Uses of honey-processed licorice polysaccharide in the preparation of products for the treatment and prevention of liver fibrosis

Abstract

This invention discloses the use of honey-processed licorice polysaccharide in the preparation of products for the treatment and prevention of liver fibrosis, belonging to the field of biomedical technology. This invention discovers that honey-processed licorice polysaccharide can effectively treat CCl4-induced liver fibrosis, with a dose-dependent effect. The efficacy of high-dose honey-processed licorice polysaccharide is comparable to that of glycyrrhizic acid, without the side effect of causing a decrease in serum potassium levels. Furthermore, honey-processed licorice polysaccharide is superior to raw licorice polysaccharide and stir-fried licorice polysaccharide in inhibiting laminin deposition. This provides a potential candidate drug for the treatment of liver fibrosis and is expected to be used in the preparation of drugs, functional foods, feed, and other products for the prevention or treatment of liver fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of honey-processed licorice polysaccharide in the preparation of products for the treatment and prevention of liver fibrosis. Background Technology

[0002] Liver fibrosis is a common pathological process in the progression of various chronic liver diseases to cirrhosis. Its core mechanism lies in the continuous activation of hepatic stellate cells (HSCs), leading to excessive deposition of extracellular matrix (ECM), primarily composed of collagen. This process is essentially the liver's repair response to various chronic injuries and is a key pathological stage in the progression to cirrhosis and liver cancer. Without timely intervention, liver fibrosis is likely to progress to cirrhosis and even end-stage liver diseases such as liver cancer. Therefore, reversing or delaying liver fibrosis is of great significance in reducing the incidence of cirrhosis and liver cancer.

[0003] From a pathogenesis perspective, liver injury and liver fibrosis are fundamentally different. Liver injury is a pathological event caused by various pathogenic factors (such as viral infection, alcohol or drug metabolites, ischemia and hypoxia) directly or through immune-mediated mechanisms (such as activation of Kupffer cells to release inflammatory factors), leading to hepatocyte degeneration, necrosis, or apoptosis. It is mainly manifested as disruption of cell membrane integrity, leakage of intracellular enzymes (such as ALT and AST), and infiltration of inflammatory cells. Liver fibrosis, on the other hand, occurs on the basis of persistent or repeated liver injury. Damaged cells and activated immune cells release pro-fibrotic factors such as transforming growth factor-β (TGF-β), which in turn activate hepatic stellate cells, causing them to transform into myofibroblasts. This leads to the excessive synthesis and deposition of ECM, mainly type I and type III collagen, forming fibrous scar tissue, resulting in abnormal liver tissue structure and affecting normal liver function. With the persistence of liver fibrosis, normal hepatocytes continuously die and undergo apoptosis, and ECM gradually accumulates and replaces the liver parenchyma, eventually leading to cirrhosis. In short, liver injury focuses on "the destruction and inflammation of hepatocytes," while liver fibrosis originates from "excessive accumulation of ECM due to post-injury repair disorder." The two are causally related but essentially different pathological stages.

[0004] Currently, there is still a lack of approved specific anti-hepatic fibrosis drugs internationally, and the core treatment strategy recommended by clinical guidelines remains etiological treatment (such as antiviral therapy and alcohol abstinence). Targeting the etiology of liver fibrosis and inhibiting HSC activation and proliferation through multiple pathways to reduce ECM deposition is the main research direction for reversing liver fibrosis. According to the Chinese "Guidelines for the Diagnosis and Treatment of Liver Fibrosis with Integrated Traditional Chinese and Western Medicine (2023 Edition)," in addition to etiological treatment, certain potential anti-fibrotic traditional Chinese medicines can be used in combination for "dual anti-therapy" (antiviral + anti-hepatic fibrosis), such as Fuzheng Huayu tablets / capsules, compound turtle shell liver-softening tablets, Anluo Huaxian pills, turtle shell decoction pills, and strong liver capsules. However, the complex composition of these compound formulas, the unclear mechanisms of action, and the difficulty in quality control limit their modernization process.

[0005] Glycyrrhizic acid, a triterpenoid saponin found in licorice, has been proven to have a clear anti-fibrotic effect. However, its clinical application has long been limited by adverse reactions such as pseudoaldosteronism, mainly manifested as hypokalemia and sodium and water retention. This has led to its contraindication in patients with hypokalemia, hypertension, heart failure, impaired renal function, or edema. Although studies have attempted to reduce side effects through structural modifications (such as developing derivatives like magnesium glycyrrhizate, diamine glycyrrhizate, and magnesium isoglycyrrhizate), the results have been unsatisfactory. Therefore, finding alternative anti-fibrotic drugs for liver disease with proven efficacy and higher safety is of significant clinical importance.

[0006] Previous studies have shown that honey-processed licorice exhibits superior hepatoprotective activity compared to raw and stir-fried licorice in liver injury models, and this advantage is closely related to the increased polysaccharide content and structural changes after processing. Further research revealed that the hepatoprotective effect of honey-processed licorice polysaccharides is superior to that of raw and stir-fried licorice polysaccharides, and its mechanism may involve antioxidant and gut microbiota regulation pathways. However, whether honey-processed licorice polysaccharides are equally effective in the liver fibrosis stage remains unclear. Summary of the Invention

[0007] To address the aforementioned shortcomings in the prior art, this invention provides the use of honey-processed licorice polysaccharide in the preparation of products for treating liver fibrosis. Research in this invention has found that honey-processed licorice polysaccharide can effectively treat CCl4-induced liver fibrosis without causing the side effect of decreased blood potassium levels.

[0008] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide the use of honey-processed licorice polysaccharide in the preparation of drugs for treating liver fibrosis.

[0009] Furthermore, honey-processed licorice polysaccharide treats or delays the pathological progression of liver fibrosis by inhibiting the deposition of laminin (LN) in liver tissue, reducing type IV collagen (C-IV), laminin (LN), and hydroxyproline (Hyp), thereby reducing the deposition of extracellular matrix, mainly collagen, in the liver.

[0010] Furthermore, liver fibrosis is CCl4-induced liver fibrosis.

[0011] Furthermore, honey-processed licorice polysaccharide does not cause a decrease in blood potassium levels.

[0012] Furthermore, the products are pharmaceuticals, functional foods, health products, nutritional products, additives, or animal feed.

[0013] Another object of the present invention is to provide a composition for the prevention and / or treatment of liver fibrosis, comprising the above-mentioned honey-processed licorice polysaccharide, and a pharmaceutically, food, or feed-acceptable carrier thereof.

[0014] Furthermore, the dosage form of the composition is tablets, pills, powders, capsules, granules, or liquids.

[0015] Another object of the present invention is to provide the use of the above-mentioned honey-processed licorice polysaccharide or composition in the preparation of products for inhibiting the deposition of laminin in liver tissue.

[0016] Another object of the present invention is to provide the use of the above-mentioned honey-processed licorice polysaccharide or composition in the preparation of products for reducing extracellular matrix deposition, primarily collagen, in the liver.

[0017] The beneficial effects of this invention are: This invention is the first to demonstrate that honey-processed licorice polysaccharide has a significant therapeutic effect on CCl4-induced liver fibrosis. Its anti-liver fibrosis effect is dose-dependent, and the efficacy of high dose (400 mg / kg) is comparable to that of glycyrrhizic acid (35 mg / kg). However, honey-processed licorice polysaccharide does not cause a decrease in blood potassium levels, thus avoiding the risk of pseudoaldosteronism caused by long-term use of glycyrrhizic acid. It is safer and therefore has a wider range of applicable populations than glycyrrhizic acid.

[0018] The present invention also found that honey-processed licorice polysaccharide is significantly superior to the other three licorice polysaccharides (raw licorice polysaccharide, stir-fried licorice polysaccharide and GFSH-processed licorice polysaccharide) in inhibiting laminin deposition, suggesting that it has potential advantages in the treatment of mid-to-late stage liver fibrosis. Attached Figure Description

[0019] Figure 1 A flowchart for animal experiments; Figure 2 The effects of different licorice polysaccharides on body weight and behavioral status in mice with liver fibrosis; Figure 3 The effect of different doses of honey-processed licorice polysaccharide on transaminase levels in mice with liver fibrosis; Figure 4 To investigate the effects of different doses of honey-processed licorice polysaccharide on the contents of C-IV, LN, and Hyp in liver tissue of mice with liver fibrosis; Figure 5 The effects of different doses of honey-processed licorice polysaccharide on liver index, liver pathology, and tissue collagen deposition in mice with liver fibrosis were investigated. Figure 6 The effects of honey-processed licorice polysaccharide and glycyrrhizic acid on serum potassium levels in mice with liver fibrosis.

[0020] Figure 7The effects of polysaccharides from different processed licorice products on liver index, liver pathology, and tissue collagen deposition in mice with liver fibrosis; Figure 8 The effects of polysaccharides from different processed licorice products on the content of C-IV, LN, and Hyp in liver tissue of mice with liver fibrosis. Detailed Implementation

[0021] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0022] Example 1 Preparation of polysaccharides 1. Pretreatment of licorice slices Take 100g of honey-processed licorice root, dry it in an oven at 55℃ until constant weight, pulverize it, and pass it through a 30-mesh sieve to obtain licorice powder. Place the powder in a Soxhlet extractor, add 4 times the amount (v / w) of anhydrous ethanol, reflux for 2 hours, and repeat once to remove lipids and pigments. After defatting, air-dry the powder in a ventilated place.

[0023] 2. Extraction of crude polysaccharides from licorice Weigh 100g of the above-mentioned licorice powder, add 8 times the amount of water (800mL), soak for 1 hour, reflux for 1.5 hours, and 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. After washing the precipitate with ethanol and n-butanol, redissolve it in water, freeze-dry, and obtain honey-processed licorice polysaccharide (HGP) freeze-dried powder.

[0024] The extraction of polysaccharides from other processed licorice products (stir-fried licorice polysaccharide (FGP), raw licorice polysaccharide (RGP), and GFSH-processed licorice polysaccharide (GGP)) was the same as that of honey-processed licorice polysaccharide.

[0025] 3. Purification of crude licorice polysaccharides The crude polysaccharide was dissolved in pure water, extracted with Sevage reagent for 20 min, centrifuged, and the supernatant was retained. This process was repeated until no turbidity was observed. The deproteinized polysaccharide solution was concentrated and dialyzed in deionized water at 4°C. After dialysis, the solution was concentrated under reduced pressure and freeze-dried to obtain honey-processed licorice polysaccharide (HGP).

[0026] Example 2 Animal Experiment 1. Laboratory animals and grouping SPF-grade male ICR mice, 6 weeks old, weighing 20-25g, were housed at the Experimental Animal Center of Capital Medical University (room temperature 25℃, humidity 40%–60%, 12h diurnal cycle). The animal experimental protocol was approved by the Animal Welfare and Ethics Committee (ethics number AEEI-2019-145).

[0027] Ten mice were randomly selected as the blank control group (Control). The remaining mice were injected intraperitoneally with 20% CCl4 peanut oil solution (2 ml / kg) twice a week. After 5 weeks, the model mice were randomly divided into the following groups (n=10 per group): Model group, low / medium / high dose honey-processed licorice polysaccharide group (HGP-L / M / H, 100 / 200 / 400 mg / kg), stir-fried licorice polysaccharide group (FGP, 200 mg / kg), raw licorice polysaccharide group (RGP, 200 mg / kg), GFSH-processed licorice polysaccharide group (GGP, 200 mg / kg), and glycyrrhizic acid group (GA, 35 mg / kg). Each treatment group was administered 0.1 ml / 10 g by gavage once daily for 14 consecutive days; the blank control group and the model group were given an equal volume of physiological saline. The experiment lasted for 7 weeks.

[0028] 2. Sample collection After the last administration, the liver was weighed, blood was drawn from the ophthalmic vein after anesthesia, the patient was euthanized by cervical dislocation, the liver was removed, washed with physiological saline, weighed, and its morphology was observed. Part of the liver was fixed in 4% paraformaldehyde, and the remaining liver and serum were flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0029] 3. Liver function and liver fibrosis marker detection The liver index is calculated using the following formula: Liver index (%) = Liver weight (g) / Body weight (g) × 100%.

[0030] 150 mg of liver was collected and homogenized with 9 times the volume of physiological saline. The supernatant was collected by centrifugation. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), type IV collagen (C-IV), laminin (LN), and hydroxyproline (Hyp) in the liver tissue were detected using a kit. C-IV reflects increased formation and destruction of the basement membrane, while LN reflects basement membrane transformation and is related to portal hypertension. Hyp is a specific amino acid that constitutes collagen; its content reflects the total amount of collagen in the liver, quantifying liver fibrosis.

[0031] 4. Histopathological examination Liver tissue was fixed in 4% paraformaldehyde for more than 24 hours, embedded in paraffin, and sectioned (5 μm thick). Hematoxylin-eosin (H&E), Sirius red, and Masson's trichrome staining were performed, and the pathological changes and collagen deposition in the liver tissue were observed under an optical microscope. Collagen volume fraction (CVF), i.e., the percentage of collagen-positive area to the total field of view, was calculated using an image analysis system.

[0032] 5. Blood potassium concentration measurement The potassium ion concentration in the plasma of mice in each group was measured according to the kit instructions.

[0033] 6. Statistical Analysis All data are expressed as mean ± standard deviation (SD). Differences between groups were assessed using one-way ANOVA. P A value <0.05 is considered statistically significant.

[0034] Example 3: The therapeutic effect of different doses of honey-processed licorice polysaccharide on CCl4-induced liver fibrosis 1. Effects on mouse body weight and behavioral status Changes in body weight of mice in each group are as follows Figure 2 As shown in the figure, A represents the trend of weight change, and B represents the result of the last weigh-in. Data are expressed as mean ± standard deviation (n=10). #Compared with the control group,### P <0.01. *Compared to the model group, *P <0.05, **P <0.01.

[0035] Compared with the blank control group, the weight gain of mice in the other groups was generally slower. With prolonged administration time, the weight gain trend of mice in each treatment group gradually approached that of the control group. Figure 2 (A). The final weighing results showed that the body weight of mice in the model group was significantly lower than that in the blank control group, and the body weight of mice in each drug-treated group was significantly higher than that in the model group. Figure 2 (B)

[0036] Behavioral observation such as Figure 2 As shown in the figure, C represents writhing response, D represents lethargy, reduced activity, and unkempt, dull fur, and E represents a tendency to huddle together. The model group mice exhibited lethargy, reduced activity, a tendency to huddle together, and unkempt, dull fur; the overall condition improved in all drug-treated groups after intervention. Figure 2 (CE).

[0037] 2. Effects on liver transaminases The results are as follows Figure 3 As shown in the figure, A is a bar chart of ALT levels in liver tissue; B is a bar chart of AST levels in liver tissue. Each group has n=6 members. #Compared with the control group, ###P<0.01. *Compared to the Mod group, *P <0.05, **P <0.01. & Compared with the HGP-M group, & P <0.05.

[0038] Compared with the blank control group, the levels of ALT and AST in the model group mice were significantly increased. P <0.01; Compared with the model group, the ALT and AST levels in mice in the medium- and high-dose honey-processed licorice polysaccharide (HGP-M, HGP-H) and glycyrrhizic acid (GA) groups were significantly reduced ( P <0.01). Significant differences were observed among the low, medium, and high dose groups (P<0.05), showing a dose-dependent trend. There was no significant difference between the high-dose honey-processed licorice polysaccharide group and the glycyrrhizic acid group, indicating that they achieved equivalent hepatoprotective efficacy. Figure 3 (A, B)

[0039] 3. Effects on liver pathology and liver index The results are as follows Figure 5 As shown in the figure. Compared with the model group, the liver index of the low-dose honey-processed licorice polysaccharide (HGP-L) group did not show significant improvement, while the liver index of the medium- and high-dose groups (HGP-M, HGP-H) and the glycyrrhizic acid group was significantly reduced. P <0.05, Figure 5 (B)

[0040] H&E staining results showed ( Figure 5 (A) The pathological changes in the HGP-L group were not significantly improved compared with the model group; the liver tissue damage in the HGP-M group was reduced; the liver lobule structure in the HGP-H group was basically intact, with only a small number of scattered inflammatory cells, and the degree of improvement was comparable to that in the GA group.

[0041] 4. Effects on collagen deposition in liver tissue (dose-dependent) The results of Masson's tricolor and Sirius red staining are as follows: Figure 5 As shown. The livers of the model group mice exhibited typical fibrotic pathological features: a large number of blue (Mason's) or bright red (Sirius red) collagen fibers were deposited around the portal area, forming fibrous septa, indicating that collagen deposition occurred in the model group and that the collagen had undergone cross-linking and maturation. Collagen deposition in the HGP-L group was slightly reduced compared to the model group, but the difference was not statistically significant; collagen deposition in the HGP-M group was significantly reduced ( P <0.05); the HGP-H group and GA group showed the most significant improvement, with collagen deposition mainly limited to the perivascular wall ( Figure 5 The results (C and D) indicate that HGP-M / H can effectively reduce collagen deposition and may also inhibit the collagen maturation process.

[0042] 5. Effects on liver fibrosis markers (dose-dependent) The results are as follows Figure 4 As shown in the figure, each group has n=6, #compared to the control group, ###P <0.01. *Compared to the Mod group, *P <0.05, **P <0.01. & Compared with the HGP group, P <0.05. At C-IV and Hyp levels, the HGP-L group showed a decreasing trend compared to the model group, but the difference was not statistically significant. The HGP-M, HGP-H, and GA groups showed significant decreases (…). P <0.05), a significant dose-dependent relationship was observed among the three dose groups, and there was no significant difference between the HGP-H group and the GA group. Regarding LN levels, there was no significant change in the HGP-L group, while the HGP-M and HGP-H groups showed significant decreases (<0.05). P <0.05).

[0043] 6. Effects of honey-processed licorice polysaccharide and glycyrrhizic acid on serum potassium levels in mice with liver fibrosis The results are as follows Figure 6 As shown, after 14 days of continuous administration, compared with the blank control group and the model group, the serum potassium concentration in the GA group mice was significantly lower ( P The value <0.05 indicates that glycyrrhizic acid has the side effect of causing hypokalemia. However, the serum potassium level in the HGP-M group was not significantly different from that in the control group, indicating that licorice polysaccharide treatment for 2 weeks did not affect serum potassium concentration and did not have the side effect of causing hypokalemia.

[0044] Example 4: Comparison of the effects of polysaccharides from different processed licorice products on mice with liver fibrosis. 1. Effects on collagen deposition in liver tissue The results of Masson's trichrome staining and Sirius red staining are as follows: Figure 7 As shown, the livers of the model group mice exhibited typical fibrotic pathological features: a large number of blue (Mason's) or bright red (Sirius red) collagen fibers were deposited around the portal area, forming fibrous septa. The degree of collagen deposition was reduced in all treatment groups, and the fibrous septa became thinner and fewer.

[0045] Quantitative results of image analysis, such as Figure 7 As shown in the figure, (C) is the area ratio of Msson staining positive regions, and (D) is the area ratio of Sirius red staining positive regions. Each group has n=6. #Compared with the control group, ###P <0.01. *Compared to the Mod group, *P <0.05, **P <0.01. The proportion of collagen-positive area in the model group was significantly higher than that in the control group ( P <0.01); compared with the model group, the collagen-positive area in each of the HGP, GGP, FGP, and RGP groups was significantly reduced ( P <0.01, Figure 7 (C, D) There were no differences among the different treatment groups, indicating that HGP, GGP, FGP, and RGP can all effectively reduce collagen deposition in the liver and may also affect the degree of collagen maturation.

[0046] 2. Effects on liver fibrosis markers The results are as follows Figure 8 As shown in the figure, (A) liver hydroxyproline level, (B) liver type IV collagen level, and (C) liver laminin level, with n=6 in each group. #Compared with the control group, ###P <0.01. *Compared to the Mod group, *P <0.05, **P <0.01. & Compared with the HGP-M group, P <0.05. Compared with the blank control group, the levels of C-IV, LN, and Hyp in the liver tissue of mice in the model group were significantly increased ( P <0.05. After intervention with different licorice polysaccharides, the levels of C-IV and Hyp in each treatment group were significantly lower than those in the model group ( P <0.05, indicating that these drugs can all alleviate liver fibrosis. While LN levels showed a decreasing trend in all treatment groups, a statistically significant difference was observed only in the HGP group ( P The value <0.05 indicates that honey-processed licorice polysaccharide may be superior to the other three licorice polysaccharides in inhibiting LN deposition.

[0047] In summary, this invention has found that honey-processed licorice polysaccharide has comparable efficacy to GA in anti-liver fibrosis and does not cause changes in blood potassium concentration. Therefore, honey-processed licorice polysaccharide not only has the effect of treating liver fibrosis, but also has the advantage of avoiding electrolyte imbalance.

[0048] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Use of honey-processed licorice polysaccharide in the preparation of products for the treatment and prevention of liver fibrosis.

2. The use according to claim 1, characterized in that, The honey-processed licorice polysaccharide treats or delays the pathological progression of liver fibrosis by inhibiting the deposition of laminin in liver tissue, reducing the levels of type IV collagen, laminin, and hydroxyproline, thereby reducing the deposition of extracellular matrix, mainly collagen, in the liver.

3. The use according to claim 1, characterized in that, The honey-processed licorice polysaccharide does not cause a decrease in blood potassium levels.

4. The use according to claim 1, characterized in that, The products mentioned are pharmaceuticals, functional foods, health products, nutritional products, additives, or feed.

5. A composition for the prevention and / or treatment of liver fibrosis, characterized in that, This includes the honey-processed licorice polysaccharide as described in claim 1, and its pharmaceutically, food, or feed-acceptable carriers.

6. The composition according to claim 5, characterized in that, The dosage form of the composition is tablets, pills, powders, capsules, granules, or liquids.

7. Use of the honey-processed licorice polysaccharide of claim 1 or the composition of claim 5 or 6 in the preparation of a product for inhibiting the deposition of laminin in liver tissue.

8. Use of the honey-processed licorice polysaccharide of claim 1 or the composition of claim 5 or 6 in the preparation of a product for reducing extracellular matrix deposition, primarily collagen, in the liver.

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