A lycoperdon pygmaeus active polysaccharide, a preparation method thereof and application thereof in preparing drugs for improving drug-induced liver injury

Abstract

The application discloses a Lyophyllum connatum active polysaccharide, a preparation method thereof and application of the Lyophyllum connatum active polysaccharide in preparation of a medicine for improving drug-induced liver injury, and relates to the technical field of biological medicines. The preparation method comprises the following steps: taking a Lyophyllum connatum fruiting body as raw material, and extracting a Lyophyllum connatum crude extract by using a water extraction and alcohol precipitation method; after the Lyophyllum connatum crude extract is subjected to a deproteinization treatment, the Lyophyllum connatum crude extract is subjected to a dialysis treatment to obtain a Lyophyllum connatum crude polysaccharide; and the Lyophyllum connatum crude polysaccharide is purified through a DEAE-52 cellulose anion exchange column and a Superdex 16 / 600 200 pg molecular sieve gel filtration column to obtain the Lyophyllum connatum active polysaccharide. The Lyophyllum connatum active polysaccharide provided by the application has a good protective effect on drug-induced liver injury, can be used for preparing polysaccharide liver-protecting agent medicines, liver-protecting auxiliary materials and functional foods, and has important economic value and market value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an active polysaccharide from lotus leaf, its preparation method, and its application in the preparation of drugs to improve drug-induced liver injury. Background Technology

[0002] The liver, as a vital metabolic and detoxification organ, suffers from complex and diverse damage mechanisms, which can be categorized into major types based on the causative factors, including metabolic, infectious, and drug-induced liver injury. Metabolic liver injury is characterized by lipid metabolism disorders, including non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (ALF). NAFLD is caused by the accumulation of excessive free fatty acids in the liver parenchyma, leading to mitochondrial dysfunction and excessive reactive oxygen species production, thereby driving hepatocyte inflammation, necrosis, and fibrosis. ALF exhibits unique ethanol metabolic toxicity; its ethanol metabolite acetaldehyde can directly damage mitochondria, inhibit fatty acid oxidation, and exacerbate liver fibrosis. In infectious liver injury, hepatotropic viruses are the primary pathogens that infect cells, causing liver damage through direct killing and inducing immune responses. Drug-induced liver injury (DILI) differs from the above two types. It is mainly caused by hepatotoxicity of drugs or their metabolites, or by hepatic allergic reactions to drugs, and is one of the most common types of adverse drug reactions. Acetaminophen (APAP) overdose is a common cause of drug-induced liver injury and is also a recognized ideal model for studying drug-induced liver injury.

[0003] Currently, the only clinically approved antidote for acetaminophen is N-acetylcysteine, but it has a limited duration of action and is accompanied by side effects such as vomiting, headache, and nausea. Therefore, it is of great significance to find potential drugs that have no toxic side effects and can improve drug-induced liver injury.

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

[0005] The purpose of this invention is to provide an active polysaccharide from *Hymenochloa crus-galli*, its preparation method, and its application in the preparation of drugs to improve drug-induced liver injury, thereby solving the problems existing in the prior art. This active polysaccharide from *Hymenochloa crus-galli* exhibits good protective effects against drug-induced liver injury and can be used to prepare polysaccharide-based liver-protective drugs, hepatoprotective excipients, and functional foods, possessing significant economic and market value.

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

[0007] This invention provides a method for preparing active polysaccharides from lotus leaf apophytes, comprising the following steps:

[0008] The crude extract of *Nelumbo nucifera* was obtained by water extraction and alcohol precipitation using the fruiting body of *Nelumbo nucifera*.

[0009] After deproteinization of the crude extract of *Heliotropium indicum*, it was subjected to dialysis to obtain crude polysaccharide of *Heliotropium indicum*.

[0010] The crude polysaccharide from *Hymenochloa crus-galli* was purified by DEAE-52 cellulose anion exchange chromatography column and Superdex 16 / 600 200 pg molecular sieve gel filtration column to obtain the active polysaccharide from *Hymenochloa crus-galli*.

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

[0012] After crushing the lotus leaf pleated umbelliferous fruiting body, water was used to extract it, resulting in an aqueous extract.

[0013] After concentrating the aqueous extract, add three times the volume of anhydrous ethanol, mix well, let stand, then centrifuge to collect the precipitate, and dry to obtain the crude extract of lotus leaf pleated umbrella.

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

[0015] Furthermore, the dialysis treatment was performed using a dialysis bag with a 3500 Da retention capacity.

[0016] The present invention also provides an active polysaccharide of lotus leaf pleated cap prepared according to the above preparation method.

[0017] The present invention also provides the application of the above-mentioned lotus leaf polysaccharide in the preparation of a drug for improving drug-induced liver injury.

[0018] The present invention also provides the application of the above-mentioned lotus leaf polysaccharide in the preparation of health products that have an auxiliary protective effect against drug-induced liver injury.

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

[0020] The present invention also provides a drug for improving drug-induced liver injury, the active ingredient of which includes the above-mentioned lotus leaf polysaccharide.

[0021] The present invention also provides a health product that has an auxiliary protective effect against drug-induced liver injury, the active ingredient of which includes the above-mentioned lotus leaf polysaccharide.

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

[0023] This invention obtains an active polysaccharide component, LDP1, from the natural fungal resource *Pheretima aspergillum*. Animal experiments have shown that this polysaccharide can improve liver tissue and cell damage, inhibit inflammatory responses, reduce the production of lipid peroxidation products, increase the diversity of intestinal flora and the relative abundance of beneficial bacteria, maintain intestinal microbiota homeostasis, and has a good protective effect against drug-induced liver injury (especially acetaminophen-induced liver injury). It can be used to prepare polysaccharide-based liver-protective drugs, hepatoprotective excipients, and functional foods, possessing significant economic and market value. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Elution curves of lotus leaf polysaccharide; where A is the DEAE-52 elution curve; B is the Superdex 16 / 600 200pg elution curve;

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

[0027] Figure 3 The glucose standard curve for LDP1;

[0028] Figure 4 The infrared spectrum of LDP1;

[0029] Figure 5 The molecular weight spectrum of LDP1;

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

[0031] Figure 7 The graphs show the effects of LDP1 on serum transaminases and oxidative stress kinases in mice. A represents serum ALT levels; B represents serum AST levels; C and D represent serum and liver GSH-Px levels, respectively; E and F represent serum and liver SOD levels, respectively; G and H represent serum and liver MDA levels, respectively; and I and J represent serum and liver CAT levels, respectively. #### P<0.0001and ### P<0.001 vs Control; **** P<0.0001and *** P<0.001and ** P<0.01 and * P < 0.05 vs Model;

[0032] Figure 8 The graph shows the detection results of inflammatory factors in mice under the action of LDP1; where 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; and G and H are statistical graphs of IL-10 levels in serum and liver, respectively. #### P<0.0001vsControl; **** P<0.0001and ** P<0.01 and * P < 0.05 vs Model;

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

[0034] Figure 10 Principal component analysis (PCoA) plot at the OUTs level;

[0035] Figure 11 A bar chart showing the relative abundance at the phylum level;

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

[0037] Figure 13 Analyze Venn diagrams for OTUs;

[0038] Figure 14 Predict abundance plots for MetaCyc;

[0039] Figure 15 A pie chart showing the classification of serum metabolites;

[0040] Figure 16 This is a graph showing the positive ion mode analysis of PCA in serum samples.

[0041] Figure 17 A graph showing the negative ion pattern of PCA in serum samples;

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

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

[0044] Figure 20 Figure showing the Venn differential metabolite analysis in mouse serum;

[0045] Figure 21 The images show the OPLS-DA analysis of mouse serum samples. A, C, and E represent the OPLS-DA analysis of the CTRL vs Model group, Model vs LDP1 group, and Model vs LDP1 group in positive ion mode, respectively. B, D, and F represent the OPLS-DA analysis of the CTRL vs Model group, Model vs LDP1 group, and Model vs LDP1 group in negative ion mode, respectively.

[0046] Figure 22 Heatmap of correlations between differential metabolites - positive ion mode;

[0047] Figure 23 Heatmap of differential metabolite correlations - negative ion pattern;

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

[0049] Figure 25 Bubble diagrams (Model and LDP1) for metabolic pathway enrichment analysis. Detailed Implementation

[0050] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0052] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0053] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0055] Example 1: Preparation of active polysaccharides from lotus leaf chrysanthemum

[0056] (1) The fruiting bodies of *Ulva spp.* were dried and pulverized. 100g of *Ulva spp.* powder was weighed and added to 3L of water at a material-to-liquid ratio of 1:30. The mixture was extracted at 89℃ for 3 hours, and the extraction was repeated 3 times. The filtrates from the three extractions were combined and concentrated to one-tenth of the original volume by heating. Three times the volume of anhydrous ethanol was added and mixed thoroughly. The mixture was then allowed to stand at 4℃ for 24 hours. After standing, the precipitate was collected by centrifugation (8000r / min, 8min) and dried to obtain the crude extract of *Ulva spp.*.

[0057] (2) After dissolving the crude extract of lotus leaf pleated umbrella sac with deionized water, add an equal volume of Sevag reagent (chloroform: n-butanol = 3:1) to mix the two thoroughly, centrifuge, discard the middle protein layer and the lower organic solvent, repeat the above steps 3 times, collect the supernatant, dialyze through a 3500 Da cutoff dialysis bag to remove small molecules, concentrate and freeze dry to obtain crude polysaccharide of lotus leaf pleated umbrella sac.

[0058] (3) Weigh 100 mg of crude polysaccharide from *Hymenochloa crus-galli* and dissolve it in 5 mL of ultrapure water. Load the solution into 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 solutions, respectively, at a rate of 1 mL / min. Collect one sample every 5 minutes. Determine the polysaccharide concentration range using the phenol-sulfuric acid method. Collect the effective fraction, concentrate by dialyzing, lyophilize, and then store in a dry place. Results are as follows: Figure 1 As shown in Figure A.

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

[0060] Example 2: Monosaccharide composition analysis of LDP1 polysaccharide from lotus leaf

[0061] (1) Sample pretreatment: Take a clean chromatographic bottle, accurately weigh 20 mg of LDP1 sample, add 2 mL of 2M trifluoroacetic acid solution, heat at 121℃ for 2 hours. Purge with nitrogen, dry, add 99.99% methanol to wash, dry again, repeat the methanol washing 2-3 times to thoroughly remove trifluoroacetic acid, add sterile water to dissolve, transfer to a chromatographic bottle for analysis.

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

[0063] Table 1. Concentration information of monosaccharide standard mixture

[0064]

[0065] (3) After filtering all the prepared sugar solutions through a 0.22 μm microporous membrane, they were detected by high performance liquid chromatography. The injection volume was 5 μL. Mobile phase A (ddH2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc), flow rate 0.5 mL / min; column temperature 30℃; elution gradient: 0 min A / B / C (95:5:0), 26 min A / B / C (85:5:10), 42 min A / B / C (85:5:10), 42.1 min A / B / C (60:0:40), 52 min A / B / C (60:40:0), 52.1 min A / B / C (95:5:0), 60 min A / B / C (95:5:0). Results are as follows. 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 lotus leaf polysaccharide LDP1

[0067] Glucose standard was dried to constant weight at 90℃. 1.000 g of the standard was accurately weighed and diluted to volume with distilled water in a volumetric flask to prepare a 10 mg / mL stock solution. Accurately pipette the stock solution to a 1 mg / mL glucose standard solution and transfer 0, 10, 20, 30, 40, 50, 60, 70, and 80 μL to tubes, respectively. Distilled water was added to a final volume of 200 μL. In a separate EP tube, 200 μL of 1 mg / mL LDP1 polysaccharide solution was added, followed by 150 μL of 5% phenol solution. The mixture was then rapidly and slowly added to 0.5 mL of concentrated sulfuric acid. After mixing, the tubes were allowed to stand for 30 minutes and cooled to room temperature. 200 μL of each sample was then transferred to a 96-well plate, and the OD was measured using a microplate reader. 490 Record the numerical values ​​according to the glucose standard curve ( Figure 3 The total sugar content of LDP1 was calculated to be 75.23%.

[0068] Example 4: Infrared Spectroscopic Analysis of Lotus Leaf Pleurotus Polysaccharide LDP1

[0069] (1) Sample preparation: 1-5 mg of accurate polysaccharide sample is placed in a vacuum drying oven (60℃, 24h) to completely remove moisture; 2 mg of dried sample and 200 mg of dried KBr powder are ground evenly in an agate mortar. The grinding process is carried out under a drying lamp to prevent moisture absorption.

[0070] (2) Tableting and Scanning: The ground mixture was pressed into thin tablets using a tablet press at a pressure of 30 MPa for approximately 30 seconds. The tablets were then removed and scanned using a Fourier transform infrared spectrometer, with the scanning range set to 4000-4000 cm⁻¹. -1 The resolution is set to 4cm. -1 The average spectrum was obtained after three sample collections. The results are as follows: Figure 4 As shown, LDP1 contains -OH stretching vibration, CH stretching vibration, C=O stretching vibration, C=H stretching vibration, and CO angle vibration, all of which are characteristic absorption peaks of polysaccharides.

[0071] Example 5: Molecular weight determination of LDP1, a polysaccharide from lotus leaf.

[0072] The polysaccharide sample was dissolved in 0.1M NaNO3 aqueous solution (containing 0.02% NaN3) to a final concentration of 1 mg / mL, and filtered through a 0.45 μm filter before analysis. Specific column and elution conditions were as follows: Ohpak SB-805HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm) gel size exclusion columns were used in series. The column temperature was 45℃, 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. Results are as follows: Figure 5 As shown, the molecular weight of this polysaccharide is 12.207 kDa.

[0073] Example 6: Methylation analysis of LDP1, a polysaccharide from lotus leaf.

[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 to react for 1 h.

[0075] (2) Add 1 mL of deionized water and 2 mL of dichloromethane, mix well, centrifuge, discard the aqueous phase, and repeat the washing with water 3 times. Take the lower dichloromethane phase and blow it dry with nitrogen. Then dissolve it in 100 μL of 2M trifluoroacetic acid, react at 121℃ for 90 min, and then evaporate it to dryness at 30℃.

[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 to 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 to mix, centrifuge, discard the aqueous phase, and repeat the washing with water 3 times. Take the lower dichloromethane phase and analyze it by GC-MS.

[0078] The results, shown in Table 2, indicate that the main glycosidic bonds in lotus leaf polysaccharide LDP1 are four types: 6-Gal, 2,6-Gal, t-Man, and t-Fuc. The molar ratios are 33.41%, 25.98%, 23.68%, and 7.67%, respectively.

[0079] Table 2 LDP1 Bond Structure Analysis Results

[0080]

[0081] The following experiments demonstrate the medicinal uses of the lotus leaf polysaccharide obtained by this invention.

[0082] Test case

[0083] 1. Experimental 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 administered normal saline by gavage. The high-, medium-, and low-dose polysaccharide administration groups were administered LDP1 by gavage at doses of 400 mg / kg, 200 mg / kg, and 100 mg / kg, respectively. This was done once daily for 21 consecutive days. On day 21, the model group was administered APAP solution at a dose of 200 mg / kg two hours after normal saline gavage. The positive control group was administered acetylcysteine ​​at a dose of 200 mg / kg one hour beforehand, followed by APAP solution at a dose of 200 mg / kg.

[0086] 2. Effects of lotus leaf polysaccharide LDP1 on body weight, organ index, and histopathological changes in mice with liver injury.

[0087] During the experiment, the growth of mice was observed daily, and they were weighed every two days. All mice were fasted for 16 hours before the last administration of medication. After the animal experiment, mice were anesthetized by intraperitoneal injection, euthanized by cervical dislocation, and approximately 1.5 mL of blood was collected from each mouse using the ocular blood sampling method. The blood was centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected and centrifuged twice to obtain serum samples, which were then stored at -80℃ for later use. After blood collection, the mice were cleaned with 75% alcohol, and the contents of the cecum were collected by dissection. These contents were sealed in cryovials, flash-frozen in liquid nitrogen, and then stored at -80℃ for later use. The liver was immediately collected, rinsed thoroughly with physiological saline, and dried with filter paper. The organ index was calculated: Organ Index (mg / g) = Organ Mass (mg) / Mouse Body Weight (g).

[0088] Mouse body weight and organ index test results are as follows Figure 6 As shown in Figures A and B, the body weight of mice in all groups showed an increasing trend. After administration of LDP1, the liver index of mice decreased significantly, with the medium dose showing better effect, and the results were similar to those of the normal group mice.

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

[0090] Fixed liver tissue was harvested and dehydrated and cleared sequentially using ethanol and xylene of different concentrations. The tissue was then embedded in paraffin and prepared into 5 μm sections. Hematoxylin and eosin (HE) staining was performed, followed by dehydration and clearing. The sections were then observed under a light microscope, photographed, and the overall condition of the liver tissue, as well as the morphology and structure of cells, the integrity of nuclear structures, and the extent of inflammatory cell infiltration, were recorded. Results are as follows: Figure 6 As shown in Figure C, the results indicate that LDP1 can improve abnormal liver tissue structure and cell necrosis.

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

[0092] Serum samples were collected from mice in each group, and the levels of ALT, AST, GSH-Px, SOD, MDA, and CAT-related cytokines in the serum were detected. Frozen liver tissue was slowly thawed, rinsed thoroughly with physiological saline, and excess water was blotted dry with filter paper. The homogenate was then mixed with 9 times its volume of physiological saline in a 5 mL centrifuge tube and ground evenly. During homogenization, the mixture was placed on ice for 3 minutes every 30 seconds to prevent protein degradation. The homogenate was centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. This process was repeated twice to obtain liver tissue homogenate. The levels of GSH-Px, SOD, MDA, and CAT-related cytokines in the mouse liver homogenate were then detected. Results are as follows: Figure 7 As shown, 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, and bring MDA levels close to normal, thus having a good regulatory effect on oxidative stress response in the liver.

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

[0094] Serum samples were collected from mice in each group, and the levels of TNF-α, IL-1β, IL-10, and IL-6-related cytokines in the serum were detected. The frozen liver tissue was taken, rinsed thoroughly with physiological saline, and excess water was blotted with filter paper. Nine times the volume of physiological saline was mixed into a 5 mL centrifuge tube and homogenized. During homogenization, the mixture was placed on ice for 3 minutes every 30 seconds to prevent protein degradation. The mixture was centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected. This process was repeated twice to obtain a liver tissue homogenate. The levels of TNF-α, IL-1β, IL-10, and IL-6-related cytokines in the mouse liver tissue were then 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 lotus leaf polysaccharide LDP1 on gut microbiota in liver-injured mice

[0096] The cecal contents collected from each group were added to sterile PBS buffer and vortexed until homogeneous. Centrifuged (12,000 rpm, 5 minutes), and the supernatant or precipitate was collected. DNA extraction was performed using a DNA extraction kit according to the manufacturer's instructions. DNA concentration and purity were analyzed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Polymerase chain reaction (PCR) amplification of the V3-V4 region of the bacterial 16S rRNA gene was performed using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Product quality was assessed using 2% agarose gel electrophoresis. Finally, the product was analyzed using a Quantus microfluorometer. TM The purified product was quantified using a fluorometer. Paired-end sequencing of cecal contents DNA was performed using the Illumina platform. Sequence denoising and clustering were performed based on the raw sequencing data. VSEARCH was used to cluster the sequences into operational taxonomic units (OTUs), with high-quality sequences defined as having 97% similarity. Species annotation was performed for OTUs within different units. Alpha and beta diversity of the samples were assessed, analyzing the diversity of species within and between habitats. Results are as follows: Figures 9-14As shown, LDP1 can improve the diversity and abundance of gut microbiota in mice; reverse the dysbiosis of Firmicutes and Bacteroidetes in the mouse gut; reduce the relative abundance of harmful bacteria 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 species composition disorder in the mouse gut; 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 lotus leaf polysaccharide LDP1 on serum metabolites in mice with liver injury

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

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

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

[0101] (1) Using a locally built database and a public database, the structure of metabolites in serum samples was identified by matching them with information such as retention time, molecular weight (molecular weight error within <10ppm), secondary fragmentation spectrum, and collision energy. The number of identified metabolites in each group of serum samples was counted, and they were classified and integrated according to their chemical classification. The results are as follows: Figure 15As shown, a total of 1018 metabolites were identified using both positive and negative ion modes. Of these, 600 were identified using the positive ion mode and 418 using the negative ion mode. All metabolites were classified into 11 superclasses. Lipids and lipid molecules were the most abundant, accounting for 33.202%, followed by organic acids and their derivatives at 25.442%. Heterocyclic compounds were the next most abundant at 11.690%, followed by aromatic compounds at 8.841%. Organometallic compounds and lignans, neolignans, and related compounds were the least abundant, each accounting for only 0.196%.

[0102] PCA analysis generates new characteristic variables by linearly combining metabolite variables with certain weights. These new main variables are then used to categorize the data into groups, removing samples with poor repeatability, and analyzing differences between samples. Results are as follows: Figures 16-17 As shown, Ri for the positive and negative ion models 2 X values ​​were 0.576 and 0.551, respectively, and the interpretability of the PCA model was greater than 0.5, indicating good stability of the system. In positive ion mode, the CTRL group and the Model group were completely separated, while the LDP1 group was close to the CTRL group. In negative ion mode, the Model group and the CTRL group were not completely separated, but the LDP1 group remained close to the CTRL group with some overlap. Metabolite samples in the model group showed changes compared to the blank control group, while after administration of LDP1, the composition of variables in the samples was closer to that of the blank control group.

[0103] OPLS-DA analysis, combined with orthogonal signal correction techniques, removes information irrelevant to classification, concentrating relevant information primarily on the first predicted component. Without reducing the model's predictive power, it effectively reduces model complexity and enhances its interpretability, thereby maximizing the observation of inter-group differences. As shown in Table 3, the R-values ​​for each group are... 2 Y, Q 2 All parameters are greater than 50%, indicating a high model fitting accuracy. The results are as follows: Figure 21 As shown, in the four groups CTRL, Model, LDP1, and PC, samples within the same group clustered on the same side, while samples between groups were clearly distinguishable, indicating that the experimental sample model was established effectively and that there were differences in metabolites among the samples in each group.

[0104] Table 3 Parameters of the OPLS-DA Model in Positive and Negative Ion Modes

[0105]

[0106] Univariate statistical analysis was performed using fold change analysis and t-tests to calculate p-values. Differential analyses were conducted on all metabolites detected in both positive and negative ion modes. A p-value < 0.05 was used as the screening criterion, and the data results were visualized using a volcano plot. The differentially expressed metabolites were compared between the Model group and the LDP1 group; the results are shown below. Figures 18-19 As shown, in positive ion mode, a total of 177 differential metabolites were detected, with 126 significantly upregulated and 51 significantly downregulated. In negative ion mode, a total of 79 differential metabolites were detected, with 49 significantly upregulated and 30 significantly downregulated.

[0107] Using strict criteria of OPLS-DAVIP>1 and P value<0.05 as important variables and significant differential metabolites, differential metabolites in the cecal contents of mice in the CTRL group and Model group, and Model group and LDP1 group were analyzed. Figure 21 Venn diagrams were used to compare the number of common and unique substances among groups. The results are as follows: Figure 20 As shown, there were 57 significantly different metabolites between the cecal contents of the CTRL group and the Model group, and 16 significantly different metabolites between the cecal contents of the Model group and the LDP1 group. There were a total of 3 significantly different metabolites between the two groups.

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

[0109] By calculating the Pearson correlation coefficients between all differentially expressed metabolites, the linear relationship between two metabolites was observed to determine whether there was a correlation. Statistical tests were then performed on the metabolite association analysis using a function. The test criteria were: a correlation coefficient approaching 1 or -1, and a p-value < 0.05, indicating a significant correlation. In the differentially expressed metabolite heatmap, metabolites with the same trend of change were positively correlated (represented in red), while those with opposite trends were negatively correlated (represented in blue). The results are as follows: Figures 22-23 As shown, metabolites positively correlated with betulinic acid, including 1-piperazinecarboxamide, 4-(3-chloro-2-pyridinyl)-n-[4-(1,1-dimethylethyl)phenyl]-, and N2-Acetyl-L-ornithine, were screened. Betulinic acid is a lupin-type pentacyclic triterpenoid compound that can reduce serum TLR-9, NF-κB, IL-18, and MDA levels in rats with an APAP-induced liver injury model. By improving the tissue redox system 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 calculate the significance level of metabolite enrichment in each pathway, thereby identifying significantly affected metabolic and signal transduction pathways. 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 each pathway, and displayed as a bubble chart. Results are as follows... Figures 24-25 As shown, a comparison between the CTRL group and the Model group revealed 20 related metabolic pathways, with the bile secretion pathway showing the highest enrichment. Administration of LDP1 suggested that changes in related metabolites might be related to propionic acid metabolism, pyrimidine metabolism, arginine biosynthesis, and ABC transporters.

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

Claims

1. The application of a lotus leaf polysaccharide in the preparation of a drug for improving drug-induced liver injury, characterized in that, The preparation method of the lotus leaf polysaccharide includes the following steps: The crude extract of *Nelumbo nucifera* was obtained by water extraction and alcohol precipitation using the fruiting body of *Nelumbo nucifera*. After deproteinization of the crude extract of *Heliotropium indicum*, it was subjected to dialysis to obtain crude polysaccharide of *Heliotropium indicum*. The crude polysaccharide from *Heliotropium indicum* was purified by DEAE-52 cellulose anion exchange chromatography column and Superdex 16 / 600 200 pg molecular sieve gel filtration column to obtain the active polysaccharide from *Heliotropium indicum*. The drug-induced liver injury mentioned is liver injury caused by acetaminophen; The molecular weight of the active polysaccharide from lotus leaf pleated capillaris is 12.207 kDa; The water extraction and alcohol precipitation method includes the following steps: After crushing the lotus leaf pleated umbelliferous fruiting body, water was used to extract it, resulting in an aqueous extract. After concentrating the aqueous extract, add three times the volume of anhydrous ethanol, mix well, let stand, then centrifuge to collect the precipitate, and dry it to obtain the crude extract of lotus leaf pleated umbrella. The ratio of crushed lotus leaf pleated umbelliferous fruiting bodies to water was 1:30; the crushed lotus leaf pleated umbelliferous fruiting bodies were repeatedly extracted with water 3 times, with the extraction temperature at 89℃ and the extraction time at 3h each time. The deproteinization treatment was performed using Sevag reagent; The dialysis treatment was performed using a dialysis bag with a retention capacity of 3500 Da; When purifying using the DEAE-52 cellulose anion exchange chromatography column, elution was performed with ultrapure water, 0.1, 0.3, and 0.5 mol / L NaCl solutions, respectively, at a rate of 1 mL / min. When purifying using the Superdex 16 / 600 200 pg molecular sieve gel filtration column, ultrapure water was used for elution at a rate of 0.5 mL / min.

2. The application of the lotus leaf polysaccharide as described in claim 1 in the preparation of health products with adjuvant protective effects against drug-induced liver injury, characterized in that, The drug-induced liver injury mentioned refers to liver injury caused by acetaminophen.

3. A drug for improving drug-induced liver injury, characterized in that, The active ingredient includes the lotus leaf polysaccharide as described in claim 1.

4. A health product with an auxiliary protective effect against drug-induced liver injury, characterized in that, The active ingredient includes the lotus leaf polysaccharide as described in claim 1.

Images