Application of honey-fried glycyrrhiza polysaccharide in prevention and / or treatment of liver injury

Abstract

The invention discloses application of honey-fried glycyrrhiza polysaccharide to prevention and / or treatment of liver injury, and belongs to the technical field of biological medicine. The honey-fried glycyrrhiza polysaccharide can treat liver injury, the curative effect of the honey-fried glycyrrhiza polysaccharide combined with glycyrrhizic acid is superior to that of glycyrrhizic acid or honey-fried glycyrrhiza polysaccharide independently used, and side effects caused by use of glycyrrhizic acid can be relieved. The invention finds that the curative effect of combined use of glycyrrhizic acid and honey-fried glycyrrhiza polysaccharide is superior to that of single use of glycyrrhizic acid or honey-fried glycyrrhiza polysaccharide and is also superior to that of compounding of glycyrrhizic acid and other types of glycyrrhiza polysaccharide, such as fried glycyrrhiza polysaccharide. It is found for the first time that the honey-fried glycyrrhiza polysaccharide has a remarkable treatment effect on CCl4-induced acute liver injury and has dose dependence, and the liver injury treatment effect of the honey-fried glycyrrhiza polysaccharide is equivalent to that of glycyrrhizic acid. In addition, the honey-fried glycyrrhiza polysaccharide can relieve the side effect that the blood potassium level is reduced when glycyrrhizic acid is used. The compound is expected to be used for preparing health-care products, functional foods or medicines capable of preventing or treating inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of honey-roasted licorice polysaccharide in preventing and / or treating liver damage. Background Art

[0002] The carbon tetrachloride (CCl4)-induced liver injury model is a classic animal model for preclinical studies of hepatoprotective efficacy. The pathogenesis of CCl4 hepatotoxicity is primarily attributed to CYP450-dependent metabolic activation, in which the parent compound is enzymatically converted into highly reactive trichloromethyl radicals (·CCl3). These reactive intermediates subsequently initiate a self-propagating lipid peroxidation chain reaction by covalently binding to polyunsaturated fatty acids in cell membranes. The resulting oxidative stress disrupts redox homeostasis, ultimately leading to mitochondrial dysfunction, depletion of antioxidant defense systems, and ultimately necrotic / apoptotic cell death in hepatocytes.

[0003] Glycyrrhizic acid preparations are first-line drugs for the treatment of hepatitis, drug-induced liver injury, and adjuvant treatment of cirrhosis. However, because glycyrrhizic acid inhibits 11β-hydroxysteroid dehydrogenase (11β-HSD2), it causes cortisol to bind to the mineralocorticoid receptor, leading to side effects such as water and sodium retention and increased potassium excretion. The clinical use of glycyrrhizic acid preparations can cause pseudoaldosteronism, leading to adverse reactions such as electrolyte imbalance (hypokalemia, hypernatremia), increased blood pressure and edema, endocrine disorders, gastrointestinal discomfort, and abnormal renal function (increased creatinine). The above reactions are all dose-related. Clinically, it is recommended to give priority to the lowest effective dose for patients with acute hepatitis. For patients with chronic liver disease who require high-dose long-term treatment, regular testing of blood potassium, blood sodium and renal function is required. At the same time, it is stipulated that glycyrrhizic acid preparations cannot be used for patients with hypokalemia, hypertension, heart failure, renal insufficiency, and edema.

[0004] The main source of glycyrrhizic acid is licorice. Licorice is the dried root and rhizome of the legume plants Glycyrrhiza uralensis Fisch., Glycyrrhiza inflate Bat. or Glycyrrhiza glabra L.. As an important Chinese medicinal material with both medicinal and edible properties, licorice is widely used in clinical practice and the food industry. The medicinal forms of licorice used in ancient books and existing standards include: raw licorice, honey-roasted licorice and stir-fried licorice. The research on the basic active substances of licorice has long focused on saponins (such as glycyrrhizic acid) and flavonoids (such as glycyrrhizin and liquiritin). Studies have shown that licorice polysaccharides have multiple biological activities such as antioxidant, anti-inflammatory, immunomodulatory, and anti-tumor. A small number of literature reports that raw licorice polysaccharides have certain hepatoprotective effects. It is worth noting that there have been no research reports on the hepatoprotective effect or efficacy of honey-roasted licorice polysaccharides in treating liver damage. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of existing products, the present invention provides a use of honey-roasted licorice polysaccharide in the preparation of a product for treating liver damage. The present invention has found that honey-roasted licorice polysaccharide can effectively treat liver damage and can also alleviate the side effect of low blood potassium level caused by the use of glycyrrhizic acid.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve the problems of existing products is:

[0007] The purpose of the present invention is to provide the use of honey-roasted licorice polysaccharide in preparing products for preventing and / or treating liver damage.

[0008] Furthermore, the effective dosage of honey-roasted licorice polysaccharide is 110-400 mg / kg.

[0009] Furthermore, the effective dosage of honey-roasted licorice polysaccharide is 110, 130, 150, 170, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 mg / kg, etc.

[0010] Furthermore, the product is a medicine, a nutritional product, a health product or a functional food.

[0011] Another object of the present invention is to provide a composition comprising glycyrrhizic acid and honey-roasted glycyrrhizic acid polysaccharide; wherein the effective amount of glycyrrhizic acid is 20 to 50 mg / kg, and the effective amount of honey-roasted glycyrrhizic acid polysaccharide is 100 to 260 mg / kg.

[0012] Furthermore, the effective amount of glycyrrhizic acid in the composition is 30-50 mg / kg, and the effective amount of honey-roasted licorice polysaccharide is 150-230 mg / kg.

[0013] Another object of the present invention is to provide a product for treating liver damage, which comprises the above-mentioned honey-roasted licorice polysaccharide, or composition.

[0014] Another object of the present invention is to provide use of the above composition in preparing a product for preventing and / or treating inflammatory diseases.

[0015] Furthermore, the product is a medicine, a nutritional product, a health product or a functional food.

[0016] Furthermore, the dosage forms of the product are tablets, pills, powders, liquids, granules, capsules and the like.

[0017] Another object of the present invention is to provide the use of honey-roasted licorice polysaccharide in preparing a product for reducing the side effects of glycyrrhizic acid.

[0018] Furthermore, honey-roasted licorice polysaccharide can reduce the side effect of lowering blood potassium levels caused by the use of glycyrrhizic acid.

[0019] Another object of the present invention is to provide a preparation capable of reducing the side effects of glycyrrhizic acid, comprising honey-roasted glycyrrhizic acid polysaccharide and a pharmaceutically or food-acceptable carrier; the amount of honey-roasted glycyrrhizic acid polysaccharide is 4 to 10 times the amount of glycyrrhizic acid.

[0020] Furthermore, the amount of honey-roasted licorice polysaccharide in the preparation is 4 to 7 times the amount of glycyrrhizic acid.

[0021] Furthermore, the effective dosage of glycyrrhizic acid is 40-50 mg / kg, and the effective dosage of honey-roasted licorice polysaccharide is 200-260 mg / kg.

[0022] Beneficial effects of the present invention:

[0023] The present invention has found that the therapeutic effect of the combined use of glycyrrhizic acid and honey-roasted licorice polysaccharide is better than that of glycyrrhizic acid or honey-roasted licorice polysaccharide used alone, and is also better than the combination of glycyrrhizic acid and other types of licorice polysaccharides, such as stir-fried licorice polysaccharide. For the first time, it was found that honey-roasted licorice polysaccharide has a significant therapeutic effect on acute liver injury induced by CCl4 and is dose-dependent, and the efficacy of honey-roasted licorice polysaccharide in treating liver injury is comparable to that of glycyrrhizic acid. In addition, glycyrrhizic acid has the side effect of lowering blood potassium levels when used, while honey-roasted licorice polysaccharide not only does not cause a decrease in blood potassium levels itself, but also can alleviate the side effect of glycyrrhizic acid that causes a decrease in blood potassium levels when used. This result provides a scientific basis for the development of drugs, functional foods or health foods for the treatment and prevention of liver injury based on honey-roasted licorice polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the experimental flow chart;

[0025] Figure 2 To investigate the efficacy of different processed liquorice decoctions on liver damage in mice;

[0026] Figure 3 To investigate the efficacy of different prepared liquorice decoctions on liver oxidative stress injury;

[0027] Figure 4 The efficacy of different active ingredients of licorice on liver damage in mice;

[0028] Figure 5 The efficacy of different active ingredients of licorice on liver oxidative stress injury;

[0029] Figure 6 The morphological and tissue changes of mouse liver after being treated with different active ingredients of licorice;

[0030] Figure 7 The efficacy of honey-roasted and stir-fried licorice polysaccharides on liver damage in mice;

[0031] Figure 8To investigate the efficacy of honey-roasted and stir-fried licorice polysaccharides on liver oxidative stress injury.

[0032] Figure 9 The effects of honey-roasted licorice polysaccharide and stir-fried licorice polysaccharide on the liver morphology and tissue changes of mice;

[0033] Figure 10 The purpose is to compare the effects of different activities of licorice on blood potassium concentration. DETAILED DESCRIPTION

[0034] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0035] 1. Experimental materials and reagents

[0036] Licorice root was purchased from Sichuan Xinhehua Chinese Medicine Pieces Co., Ltd., China. Bifendate tablets (batch number: 210304) were purchased from Beijing United Pharmaceutical Factory (Beijing, China). Honey was purchased from Beijing Tongrentang Pharmacy (Beijing, China). Carbon tetrachloride (batch number: C12773728) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Edible-grade Luhua peanut oil was used. Aspartate aminotransferase (AST, C009-2-1), alanine aminotransferase (ALT, C010-2-1), malondialdehyde (MDA, A003-4-1), glutathione (GSH, A006-2-1), and superoxide dismutase (SOD, A001-3-2) assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A BCA protein quantification kit (WB6501, Yongtai Xingcheng, Beijing, China) was used. 4% paraformaldehyde tissue fixative (G1101-3ML) and hematoxylin eosin (HE) staining kit (G1076-500ML) were purchased from Wuhan Sewell Biotechnology Co., Ltd. (Hubei, China).

[0037] 2. Experimental Animals

[0038] Seventy male SPF-grade ICR mice, 6 weeks old, weighing 20–25 g, were housed at the Capital Medical University Laboratory Animal Center in an environment with a room temperature of 25°C, a humidity of 40%–60%, and a 12-h day / night cycle. The animals had free access to food and water. All procedures and studies were approved by the Institutional Animal Care and Use Committee of Capital Medical University and were conducted in accordance with the Guide for Institutional Animal Care and Use of Laboratory Animals adopted and promulgated by the National Institutes of Health (Ethics Number AEEI-2019-145).

[0039] Example 1 Therapeutic effects of different processed liquorice products on CCl4-induced liver damage

[0040] 1. Preparation of decoctions of different processed licorice products

[0041] Weigh 100g each of raw licorice, honey-roasted licorice, and stir-fried licorice, add 800ml of water (8 times the volume), soak for 30 minutes, and reflux for 30 minutes. Collect the decoction, add 700ml of water (7 times the volume), and reflux again for 30 minutes. Combine the two decoctions, concentrate, and dilute to 100ml to obtain a 1g / ml decoction of each processed licorice product. Aliquot the decoction and store in a 4°C refrigerator until needed.

[0042] 2. Experimental Animal Grouping, Modeling and Drug Administration

[0043] Animal experiment procedures Figure 1 After one week of adaptive feeding, 70 mice were randomly divided into a control group (Control), a model group (Model), a positive drug bifendate group (Bif, 0.2 g / kg), honey-roasted licorice root (H, 7.5 g / kg), stir-fried licorice root (F, 7.5 g / kg), raw licorice root (R, 7.5 g / kg), and a honey group (honey, 1.9 g / kg), with 10 mice in each group. On Days 1 and 7 of the experimental process, all mice except the control group were intraperitoneally administered with a 0.3% carbon tetrachloride-peanut oil solution (10 ml / kg) to induce a chronic liver injury mouse model. The control group received an equal volume of peanut oil intraperitoneally. Starting on Day 2, mice in each treatment group were gavaged with the corresponding drug (10 ml / kg), while the control and model groups were gavaged with an equal volume of sterile saline once daily for 11 consecutive days. One hour after the last drug administration on Day 12, the mice were weighed, anesthetized, and blood and liver samples were collected. Blood was centrifuged at 4°C to obtain serum. A portion of the liver was fixed in 4% paraformaldehyde, and the remaining liver and serum were quickly frozen in liquid nitrogen and stored at −80°C for subsequent experiments.

[0044] 3. Index test

[0045] (1) Liver index was calculated according to the following formula, and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using commercial kits. Figure 2 A, B, C. Figure 2Control, control group; Modle, model group; Bif, bifendate group; R, raw licorice root; F, stir-fried licorice root; H, honey-roasted licorice root; Honey, honey group. n = 6 per group. # Compared with the control group, #P < 0.05, ##P < 0.01. * Compared with the Mod group, *P < 0.05, **P < 0.01. &P < 0.05, &&P < 0.01. (C) No significant difference between the Bif group and the H group

[0046] Liver index (%) = liver weight (g) / body weight (g) × 100%

[0047] Compared with the control group, the liver index and ALT and AST levels in the model group were significantly increased (P<0.05) ( Figure 2 B, C) showed that the CCl4-induced liver injury model in mice was successfully established. Compared with the model group, the liver ALT and AST levels in groups R, F, and especially group H were significantly reduced. Compared with groups R and F, the liver index in group H was significantly reduced (P<0.05), and the ALT and AST levels were also significantly reduced ( Figure 2 B, C) (P<0.01). It is also worth noting that among all the licorice-treated groups, only group H had a significant decrease in liver index ( Figure 2 A), indicating that honey-roasted licorice is better than other licorice to alleviate liver damage caused by CCl4. Therefore, although all licorice decoctions showed the effect of alleviating liver damage, honey-roasted licorice was significantly more effective than raw licorice or stir-fried licorice (R, F).

[0048] (2) Liver histopathological examination

[0049] To examine the pathological changes in liver tissue, tissue sections from different parts of the liver were fixed with 4% paraformaldehyde for at least 24 hours, washed with PBS, and embedded in paraffin. 5-μm sections were stained with hematoxylin and eosin (H&E) according to standard procedures. The degree of liver tissue necrosis was assessed under a light microscope. Figure 2 D, E.

[0050] The appearance of the liver showed that compared with the Con group, the liver of the Mod group showed obvious changes in texture and color. Granular areas appeared on the surface of the liver, and the color changed from bright red to dull ( Figure 2 Compared with the model group, each drug-treated group showed different degrees of improvement. The area of ​​the granular region of mice in groups F and R was significantly reduced, but there were still obvious granules on the surface. However, the surface of the liver of mice in group H was smooth and flat, with no granules observed. The entire liver was closer to the state of the blank control group ( Figure 2 D).

[0051] Figure 2E shows the results of hematoxylin-eosin (H&E) staining of liver tissues in different groups (×100). The blue arrows in the figure indicate specific pathological changes in the liver tissue, such as inflammatory cell infiltration, hepatocyte degeneration or necrosis. The hepatocytes in the Con group were arranged tightly in a typical radial pattern, with intact structure, and no vacuolar degeneration or inflammatory cell infiltration was observed. In contrast, the hepatic cords in the Mod group were disordered and necrotic; and a large area of ​​inflammatory cell infiltration appeared (the area indicated by the blue arrow). All drug-treated groups alleviated the above-mentioned problems of mice to varying degrees, and the area of ​​inflammatory cell infiltration was significantly reduced. It is worth noting that compared with the F and R groups, the hepatocytes in the H group were regularly distributed and tightly arranged, with almost no inflammatory cell infiltration, which was closer to the cell state of the Con group.

[0052] In summary, different processed licorice can alleviate CCl4-induced liver damage in mice to a certain extent. The effect of honey-roasted licorice (7.5g / kg) is significantly better than that of stir-fried licorice and raw licorice, while the effects of stir-fried licorice and raw licorice are comparable.

[0053] 4. Advantages of honey-roasted licorice in reducing liver oxidative stress damage

[0054] In order to examine the effect of honey-roasted licorice on CCl4-induced oxidative stress in mice, the levels of MDA, SOD and GSH in liver tissue were determined. Figure 3 Figures (A) Histogram of GSH levels in liver tissue; (B) Histogram of SOD levels in liver tissue; (C) Histogram of MDA levels in liver tissue. n = 6 per group. #Compared with the control group, ###P < 0.001. *Compared with the Mod group, **P < 0.01, ***P < 0.001. &P < 0.05, &P < 0.01. No significant differences were found between the Bif group and the H group.

[0055] like Figure 3 As shown in (AC), the water decoctions of different licorice preparations significantly reduced MDA levels (P < 0.01) and increased the levels of endogenous antioxidants GSH and SOD (P < 0.01). However, compared with the R and F groups, the H group showed a significant decrease in MDA levels and a significant increase in GSH and SOD levels (P < 0.05). These results suggest that honey-roasted licorice is more effective than other licorice preparations in reducing CCL4-induced oxidative stress.

[0056] Example 2 Effects of different licorice polysaccharides on CCl4-induced chronic liver injury

[0057] 1. Preparation of polysaccharides

[0058] Take appropriate amount of honey-roasted licorice and stir-fried licorice powder, weigh 100g of powder, add 8 times the amount of water (800ml), soak for 1h, reflux extraction for 1.5h, collect the decoction, add 7 times the amount of water (700ml) to the residue and reflux again for 1.5 hours, combine the two decoctions, concentrate to 100ml, add 375ml of 95% ethanol, precipitate overnight, and collect the precipitate. The precipitate is a crude polysaccharide. After washing with ethanol, the solvent is evaporated and redissolved in water. The sample is freeze-dried to obtain honey-roasted licorice polysaccharide (HGP) and stir-fried licorice polysaccharide (FGP), with freeze-dried powder yields of 4% and 2% respectively. The purity of the polysaccharide is 36% as determined by the anthranone-concentrated sulfuric acid method.

[0059] 2. Animal experiments

[0060] (1) Animal experimental grouping and treatment

[0061] The experimental process is as follows Figure 1 After one week of adaptive feeding with a standard maintenance diet, 90 mice were randomly divided into a control group (Control), a model group (Model), a positive drug bifendate group (Bif, 0.2 g / kg), low-, medium-, and high-dose honey-roasted licorice polysaccharide groups (GP-L / GP-M / GP-H, 100 / 200 / 400 mg / kg), a glycyrrhizic acid group (GA, 50 mg / kg), and a glycyrrhizic acid plus honey-roasted licorice polysaccharide group (HGP-GA, GA: 50 mg / kg, HGP: 200 mg / kg), with 10 mice in each group. The animal modeling method, drug administration process, and sample collection were the same as described above.

[0062] (2) Liver function assessment

[0063] Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured. Figure 4 Figure 1 shows (A) ALT levels in liver tissue; (B) AST levels in liver tissue. n = 6 per group. #Compared with the control group, ###P < 0.01. *Compared with the Mod group, **P < 0.01, ***P < 0.001. &P < 0.05, &&P < 0.01.

[0064] like Figure 4 As shown in Figure 2, compared with the control group, the ALT and AST levels in the liver of the model group were significantly increased (P<0.001) ( Figure 4 A, B). Compared with the model group, the ALT and AST levels in the medium and high doses of honey-roasted licorice polysaccharide groups were significantly reduced (P<0.05, P<0.01), indicating that honey-roasted licorice polysaccharide has a certain effect in treating liver damage, and the effect is dose-dependent. At the same time, the effect of high doses of honey-roasted licorice polysaccharide is equivalent to that of glycyrrhizic acid ( Figure 4A, B), In a CCl4-induced chronic liver injury model, high-dose honey-roasted licorice polysaccharide can achieve an enzyme-lowering effect comparable to that of glycyrrhizic acid.

[0065] It is worth noting that the combined administration of medium-dose glycyrrhizic acid and glycyrrhizic acid (HGP-GA) was more effective than glycyrrhizic acid (GA) alone (P < 0.05, Figure 4 A, B), and was also stronger than the single medium-dose honey-roasted glycyrrhizic acid polysaccharide (GP-M) (P<0.05). These results suggest that in the CCl4-induced chronic liver injury model, the therapeutic effect of the combination of honey-roasted glycyrrhizic acid and glycyrrhizic acid is superior to that of glycyrrhizic acid alone or high-dose honey-roasted glycyrrhizic acid alone.

[0066] (3 The results of determination of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) in liver tissue are shown in Figure 5 Figures (A) Histogram of liver tissue GSH levels; (B) Histogram of liver tissue SOD levels; (C) Histogram of liver tissue MDA levels. n = 6 per group. #Compared with the control group, ###P < 0.001. *Compared with the Mod group, *P < 0.05, **P < 0.01, ***P < 0.001. &P < 0.05, &&P < 0.01. (A)(B) There were no significant differences between the GP-L / M / H groups, nor between the HGP-GA group and the H group. (C) There were no significant differences between the GP-L group and the GP-M group.

[0067] like Figure 5 As shown in the figure, compared with the blank group, MDA in the liver tissue of the Mod group was significantly increased, while SOD and GSH were significantly decreased. Compared with the Mod group, the three doses of honey-roasted licorice polysaccharides (HGP-L / M / H) significantly decreased MDA, while GSH was significantly increased. SOD in the HGP-M / H group was significantly increased ( Figure 5 AC) (P<0.05), indicating that honey-roasted licorice polysaccharide has a certain effect on improving liver tissue oxidative stress damage, and the effect is dose-dependent. At the same time, the effect of high-dose honey-roasted licorice polysaccharide is equivalent to that of glycyrrhizic acid, indicating that in the CCl4-induced liver injury model, high-dose honey-roasted licorice polysaccharide can achieve the same effect as glycyrrhizic acid in improving liver tissue oxidative stress damage.

[0068] It is worth noting that the therapeutic effect of the combination of medium-dose glycyrrhizic acid and glycyrrhizic acid (HGP-GA) was significantly stronger than that of glycyrrhizic acid and glycyrrhizic acid alone ( Figure 5 AC, P < 0.05). The results suggest that in the CCl4-induced liver injury model, the therapeutic effect of the combination of honey-roasted licorice polysaccharide and glycyrrhizic acid is superior to that of glycyrrhizic acid alone or high-dose honey-roasted licorice polysaccharide alone.

[0069] (4) Liver histopathological examination: evaluate the degree of liver tissue necrosis. The results are shown in Figure 6 (A) Liver pathological observation; (B) Based on H&E staining (100x original magnification), different active ingredients in licorice and honey-fried licorice decoction can alleviate CCL4-induced liver injury. Areas of severe liver necrosis and inflammatory cell infiltration are marked with arrows. Scale bar: 50 μm.

[0070] Figure 6 (A) Shows the macroscopic morphological changes in the livers of mice in the different treatment groups. Compared to the Con group, the livers in the Mod group showed significant changes in texture and color, with granular areas appearing on the liver surface and the color shifting from bright red to a duller color. Each treatment group showed varying degrees of improvement. Compared to the other treatment groups, the liver morphology of HGP-GA was closer to that of the blank group.

[0071] Figure 6 (B) Hematoxylin and eosin (H&E) staining of liver tissue from different groups (×100). In the Con group, hepatocytes showed a typical radially dense arrangement, with intact structure, and no vacuolar degeneration or inflammatory cell infiltration. In contrast, in the Mod group, hepatic cords were disorganized, necrotized, and fibrotic, with extensive inflammatory cell infiltration (indicated by blue arrows). Honey-roasted licorice polysaccharide (L / M / H) showed a dose-dependent improvement in liver pathological changes. Hepatocyte regeneration and gradually denser arrangement were observed, and the area of ​​inflammatory cell infiltration in the portal tract was reduced compared to the Mod group. The high-dose honey-roasted licorice polysaccharide group showed a very small area of ​​inflammatory cell infiltration, comparable to that of the GA group. In the HGP-GA group, inflammatory cell infiltration was essentially unobservable, and the integrity of hepatic lobule reconstruction was closer to that of the Con group. Pathological results suggest that the different treatment groups may produce a synergistic therapeutic effect by regulating hepatocyte regeneration, inhibiting inflammatory responses, and improving fibrosis progression.

[0072] The above results showed that in the CCl4-induced liver injury model, the therapeutic effect of high-dose honey-roasted licorice polysaccharide was comparable to that of glycyrrhizic acid, and the therapeutic effect of medium-dose honey-roasted licorice polysaccharide and glycyrrhizic acid combined (HGP-GA) was significantly stronger than that of glycyrrhizic acid and honey-roasted licorice polysaccharide alone.

[0073] Example 3: Detection of the therapeutic effects of honey-roasted licorice and stir-fried licorice polysaccharides on chronic liver injury induced by CCl4

[0074] 1. Grouping and treatment of experimental animals

[0075] The experimental process is as follows Figure 1After one week of adaptive feeding with a standard maintenance diet, 60 mice were randomly divided into a control group (Control), a model group (Modle), a positive drug bifendate group (Bif, 0.2 g / kg), a glycyrrhizic acid-co-administered honey-roasted glycyrrhizic acid polysaccharide group (HGP-GA: 50 mg / kg GA + 200 mg / kg HGP), and a glycyrrhizic acid-co-administered stir-fried glycyrrhizic acid polysaccharide (FGP) low- and medium-dose groups (FGP-GA-L / M, GA: 50 mg / kg, FGP: 150 / 200 mg / kg), with 10 mice in each group. The animal modeling methods, dosing procedures, and sample collection were the same as those for the same family members.

[0076] 2. Liver function assessment

[0077] (1) The experimental method for liver function assessment is the same as 1.3.2

[0078] Liver index, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity results are shown in Figure 7 Figures: (A) Histogram of ALT levels in liver tissue; (B) Histogram of AST levels in liver tissue; (C) Histogram of liver index. n = 6 per group. #Compared with the control group, ###P < 0.01. *Compared with the Mod group, **P < 0.01, ***P < 0.001. &P < 0.05, &&P < 0.01.

[0079] Compared with the control group, the levels of ALT and AST in the liver of the model group were significantly increased (P<0.001) ( Figure 7 A, B). Compared with the model group, the ALT and AST levels in the HGP-GA group and the FGP-GA-L / M group were significantly decreased (P<0.01). Compared with the FGP-GA-L / M group, the ALT and AST levels in the HGP-GA group were significantly decreased (P<0.05, Figure 7 A, B). The results showed that the above drug combinations were effective in treating chronic liver damage caused by CCl4, and HGP-GA was more effective than FGP-GA-L / H.

[0080] In addition, the liver index of the Mod group was significantly higher than that of the Con group (P<0.001). Compared with the Mod group, the liver index of each group containing glycyrrhizic acid (GA) was significantly reduced (P<0.05) ( Figure 7 C).

[0081] (2) The results of GSH / SOD and MDA content in mouse liver are shown in Figure 8Figures: (A) GSH level in liver tissue; (B) SOD level in liver tissue; (C) MDA level in liver tissue. n = 6 per group. #Compared with the control group, ###P < 0.001. *Compared with the Mod group, *P < 0.05, **P < 0.01, ***P < 0.001. &P < 0.05, &&P < 0.01.

[0082] Compared with the Con group, MDA in the liver tissue of the Mod group was significantly increased, while SOD and GSH were significantly decreased, indicating that CCl4 caused oxidative stress damage to the liver of mice (P<0.001, Figure 8 AC). Compared with the Mod group, the GSH and SOD contents in the liver of mice in each drug-treated group were significantly increased, and the MDA content in the liver was significantly decreased (P<0.01, Figure 8 AC). It is worth noting that compared with FGP-GA-L / M, the SOD level in the liver of mice in the HGP-GA group was significantly increased (P<0.05), while the MDA level was significantly decreased (P<0.05, P<0.001, Figure 8 BC). This indicates that HGP-GA has a more significant effect on improving oxidative stress damage than FGP-GA. This indicates that honey-roasted licorice polysaccharide HGP has a significant synergistic effect when used in combination with GA, and its efficacy is significantly better than the combination of stir-fried licorice polysaccharide FGP and GA.

[0083] (3) The results of liver histopathological examination are shown in Figure 9 In the figure, (A) liver pathological observation; (B) based on H&E staining (100x original magnification), the combination of glycyrrhizic acid with honey-roasted licorice polysaccharide and stir-fried licorice polysaccharide improved CCl4-induced chronic liver injury to varying degrees. Areas of severe liver necrosis and inflammatory cell infiltration are marked with arrows. Scale bar: 50 μm.

[0084] Figure 9 (A) shows the macroscopic morphological changes of the livers of mice in different treatment groups. It can be seen that compared with the Con group, the texture and color of the liver in the Mod group have changed significantly. The liver in the model group is granular, and the color changes from bright red to dull. Each intervention group showed different degrees of improvement. Among them, the liver tissue surface of the HGP-GA group, the drug-treated group, returned to a smooth state, and its state was closer to the normal Con group. It is worth noting that compared with the HGP-GA group, a small amount of granularity can still be seen on the livers of the two FGP-GA groups. Therefore, through liver morphological observation, it can be preliminarily inferred that the honey-roasted licorice polysaccharide HGP is better than the stir-fried licorice polysaccharide FGP in improving liver pathological morphology.

[0085] H&E staining results ( Figure 9B) shows: the liver tissue structure of mice in the Con group is intact, and the hepatocytes are tightly arranged. Compared with the Con group, the liver tissue of mice in the Mod group is loose, the hepatic cords are disordered, the intercellular spaces are enlarged, and large areas of inflammatory cell infiltration can be seen (indicated by blue arrows). Compared with the Mod group, the liver tissue of mice in each drug-treated group is gradually tightly arranged, the intercellular spaces are reduced, and the density of inflammatory cell infiltration approaches the normal range. Among them, the HGP-GA group can clearly observe a decrease in fatty degeneration of hepatocytes, and the inflammatory cell infiltration around the central vein is basically close to that of the Con group. Compared with the HGP-GA group, the two FGP-GA groups still have large areas of inflammatory infiltration, and fatty degeneration vacuoles of hepatocytes are still present.

[0086] The above results show that the synergistic administration of glycyrrhizic acid and glycyrrhizic acid polysaccharides can effectively improve hepatocyte inflammatory infiltration and promote hepatocyte regeneration, playing a synergistic role. However, not all types of glycyrrhizic acid polysaccharides can produce a synergistic effect with glycyrrhizic acid. The above tests show that only honey-roasted glycyrrhizic acid polysaccharides can produce a synergistic effect with glycyrrhizic acid. When stir-fried glycyrrhizic acid polysaccharides are used together, there is no synergistic effect.

[0087] Example 4 Effects of different active ingredients of honey-roasted licorice on blood potassium concentration in mice

[0088] Take 50 μl of serum from each of the blank group, GP-M / H group, GA group, and HGP-GA group in Example 2. Test the blood potassium concentration according to the instructions of the reagent. The results are shown in the table. Figure 10 In the figure, *<0.05, **p<0.01, n=5.

[0089] like Figure 10 As shown in the results, after 12 consecutive days of drug administration, the blood potassium concentration of mice was significantly reduced compared with the blank group, GA group, and HGP-GA group (p<0.05, P<0.01), indicating that the above drugs can cause a decrease in the blood potassium level of mice, verifying the clinical side effect of glycyrrhizic acid causing hypokalemia. However, there was no significant difference between medium and high doses of licorice polysaccharide, indicating that licorice polysaccharide does not cause a decrease in blood potassium concentration.

[0090] Compared to the GA group, the HGP-GA group showed significantly higher blood potassium levels. Furthermore, HGP-GA is more effective than GA in treating liver damage, meaning that the dosage of HGP-GA can be reduced to achieve the same therapeutic effect. Therefore, the combined use of HGP-GA and GP-H, while achieving the same therapeutic effect as GA, can mitigate the side effect of glycyrrhizic acid-induced hypokalemia.

[0091] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Use of honey-roasted licorice polysaccharide in preparing products for preventing and / or treating liver damage.

2. The use according to claim 1, characterized in that The effective dosage of the honey-roasted licorice polysaccharide is 110-400 mg / kg.

3. The use according to claim 1, characterized in that The product is a medicine, a nutritional product, a health product or a functional food.

4. A composition, characterized in that The composition comprises glycyrrhizic acid and honey-roasted glycyrrhizic acid polysaccharide; the effective dosage of the glycyrrhizic acid is 20-50 mg / kg, and the effective dosage of the honey-roasted glycyrrhizic acid polysaccharide is 100-260 mg / kg.

5. A product for preventing and / or treating liver damage, characterized in that: The invention comprises the honey-roasted licorice polysaccharide as claimed in claim 1 or the composition as claimed in claim 4.

6. Use of the composition according to claim 4 in preparing a product for preventing and / or treating inflammatory diseases.

7. The use according to claim 6, characterized in that The product is a medicine, a nutritional product, a health product or a functional food.

8. The use according to claim 6 or 7, characterized in that The dosage form of the product is tablet, pill, powder, liquid, granule or capsule.

9. Use of the honey-roasted licorice polysaccharide according to claim 1 in preparing a product for reducing the side effects of glycyrrhizic acid, characterized in that: Honey-roasted licorice polysaccharide can reduce the side effect of lowering blood potassium levels caused by the use of glycyrrhizic acid.

10. A composition capable of reducing the side effects of glycyrrhizic acid, characterized in that: The preparation comprises honey-roasted licorice polysaccharide and a pharmaceutically or food-acceptable carrier; the dosage of the honey-roasted licorice polysaccharide is 4 to 10 times the dosage of glycyrrhizic acid.

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