Lotus leaf lyophyllum decastes active polysaccharide, preparation method thereof and application of lotus leaf lyophyllum decastes active polysaccharide in preparation of medicine for improving medicine-induced liver injury

Abstract

The invention discloses a lyophyllum decastes active polysaccharide, a preparation method thereof and application of the lyophyllum decastes active polysaccharide in preparation of drugs for improving drug-induced liver injury, and relates to the technical field of biological medicines. The preparation method comprises the following steps: with lyophyllum decastes sporocarp as a raw material, extracting by adopting a water extraction and alcohol precipitation method to obtain a lyophyllum decastes crude extract; performing deproteinization treatment on the lyophyllum decastes crude extract, and performing dialysis treatment to obtain lyophyllum decastes crude polysaccharide; and purifying the crude polysaccharide of the lyophyllum decastes through a DEAE-52 cellulose anion exchange chromatography column and a Superdex 16 / 600200pg molecular sieve gel filtration column respectively to obtain the active polysaccharide of the lyophyllum decastes. The lotus leaf lyophyllum decastes active polysaccharide provided by the invention has a good protection effect on drug-induced liver injury, can be used for preparing polysaccharide liver protection agent drugs, liver protection auxiliary materials and functional foods, and has important economic value and market value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an active polysaccharide from Lycopodiella pleurotus ostreatus, a preparation method thereof, and application thereof in preparing a medicament for improving drug-induced liver injury. Background Art

[0002] As a crucial metabolic and detoxification organ in the human body, the liver is subject to complex and diverse mechanisms of injury. Based on the causative factors, it can be categorized into metabolic, infectious, and drug-induced types. Metabolic liver injury, characterized by lipid metabolism disorders, includes nonalcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (ANFLD). The former is characterized by the accumulation of excessive free fatty acids in the liver parenchyma, triggering mitochondrial dysfunction and excessive production of reactive oxygen species, thereby driving hepatocyte inflammatory necrosis and fibrosis. The latter exhibits unique ethanol metabolic toxicity, with its ethanol metabolite, acetaldehyde, directly damaging mitochondria, inhibiting fatty acid oxidation and aggravating liver fibrosis. Among infectious liver injury types, hepatotropic viruses are the primary pathogens that infect cells, causing liver damage through direct cell death and inducing immune responses. Drug-induced liver injury (DILI) differs from these two types in that it is primarily caused by hepatocellular toxicity induced by drugs or their metabolites, or by a hypersensitivity reaction of the liver to the drug. It is one of the most common adverse drug reactions. Among them, acetaminophen (APAP) overdose is a common cause of drug-induced liver injury and is also recognized as an ideal model for studying drug-induced liver injury.

[0003] Currently, the only clinically approved antidote for acetaminophen is N-acetylcysteine, but it has a certain time limit and is accompanied by side effects such as vomiting, headache, and nausea. Therefore, it is of great significance to find potential drugs that are non-toxic and can improve drug-induced liver injury.

[0004] Polysaccharides from edible and medicinal fungi have become an important source for natural drug development due to their minimal toxicity, low immunogenicity, and significant pharmacological effects. The nutrient-rich mushroom (Lyophyllum decastes) possesses high edible and medicinal value. Its polysaccharides exhibit a variety of pharmacological activities, including anti-tumor, lipid-lowering, and immune-enhancing activities. However, there are currently no reports on whether polysaccharides from L. decastes can protect against acetaminophen-induced liver damage or inhibit its hepatotoxic effects. Therefore, in-depth research and development of the medicinal properties of L. decastes is of great significance. Summary of the Invention

[0005] The present invention aims to provide an active polysaccharide from L. shimejiensis, a preparation method thereof, and its use in the preparation of a drug for ameliorating drug-induced liver injury, thereby overcoming the aforementioned problems of the prior art. This active polysaccharide from L. shimejiensis has a strong protective effect against drug-induced liver injury and can be used to prepare polysaccharide liver protective drugs, liver-protecting excipients, and functional foods, possessing significant economic and market value.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing active polysaccharides from Lyophyllum nucifera, comprising the following steps:

[0008] The fruiting bodies of Lycopodiella oleracea were used as raw materials and the crude extract of Lycopodiella oleracea was obtained by water extraction and alcohol precipitation.

[0009] Deproteinizing the crude extract of Lycopodiella cernua and then performing a dialysis treatment to obtain crude polysaccharide of Lycopodiella cernua;

[0010] The crude polysaccharide of the lotus leaf Lycopodiella oleracea is purified by a DEAE-52 cellulose anion exchange chromatography column and a Superdex 16 / 600 200 pg molecular sieve gel filtration column to obtain the active polysaccharide of the lotus leaf Lycopodiella oleracea.

[0011] Furthermore, the water extraction and alcohol precipitation method comprises the following steps:

[0012] After crushing the fruiting body of the lotus leaf parasol, extracting it with water to obtain a water extract;

[0013] After concentrating the water extract, three times the volume of anhydrous ethanol was added, mixed evenly and allowed to stand, and then centrifuged to collect the precipitate, and dried to obtain the crude extract of the lotus leaf parasol mushroom.

[0014] Furthermore, the deproteinization treatment is performed using Sevag reagent.

[0015] Furthermore, a 3500Da cut-off dialysis bag is used for the dialysis treatment.

[0016] The present invention also provides an active polysaccharide of Lycopodiella oleracea prepared according to the above preparation method.

[0017] The present invention also provides the use of the active polysaccharide of Lyophyllum nucifera in preparing a medicine for improving drug-induced liver injury.

[0018] The present invention also provides the use of the active polysaccharide of Lycopodiella nucifera in the preparation of a health product having an auxiliary protective effect on drug-induced liver injury.

[0019] Furthermore, the drug-induced liver injury is liver injury caused by acetaminophen.

[0020] The present invention also provides a medicine for improving drug-induced liver injury, wherein the active ingredient includes the above-mentioned active polysaccharide of Lyophyllum nucifera.

[0021] The present invention also provides a health product with auxiliary protective effect on drug-induced liver damage, wherein the active ingredient includes the above-mentioned active polysaccharide of Lyophyllum nucifera.

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

[0023] The present invention obtains an active polysaccharide component, LDP1, from the natural fungus resource, Lycopodiella pleurotifolia. Animal experiments have also shown that this polysaccharide can improve liver tissue and cell damage, inhibit inflammatory responses, reduce the production of lipid peroxidation products, increase intestinal flora diversity and the relative abundance of beneficial bacteria, maintain intestinal microbial homeostasis, and exhibit a protective effect against drug-induced liver injury (particularly acetaminophen-induced liver injury). LDP1 can be used to prepare polysaccharide liver protective drugs, liver-protecting excipients, and functional foods, possessing significant economic and market value. 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0025] Figure 1 The elution curves of polysaccharide from lotus leaf are shown in Figure 1. A is the elution curve of DEAE-52; B is the elution curve of Superdex16 / 600200pg;

[0026] Figure 2 This is the monosaccharide composition analysis diagram of LDP1;

[0027] Figure 3 is the glucose standard curve of LDP1;

[0028] Figure 4 is the infrared spectrum of LDP1;

[0029] Figure 5 is the molecular weight spectrum of LDP1;

[0030] Figure 6 Figures 2 and 3 show the body weight, organ indexes, and histopathological sections of mice under the action of LDP1; A is the trend graph of mouse body weight; B is the statistical graph of liver index (###P<0.001 vs Control; ** P < 0.01and *P<0.05 vs Model); C is a microscopic observation of pathological changes in mouse liver tissue (HE, ×400);

[0031] Figure 7 The test results of the effect of LDP1 on serum transaminases and oxidative stress kinases in mice are shown in Figure 1. A is a statistical graph showing serum ALT levels; B is a statistical graph showing serum AST levels; C and D are statistical graphs showing serum and liver GSH-Px levels, respectively; E and F are statistical graphs showing serum and liver SOD levels, respectively; G and H are statistical graphs showing serum and liver MDA levels, respectively; I and J are statistical graphs showing serum and liver CAT levels, respectively. #### P < 0.0001and ### P < 0.001 vs Control; **** P < 0.0001and *** P < 0.001and ** P < 0.01and * P < 0.05 vs Model;

[0032] Figure 8 Figure 1 is a graph showing the detection results of inflammatory factors in mice under the action of LDP1; A and B are statistical graphs of TNF-α levels in serum and liver, respectively; C and D are statistical graphs of IL-1β levels in serum and liver, respectively; E and F are statistical graphs of IL-6 levels in serum and liver, respectively; G and H are statistical graphs of IL-10 levels in serum and liver, respectively; #### P < 0.0001 vs Control; **** P < 0.0001and ** P < 0.01and * P < 0.05 vs Model;

[0033] Figure 9 is the box plot of Alpha diversity index;

[0034] Figure 10 is the principal component analysis (PCoA) graph at the OUTs level;

[0035] Figure 11 is the relative abundance histogram at the phylum level;

[0036] Figure 12 Heatmap of species composition at the genus level;

[0037] Figure 13 Venn diagrams were analyzed for OTUs;

[0038] Figure 14 Predict abundance maps for MetaCyc;

[0039] Figure 15 Pie chart of serum metabolite classification;

[0040] Figure 16 This is the PCA positive ion mode analysis diagram of serum samples;

[0041] Figure 17 This is the PCA negative ion mode analysis diagram of serum samples;

[0042] Figure 18 Univariate volcano plot for differential metabolites - positive ion mode;

[0043] Figure 19 Univariate volcano plot for differential metabolites - negative ion pattern;

[0044] Figure 20 This is the Venn analysis diagram of differential metabolites in mouse serum;

[0045] Figure 21 OPLS-DA analysis of mouse serum samples; A, C, and E are OPLS-DA analysis graphs of the CTRLvs Model group, Modelvs LDP1 group, and Model vs LDP1 group in positive ion mode, respectively; B, D, and F are OPLS-DA analysis graphs of the CTRLvsModel group, Model vs LDP1 group, and Model vs LDP1 group in negative ion mode, respectively;

[0046] Figure 22 Correlation heat map of differential metabolites - positive ion mode;

[0047] Figure 23 is the heat map of correlation of differential metabolites-negative ion mode;

[0048] Figure 24 Bubble diagram for enrichment analysis of mouse metabolic pathways (CTRL and Model);

[0049] Figure 25 Bubble diagram for metabolic pathway enrichment analysis (Model and LDP1). DETAILED DESCRIPTION

[0050] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0051] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0052] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0053] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0054] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0055] Example 1 Preparation of active polysaccharide from Lycopodiella oleracea

[0056] (1) The fruiting bodies of the lotus leaf parasol were dried and crushed. 100 g of the lotus leaf parasol powder was weighed and added to 3 L of water at a solid-liquid ratio of 1:30. The extract was then extracted at 89°C for 3 h. The extraction was repeated three times. The three filtrates were combined and concentrated by heating to one-tenth of the original volume. Three times the volume of anhydrous ethanol was added and mixed evenly. The extract was then allowed to stand at 4°C for 24 h. After standing, the extract was centrifuged (8000 r / min, 8 min) to collect the precipitate, which was then dried to obtain the lotus leaf parasol crude extract.

[0057] (2) After dissolving the crude extract of P. pleurotus ostreatus with deionized water, an equal volume of Sevag reagent (chloroform: n-butanol = 3:1) was added, the two were thoroughly mixed, centrifuged, and the middle protein layer and the lower organic solvent were discarded. The above steps were repeated 3 times, and the supernatant was collected. After dialyzing through a 3500Da cutoff dialysis bag to remove small molecules, the supernatant was concentrated and freeze-dried to obtain the crude polysaccharide of P. pleurotus ostreatus.

[0058] (3) Weigh 100 mg of crude polysaccharide from the lotus leaf parasol and dissolve it in 5 mL of ultrapure water. Load the sample onto a DEAE-52 cellulose anion exchange chromatography column (2.6 cm × 20 cm). Elute with ultrapure water, 0.1, 0.3, and 0.5 mol / L NaCl solution, respectively, at an elution rate of 1 mL / min. Collect one test tube every 5 minutes. Determine the polysaccharide presence interval using the phenol-sulfuric acid method, collect the effective fraction, concentrate by dialyzing, freeze-dry, and store in a dry place. The results are as follows: Figure 1 As shown in A.

[0059] (4) 100 mg of polysaccharide purified by DEAE-52 cellulose anion exchange chromatography column was dissolved in 6 mL of ultrapure water and loaded onto a molecular sieve gel filtration column Superdex 16 / 600 200 pg (1.6 cm × 60 cm). The column was eluted with ultrapure water at a rate of 0.5 mL / min, and one tube was collected every 3 minutes. The polysaccharide was determined in the presence range, dialyzed and concentrated, freeze-dried and stored, and named LDP1. The results are shown in the figure below. Figure 1 As shown in B.

[0060] Example 2 Analysis of Monosaccharide Composition of Polysaccharide LDP1 from Lotus Leaf

[0061] (1) Sample pretreatment: Accurately weigh 20 mg of LDP1 sample into a clean chromatographic vial, add 2 mL of 2 M trifluoroacetic acid solution, and heat at 121°C for 2 hours. Purge with nitrogen, dry, then rinse with 99.99% methanol, dry again, and repeat the methanol rinse 2-3 times to completely remove the trifluoroacetic acid. Dissolve the sample in sterile water and transfer to a chromatographic vial for analysis.

[0062] (2) Preparation of standard substances: Accurately weigh fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, and guluronic acid, and add deionized water to prepare a 10 mg / mL standard solution. Then, take an appropriate amount of the standard solution and mix them to prepare a standard solution with a maximum index concentration of 60 μg / mL, 50 μg / mL, or 40 μg / mL. Prepare the standard solution according to the following concentration gradient.

[0063] Table 1 Concentration information of monosaccharide standard mixture

[0064]

[0065] (3) All prepared sugar solutions were filtered through a 0.22 μm microporous membrane and then analyzed by high performance liquid chromatography (HPLC). The injection volume was 5 μL. Mobile phase A (ddH2O), mobile phase B (0.1M NaOH), mobile phase C (0.1MNaOH, 0.2MNaAc), flow rate 0.5mL / min; column temperature 30℃; elution gradient: 0min A phase / B phase / C phase (95:5:0), 26min A phase / B phase / C phase (85:5:10), 42min A phase / B phase / C phase (85:5:10), 42.1min A phase / B phase / C phase (60:0:40), 52min A phase / B phase / C phase (60:40:0), 52.1min A phase / B phase / C phase (95:5:0), 60min A phase / B phase / C phase (95:5:0). The results are shown in Figure 5. Figure 2 As shown, LDP1 is mainly composed of galactose, mannose, fucose, and glucose, with molar percentages of 63.60%, 18.78%, 15.34%, and 2.28%, respectively.

[0066] Example 3 Determination of total sugar content of polysaccharide LDP1 from lotus leaf

[0067] The glucose standard was dried at 90°C to constant weight, 1.000 g was accurately weighed, and distilled water was added to a volumetric flask to make the prepared 10 mg / mL standard stock solution. After accurate aspiration, the volume was adjusted to 1 mg / mL glucose standard solution. 0, 10, 20, 30, 40, 50, 60, 70, and 80 μL were respectively transferred to the tube, and distilled water was added to 200 μL. A separate EP tube was taken, 200 μL of 1 mg / mL LDP1 polysaccharide solution was added, and 150 μL of 5% phenol solution was added to each tube. The mixture was mixed, and 0.5 mL of concentrated sulfuric acid was added quickly and slowly. After mixing, the mixture was allowed to stand for 30 min to cool to room temperature. 200 μL of each sample was placed in a 96-well plate and the OD value was detected by enzyme marker. 490 The numerical values are recorded according to the glucose standard curve ( Figure 3 ) The total sugar content of LDP1 was calculated to be 75.23%.

[0068] Example 4 Infrared spectral analysis of polysaccharide LDP1 from lotus leaf

[0069] (1) Sample processing: 1-5 mg of polysaccharide sample was placed in a vacuum drying oven (60°C, 24 h) to completely remove moisture; 2 mg of the dried sample was evenly ground with 200 mg of dry KBr powder in an agate mortar. The grinding process was carried out under a dry lamp to prevent moisture absorption.

[0070] (2) Tablet pressing and scanning: The ground mixture was pressed into thin sheets using a tablet press with a pressure of 30 MPa for about 30 seconds. The tablets were taken out and placed in a Fourier transform infrared spectrometer for scanning with a scanning range of 4000–400 cm -1 , the resolution is set to 4cm -1 The sample was collected 3 times and the average spectrum was obtained. Figure 4 As shown, it shows that LDP1 contains -OH stretching vibration, C-H stretching vibration, C=O stretching vibration, C=H stretching vibration and CO angle vibration, which are all characteristic absorption peaks of polysaccharides.

[0071] Example 5 Molecular Weight Detection of Polysaccharide LDP1 from Lotus Leaf

[0072] The polysaccharide sample was dissolved in 0.1M NaNO3 aqueous solution (containing 0.02% NaN3) with a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm before being tested on the machine. The specific chromatographic column and elution conditions were: gel exclusion chromatography columns Ohpak SB-805HQ (300×8mm) and Ohpak SB-803HQ (300×8mm) were connected in series. The column temperature was 45°C, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min. The results are shown in Figure 2. Figure 5 As shown, the molecular weight of the polysaccharide is 12.207 kDa.

[0073] Example 6 Methylation Analysis of Polysaccharide LDP1 from Lycopodiella oleracea

[0074] (1) Accurately weigh 3 mg of LDP1 sample, dissolve it in 500 μL of dimethyl sulfoxide, add 1 mg of NaOH, incubate for 30 min, and then add 50 μL of iodomethane solution and react for 1 h.

[0075] (2) Add 1 mL of deionized water and 2 mL of dichloromethane, mix well, and centrifuge. Discard the aqueous phase and repeat the water wash three times. Aspirate the lower dichloromethane phase and blow dry with nitrogen. Dissolve in 100 μL of 2 M trifluoroacetic acid, react at 121°C for 90 min, and evaporate to dryness at 30°C.

[0076] (3) Add 50 μL of 2M ammonia and 1M NaBD4, mix well, react at room temperature for 2.5 h, add 20 μL of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL of methanol, and blow dry with nitrogen.

[0077] (4) Add 250 μL of acetic anhydride, vortex mix, and react at 100°C for 2.5 h. Add 1 mL of water and let stand for 10 min. Add 500 μL of dichloromethane, vortex mix, centrifuge, discard the aqueous phase, and repeat the water wash three times. Remove the dichloromethane phase and analyze it on a GC-MS machine.

[0078] The results are shown in Table 2, indicating that the main glycosidic bonds of the polysaccharide LDP1 from the lotus leaf are 6-Gal, 2,6-Gal, t-Man, and t-Fuc, with molar ratios of 33.41%, 25.98%, 23.68%, and 7.67%.

[0079] Table 2 Analysis results of LDP1 bonding structure

[0080]

[0081] The following experiments demonstrate the medical uses of the polysaccharide obtained from the lotus leaf.

[0082] Test example

[0083] 1. Trial Grouping

[0084] Sixty 6-week-old male Kunming mice were randomly divided into six groups: blank control group (Ctrl), model group (Model), positive group (PC), high-dose polysaccharide administration group (LDP1-H), medium-dose polysaccharide administration group (LDP1-M) and low-dose polysaccharide administration group (LDP1-L).

[0085] The blank control group, model group, and positive control group were gavaged with normal saline. The high-, medium-, and low-dose polysaccharide groups were gavaged with LDP1 at doses of 400 mg / kg, 200 mg / kg, and 100 mg / kg, respectively. The treatment was administered once daily for 21 consecutive days. On the 21st day, the model group was gavaged with normal saline 2 hours before being gavaged with 200 mg / kg of APAP solution. The positive control group was gavaged with 200 mg / kg of acetylcysteine 1 hour beforehand, followed by 200 mg / kg of APAP solution.

[0086] 2. Effects of LDP1 polysaccharide from lotus leaf on body weight, organ indexes and histopathology in mice with liver injury

[0087] During the experiment, the growth of mice was observed every day and their weights were weighed every two days. All mice were fasted for 16 hours before the last administration. After the animal experiment, the mice were anesthetized by intraperitoneal injection and killed by cervical dislocation. About 1.5 mL of blood was collected from each mouse by eyeball bleeding. Centrifuge at 4000 r / min for 10 minutes, take the supernatant, centrifuge twice, and obtain serum samples, which were stored at -80°C for later use. After blood collection, the mice were cleaned with 75% alcohol, and the contents of the mouse cecum were collected by dissection, sealed with cell cryopreservation tubes, quickly frozen with liquid nitrogen, and then stored at -80°C for later use. The liver was immediately collected, rinsed with physiological saline, and excess water was absorbed with filter paper. The organ index was calculated by weighing: organ index (mg / g) = organ mass (mg) / mouse body weight (g).

[0088] The results of mouse body weight and organ index tests are as follows Figure 6 As shown in A and B, the body weight of mice in each group showed an upward trend. After administration of LDP1, the liver index of mice decreased significantly, with the medium dose having a better effect, tending to be similar to that of mice in the normal group.

[0089] The liver tissue was divided into two halves, quickly frozen in liquid nitrogen, and stored at -80°C for later use, while the other half was fixed with 4% paraformaldehyde for storage.

[0090] The fixed liver tissue was dehydrated and transparentized with different concentrations of ethanol and xylene, embedded in paraffin, and made into 5μm sections. Hematoxylin and eosin were used for HE staining. After dehydration and transparency, each group of sections was observed under an optical microscope, photographed, and the overall condition of the liver tissue, the structural morphology of the cells, the integrity of the nuclear structure, and the infiltration of inflammatory cells were recorded. Figure 6 As shown in middle C, the results showed that LDP1 could improve liver tissue structural abnormalities and cell necrosis.

[0091] 3. Effects of LDP1 polysaccharide from lotus leaf on serum transaminases and oxidative stress kinases in mice with liver injury

[0092] Serum samples were taken from each group of mice to detect the levels of ALT, AST, GSH-Px, SOD, MDA and CAT related cytokines in the serum. The frozen liver tissue was slowly thawed, rinsed with normal saline, and excess water was absorbed with filter paper. It was mixed with 9 times the volume of normal saline and ground evenly in a 5mL centrifuge tube. During the homogenization, it was placed on ice for 3 minutes every 30 seconds to prevent protein degradation. Centrifuged at 12000rpm for 5 minutes, the supernatant was taken, and repeated twice to obtain liver tissue homogenate. The levels of GSH-Px, SOD, MDA, and CAT related cytokines in the mouse liver homogenate were detected. The results are as follows Figure 7 As shown in the results, LDP1 can reduce the levels of ALT and AST in mouse serum, significantly enhance the activity of GSH-Px, SOD, and CAT in mouse serum and liver, make the MDA level close to normal, and has a good regulatory effect on the oxidative stress response in the liver.

[0093] 4. Effects of LDP1 polysaccharide from lotus leaf on inflammatory factors in mice with liver injury

[0094] Serum samples were taken from mice in each group to detect the levels of TNF-α, IL-1β, IL-10 and IL-6 related cytokines in the serum. The frozen liver tissue was taken, rinsed with saline, and excess water was absorbed with filter paper. It was mixed with 9 times the volume of saline and ground evenly in a 5mL centrifuge tube. During the homogenization, it was placed on ice for 3 minutes every 30 seconds to prevent protein degradation. Centrifuged at 12000rpm for 5 minutes, the supernatant was taken, and repeated twice to obtain liver tissue homogenate, and the levels of TNF-α, IL-1β, IL-10 and IL-6 related cytokines in mouse liver tissue were detected. The results are as follows Figure 8 As shown, LDP1 can reduce the release of TNF-α, IL-1β, IL-6 and IL-10 in mice, thereby inhibiting the body's inflammatory response.

[0095] 5. Effects of LDP1 polysaccharide from lotus leaf on intestinal flora in mice with liver injury

[0096] The cecal contents collected from each group were added to sterile PBS buffer and vortexed to mix well. Centrifugation was performed (12,000 rpm, 5 minutes). The supernatant or precipitate was collected and DNA was extracted using a DNA extraction kit according to the manufacturer's instructions. DNA concentration and purity were analyzed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The V3-V4 region of the bacterial 16S rRNA gene was amplified by polymerase chain reaction (PCR) using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Product quality was detected by 2% agarose gel electrophoresis. Finally, the product was analyzed using a micro-fluorimeter Quantus. TM Fluorometer was used to quantify the purified product. Paired-end sequencing of cecal content DNA was performed using the Illumina platform. Sequence denoising and clustering were performed based on the results of the original sequencing data. VSEARCH was used to cluster the sequences into operational taxonomic units (OTUs), and high-quality sequences were based on a similarity of 97%. Species annotation was performed on the OTUs of different units. The alpha diversity and beta diversity of the samples were evaluated, and the diversity of species within and between habitats was analyzed respectively. The results are shown in Figure 2. Figures 9-14As shown, LDP1 can improve the diversity and richness of intestinal flora in mice; reverse the imbalance of Firmicutes and Bacteroidetes flora in the mouse intestine; reduce the relative abundance of harmful bacterial genera such as Bacteroides, Prevotella, and Sutterella, and increase the abundance of Oscillospira and Roseburia, which play a beneficial role in various liver diseases; alleviate the disorder of species composition in the mouse intestine; at the same time, LDP1 may alleviate APAP-induced acute liver injury by regulating the metabolite betulinic acid and ABC transporter pathway.

[0097] 6. Effects of LDP1 polysaccharide from lotus leaf on serum metabolites in mice with liver injury

[0098] Take 50 μL of serum and place it in a 1.5 mL centrifuge tube. Add 500 μL of water and 100 mg of glass beads, homogenize for 1 minute, centrifuge at 4°C 12000 rpm for 10 minutes, take 200 μL of supernatant, add 100 μL of 15% phosphoric acid, and then add 20 μL of 375 μg / mL internal standard (4-methylvaleric acid) solution and 280 μL of ether and homogenize for 1 minute. Centrifuge at 4°C 12000 rpm for 10 minutes, and take the supernatant for testing.

[0099] Samples were analyzed using a Thermo Trace 1300 gas phase system with an Agilent HP-INNOWAX capillary column (30 m × 0.25 mm ID × 0.25 μm). Split injection was performed with a 1 μL injection volume and a split ratio of 10:1. The inlet temperature was 250°C, the ion source temperature was 300°C, and the transfer line temperature was 250°C. The temperature program started at 90°C, then increased at 10°C / min to 120°C, then at 5°C / min to 150°C, and finally increased at 25°C / min to 250°C and held for 2 min. Helium was used as the carrier gas at a flow rate of 1.0 mL / min.

[0100] The samples were separated by a Thermo Trace 1300 gas chromatography system and analyzed by mass spectrometry using a Thermo ISQ 7000 mass spectrometer with an electron impact ionization (EI) source, SIM scanning mode, and an electron energy of 70 eV.

[0101] (1) Using local self-built databases and public databases, we searched the databases and matched the metabolites with the retention time, molecular mass (molecular mass error within <10ppm), secondary fragmentation spectra, collision energy and other information in the database to identify the metabolites in the serum samples. We counted the number of metabolites identified in each serum sample group and classified them according to their chemical classification. The results are as follows: Figure 15As shown, a total of 1,018 metabolites were identified using a combination of positive and negative ion modes, with 600 identified in positive ion mode and 418 in negative ion mode. All metabolites were divided into 11 superclasses, with lipids and lipoid molecules accounting for the highest proportion, at 33.202%, followed by organic acids and their derivatives, at 25.442%. Heterocyclic compounds accounted for 11.690%, followed by aromatic compounds at 8.841%. The lowest proportions were organometallic compounds and lignans, neolignans, and related compounds, each accounting for only 0.196%.

[0102] PCA analysis generates new characteristic variables by linearly combining metabolite variables according to certain weights. The data of each group are classified by the main new variables, samples with poor repeatability are removed, and the differences between samples are analyzed. Figure 16-17 As shown, the R 2 The x-values were 0.576 and 0.551, respectively, and the PCA model interpretability was greater than 0.5, indicating good stability of the system. In positive ion mode, the CTRL and Model groups were completely separated, while the LDP1 group and CTRL group were close. In negative ion mode, the Model and CTRL groups were not completely separated, but the LDP1 and CTRL groups remained close and had some overlap. Metabolite samples in the Model group changed compared to the blank control group, while after LDP1 administration, the variable composition of the samples was closer to that of the blank control group.

[0103] OPLS-DA analysis combined with orthogonal signal correction technology removes information irrelevant to the classification, so that the relevant information is mainly concentrated on the first prediction component. Without reducing the predictive ability of the model, it effectively reduces the complexity of the model and enhances the explanatory power of the model, thereby maximizing the difference between the groups. As shown in Table 3, the R 2 Y, Q 2 The parameters are all greater than 50%, and the model fitting accuracy is high. Figure 21 As shown in the figure, in the four groups of CTRL, Model, LDP1, and PC, samples in the same group clustered on the same side, while samples between groups were clearly distinguished, indicating that the experimental sample model was effectively established and that there were differences in the metabolites of samples in each group.

[0104] Table 3 OPLS-DA model parameters for positive and negative ion modes

[0105]

[0106] Univariate statistical analysis was performed using the fold variation analysis method and T-test to calculate the P value. Differential analysis was performed on all metabolites detected in the positive and negative ion modes. The P value < 0.05 was used as the screening criterion, and the data results were visualized in the form of a volcano plot. The differential metabolites of the Model group and the LDP1 group were screened and compared. The results are as follows Figure 18-19 As shown in the figure, in the positive ion mode, a total of 177 differential metabolites were detected, of which 126 were significantly upregulated and 51 were significantly downregulated. In the negative ion mode, a total of 79 differential metabolites were detected, of which 49 were significantly upregulated and 30 were significantly downregulated.

[0107] Using strict OPLS-DAVIP>1 and P value<0.05 as the screening criteria for important variables and significant differential metabolites, the differential metabolites in the cecal contents of mice in the CTRL group and the Model group, and the Model group and the LDP1 group were analyzed ( Figure 21 ), and the number of common and unique substances between groups was compared using a Venn diagram. Figure 20 As shown, there were 57 significantly different metabolites between the cecal contents of mice in the CTRL group and the Model group, and 16 significantly different metabolites between the cecal contents of mice in the Model group and the LDP1 group, and there were 3 significantly different metabolites in total between the two groups.

[0108] (2) Correlation and pathway analysis of differential metabolites

[0109] By calculating the Pearson correlation coefficient between all differential metabolites, and observing the linear relationship between the two metabolites, we can determine whether there is a correlation between the two. At the same time, we use the function to perform statistical tests on the metabolite association analysis. The test standard is: if the correlation coefficient tends to 1 or -1, and Pvalue < 0.05, there is a significant correlation. In the differential metabolite heat map, if the metabolite change trends are the same, it is a positive correlation, represented by red; if the change trends are opposite, it is a negative correlation, represented by blue. The results are as follows Figure 22-23 As shown in the results, betulinic acid was screened for metabolites positively correlated with betulinic acid: 1-piperazinecarboxamide, 4-(3-chloro-2-pyridinyl)-n-[4-(1,1-dimethylethyl)phenyl]-, and N2-Acetyl-L-ornithine. Betulinic acid, a lupane-type pentacyclic triterpenoid, can reduce serum TLR-9, NF-κB, IL-18, and MDA levels in rats in an APAP-induced liver injury model. By improving tissue redox systems and reducing lipid peroxidation in the liver, it has a protective effect against non-alcoholic fatty liver disease.

[0110] KEGG enrichment analysis uses Fisher's exact test to analyze and calculate the significance level of metabolite enrichment in each pathway, thereby determining the metabolic and signal transduction pathways that are significantly affected. Based on the KEGG enrichment results, the degree of enrichment is measured by combining the Rich factor, FDR value, and the number of metabolites enriched in this pathway, and a bubble chart is displayed. The results are as follows Figure 24-25 As shown in the figure, 20 related metabolic pathways were found after comparing the CTRL group with the Model group, among which the bile secretion pathway was the most enriched pathway. After administration of LDP1, changes in related metabolites were suggested to be related to propionate metabolism, pyrimidine metabolism, arginine biosynthesis, and ABC transporters.

[0111] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing active polysaccharides from Lycopodiella oleracea, characterized in that: The following steps are involved: The fruiting bodies of Lycopodiella oleracea were used as raw materials and the crude extract of Lycopodiella oleracea was obtained by water extraction and alcohol precipitation. Deproteinizing the crude extract of Lycopodiella cernua and then performing a dialysis treatment to obtain crude polysaccharide of Lycopodiella cernua; The crude polysaccharide of the lotus leaf Lycopodiella oleracea is purified by a DEAE-52 cellulose anion exchange chromatography column and a Superdex 16 / 600 200 pg molecular sieve gel filtration column to obtain the active polysaccharide of the lotus leaf Lycopodiella oleracea.

2. The preparation method according to claim 1, characterized in that The water extraction and alcohol precipitation method comprises the following steps: After crushing the fruiting body of the lotus leaf parasol, extracting it with water to obtain a water extract; After concentrating the water extract, three times the volume of anhydrous ethanol was added, mixed evenly and allowed to stand, and then centrifuged to collect the precipitate, and dried to obtain the crude extract of the lotus leaf parasol mushroom.

3. The preparation method according to claim 1, characterized in that The deproteinization treatment was performed using Sevag reagent.

4. The preparation method according to claim 1, characterized in that The dialysis treatment was performed using a 3500Da cut-off dialysis bag.

5. An active polysaccharide from Lycopodiella oleracea prepared by the method according to any one of claims 1 to 4.

6. Use of the active polysaccharide of Lycopodiella pleurotus ostreatus as claimed in claim 5 in the preparation of a drug for improving drug-induced liver injury.

7. Use of the active polysaccharide of Lycopodiella pleurotus ostreatus as claimed in claim 5 in the preparation of a health product having an auxiliary protective effect against drug-induced liver injury.

8. The use according to claim 6 or 7, characterized in that The drug-induced liver injury is liver injury caused by acetaminophen.

9. A drug for improving drug-induced liver injury, characterized in that: The active ingredient comprises the active polysaccharide from the lotus leaf mushroom according to claim 5.

10. A health product with auxiliary protective effect on drug-induced liver injury, characterized in that: The active ingredient comprises the active polysaccharide from the lotus leaf mushroom according to claim 5.

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