Application of Polysaccharide from Lotus Leaf in Preparation of Drugs for Improving Alcoholic Liver Injury

Abstract

The invention discloses the application of L. pleurotus polysaccharide in the preparation of a drug for improving alcoholic liver damage, and relates to the field of biomedicine technology. The invention finds that L. pleurotus polysaccharide can effectively protect the liver cell structure, inhibit liver swelling caused by alcohol, and reduce the liver index; downregulate the activity of liver cell damage-related biological enzymes aspartate aminotransferase and alanine aminotransferase in serum; improve the activity of liver antioxidant enzymes, inhibit the generation of hepatocyte oxidation product malondialdehyde, and reduce the oxidative stress damage of the liver of alcoholic liver mice; at the same time, L. pleurotus polysaccharide can also reduce the levels of triglycerides and fatty acids in the liver and serum, effectively improve lipid metabolism disorders, and reduce lipid accumulation; in addition, L. pleurotus polysaccharide can reduce the release of inflammatory factors such as interleukin 6, interleukin 1β and tumor necrosis factor α, promote the body's inflammation level to restore the balance state, reduce liver inflammation, and thus improve the damage of alcohol to liver tissue.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of Lycopodiella pleurotus polysaccharide in preparing medicines for improving alcoholic liver damage. Background Art

[0002] Alcoholic liver damage is a syndrome caused by long-term or heavy drinking, with inflammation as the main response and involving multiple organs of the body. According to statistics, there are about 2 billion people drinking alcohol worldwide, of which no less than 7.5 million people abuse alcohol, and more than 50% of cirrhosis-related mortality can be attributed to alcohol. Currently, treatments for alcoholic liver damage include alcohol withdrawal, nutritional supplements, and drug-assisted therapy. Although existing treatments can control the progression of alcoholic liver disease, patient compliance, side effects of some drugs, and high prices also hinder the treatment effect.

[0003] L. pleurotus, also known as antler mushroom, belongs to the phylum Basidiomycetes, class Agaricaceae, order Agaricales, family Lepiophyllum, and genus Lepiophyllum. It is a new variety of edible and medicinal mushrooms. It is delicious, has a unique taste, and is rich in nutrients. It is very popular in the consumer market. Studies have found that the fruiting bodies of L. pleurotus are rich in protein, rich in amino acids, and have a high proportion of essential amino acids. It is a high-quality protein food. In addition, L. pleurotus has a low fat content and a variety of vitamins and minerals. Modern pharmacological studies have found that L. pleurotus polysaccharide is one of its main active ingredients, which has anti-tumor and immune-enhancing biological activities. At present, there is no report on the application of L. pleurotus polysaccharide in improving alcoholic liver damage. Summary of the invention

[0004] The purpose of the present invention is to provide an application of polysaccharide of lotus leaf in the preparation of a drug for improving alcoholic liver damage, so as to solve the problems existing in the above-mentioned prior art. The present invention has found that polysaccharide of lotus leaf has the effect of improving alcoholic liver damage and has broad market application prospects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides application of Lycopodiella pleurotus polysaccharide in preparing a medicine for improving alcoholic liver damage.

[0007] Furthermore, the drug improves alcoholic liver damage by protecting the liver cell structure and inhibiting liver swelling caused by alcohol.

[0008] Furthermore, the drug improves alcoholic liver damage by downregulating the activity of biological enzymes related to liver cell damage in serum.

[0009] Furthermore, the drug improves alcoholic liver damage by increasing the activity of liver antioxidant enzymes, inhibiting the generation of malondialdehyde, an oxidation product of liver cells, and alleviating oxidative stress damage to the liver.

[0010] Furthermore, the drug improves alcoholic liver damage by reducing the release of inflammatory factors and alleviating liver inflammation.

[0011] Furthermore, the preparation method of the polysaccharide of Lycopodiella cernua comprises the following steps:

[0012] The fruiting bodies of Lycopodiella cernua are added with an aqueous solution containing EDTA-2Na, subjected to ultrasonic treatment, and then subjected to water extraction treatment to obtain an extract;

[0013] After concentrating the extract, adding anhydrous ethanol for precipitation, and then centrifuging to obtain the precipitate, thus obtaining the crude polysaccharide of Lycopodiella cernua;

[0014] The crude polysaccharide of Lycopodiella cernua is subjected to protein removal and dialysis treatment to obtain the polysaccharide of Lycopodiella cernua.

[0015] The present invention also provides a medicine for improving alcoholic liver damage, wherein the active ingredient comprises polysaccharide from Lycopodiella cernua

[0016] Furthermore, the preparation method of the polysaccharide of Lycopodiella cernua comprises the following steps:

[0017] The fruiting bodies of Lycopodiella cernua are added with an aqueous solution containing EDTA-2Na, subjected to ultrasonic treatment, and then subjected to water extraction treatment to obtain an extract;

[0018] After concentrating the extract, adding anhydrous ethanol for precipitation, and then centrifuging to obtain the precipitate, thus obtaining the crude polysaccharide of Lycopodiella cernua;

[0019] The crude polysaccharide of Lycopodiella cernua is subjected to protein removal and dialysis treatment to obtain the polysaccharide of Lycopodiella cernua.

[0020] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0021] Furthermore, the auxiliary materials include emulsifiers, fillers, binders, disintegrants, colorants and / or solubilizers.

[0022] The present invention discloses the following technical effects:

[0023] The present invention finds that the polysaccharide of L. pleurotus has the effect of improving alcoholic liver damage, which is specifically embodied in the following aspects: the polysaccharide of L. pleurotus can effectively protect the liver cell structure, inhibit the liver swelling caused by alcohol, and reduce the liver index; down-regulate the activity of the liver cell damage-related biological enzymes aspartate aminotransferase and alanine aminotransferase in the serum; improve the activity of liver antioxidant enzymes, inhibit the generation of malondialdehyde, an oxidation product of liver cells, and reduce the oxidative stress damage of the liver of alcoholic liver mice; at the same time, the polysaccharide of L. pleurotus can also reduce the levels of triglycerides and fatty acids in the liver and serum, effectively improve lipid metabolism disorders, and reduce lipid accumulation; in addition, the polysaccharide of L. pleurotus can reduce the release of inflammatory factors such as interleukin-6, interleukin-1β and tumor necrosis factor α, promote the body's inflammation level to return to a balanced state, reduce liver inflammation, and thus improve the damage of alcohol to liver tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 Observation pictures of pathological sections of liver tissues of mice in each experimental group of Example 1 (400×); A: blank group; B: model group; C: positive group; D: low-dose group; E: medium-dose group; F: high-dose group; in A to F, the scale bars are all 50 μm;

[0026] Figure 2 The statistical diagram of serum alanine aminotransferase levels in mice in each experimental group of Example 1; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, ** P < 0.01 indicates extremely significant difference;

[0027] Figure 3 The statistical diagram of serum aspartate aminotransferase content of mice in each experimental group of Example 1; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, ** P < 0.01 indicates extremely significant difference;

[0028] Figure 4 The statistical diagram of the total cholesterol content in the liver of mice in each experimental group of Example 1; compared with the blank group, # P<0.05 was considered significant difference. ##P<0.01 was considered to be extremely significant. Compared with the model group, ** P < 0.01 indicates extremely significant difference;

[0029] Figure 5 The statistical diagram of triglyceride content in the liver of mice in each experimental group of Example 1; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference. ** P < 0.01 indicates extremely significant difference;

[0030] Figure 6 The statistical diagram of the low-density lipoprotein cholesterol content in the liver of mice in each experimental group of Example 1; compared with the blank group, # P<0.05 was considered significant difference; compared with the model group, ** P < 0.01 indicates extremely significant difference;

[0031] Figure 7 The statistical diagram of the high-density lipoprotein cholesterol content in the liver of mice in each experimental group of Example 1; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, ** P < 0.01 indicates extremely significant difference;

[0032] Figure 8 The statistical diagram of catalase content in the liver of mice in each experimental group of Example 1; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference. ** P < 0.01 indicates extremely significant difference;

[0033] Fig. 9 The above is a statistical chart of the superoxide dismutase content in the liver of mice in each experimental group of Example 1; compared with the blank group, # P<0.05 was considered significant difference; compared with the model group, ** P < 0.01 indicates extremely significant difference;

[0034] Fig.10 The statistical diagram of glutathione peroxidase content in the liver of mice in each experimental group of Example 1; compared with the blank group, ## P<0.01 was considered to be extremely significant. Compared with the model group, ** P < 0.01 indicates extremely significant difference;

[0035] Fig.11The statistical diagram of the malondialdehyde content in the liver of mice in each experimental group of Example 1; compared with the blank group, ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference. ** P < 0.01 indicates extremely significant difference;

[0036] Fig.12 The statistical diagram of interleukin-6 content in the liver of mice in each experimental group of Example 1; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference. ** P < 0.01 indicates extremely significant difference;

[0037] Fig.13 The statistical diagram of interleukin-1β content in the liver of mice in each experimental group of Example 1; compared with the blank group, ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference. ** P < 0.01 indicates extremely significant difference;

[0038] Fig.14 The statistical diagram of tumor necrosis factor α content in the liver of mice in each experimental group of Example 1; compared with the blank group, # P<0.05 was considered significant difference. ## P < 0.01 indicates extremely significant difference;

[0039] Fig.15 Observation pictures of pathological sections of liver tissues of mice in each experimental group of Example 2 (400×); A: blank group; B: model group; C: positive group; D: low-dose group; E: medium-dose group; F: high-dose group;

[0040] Fig.16 The statistical diagram of serum alanine aminotransferase levels in mice in each experimental group of Example 2; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference. ** P < 0.01 indicates extremely significant difference;

[0041] Fig.17 The statistical diagram of serum aspartate aminotransferase content of mice in each experimental group of Example 2; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, **P < 0.01 indicates extremely significant difference;

[0042] Fig.18 The statistical diagram of the total cholesterol content in the liver of mice in each experimental group of Example 2; compared with the blank group, # P<0.05 was considered significant difference; compared with the model group, * P<0.05 was considered significant difference;

[0043] Fig.19 The statistical diagram of triglyceride content in the liver of mice in each experimental group of Example 2; compared with the blank group, ## P<0.01 was considered to be extremely significant. Compared with the model group, ** P < 0.01 indicates extremely significant difference;

[0044] Fig. 20 The statistical diagram of the low-density lipoprotein cholesterol content in the liver of mice in each experimental group of Example 2; compared with the blank group, ## P<0.01 was considered to be extremely significant. Compared with the model group, ** P < 0.01 indicates extremely significant difference;

[0045] Fig.21 This is a statistical chart of the high-density lipoprotein cholesterol content in the liver of mice in each experimental group of Example 2; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference. ** P < 0.01 indicates extremely significant difference;

[0046] Fig. 22 The statistical diagram of catalase content in the liver of mice in each experimental group of Example 2; compared with the blank group, ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference. ** P < 0.01 indicates extremely significant difference;

[0047] Fig.23 The statistical diagram of superoxide dismutase content in the liver of mice in each experimental group of Example 2; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, ** P < 0.01 indicates extremely significant difference;

[0048] Fig.24 The statistical diagram of glutathione peroxidase content in the liver of mice in each experimental group of Example 2; compared with the blank group, ## P<0.01 was considered to be extremely significant. Compared with the model group,* P<0.05 was considered significant difference. ** P < 0.01 indicates extremely significant difference;

[0049] Fig.25 The statistical diagram of the malondialdehyde content in the liver of mice in each experimental group of Example 2; compared with the blank group, ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference;

[0050] Fig.26 The statistical diagram of interleukin-6 content in the liver of mice in each experimental group of Example 2; compared with the blank group, # P<0.05 was considered significant difference; compared with the model group, * P<0.05 was considered significant difference. ** P < 0.01 indicates extremely significant difference;

[0051] Fig. 27 The statistical diagram of the interleukin-1β content in the liver of mice in each experimental group of Example 2; compared with the blank group, # P<0.05 was considered significant difference. ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference;

[0052] Fig.28 The statistical diagram of tumor necrosis factor α content in the liver of mice in each experimental group of Example 2; compared with the blank group, ## P<0.01 was considered to be extremely significant. Compared with the model group, * P<0.05 was considered significant difference. ** P<0.01 indicated extremely significant difference. DETAILED DESCRIPTION

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0055] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0056] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0057] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0058] The polysaccharide of Lycopodiella cernua used in the following examples was prepared according to the following method:

[0059] (1) According to the solid-liquid ratio of 1 g: 22 mL, the lotus leaf pleurotus erythrorhizon fruiting body (provided by Guizhou Jianrong Biotechnology Co., Ltd.) was added with a 1 wt% EDTA-2Na (disodium ethylenediaminetetraacetic acid) aqueous solution, and ultrasonically treated at a power of 100 W for 30 min, and then water-extracted at 98° C. for 3 h to obtain an extract;

[0060] (2) After concentrating the extract, adding anhydrous ethanol for precipitation, and then centrifuging to obtain the precipitate, thereby obtaining the crude polysaccharide of Lycopodiella cernua;

[0061] (3) The crude polysaccharide of Lycopodiella cernua is subjected to deproteinization and dialysis treatment to obtain the polysaccharide of Lycopodiella cernua.

[0062] Example 1

[0063] 1. Animal Experiment Modeling and Grouping and Drug Administration

[0064] After one week of adaptive feeding, KM mice were randomly divided into a blank group, a model group, a positive group and three polysaccharide groups (low-dose group, medium-dose group and high-dose group), with 15 mice in each group. The blank group and the model group mice were gavaged with 0.10mL / 10g normal saline per mouse; the low-dose group, the medium-dose group and the high-dose group dissolved the polysaccharide in distilled water and gavaged at 0.10mL / 10g, with the dosage of 100, 200 and 400mg / kg respectively; the positive group dissolved the bifendate powder in distilled water and gavaged at 0.10mL / 10g, with the dosage of 150mg / kg (converted according to the daily dosage of adults). The dosage used in each group was safe and had no toxic side effects. During the period, all mice were free to eat and drink water, the weight of the mice was weighed every three days, and the condition of the mice was observed, and the eating and drinking conditions were recorded. The experimental medication lasted for 7 days. Four hours after administration on the 7th day, the model group, low-dose group, medium-dose group, high-dose group and positive group were gavaged with 0.15 mL / 10 g of 56° Beijing Red Star Erguotou, respectively, and the blank group was given 0.15 mL / 10 g of normal saline.

[0065] 2. Animal Handling and Sample Collection

[0066] After the mouse experiment, the mice were fasted but not watered for 12 hours. All mice were weighed, blood was collected from the eyeballs, and the neck was suddenly dead and dissected. The collected whole blood of the mice was collected in a sterile RNAse-free tube and centrifuged twice (3500rpm) at 4°C for 10 minutes each time, and the supernatant was taken. The serum was stored in an ultra-low temperature refrigerator at -80°C until detection. The heart, lungs, liver, kidneys, spleen and part of the lungs obtained by dissection were fixed in 10% neutral formalin buffer, and the other part was placed in a sterile RNAse-free tube and then quickly frozen with liquid nitrogen, and also stored at -80°C for use. The feces were carefully collected from the mouse colon, stored in a sterile RNAse-free tube, and then quickly frozen with liquid nitrogen, and also stored at -80°C for use.

[0067] 3. Polysaccharide from lotus leaf improves liver tissue morphology in mice with acute alcoholic liver injury

[0068] Mouse liver tissue sections were stained with hematoxylin-eosin method and microscopic observation was shown in Figure 2. Figure 1 As shown. Figure 1It can be found that a small amount of hepatocyte fatty degeneration can be seen in the liver tissue of the blank group mice, and small round vacuoles appear in the cytoplasm; a small number of hepatocytes have irregular nuclear morphology; and no obvious inflammatory cell infiltration is observed. In the liver tissue of the model group, a large number of hepatocytes with watery degeneration, enlarged cell bodies, loose and lightly stained cytoplasm, a large number of hepatic sinusoids dilation, abnormalities in the portal area, and inflammatory cell infiltration can be seen. Compared with the model group, the three polysaccharide groups and the positive group have improved, the degree of hepatocyte necrosis has been reduced, and the number of lipid droplets has been significantly reduced. From the results of mouse liver slices, it can be preliminarily judged that the consumption of L. pleurotus is relatively safe and effective, and has a good protective effect on the liver.

[0069] 4. Polysaccharide from lotus leaf improves the levels of alanine aminotransferase and aspartate aminotransferase in mice with acute alcoholic liver injury

[0070] The levels of alanine aminotransferase and aspartate aminotransferase in mouse serum samples were detected using Nanjing Jiancheng kit. The results are shown in Figure 2 and Figure 3 The results showed that compared with the blank group, the serum alanine aminotransferase level in the model group mice increased significantly (P<0.05), while the aspartate aminotransferase level increased extremely significantly (P<0.01), indicating that the model of alcoholic liver injury mice was successfully established. Compared with the model group, the serum alanine aminotransferase and aspartate aminotransferase levels in the high-dose group and the positive group mice decreased extremely significantly (P<0.01).

[0071] 5. Polysaccharide from lotus leaf improves lipid metabolism in mice with acute alcoholic liver injury

[0072] The total cholesterol, triglyceride, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in mouse liver were detected using Nanjing Jiancheng kit. The results are shown in Figure 4-Figure 7 . The results showed that compared with the blank group, the levels of total cholesterol and triglyceride in the liver of the model group mice were extremely significantly increased (P<0.01), low-density lipoprotein cholesterol was significantly increased (P<0.05), and high-density lipoprotein cholesterol was extremely significantly decreased (P<0.01). Compared with the model group, the total cholesterol and low-density lipoprotein cholesterol levels of mice in the polysaccharide groups and the positive group were extremely significantly decreased after oral administration of different doses (P<0.01). After treatment in the positive group and the high-dose group, the triglyceride content of mice was extremely significantly decreased (P<0.01), and the high-density lipoprotein cholesterol content was extremely significantly increased. The above results show that the polysaccharide of the lotus leaf parasol has a good regulatory effect on the lipid metabolism in the liver of mice with alcoholic liver injury.

[0073] 6. Polysaccharide from lotus leaf improves antioxidant enzyme activity in mice with acute alcoholic liver injury

[0074] The contents of catalase, superoxide dismutase and glutathione peroxidase in mouse liver were detected using Nanjing Jiancheng kit. The results are shown in Figure 8-Figure 10 . The results showed that compared with the blank group, the levels of catalase and glutathione peroxidase in the liver of mice in the model group decreased significantly (P<0.01), and the activity of superoxide dismutase decreased significantly (P<0.05). Compared with the model group, the levels of catalase and glutathione peroxidase in the liver of mice in the high-dose group and the positive group decreased significantly (P<0.01). This shows that the polysaccharide of lotus leaf can effectively improve the oxidative stress level of mice with alcoholic liver injury.

[0075] 7. Polysaccharide from lotus leaf improves the production of oxidative products in mice with acute alcoholic liver injury

[0076] The content of malondialdehyde in mouse liver was detected using the Nanjing Jiancheng kit. Fig.11 The results showed that compared with the blank group, the level of malondialdehyde in the liver of the model group mice was significantly increased (P<0.01), and compared with the model group, the level of malondialdehyde in the liver of the high-dose group and the positive group mice was significantly decreased (P<0.01). This shows that the polysaccharide of lotus leaf can effectively improve the oxidative stress level of mice with alcoholic liver injury.

[0077] 8. Polysaccharide from lotus leaf improves inflammation level in mice with acute alcoholic liver injury

[0078] The double antibody sandwich method was used to determine the contents of interleukin-6, interleukin-1β and tumor necrosis factor-α in mouse liver samples using the Jingmei kit. The results are shown in Figure 12-14 . The results showed that compared with the blank group mice, the levels of interleukin 6, interleukin 1β and tumor necrosis factor α in the liver of the model group mice were significantly increased (P<0.01), indicating that alcoholic liver injury can induce liver inflammation in mice. Compared with the model group, the content of interleukin 6 in the liver of the positive group and the high-dose group mice was significantly reduced (P<0.01), and the level of interleukin 1β in the liver of the high-dose group mice was also significantly reduced (P<0.01). The above results show that the polysaccharide of lotus leaf has a good regulatory effect on the level of inflammatory factors in liver cells of mice with alcoholic liver injury.

[0079] In summary, the above experimental results show that the polysaccharide of lotus leaf Lycopodiella pleurotus has the effect of improving acute alcoholic liver injury.

[0080] Example 2

[0081] 1. Animal Experiment Modeling and Grouping and Drug Administration

[0082] After one week of adaptive feeding, KM mice were randomly divided into a blank group, a model group, a positive group and three polysaccharide groups (low-dose group, medium-dose group and high-dose group), with 15 mice in each group. The blank group and the model group mice were gavaged with 0.10mL / 10g normal saline per mouse; the low-dose group, the medium-dose group and the high-dose group dissolved the polysaccharide in distilled water and gavaged at 0.10mL / 10g, with the dosage of 100, 200 and 400mg / kg respectively; the positive group dissolved the bifendate powder in distilled water and gavaged at 0.10mL / 10g, with the dosage of 150mg / kg (converted according to the daily dosage of adults). The dosage used in each group was safe and had no toxic side effects. During the period, all mice were free to eat and drink water, the weight of the mice was weighed every three days, and the condition of the mice was observed, and the eating and drinking conditions were recorded. The experimental medication lasted for 21 days. Four hours after administration on the 21st day, the model group, low-dose group, medium-dose group, high-dose group and positive group were gavaged with 0.10 mL / 10 g of 56° Beijing Red Star Erguotou, respectively, and the blank group was given 0.10 mL / 10 g of normal saline.

[0083] 2. Animal Handling and Sample Collection

[0084] After the mouse experiment, the mice were fasted but not watered for 12 hours. All mice were weighed, blood was collected from the eyeballs, and the neck was suddenly dead and dissected. The collected whole blood of the mice was collected in a sterile RNAse-free tube and centrifuged twice (3500rpm) at 4°C for 10 minutes each time, and the supernatant was taken. The serum was stored in an ultra-low temperature refrigerator at -80°C until detection. Part of the heart, lungs, liver, kidneys, spleen and lungs obtained by dissection were fixed in 10% neutral formalin buffer, and the other part was placed in a sterile RNAse-free tube and then quickly frozen in liquid nitrogen, and also stored at -80°C for use. Feces were carefully collected from the mouse colon, stored in a sterile RNAse-free tube, and then quickly frozen in liquid nitrogen, and also stored at -80°C for use.

[0085] 3. Polysaccharide from lotus leaf improves liver tissue morphology in mice with subacute alcoholic liver injury

[0086] Mouse liver tissue sections were stained with hematoxylin-eosin method and microscopic observation was shown in Figure 2. Fig.15As shown, the results showed that the liver tissue cells of the blank group mice were closely arranged, the hepatocytes were uniform, the hepatocyte plate boundaries were clear, and the cell nuclei were of the same size. In the model group, obvious small lipid droplets were formed in the liver tissue cells, which were widely distributed, with obvious vacuoles, unclear hepatocyte plates, blurred hepatocyte boundaries, and hepatocytes of different sizes. A small amount of blood was visible in some hepatic sinusoids, and some hepatocyte nuclei were clear and sparsely chromatin. There was no obvious inflammatory cell infiltration as a whole, and no obvious necrotic area was observed in the hepatocytes. Compared with the model group, the three polysaccharide groups and the positive group were improved, and the number of lipid droplets was significantly reduced. From the results of mouse liver sections, it can be preliminarily judged that the administration of lotus leaf polysaccharide is relatively safe and effective, and has a good protective effect on the liver.

[0087] 4. Polysaccharide from lotus leaf improves the levels of alanine aminotransferase and aspartate aminotransferase in mice with subacute alcoholic liver injury

[0088] The levels of alanine aminotransferase and aspartate aminotransferase in mouse serum samples were detected using Nanjing Jiancheng kit. The results are shown in Fig.16 and Fig.17 The results showed that compared with the blank group, the serum levels of alanine aminotransferase and aspartate aminotransferase in the model group mice were significantly increased (P<0.01), indicating that the model of alcoholic liver injury mice was successfully established. Compared with the model group, the serum levels of alanine aminotransferase and aspartate aminotransferase in the medium-dose group, high-dose group and positive group mice were significantly decreased (P<0.01).

[0089] 5. Polysaccharide from lotus leaf improves lipid metabolism in mice with subacute alcoholic liver injury

[0090] The total cholesterol, triglyceride, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in mouse liver were detected using Nanjing Jiancheng kit. The results are shown in Figure 18-Figure 21 . The results showed that compared with the blank group, the total cholesterol level in the liver of the model group mice was significantly increased (P<0.05), the triglyceride and low-density lipoprotein cholesterol levels were extremely significantly increased (P<0.01), and the high-density lipoprotein cholesterol was extremely significantly decreased (P<0.01). Compared with the model group, the total cholesterol content of the medium and high-dose groups was significantly reduced after treatment (P<0.05), the triglyceride and low-density lipoprotein cholesterol content was extremely significantly reduced (P<0.01), and the high-density lipoprotein cholesterol in the positive group and the high-dose group was extremely significantly increased (P<0.05). This shows that the polysaccharide of the lotus leaf parasol has a good regulatory effect on the lipid metabolism in the liver of mice with alcoholic liver injury.

[0091] 6. Lotus leaf polysaccharide improves the antioxidant enzyme activity in mice with subacute alcoholic liver injury

[0092] The contents of catalase, superoxide dismutase and glutathione peroxidase in mouse liver were detected using Nanjing Jiancheng kit. The results are shown in Figure 22-Figure 24 The results showed that compared with the blank group, the activities of catalase, superoxide dismutase and glutathione peroxidase in the liver of the model group mice were significantly reduced (P<0.01). After treatment with L. pleurotus polysaccharide, the levels of catalase and glutathione peroxidase were significantly increased (P<0.01), indicating that L. pleurotus polysaccharide can effectively improve the oxidative stress level of mice with alcoholic liver injury.

[0093] 7. Polysaccharide from lotus leaf improves the production of oxidative products in mice with subacute alcoholic liver injury

[0094] The content of malondialdehyde in mouse liver was detected using the Nanjing Jiancheng kit. Fig.25 The results showed that compared with the blank group, the level of malondialdehyde in the liver of the model group mice was extremely significantly increased (P<0.01). After treatment with L. pleurotus polysaccharide, the level of malondialdehyde was significantly reduced (P<0.05), indicating that L. pleurotus polysaccharide can effectively improve the oxidative stress level of mice with alcoholic liver injury.

[0095] 8. Polysaccharide from lotus leaf improves inflammation level in mice with subacute alcoholic liver injury

[0096] The double antibody sandwich method was used to determine the contents of interleukin-6, interleukin-1β and tumor necrosis factor-α in mouse liver samples using the Jingmei kit. The results are shown in Figure 26-Figure 28 . The results showed that compared with the blank group mice, the level of interleukin 6 in the liver of the model group mice was significantly increased (P<0.05), while the levels of interleukin 1β and tumor necrosis factor α were extremely significantly increased (P<0.01), indicating that alcoholic liver injury can induce liver inflammation in mice. Compared with the model group, the levels of interleukin 6 and interleukin 1β in the liver of mice in the medium and high dose groups were significantly reduced (P<0.05); the level of tumor necrosis factor α in the liver of mice in the positive group and high dose group was extremely significantly reduced (P<0.01). The above results show that the polysaccharide of lotus leaf has a good regulatory effect on the level of inflammatory factors in liver cells of mice with alcoholic liver injury.

[0097] In summary, the above experimental results show that the polysaccharide of lotus leaf Lycopodiella pleurotus has the effect of improving subacute alcoholic liver injury.

[0098] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of polysaccharide from lotus leaf in the preparation of a drug for improving alcoholic liver damage, characterized in that: The preparation method of the polysaccharide of Lycopodiella cernua comprises the following steps: The fruiting bodies of Lycopodiella cernua are added with an aqueous solution containing EDTA-2Na, subjected to ultrasonic treatment, and then subjected to water extraction treatment to obtain an extract; After concentrating the extract, adding anhydrous ethanol for precipitation, and then centrifuging to obtain the precipitate, thus obtaining the crude polysaccharide of Lycopodiella cernua; The crude polysaccharide of Lycopodiella cernua is subjected to protein removal and dialysis treatment to obtain the polysaccharide of Lycopodiella cernua.

2. The use according to claim 1, characterized in that: The drug improves alcoholic liver damage by protecting liver cell structure and inhibiting alcohol-induced liver swelling.

3. The use according to claim 1, characterized in that: The drug improves alcoholic liver damage by downregulating the activity of biological enzymes related to liver cell damage in serum.

4. The use according to claim 1, characterized in that: The drug improves alcoholic liver damage by increasing the activity of liver antioxidant enzymes, inhibiting the generation of malondialdehyde, an oxidation product of liver cells, and alleviating oxidative stress damage of the liver.

5. The use according to claim 1, characterized in that: The drug improves alcoholic liver damage by reducing the release of inflammatory factors and alleviating liver inflammation.

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