Preparation method of cordyceps sobolifera polysaccharide and application of cordyceps sobolifera polysaccharide in relieving acute chemical liver injury

Abstract

The invention discloses a preparation method of cordyceps sobolifera polysaccharide and application of the cordyceps sobolifera polysaccharide in relieving acute chemical liver injury. Monosaccharide of the cordyceps sobolifera polysaccharide (abbreviated as CCPW) is composed of glucose, mannose and galactose according to the molar ratio of 8.918: 1: 0.356, and the relative molecular mass is 2.62 * 10 < 4 > Da. Efficacy experiments show that the cordyceps sobolifera homogeneous polysaccharide CCPW can significantly inhibit increase of ALT and AST in a CCl4-induced acute chemical liver injury model mouse, effectively improve the degree of liver cell disruption and swelling, relieve liver inflammatory response, relieve CCl4-induced mouse liver tissue injury, protect mouse intestinal barriers, recover intestinal microbial balance and have obvious liver protection activity. The cordyceps sobolifera polysaccharide CCPW is clear in composition and remarkable in effect, and has wide development and application prospects in the aspect of relieving acute chemical liver injury.

Description

Technical Field

[0001] The invention belongs to the field of preparation and application of polysaccharide extracts, and specifically relates to a preparation method of Cordyceps sinensis polysaccharide and application of the polysaccharide in alleviating acute chemical liver damage. Background Art

[0002] Isaria cicadae Miq belongs to the Ascomycetes, Myxomycetes, Cordyceps, also known as golden cicada flower or big cordyceps. Isaria cicadae Miq has a wide range of biological activities and potential benefits, such as anti-inflammatory and antioxidant, analgesic and sedative, anti-tumor and lipid metabolism regulation. In 2021, the artificially cultivated fruiting bodies of Isaria cicadae were included in the scope of new food ingredients, showing its huge development potential in the fields of food, health products and medicines.

[0003] Acute liver injury (ALI) refers to a disease that causes a sharp deterioration of liver function within 2 weeks after direct or indirect exposure to various liver damage factors. As one of the common types of liver injury, ALI seriously threatens people's lives and health and has become a major public health issue. The causes of liver injury are complex, and there are great differences in hepatocyte apoptosis and its pathophysiological effects, which can be regulated by different causes, mechanisms and regulation. 4 It is a classic chemical hepatotoxic agent. Under the action of liver microsomal enzymes, it changes the permeability of the plasma membrane, causing liver cells to be attacked by a series of proinflammatory cytokines and lipid peroxidation, leading to protein synthesis disorders and lipid metabolism disorders, and further causing a variety of typical liver injury manifestations. It is the most effective and most commonly used chemical inducer of liver injury. At present, liver-protecting drugs occupy an important position in the treatment of acute liver injury, but existing clinical drugs such as silymarin and polyene phosphatidylcholine have limited efficacy, put a burden on the patient's kidneys, and cannot be used for a long time. Therefore, the development of effective drugs and new targets for the treatment of ALI is a pain point and difficulty that needs to be solved urgently in this field. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for preparing Cordyceps sinensis polysaccharide and its application in alleviating acute chemical liver injury.

[0005] Studies have shown that natural polysaccharides can affect the development of liver injury processes, have good anti-liver injury potential, and have fewer adverse reactions and side effects. As one of the main active ingredients of Cordyceps sinensis, Cordyceps sinensis polysaccharides have been shown to have kidney protection, immunomodulatory, antioxidant, and anti-diabetic effects. The applicant's research team previously found that Cordyceps sinensis polysaccharides have the effect of preventing renal interstitial fibrosis, but there are no relevant reports in the literature on the treatment of acute chemical liver injury with its uniform polysaccharide. The applicant prepared a new Cordyceps sinensis uniform polysaccharide CCPW with a specific composition and ratio, and proved that it has excellent activity in alleviating acute chemical liver injury by evaluating indicators such as ALT and AST activity, liver tissue pathology, inflammatory cytokines, and intestinal balance in ALI mice.

[0006] The novel Cordyceps sinensis polysaccharide CCPW with a specific composition and ratio is purified by DEAE-Sepharose Fast Flow chromatography technology. CCPW can reduce the levels of ALT and AST in model mice, significantly reduce liver inflammatory response, repair the intestinal barrier and restore the balance of intestinal microorganisms, and effectively alleviate CCl 4 Induced acute chemical liver injury. The Cordyceps sinensis polysaccharide CCPW prepared by the present invention is an ideal candidate preparation for protecting acute chemical liver injury.

[0007] The preparation method of Cordyceps sinensis polysaccharide of the present invention comprises the following steps:

[0008] Step 1: crush the fruiting body of Cordyceps sinensis, add 95vt% ethanol at a solid-liquid ratio of 1g:20mL, soak in a 70℃ water bath for 6 hours, filter and dry, and pass through an 80-mesh sieve to obtain pretreated Cordyceps sinensis powder;

[0009] Step 2: adding pure water to the Cordyceps sinensis powder obtained in step 1 at a solid-liquid ratio of 1 g:40 mL, extracting at 75° C. for 2-3 hours, repeating the extraction 3-4 times, and combining the extracts;

[0010] Step 3: Concentrate the extract obtained in step 2 to 1 / 250 of the original volume, centrifuge at 4000 rpm, collect the supernatant, add 4 times the volume of 95% ethanol, stir thoroughly and let stand at 4°C overnight, and collect the precipitate by centrifugation;

[0011] Step 4: Take the sample obtained in step 3, add pure water to dissolve, prepare a 20 mg / mL polysaccharide solution, prepare Sevag reagent with chloroform and n-butanol in a volume ratio of 4:1, add an equal volume of polysaccharide solution, shake vigorously, centrifuge to discard the middle layer protein, and repeat this operation until there is no obvious protein layer;

[0012] Step 5: The sample obtained in step 4 was separated and purified by DEAE-Sepharose Fast Flow ion exchange chromatography column. The pretreated DEAE-Sepharose Fast Flow filler was stirred evenly and loaded into the chromatography column (35×400mm) along a glass rod. The filler was eluted with double distilled water for equilibrium, and then the compacted filler was eluted with 0.5 mol / L NaCl solution. Finally, the NaCl solution was replaced by double distilled water at a flow rate of 2-3 mL / min. The components were collected, concentrated and freeze-dried to obtain purified Cordyceps sinensis polysaccharide.

[0013] The Cordyceps sinensis polysaccharide of the present invention is abbreviated as CCPW, and the monosaccharide composition and molar ratio are glucose: mannose: galactose = 8.918: 1: 0.356, and the relative molecular mass is 2.62×10 4 Da.

[0014] The invention discloses an application of the Cordyceps sinensis polysaccharide in preparing a food, a health product or a pharmaceutical preparation having liver protection activity and relieving acute chemical liver damage.

[0015] The Cordyceps sinensis polysaccharide of the present invention has a repairing effect on acute chemical liver damage, especially CCl 4 Caused liver damage.

[0016] The present invention is the first to prepare a novel uniform polysaccharide CCPW with the activity of alleviating acute chemical liver injury from Cordyceps sinensis, the preparation process is green and safe, the ingredients are clear, and long-term use has no toxic side effects on the body.

[0017] The polysaccharide has good liver protection activity, can significantly reduce the ALT and AST levels of ALI mice, improve the degree of liver cell fragmentation and swelling, reduce liver inflammatory response, and alleviate the symptoms caused by CCl 4 Induced liver tissue damage in mice while restoring intestinal microbial balance.

[0018] The Cordyceps sinensis polysaccharide CCPW of the present invention has a clear composition and significant efficacy, and has broad development and application prospects in the research on the preparation of candidate preparations for protecting the liver and alleviating acute chemical liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a molecular weight distribution diagram of Cordyceps sinensis polysaccharides. Figure A is a molecular weight distribution diagram of Cordyceps sinensis crude polysaccharides, and Figure B is a molecular weight distribution diagram of Cordyceps sinensis uniform polysaccharide CCPW.

[0020] Figure 2 This is the monosaccharide composition diagram of Cordyceps sinensis polysaccharide CCPW.

[0021] Figure 3 This is the H&E stained section of liver tissue pathology of ALI mice.

[0022] Figure 4 This is the effect of Cordyceps sinensis polysaccharide CCPW on the levels of ALT (Figure A) and AST (Figure B) in ALI mice.

[0023] Figure 5 Figure 3 shows the effects of Cordyceps sinensis polysaccharide CCPW on the levels of inflammatory factors in ALI mice, where A, B, and C are the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, and D is the level of anti-inflammatory factor IL-10.

[0024] Figure 6 , Figure 7 This study is the effect of Cordyceps sinensis polysaccharide CCPW on the expression of intestinal tight junction proteins Occludin and ZO-1 in ALI mice.

[0025] Figure 8-10 This study is the effect of Cordyceps sinensis polysaccharide CCPW on the intestinal flora of ALI mice.

[0026] Figure 8 (A) is a box plot of the Chao1 index differences among groups; (B) is PCoA analysis.

[0027] Fig. 9 Stacked bar chart of relative abundance of species at the genus level based on ASVs.

[0028] Fig.10 Heatmap of relative abundance of species at the genus level. DETAILED DESCRIPTION

[0029] The present invention is further described below with reference to specific embodiments. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

[0030] The cicada fungus used in the present invention is a cicada fungus (i.e., cicada spore) (Isaria cicadae Miq.) strain inoculated on a culture medium for artificial cultivation, and the fruiting body (also known as cicada spore body) product (artificial cultivation) is made through processes such as harvesting the stroma (i.e., spore stalk bundle) and drying, and is provided by the manufacturer "Anhui Cordyceps Source Biotechnology Co., Ltd."

[0031] Example 1: Preparation of Cordyceps sinensis polysaccharide CCPW

[0032] Step 1: crush the fruiting body of Cordyceps sinensis, add 95% ethanol at a solid-liquid ratio of 1 g:20 mL, soak in a 70°C water bath for 6 h, filter and dry, and pass through an 80-mesh sieve to obtain pretreated Cordyceps sinensis powder;

[0033] Step 2: adding pure water to the Cordyceps sinensis powder obtained in step 1 at a solid-liquid ratio of 1 g:40 mL, extracting at 75° C. for 2-3 hours, repeating the extraction 3-4 times, and combining the extracts;

[0034] Step 3: Concentrate the extract obtained in step 2 to 1 / 250 of the original volume, centrifuge at 4000 rpm, collect the supernatant, add 4 times the volume of 95% ethanol, stir thoroughly and let stand at 4°C overnight, and collect the precipitate by centrifugation;

[0035] Step 4: Take the sample obtained in step 3, add pure water to dissolve, prepare a 20 mg / mL polysaccharide solution, prepare Sevag reagent with chloroform and n-butanol in a volume ratio of 4:1, add an equal volume of sugar solution, shake vigorously, centrifuge to discard the middle layer protein, and repeat this operation until there is no obvious protein layer;

[0036] Step 5: The sample obtained in step 4 was separated and purified by DEAE-Sepharose Fast Flow ion exchange chromatography column. The pretreated DEAE-Sepharose Fast Flow filler was stirred evenly and loaded into the chromatography column (35×400mm) along a glass rod. The filler was eluted with double distilled water for equilibrium, and then the compacted filler was eluted with 0.5 mol / L NaCl solution. Finally, the NaCl solution was replaced by double distilled water at a flow rate of 2-3 mL / min. The components were collected, concentrated and freeze-dried to obtain purified Cordyceps sinensis polysaccharide CCPW.

[0037] Example 2: Molecular weight determination, monosaccharide composition and infrared spectroscopy analysis of Cordyceps sinensis polysaccharide CCPW

[0038] (1) Molecular weight determination

[0039] Cordyceps sinensis crude polysaccharide, CCPW and glucan standards (T10, T50, T500, T1000, T2000) were prepared into 3 mg / mL solutions with double distilled water and determined using an Agilent HPLC-evaporative light scattering detector. The chromatographic column was TSK-GelG6000PWXL, the mobile phase was deionized water, and the carrier gas was N 2 , gas flow rate was 2.5 L / min, injection volume was 10 μL. The logarithm of the relative molecular mass (Lg Mw) and retention time (Rt) of the standard sample was used as the standard curve to measure the molecular weight range of Cordyceps sinensis crude polysaccharide and CCPW. The results are shown in Figure 1 As shown in the figure, the crude polysaccharide of Cordyceps sinensis is composed of peaks with retention times of 16.928 min and 19.181 min; CCPW is a single absorption peak with a retention time of 17.143 min. The molecular weight of CCPW is 2.62×10 4 Da.

[0040] (2) Monosaccharide composition analysis

[0041] The monosaccharide composition of the sample after CCPW acid hydrolysis and pre-column PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization was determined by HPLC-DAD (Agilent high performance liquid chromatography-diode array detector). Take 8 mg of CCPW and dissolve it in 5 mL of 2 mol / L trifluoroacetic acid. Seal the tube with nitrogen and place it in an oil bath at 110℃ for 8 h. Add appropriate amount of methanol several times and evaporate it to pH 7. Add 1 mL of deionized water for later use. Take 1 mL of 3 mg / mL standard monosaccharide solution and 1 mL of acid-hydrolyzed CCPW solution, add NaOH (0.5 mL, 0.3 mol / L) and PMP solution (0.5 mL, 0.5 mol / L) in sequence, shake and mix, and react in a 70℃ water bath for 1 h for PMP pre-column derivatization. Neutralize it with HCl (0.5 mL, 0.3 mol / L) to neutrality, then extract it with chloroform three times to remove excess PMP, and take the aqueous phase through a 0.22 μm filter membrane and use HPLC-DAD for detection. Figure 2 The results show that the monosaccharide composition and molar ratio of CCPW are glucose: mannose: galactose = 8.918:1:0.356.

[0042] Example 3: Protective effect of Cordyceps sinensis polysaccharide CCPW on mice with acute chemical liver injury

[0043] SPF C57BL / 6 mice (male, 6-8 weeks, 18-22 g) were selected and provided by Hefei Qingyuan Biotechnology Co., Ltd., animal license number: SCXK (Zhejiang) 2024-0004. All mice were randomly divided into 7 groups after adaptive feeding, namely NC group, MC group, Cordyceps sinensis crude polysaccharide group, CCPW low-dose group (CCPW-L), CCPW medium-dose group (CCPW-M), CCPW high-dose group (CCPW-H), and positive control group (POS). CCPW-L, CCPW-M, and CCPW-H were given 100 mg / kg∙d, 200 mg / kg∙d, and 400 mg / kg∙d of Cordyceps sinensis polysaccharide CCPW, respectively, the Cordyceps sinensis crude polysaccharide group was given 200 mg / kg∙d of Cordyceps sinensis crude polysaccharide, the POS group was given 100 mg / kg∙d, and the NC group and MC group were given an equal volume of pure water, gavage once a day for 14 consecutive days. Two hours after the last administration, mice in the MC group, Cordyceps sinensis crude polysaccharide group, POS group (silymarin), CCPW-L, CCPW-M, and CCPW-H groups were intraperitoneally injected with 1% CCl 4 Corn oil solution 10 mL / kg. After fasting for 24 h, fresh feces, eyeball blood and liver were collected from mice.

[0044] The degree of liver injury was analyzed by hematoxylin-eosin (H&E) staining. The liver tissue was placed in 4% paraformaldehyde, dehydrated, paraffin-embedded and sectioned, and stained with H&E. The pathological changes of liver tissue were observed under an optical microscope. The liver function of ALI mice was evaluated by detecting the levels of ALT and AST in serum. The upper serum was collected by centrifugation at 5000 rpm for 10 min, and the serum ALT and AST levels were determined according to the instructions of the kit (Solebo, Beijing, China). The liver inflammation of ALI mice was evaluated by detecting the inflammatory factors TNF-α, IL-6, IL-1β and IL-10. The expression of intestinal tight junction proteins Occludin and ZO-1 was detected by immunohistochemistry. The determination of 16S rDNA of the mouse intestinal microbiota genome was completed by Shanghai Ouyi Biomedical Technology Co., Ltd.

[0045] Figure 3 This is the H&E staining section of liver tissue pathology of ALI mice. As shown in the figure, the liver lobule structure of mice in the NC group was clear and complete, and the hepatocyte cords were arranged in a regular radial pattern. The hepatocytes of mice in the MC group were swollen and degenerated, and the cytoplasm was loose. Compared with the MC group, the Cordyceps sinensis crude polysaccharide group, CCPW-L group, CCPW-M group and CCPW-H group were able to reduce the degree of liver damage, improve cell swelling and inflammatory cell infiltration, etc. Among them, the hepatocyte cords in the CCPW-H group were arranged most completely, and the structure was relatively clear, which was similar to the effect of the POS group.

[0046] Figure 4 The effect of Cordyceps sinensis polysaccharide CCPW on AST and ALT levels in ALI mice. Compared with the Contro1 group, the ALT and AST levels in the serum of the MC group mice were significantly increased. Compared with the MC group, the ALT and AST levels were reduced to varying degrees in each administration group, and showed a certain dose dependence. Among them, the CCPW-H group had the best improvement effect, which was close to the effect of the POS group, indicating that CCPW can play a liver-protective role in ALI mice.

[0047] Figure 5 Figure 2 shows the effect of Cordyceps sinensis polysaccharide CCPW on the levels of inflammatory factors in ALI mice, where A, B, and C are the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, and D is the level of anti-inflammatory factor IL-10. Figure 5 As shown, CCl 4 After induction, the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in liver tissue increased significantly, and the level of anti-inflammatory factor IL-10 decreased significantly. Compared with POS, CCPW reduced the levels of pro-inflammatory factors and increased the levels of anti-inflammatory factors to a greater extent. This indicates that CCPW can alleviate liver inflammatory response by more effectively inhibiting the expression of pro-inflammatory factors and promoting the expression of anti-inflammatory factors. 4It exerted a stronger protective effect than the positive control drug (silymarin) against acute chemical liver injury induced by silymarin.

[0048] Figure 6 , 7 This is an analysis of the expression of tight junction proteins Occludin and ZO-1 in the intestine of ALI mice treated with Cordyceps sinensis polysaccharide CCPW. The figure shows that the expression of Occludin and ZO-1 in the intestinal epithelial cell membrane and the cytoplasmic region proximal to the membrane in the MC group showed a downward trend, suggesting that in CCl 4 In the induced ALI mice, intestinal barrier damage occurred, and the NC group and CCPW group showed different degrees of positive expression, especially CCPW-H showed more brown-yellow staining. The Image Plus software was used to semi-quantitatively measure the cumulative absorbance of the slices. The results showed that compared with the MC group and the POS group, CCPW administration could significantly increase the expression of Occludin and ZO-1 in the ileum tissue, enhance the closure of the intestinal barrier, and repair intestinal barrier damage.

[0049] Figure 8-10 The effect of Cordyceps sinensis polysaccharide CCPW on the intestinal flora of ALI mice, where 8-A is the Chao1 index box plot between groups; 8-B is the PCoA analysis; Fig. 9 Stacked bar chart of relative abundance of species at the genus level based on ASV; Fig.10 Heatmap of relative abundance of species at the genus level.

[0050] Depend on Figure 8 In Figure A, the intestinal microbial richness index of ALI mice was reduced in the MC group, indicating that CCl 4 The microbial structure was destroyed, and the administration of CCPW could reverse this phenomenon and increase the abundance of intestinal flora in ALI mice; Figure B shows that there were significant differences in the composition of intestinal microbiota in the feces of mice in each group, and the intestinal flora composition of the NC group and the MC group were independently clustered, indicating that the intestinal microbiota would change significantly during liver injury. The administration of CCPW also caused a significant separation of the intestinal microbiota. Except for some overlap between the CCPW-L group and the MC group, there were significant differences among the MC group, the CCPW-M group, and the CCPW-H group. Fig. 9 , 10 As shown in the figure, compared with the MC group, the drug-treated group increased the species abundance of Muribaculaceae and Bacteroides, and reduced the species abundance of Lachnospiraceae_NK4A136_group and Escherichia-Shigella. This indicates that CCPW can shift the intestinal microbiota structure of ALI mice to that of NC mice, improve the abundance of related bacteria at the genus level, and alleviate CCl4 Induced intestinal flora disorder and regulation of body health.

Claims

1. A method for preparing Cordyceps sinensis polysaccharide, characterized in that The steps include: Step 1: crush the fruiting bodies of Cordyceps sinensis, add 95vt% ethanol, soak in a 70°C water bath for 6 h, filter and dry, and pass through an 80-mesh sieve to obtain pretreated Cordyceps sinensis powder; Step 2: adding pure water to the Cordyceps sinensis powder obtained in step 1 for extraction; Step 3: Concentrate the extract obtained in step 2 to 1 / 250 of the original volume, collect the supernatant by centrifugation, add 4 times the volume of 95vt% ethanol, stir thoroughly, let stand at 4°C for 8-12h, and collect the precipitate by centrifugation; Step 4: Take the sample obtained in step 3, add pure water to dissolve it, and prepare a polysaccharide solution; prepare Sevag reagent with chloroform and n-butanol in a volume ratio of 4:1, add an equal volume of the polysaccharide solution, shake vigorously, centrifuge to discard the middle layer protein, and repeat this operation until there is no obvious protein layer; Step 5: Separate and purify the sample obtained in step 4 using a DEAE-Sepharose Fast Flow ion exchange chromatography column, elute and collect the components, and concentrate and freeze-dry to obtain purified Cordyceps sinensis polysaccharide.

2. The preparation method according to claim 1, characterized in that: In step 1, 95vt% ethanol was added at a solid-liquid ratio of 1 g:20 mL.

3. The preparation method according to claim 1, characterized in that: In step 2, the extraction temperature is 75° C., the extraction time is 2-3 h, the extraction is repeated 3-4 times, and the extracts are combined.

4. The preparation method according to claim 1, characterized in that: In step 4, the concentration of the prepared polysaccharide solution is 20 mg / mL.

5. The preparation method according to claim 1, characterized in that: In step 5, when a DEAE-Sepharose Fast Flow ion exchange chromatography column is used for separation and purification, the pretreated DEAE-Sepharose Fast Flow filler is stirred evenly and then loaded into the chromatography column along a glass rod, eluted with double distilled water for equilibrium, and then the compacted filler is eluted with a 0.5 mol / L NaCl solution, and finally the NaCl solution is replaced by double distilled water at a flow rate of 2-3 mL / min.

6. The Cordyceps sinensis polysaccharide prepared according to any one of claims 1 to 5, characterized in that: The monosaccharide composition and molar ratio of the Cordyceps sinensis polysaccharide are glucose: mannose: galactose = 8.918: 1: 0.356, and the relative molecular mass is 2.62×10 4 Da.

7. Use of the Cordyceps sinensis polysaccharide according to claim 6 in the preparation of foods, health products or pharmaceutical preparations having liver-protecting activity and alleviating acute chemical liver damage.

8. The use according to claim 7, characterized in that: The acute chemical liver injury is liver injury caused by CCl4.

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