum can alleviate the weight loss of mice caused by alcohol.
[0057] II. Effect of the fermented liquid from the fibrous roots of Polygonatum sibiricum on the histopathology of the mouse liver
[0058] 1. Experimental method
[0059] Take a part of the mouse liver tissue, fix it in 10% neutral formalin fixative, dehydrate it routinely, embed it in paraffin, section it, then stain the sections using an HE staining kit, and finally observe the morphological changes of the liver tissue with an optical microscope.
[0060] 2. Experimental results
[0061] As Figure 2 shown, the hepatocytes of the mice in the blank control group were radially arranged around the central vein, forming clear hepatic cords. Compared with the blank control group, the liver tissue of the mice in the model group had obvious damage, the hepatic cords were disordered, and obvious fatty degeneration occurred in the mouse liver, mainly showing diffuse vacuolar degeneration of hepatocytes and infiltration of a small amount of inflammatory cells; compared with the model group, the fatty degeneration and lipid accumulation of hepatocytes in the liver tissue of the mice in the low-dose and high-dose groups of the fermented liquid of Polygonatum odoratum var. pluriflorum roots were significantly reduced. In particular, the cell morphology of the liver tissue of the mice in the high-dose group was similar to that of the liver tissue of the mice in the blank control group, indicating that the fermented liquid of Polygonatum odoratum var. pluriflorum roots has the effect of improving the damage of the liver tissue structure caused by alcohol in mice.
[0062] III. Effects of the fermented liquid of Polygonatum odoratum var. pluriflorum roots on the contents of ALT and AST in the serum of mice
[0063] 1. Experimental method
[0064] Place the obtained mouse blood samples at room temperature for 2 h, then centrifuge at 4 °C and 5000×g for 15 min to obtain serum, and store the serum in aliquots at -80 °C before subsequent analysis and detection. Then use an automatic biochemical analyzer to detect the activities of ALT and AST in the serum respectively to estimate alcohol-induced liver injury.
[0065] 2. Experimental results
[0066] After hepatocytes are damaged by alcohol, ALT and AST in the cytoplasm will be released into the systemic circulation. Therefore, measuring the levels of ALT and AST in the serum is widely used to evaluate the risk of alcoholic liver disease. To evaluate the effect of the fermented liquid of Polygonatum odoratum var. pluriflorum roots on the liver function of mice, the changes in the contents of ALT and AST in the serum of mice were measured in this study. As Figure 3 shown, compared with the blank control group, the contents of ALT and AST in the serum of the mice in the model group were significantly increased (P<0.001), indicating that the mice showed symptoms of alcoholic liver injury; compared with the model group, the contents of ALT and AST in the serum of the mice in the low-dose and high-dose groups of the fermented liquid of Polygonatum odoratum var. pluriflorum roots were significantly decreased, indicating that the fermented liquid of Polygonatum odoratum var. pluriflorum roots can reduce the damage of alcohol to mouse hepatocytes, lower the levels of ALT and AST in the serum, and show a certain correlation with the dose of the fermented liquid of Polygonatum odoratum var. pluriflorum roots, indicating that the fermented liquid of Polygonatum odoratum var. pluriflorum roots has the effect of improving the abnormal liver function caused by alcohol in mice.
[0067] IV. Effects of the Fermentation Broth of Polygonatum odoratum Root Fibers on the Contents of TC and TG in Mouse Serum
[0068] 1. Experimental Method
[0069] The obtained mouse blood samples were placed at room temperature for 2 h, and then centrifuged at 4°C and 5000×g for 15 min to obtain serum. The serum was aliquoted and stored at -80°C before subsequent analysis and detection. Then, an automatic biochemical analyzer was used to detect the contents of TC and TG respectively to evaluate the effect of alcohol on lipid metabolism in mice.
[0070] 2. Experimental Results
[0071] The liver is a key hub for many physiological processes, including energy metabolism, fatty acid metabolism, immune regulation, bile acid biosynthesis, alcohol metabolism, and inflammatory responses. Excessive alcohol consumption may lead to liver-related dysfunctions, among which lipid metabolism disorder is one of the manifestations of alcoholic liver disease. Excessive alcohol consumption promotes the synthesis of TC and TG in the liver, increases the liver lipid content, and thus promotes the deterioration of alcoholic liver disease. As Figure 4 shown, compared with the blank control group, the contents of TC and TG in the serum of the model group mice were significantly increased (P<0.001), indicating that alcohol induced lipid metabolism disorder in the model group mice; compared with the model group, the contents of TC and TG in the serum of the low-dose and high-dose groups of the fermentation broth of Polygonatum odoratum root fibers were significantly decreased, indicating that the fermentation broth of Polygonatum odoratum root fibers could improve the disorder of blood lipid metabolism in mice and relieve the abnormal blood lipid level caused by alcohol.
[0072] V. Effects of the Fermentation Broth of Polygonatum odoratum Root Fibers on the Contents of TC and TG in Mouse Serum
[0073] 1. Experimental Method
[0074] The corresponding detection kits were used to determine the activities of SOD, GSH-Px, and CAT and the content of MDA in the serum to evaluate the oxidative stress response induced by alcohol in the serum.
[0075] 2. Experimental Results
[0076] Oxidative stress plays an important role in alcoholic liver disease. Alcohol-induced liver tissue injury is always accompanied by abnormal oxidative stress. Improving oxidative stress is an important means to improve alcoholic liver disease. MDA, GSH, SOD, as biomarkers of oxidative stress, are often important indicators reflecting the degree of liver oxidative damage. The activities of SOD, CAT, and GSH-Px reflect the ability to scavenge reactive oxygen species, while the content of MDA reflects the level of lipid peroxidation. As Figure 5 and Figure 6As shown, compared with the blank control group, the activities of SOD, GSH-Px, and CAT in the serum of mice in the model group decreased significantly (P<0.05), and the content of MDA increased significantly (P<0.001), indicating that alcohol induced an oxidative stress response in the model group of mice. Compared with the model group, the activities of SOD, GSH-Px, and CAT in the serum of mice in the low-dose and high-dose groups of Polygonatum cirrhifolium (Wall.) Royle root fermented liquid increased (P<0.05), and the content of MDA in the serum decreased (P<0.05), indicating that while reducing the content of harmful substance MDA, Polygonatum cirrhifolium (Wall.) Royle root fermented liquid can increase the activities or regeneration of SOD, CAT, and GSH-Px to enhance the body's ability to scavenge free radicals and reduce oxidative stress damage, thereby achieving a protective effect on the liver. Polygonatum cirrhifolium (Wall.) Royle root fermented liquid has the effect of improving the oxidative stress response caused by alcohol in the body.
[0077] VI. Effects of Polygonatum cirrhifolium (Wall.) Royle Root Fermented Liquid on the Intestines of Mice
[0078] 1. Experimental Method
[0079] The obtained mouse blood samples were placed at room temperature for 2 h, then centrifuged at 4°C and 5000×g for 15 min to obtain serum, and the serum was aliquoted and stored at -80°C before subsequent analysis and detection. An ELISA kit was used to detect the LPS content in the serum to evaluate the effect of alcohol on the intestinal barrier function of mice.
[0080] Take a part of the mouse intestinal tissue, fix it in 10% neutral formalin fixative, dehydrate it conventionally, embed it in paraffin, section it, then stain the sections using an HE staining kit and an AB-PAS staining kit, and finally observe the morphological changes of the intestinal tissue with an optical microscope.
[0081] Excessive alcohol consumption damages the integrity of the intestinal barrier by disrupting tight junctions, leading to the transfer of intestinal LPS to the liver tissue, triggering a body inflammatory response, and exacerbating alcohol-induced liver injury. This study evaluated the tissue barrier function of mice based on the histopathological detection of mouse ileum tissue and the detection and analysis of serum LPS content. The results are as Figure 7 and Figure 8As shown in the figure. The HE staining results showed that compared with the mice in the blank control group, the ileal villi of the mice in the model group were significantly loose and disordered, indicating that alcohol intake changed the intestinal morphology. After intervention with low-dose and high-dose fermented liquid of Polygonatum odoratum var. pluriflorum roots, the disorder was alleviated. The AB-PAS staining results showed that compared with the mice in the blank control group, the content of mucin in the ileum of the mice in the model group was significantly reduced, and the situation improved after intervention with low-dose and high-dose fermented liquid of Polygonatum odoratum var. pluriflorum roots. At the same time, compared with the blank control group, the content of serum LPS in the mice in the model group was significantly increased (P<0.001), suggesting that alcohol caused intestinal barrier damage in mice; compared with the model group, the content of LPS in the serum of the mice in the low-dose and high-dose groups of fermented liquid of Polygonatum odoratum var. pluriflorum roots decreased significantly, indicating that the fermented liquid of Polygonatum odoratum var. pluriflorum roots could alleviate the intestinal barrier dysfunction in mice, and there was a certain correlation with the dose of the fermented liquid of Polygonatum odoratum var. pluriflorum roots, indicating that the fermented liquid of Polygonatum odoratum var. pluriflorum roots had the effect of improving the abnormal intestinal barrier function caused by alcohol in mice.
[0082] VII. Protective effect of fermented liquid of Polygonatum odoratum var. pluriflorum roots on intestinal tight junctions in mice
[0083] 1. Experimental method
[0084] Total RNA of liver and intestinal tissues was extracted using Trizol reagent, and the purity of RNA was evaluated according to the ratio of 260nm / 280nm. Subsequently, the RNA was reverse transcribed into complementary DNA (cDNA) using a cDNA reverse transcription kit, and then the synthesized cDNA was subjected to real-time fluorescence quantitative PCR (Quantitative Real-time PCR, RT-qPCR) using SYBR Green. GAPDH was used as a housekeeping gene to quantify the relative expression levels of other genes, and the results were analyzed using the 2 -ΔΔCT method, and the specific primers are listed in Table 3.
[0085] Table 3 Primer sequences for real-time PCR
[0086]
[0087] 2. Experimental results
[0088] Ethanol-induced intestinal barrier dysfunction plays a crucial role in the progression of alcoholic liver disease. Intestinal epithelial tight junctions are considered key regulators of intestinal mucosal permeability. Therefore, regulating the expression of related tight junction proteins or regulating their functions will positively affect intestinal barrier function. As Figure 9As shown, in this experiment, the mRNA expression levels of intestinal tight junction proteins were detected by RT-qPCR. The results showed that compared with the blank control group, the expression levels of Claudin-1, Occludin, and ZO-1 in the ileum tissues of the model group mice were significantly decreased (P<0.001), indicating that alcohol caused abnormal gene expression of the tight junction proteins Claudin-1, Occludin, and ZO-1 in mice. Compared with the model group, the gene expression levels of the intestinal tight junction proteins Claudin-1, Occludin, and ZO-1 in the low-dose and high-dose groups of the fermented liquid of Polygonatum odoratum (Mill.) Druce roots increased, indicating that administration of the fermented liquid of Polygonatum odoratum (Mill.) Druce roots could improve alcohol-induced intestinal tight junction damage. The above results indicate that the fermented liquid of Polygonatum odoratum (Mill.) Druce roots can effectively improve alcohol-induced intestinal damage in mice and maintain intestinal barrier function.
[0089] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn the basic creative concept.
[0090] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and deformations.
Claims
1. A method for preparing a drug for treating alcoholic liver damage by fermentation liquid of polygonatum sibiricum root, characterized in that: The medicine has the fermentation liquid of polygonatum sibiricum root as the only active ingredient, and the fermentation liquid of polygonatum sibiricum root is obtained by inoculating lactic acid bacteria into the homogenate of polygonatum sibiricum root for fermentation.
2. The use of the fermented broth of polygonatum sibiricum root in the preparation of a drug for treating alcoholic liver damage according to claim 1, characterized in that: The drug is used to improve liver tissue structure damage.
3. The use of the fermented broth of polygonatum sibiricum root in the preparation of a drug for treating alcoholic liver damage according to claim 1, characterized in that: The drug further comprises pharmaceutically acceptable excipients.
4. The use of the fermented broth of polygonatum sibiricum root in the preparation of a drug for treating alcoholic liver damage according to claim 3, characterized in that: The auxiliary material is sodium bicarbonate, magnesium stearate or carboxymethyl cellulose.
5. A method for preparing a fermented liquid of Polygonatum sibiricum root, characterized in that: The steps include: Wash fresh Polygonatum cyrtonema, trim the fibrous roots, and homogenize the fibrous roots at a speed of 8000 r / min to 10000 r / min for 3 min to 7 min to obtain a Polygonatum cyrtonema fibrous root homogenate; The lactic acid bacteria were fermented to a concentration of 1×10 7 CFU / mL~1×10 9 CFU / mL of lactic acid bacteria liquid, adding the lactic acid bacteria liquid to fresh polygonatum sibiricum root homogenate at an inoculation rate of 3% to 7%, culturing at 34°C to 40°C for 3d to 5d, and obtaining the polygonatum sibiricum root fermentation liquid of claim 1.
6. The method for preparing the fermented liquor of polygonatum sibiricum root according to claim 5, characterized in that: The lactic acid bacteria is Lactobacillus plantarum.
7. The method for preparing the fermented liquor of polygonatum sibiricum root according to claim 6, characterized in that: The preparation process of the lactic acid bacteria liquid is as follows: inoculating the lactic acid bacteria into a sterile liquid MRS culture medium and culturing at 34° C. to 40° C. for 18 h to 36 h.
8. A fermentation liquid of Polygonatum sibiricum fibrous roots obtained by the preparation method according to any one of claims 5 to 7.