Ganoderma lucidum mutagenic strain active polysaccharide, and preparation method and application thereof

Abstract

This invention discloses an active polysaccharide from a Ganoderma lucidum mutant strain, its preparation method, and its application, relating to the field of biomedical technology. The preparation method includes extracting the mycelium of the mutant Ganoderma lucidum strain JLAU 210928 using a water extraction and alcohol precipitation method to prepare the active polysaccharide. The mutant Ganoderma lucidum strain JLAU 210928 was deposited on September 30, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 23283. The active polysaccharide from the mutant Ganoderma lucidum strain provided by this invention exhibits significantly superior efficacy compared to ordinary strains in alleviating drug-induced liver injury, more effectively reducing drug-induced liver damage, enhancing the liver's self-repair ability, and providing stronger protection for liver health.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an active polysaccharide derived from a Ganoderma lucidum mutant strain, its preparation method, and its application. Background Technology

[0002] Drug-induced liver injury (DILI) refers to a condition caused by hepatotoxicity or hepatic hypersensitivity to drugs, resulting from the use of drugs or their metabolites. It is one of the most common types of adverse drug reactions. In recent years, the incidence of liver injury caused by overdose of acetaminophen has been gradually increasing and has become a major factor leading to acute liver failure.

[0003] Currently, the prevention and treatment of drug-induced liver injury mainly focus on drug discontinuation and symptomatic relief, lacking specific treatment methods. Natural products, due to their high safety and broad bioactivity, have attracted significant attention in the field of liver protection. Ganoderma lucidum, a traditional and valuable medicinal fungus, contains polysaccharides that have been proven to possess various physiological activities, showing potential in liver protection. However, the efficacy of polysaccharides from common Ganoderma lucidum strains in alleviating drug-induced liver injury still has room for improvement, failing to meet the clinical and market demand for highly effective hepatoprotective substances. Therefore, further exploration of functional components with superior activity in Ganoderma lucidum resources is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide an active polysaccharide from a Ganoderma lucidum mutant strain, its preparation method, and its application, thereby solving the problems existing in the prior art. The active polysaccharide from the Ganoderma lucidum mutant strain provided by this invention exhibits significantly superior efficacy compared to ordinary strains in alleviating drug-induced liver injury, and can more effectively reduce drug-induced liver damage.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing active polysaccharides from Ganoderma lucidum mutant strains, comprising the steps of extracting mycelia of Ganoderma lucidum mutant strain JLAU 210928 by water extraction and alcohol precipitation to prepare the active polysaccharides from the Ganoderma lucidum mutant strains;

[0007] The mutant Ganoderma lucidum strain JLAU 210928 was deposited on September 30, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.23283.

[0008] Furthermore, the specific steps of the water extraction and alcohol precipitation method include:

[0009] The mycelium of the mutant Ganoderma lucidum strain JLAU 210928 was mixed with water and then stirred for extraction. The supernatant was obtained by centrifugation.

[0010] After concentrating the supernatant, anhydrous ethanol was added for precipitation treatment to obtain a precipitate;

[0011] The precipitate was subjected to dialysis to obtain the active polysaccharide of the Ganoderma lucidum mutant strain.

[0012] Furthermore, the stirring extraction temperature is 90°C, and the time is 3 hours.

[0013] Further, the mycelium of the mutant Ganoderma lucidum strain JLAU 210928 was mixed with water at a mass ratio of 1:30.

[0014] Furthermore, the concentration involves concentrating the supernatant to 1 / 4 of its original volume.

[0015] The present invention also provides an active polysaccharide of Ganoderma lucidum mutant strain prepared according to the above preparation method.

[0016] The present invention also provides the application of the above-mentioned Ganoderma lucidum mutant strain active polysaccharide in the preparation of a drug to alleviate drug-induced liver injury.

[0017] The present invention also provides a drug for alleviating drug-induced liver injury, the active ingredient of which includes the above-mentioned active polysaccharide of Ganoderma lucidum mutant strain.

[0018] This invention also provides the application of the above-mentioned Ganoderma lucidum mutant strain active polysaccharide in the preparation of health products with auxiliary protective effect against chemically induced liver damage.

[0019] The present invention also provides a health product that has an auxiliary protective effect against chemically induced liver damage, the active ingredient of which includes the above-mentioned active polysaccharide from Ganoderma lucidum mutant strains.

[0020] The present invention discloses the following technical effects:

[0021] This invention provides an active polysaccharide from a Ganoderma lucidum mutant strain, which exhibits significantly superior efficacy compared to ordinary strains in alleviating drug-induced liver injury. It can more effectively reduce drug-induced liver damage, enhance the liver's self-repair ability, and provide stronger protection for liver health. This invention not only opens up new avenues for the development and utilization of Ganoderma lucidum and its products, providing a novel polysaccharide material with superior performance, but also lays an important foundation for the research and development of novel hepatoprotective drugs, adjuvant therapy preparations, and functional foods. It possesses both outstanding economic value and broad market application potential, and is of positive significance for promoting the upgrading and development of related industries. Attached Figure Description

[0022] 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.

[0023] Figure 1 The monosaccharide composition spectrum of active polysaccharides from Ganoderma lucidum mutagenesis strains;

[0024] Figure 2 Infrared spectrum of active polysaccharides from Ganoderma lucidum mutagenesis strain;

[0025] Figure 3 Molecular weight profile of active polysaccharides from Ganoderma lucidum mutagenesis strains;

[0026] Figure 4 The images show liver pathological sections of mice in each group; where A: blank group (CON); B: model group (MOD); C: Ganoderma lucidum wild-type strain polysaccharide administration group (GLP1); D: Ganoderma lucidum mutagenesis active polysaccharide administration group (GLP2).

[0027] Figure 5 The graph shows the results of ALT level detection in the serum of mice in each group;

[0028] Figure 6 The graph shows the results of AST level detection in the serum of mice in each group;

[0029] Figure 7 The graph shows the results of IL-6 level detection in the serum of mice in each group;

[0030] Figure 8 The graph shows the results of IL-1β level detection in the serum of mice in each group;

[0031] Figure 9 The graph shows the detection results of TNF-α levels in the serum of mice in each group;

[0032] Figure 10 The graph shows the results of IL-10 level detection in the serum of mice in each group. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Example 1

[0039] The preparation method of the mutagenic Ganoderma lucidum strain JLAU 210928 is as follows:

[0040] (1) Preparation of slant culture medium: Wash 500g of potatoes and cut them into 2cm cubes. Add 1000ml of water and boil the potatoes. After filtering, add 30g / L glucose, 2g / L peptone, 3g / L potassium dihydrogen phosphate, 1.5g / L magnesium sulfate and 20g / L agar.

[0041] (2) Inoculation: While the prepared culture medium solution is still hot, dispense it into test tubes, filling the tubes to one-fifth full. Tightly plug the tubes with cotton plugs and sterilize them in an autoclave at 121°C for 40 minutes. After opening the lid, remove the tubes and place them on a slant while still hot. After 10 hours, operate in a sterile operating table to inoculate 3 mm-sized pieces of Ganoderma lucidum mother culture into the culture tubes and incubate them in an incubator at 25°C for 15 days.

[0042] (3) Mutagenesis: The original strain with good growth was carried into the Shenzhou-11 spacecraft on October 17, 2016 and returned to the ground on November 18, 2016. After strain screening, the mutagenic Ganoderma lucidum strain JLAU 210928 was obtained.

[0043] The mutant Ganoderma lucidum strain JLAU 210928 was deposited on September 30, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It was classified as Ganoderma lucidum and its accession number is CGMCC No. 23283.

[0044] Example 2

[0045] Preparation of active polysaccharides from Ganoderma lucidum mutagen strains:

[0046] The upper layer of mycelia from a culture dish containing mycelia of the mutagenic Ganoderma lucidum strain JLAU 210928 was scraped off. Distilled water was added at a material-to-liquid ratio of 1:30, and the mixture was placed in a water bath at 90°C with stirring for 3 hours. The supernatant was collected by centrifugation and concentrated to 1 / 4 of its original volume. Three volumes of anhydrous ethanol were added, and the mixture was allowed to stand overnight to precipitate. The supernatant was then discarded. The precipitate was dissolved in purified water and dialyzed against running water for 24 hours using a 3500 Da dialysis bag. The retentate was then concentrated and freeze-dried to obtain the active polysaccharides from the mutagenic Ganoderma lucidum strain.

[0047] Example 3

[0048] Monosaccharide composition analysis of active polysaccharides from Ganoderma lucidum mutagen strains:

[0049] Weigh 5 mg of the active polysaccharide sample from the Ganoderma lucidum mutant strain and place it in a clean chromatographic vial. Add 1 mL of 2M TFA solution and heat at 121℃ for 2 hours. Purge with nitrogen and dry. Wash with 99.99% methanol, then dry again. Repeat the methanol washing twice. Dissolve in sterile water and transfer to a chromatographic vial for analysis. Analyze the monosaccharide components using a Thermo ICS5000 ion chromatography system (ICS5000, ThermoFisher Scientific, USA) with an electrochemical detector. Results are as follows: Figure 1 As shown, the active polysaccharide of the Ganoderma lucidum mutant strain exists as a glucan, mainly composed of glucose, galactose and mannose, with molar percentages of 97.90%, 1.44% and 0.66%, respectively.

[0050] Example 4

[0051] Infrared spectral analysis of active polysaccharides from Ganoderma lucidum mutagen strains:

[0052] (1) Sample preparation: 5 mg of Ganoderma lucidum mutagenesis active polysaccharide sample was placed in a vacuum drying oven (60℃, 24h) to completely remove moisture; 2 mg of the dried sample and 200 mg of dried KBr powder were ground evenly in an agate mortar. The grinding process was carried out under a drying lamp to prevent moisture absorption.

[0053] (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 a scanning range of 4000-4000 cm⁻¹. -1 The resolution is set to 4cm. -1 The average spectrum was obtained after three sample collections.

[0054] The results are as follows Figure 2 As shown, the absorption peak at 3390 cm⁻¹ is caused by the OH stretching vibration. This is because, as polyhydroxy compounds, the numerous hydrogen bonds between carbohydrate molecules overlap to form a broad absorption band at 2927 cm⁻¹. -1 The absorption peaks are caused by the CH stretching vibration; these two locations are characteristic absorption peaks of polysaccharides, at 1643 cm⁻¹. -1 The nearby peak is the characteristic absorption peak of C=O, at 1365 cm⁻¹. -1 The absorption peak at 1153 cm⁻¹ is the frequency of the angular vibration of CH₄. -1 Corresponding to the COC and -OH bending vibrations in the pyran structure, 1027 cm -1 The nearby absorption peak indicates the presence of a pyranose ring, 927 cm⁻¹. -1 and 850cm -1 The presence of an absorption peak at 761 cm⁻¹ indicates that the active polysaccharides of the Ganoderma lucidum mutant strain may simultaneously contain α- and β-configurations of glycosidic bonds. -1 This is a characteristic absorption peak of mannose, consistent with the results of monosaccharide structure detection.

[0055] Example 5

[0056] Molecular weight determination of active polysaccharides from Ganoderma lucidum mutant strains

[0057] The active polysaccharide sample from the Ganoderma lucidum mutagenesis strain 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. Gel chromatography-differential imaging-multi-angle laser light scattering (GPC-RI-MALS) was used for on-site detection. 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 in series. Column temperature was 45℃, injection volume was 100 μL, mobile phase A (0.02% NaN3, 0.1M NaNO3), flow rate was 0.6 mL / min, and elution gradient was isocratic for 75 min. The chromatographic data were processed using ASTRA 6.1 software to obtain the absolute molecular weight analysis chromatogram of the sample. Results are shown below. Figure 3 As shown, the molecular weight of the active polysaccharide of this Ganoderma lucidum mutant strain is 6.351 kDa.

[0058] Comparative Example 1

[0059] Preparation of polysaccharides from wild-type Ganoderma lucidum strains:

[0060] The method is the same as in Example 2, except that the mutagenic Ganoderma lucidum strain JLAU 210928 is replaced with the Ganoderma lucidum mother strain (i.e., wild-type Ganoderma lucidum strain) from Example 1.

[0061] The following experiments demonstrate the medicinal uses of the active polysaccharides from the Ganoderma lucidum mutant strains obtained in this invention.

[0062] Example 1 of effect verification

[0063] 1. Experimental Grouping

[0064] Forty 6-week-old male Kunming mice were randomly divided into four groups: a blank control group (CON), a model group (MOD), a Ganoderma lucidum wild-type strain polysaccharide administration group (GLP1), and a Ganoderma lucidum mutagenesis active polysaccharide administration group (GLP2). GLP1 and GLP2 groups were administered the corresponding polysaccharide solution at a dose of 250 mg / kg via gavage daily for 21 consecutive days; the other groups were administered physiological saline. One hour after the last administration, except for the blank group, the other groups were administered acetaminophen (250 mg / kg) via gavage.

[0065] During the experiment, the mice's growth 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, the mice were anesthetized via 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 stored at -80℃ for later use. Immediately after blood collection, the liver was collected and rinsed thoroughly with physiological saline.

[0066] 2. Effects of Ganoderma lucidum polysaccharides on liver injury in mice

[0067] (1) Liver pathology examination

[0068] Liver tissue was fixed with 10% paraformaldehyde solution, embedded in paraffin sections, and prepared into 5 μm thin sections. Hematoxylin-eosin staining was used, and the overall condition of the liver tissue, cell structure and morphology, and inflammatory infiltration were observed under a light microscope. Results are as follows: Figure 4 As shown, compared to the control group, the model group exhibited severe abnormalities in liver tissue structure, with extensive hemorrhage and numerous erythrocytes visible in the field of view (blue arrows); a small number of hepatocytes showed fatty degeneration with round lipid droplets in the cytoplasm (green arrows); a small number of hepatocytes showed necrosis, with fragmented nuclei, condensation, and deep staining (red arrows); and a small number of inflammatory cell infiltrations were observed in the tissue (black arrows). Compared to the model group, the wild-type Ganoderma lucidum strain polysaccharide treatment group showed moderate abnormalities in liver tissue structure, with localized hemorrhage and a small number of erythrocytes visible in the field of view (blue arrows); a small number of hepatocytes showed fatty degeneration with round lipid droplets in the cytoplasm (green arrows). In contrast, the Ganoderma lucidum mutant strain active polysaccharide treatment group showed mild abnormalities in liver tissue structure, with individual hepatocytes showing edema and pale cytoplasm (yellow arrows); the hepatic sinusoids were uniform in size without dilation, and the hepatic cords were neatly and tightly arranged; no obvious inflammatory cell infiltration was observed in the tissue.

[0069] (2) Liver function index testing

[0070] After dilution, the collected mouse serum was used to measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The results are as follows: Figures 5-6 As shown in the figure. The results showed that the serum ALT and AST levels in the model group mice were sharply increased compared with the control group. After administration of Ganoderma lucidum polysaccharides, both types of Ganoderma lucidum polysaccharides could improve lipid metabolism function and reduce serum ALT and AST levels in mice. Compared with the effect of Ganoderma lucidum wild-type strain polysaccharide administration, the effect of Ganoderma lucidum mutant strain active polysaccharide was more significant, and it brought the ALT level in mice closer to that of the control group.

[0071] (3) Detection of inflammatory factor levels

[0072] Inflammatory response is a crucial factor in APAP-induced liver injury. The levels of inflammatory factors IL-6, IL-10, IL-1β, and TNF-α in mouse serum were measured, and the results are as follows: Figures 7-10As shown, under APAP induction, the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the serum of model group mice increased, while the level of anti-inflammatory factor IL-10 decreased. After administration of Ganoderma lucidum mutant mycelial polysaccharide, the levels of anti-inflammatory factor IL-10 in mice showed an increasing trend; while the expression levels of IL-1β, IL-6, and TNF-α in mouse serum decreased. Compared with the effect of Ganoderma lucidum wild-type strain polysaccharide administration, the active polysaccharide of Ganoderma lucidum mutant strain can more significantly reduce the inflammatory response in mice with liver injury and prevent the aggravation of liver injury in mice.

[0073] 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 an active polysaccharide from a Ganoderma lucidum mutant strain in the preparation of drugs for alleviating drug-induced liver injury or health products with adjuvant protective effects against chemically induced liver injury, characterized in that, The method for preparing the active polysaccharide of the Ganoderma lucidum mutant strain includes the step of extracting the mycelium of the mutant Ganoderma lucidum strain JLAU 210928 by water extraction and alcohol precipitation to prepare the active polysaccharide of the Ganoderma lucidum mutant strain. The mutant Ganoderma lucidum strain JLAU 210928 was deposited on September 30, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.23283.

2. The application according to claim 1, characterized in that, The specific steps of the water extraction and alcohol precipitation method include: The mycelium of the mutant Ganoderma lucidum strain JLAU 210928 was mixed with water and then stirred for extraction. The supernatant was obtained by centrifugation. After concentrating the supernatant, anhydrous ethanol was added for precipitation treatment to obtain a precipitate; The precipitate was subjected to dialysis to obtain the active polysaccharide of the Ganoderma lucidum mutant strain.

3. The application according to claim 2, characterized in that, The stirring extraction was performed at a temperature of 90 °C for 3 h.

4. The application according to claim 2, characterized in that, The mycelium of the mutant Ganoderma lucidum strain JLAU 210928 was mixed with water at a mass ratio of 1:

30.

5. The application according to claim 2, characterized in that, The concentration involves concentrating the supernatant to 1 / 4 of its original volume.

6. A drug for relieving drug-induced liver injury, characterized in that, The active ingredient includes the active polysaccharide of Ganoderma lucidum mutant strain as described in any one of claims 1-5.

7. A health product with an auxiliary protective effect against chemically induced liver injury, characterized in that, The active ingredient includes the active polysaccharide of Ganoderma lucidum mutant strain as described in any one of claims 1-5.

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