Application of jonquil glucoside in preparation of medicine for treating metabolism-related fatty liver disease

Abstract

The invention belongs to the technical field of biological pharmacy, and provides an application of jonquil glucoside in preparation of a medicine for treating a metabolism-related fatty liver disease, and the application is that the jonquil glucoside is applied to preparation of the medicine for treating the metabolism-related fatty liver disease. The treatment effect of the jonquil glycoside on MASLD is evaluated by adopting a method for establishing a fatty liver model through induction of HFD high-fat, high-fructose and high-cholesterol feed. Results show that the jonquil glucoside can reduce the liver weight by inhibiting weight gain, reduce the content of TC and TG in serum, improve the conditions of liver inflammation and fat change, reduce the oxidative stress level of liver tissue and restore SIRT1 protein expression of the liver tissue to achieve the purpose of treating MASLD.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology and relates to a longevity flower glycoside, specifically to the application of a longevity flower glycoside in the preparation of a drug for treating metabolic-related fatty liver disease. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a multi-system metabolic stress-induced liver injury. Its spectrum includes simple non-alcoholic fatty liver, non-alcoholic steatohepatitis (NASH), and associated liver fibrosis and cirrhosis, ultimately progressing to hepatocellular carcinoma. Following a name change in consensus statements developed by global multidisciplinary expert groups in 2020 and 2022, NAFLD has now been redefined as the more representative metabolic dysfunction-associated steatotic liver disease (MASLD).

[0003] Epidemiological studies show that the prevalence of MASLD is increasing year by year, and it has now become the leading cause of chronic liver disease worldwide, posing a significant impact on human health and imposing a huge economic burden on public health systems. Notably, the patient population with MASLD is becoming increasingly younger, indicating that more and more patients may suffer from MASLD and its related complications long-term. Furthermore, recent studies have shown that even mild fatty liver disease can significantly increase mortality from various factors, and the severity of this risk is closely related to disease progression.

[0004] The current treatments for MASLD present significant challenges, and existing therapies fail to provide satisfactory intervention strategies. In recent years, some progress has been made in drug development, including GLP-1 receptor agonists (such as smegglutide), farnesol X receptor agonists (such as obeticholic acid), and PPARα / δ dual agonists (such as Elafibranor, Resmetirom, Centicriviroc, and selonsertib). However, in clinical practice, these drugs have shown limitations to varying degrees, including significant adverse reactions and limited efficacy in addressing the systemic metabolic problems associated with MASLD. Therefore, exploring innovative treatment options and shifting from single-cause control to multi-mechanism combined intervention strategies may offer greater assistance in the development of MASLD treatments.

[0005] In this context, traditional Chinese medicine, with its systemic and low-toxicity therapeutic advantages, is receiving increasing attention from scholars both domestically and internationally in the prevention and treatment of MASLD. Some natural drug-derived compounds exhibit multi-target, systemic therapeutic potential.

[0006] Longevity flower glycoside is a compound isolated from the leaves of Elaeagnus pungens, with the chemical name (2R,3E)-4-[(1S)-1-Hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-1-yl]-3-buten-2-ylβ-D-glucopyranoside. It is found in plants such as vine tea and tea tree roots.

[0007] Existing research suggests that Kalanchoe glycosides may play an antiviral role in the intervention and treatment of viral hepatitis. However, research on its effects on metabolic diseases such as fatty liver is still lacking. Therefore, this invention uses a high-fat, high-fructose, and high-cholesterol (HFD) diet to induce a fatty liver model, evaluates the therapeutic effect of Kalanchoe glycosides on MASLD, explores the effects of Kalanchoe glycosides on weight reduction, lipid reduction, and anti-inflammation, and proposes its application in the preparation of drugs for metabolic diseases such as fatty liver, providing a new approach and means for the treatment of MASLD. Summary of the Invention

[0008] The purpose of this invention is to provide an application of Kalanchoe glycoside in the preparation of a drug for treating metabolic-associated fatty liver disease (MASLD). By applying Kalanchoe glycoside to the preparation of a drug for treating MASLD, a new approach and means is provided for the treatment of MASLD.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] This invention provides the application of longevity flower glycoside in the preparation of a drug for treating metabolic-related fatty liver disease.

[0011] Preferably, the longevity glycoside is used as the sole active ingredient or in combination with other drugs to treat metabolic-related fatty liver disease.

[0012] Preferably, the drug is used to lower blood lipids by reducing the levels of TC and TG in serum.

[0013] Preferably, the drug is used to reduce the levels of ALT and AST in liver tissue to promote liver function recovery.

[0014] Preferably, the drug is used to improve liver inflammation and steatosis.

[0015] Preferably, the drug is used to reduce the level of oxidative stress in liver tissue.

[0016] Preferably, the drug is used to restore the expression level of SIRT1 protein in liver tissue.

[0017] Preferably, the drug comprises kaempferol glycoside and medically approved excipients.

[0018] Preferably, the drug includes various acceptable dosage forms.

[0019] Preferably, the preparation is an injection, pill, capsule, granule, tablet, or oral liquid.

[0020] The beneficial effects of this invention are:

[0021] This invention employs a high-fat, high-fructose, and high-cholesterol (HFD) diet to induce a fatty liver model, explores the effects of *Lysimachia foenum-graecum* glycosides on weight reduction, lipid reduction, and anti-inflammation, and evaluates the therapeutic effects of *Lysimachia foenum-graecum* glycosides on fatty liver and other metabolic diseases. Results show that *Lysimachia foenum-graecum* glycosides can reduce HFD-induced weight and liver weight gain in mice, lower the levels of blood lipids and liver function indicators TG, TC, ALT, and AST, and improve hepatic steatosis and inflammatory response to achieve the therapeutic goal of MASLD. Attached Figure Description

[0022] Figure 1 The results show the effects of Kalanchoe glycosides on body weight and liver weight in HFD-induced MASLD mice (A shows the changes in body weight of HFD-induced MASLD mice with Kalanchoe glycosides, and B shows the changes in liver weight of HFD-induced MASLD mice with Kalanchoe glycosides; where CON represents the normal group, HFD represents the model group, LROS represents the low-dose Kalanchoe glycoside treatment group, MROS represents the medium-dose Kalanchoe glycoside treatment group, HROS represents the high-dose Kalanchoe glycoside treatment group, and PPC represents the polyene phosphatidylcholine positive control group). * P < 0.05 *** P < 0.001 compared with the CON group; * P < 0.05 ** P < 0.01, *** P < 0.001 (compared with the HFD group);

[0023] Figure 2 The following graph shows the effect of Kalanchoe glycosides on blood lipids in HFD-induced MASLD mice (A shows the TG content of blood lipids in HFD-induced MASLD mice, and B shows the TC content of blood lipids in HFD-induced MASLD mice; where CON represents the normal group, HFD represents the model group, LROS represents the low-dose Kalanchoe glycoside treatment group, MROS represents the medium-dose Kalanchoe glycoside treatment group, HROS represents the high-dose Kalanchoe glycoside treatment group, and PPC represents the polyene phosphatidylcholine positive control group). ***P < 0.001, compared with group CON; * P < 0.05 ** P < 0.01, *** P < 0.001 (compared with the HFD group);

[0024] Figure 3 The following figures illustrate the effects of Kalanchoe glycosides on liver function in HFD-induced MASLD mice: (A shows the ALT content in the liver of HFD-induced MASLD mice, and B shows the AST content in the liver of HFD-induced MASLD mice; CON represents the normal group, HFD the model group, LROS the low-dose Kalanchoe glycoside treatment group, MROS the medium-dose Kalanchoe glycoside treatment group, HROS the high-dose Kalanchoe glycoside treatment group, and PPC the polyene phosphatidylcholine positive control group). *** P < 0.001, compared with group CON; * P < 0.05 *** P < 0.001 (compared with the HFD group);

[0025] Figure 4 The figure shows the effect of Kalanchoe glycosides on the liver tissue pathology of HFD-induced MASLD mice in this invention (the figure shows the HE cell staining results and Oil Red O staining results (200×); where CON is the normal group, HFD is the model group, LROS is the low-dose Kalanchoe glycoside treatment group, MROS is the medium-dose Kalanchoe glycoside treatment group, HROS is the high-dose Kalanchoe glycoside treatment group, and PPC is the polyene phosphatidylcholine positive control group).

[0026] Figure 5 The figure shows the effect of Kalanchoe glycosides on the level of oxidative stress in liver tissue of HFD-induced MASLD mice (where CON is the normal group, HFD is the model group, LROS is the low-dose Kalanchoe glycoside treatment group, MROS is the medium-dose Kalanchoe glycoside treatment group, HROS is the high-dose Kalanchoe glycoside treatment group, and PPC is the polyene phosphatidylcholine positive control group). *** P < 0.001, compared with group CON; ** P < 0.01, *** P < 0.001 (compared with the HFD group);

[0027] Figure 6The following figures show the effects of Kalanchoe glycosides on SIRT1 protein expression in the liver tissue of HFD-induced MASLD mice (A is the Western Blot result of Kalanchoe glycosides on SIRT1 protein expression in HFD-induced MASLD mice, and B is the relative expression result of SIRT1 protein; where CON is the normal group, HFD is the model group, LROS is the low-dose Kalanchoe glycoside treatment group, MROS is the medium-dose Kalanchoe glycoside treatment group, HROS is the high-dose Kalanchoe glycoside treatment group, and PPC is the polyene phosphatidylcholine positive control group). *** P < 0.001, compared with group CON; ** P < 0.01, *** P < 0.001 (compared with the HFD group). Detailed Implementation

[0028] This invention provides the application of longevity flower glycoside in the preparation of a drug for treating metabolic-related fatty liver disease.

[0029] The present invention does not have any special requirements on the source of the longevity flower glycoside. In specific embodiments of the present invention, it can be extracted and isolated from the leaves of Elaeagnus angustifolia or purchased directly.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] Application Examples

[0034] This invention uses a high-fat diet to induce HFD to establish a fatty liver model, and verifies the therapeutic effect of longevity flower glycosides on MASLD.

[0035] It should be noted that the longevity flower glycosides used in this invention were commercially purchased (purity 95.0%, catalog number PS2796, Chengdu Pusi), and the amount used is the amount converted to 100% purity.

[0036] 1. Experimental Methods

[0037] 1.1. Modeling and Grouping

[0038] Male normal C57BL / 6 mice aged 6-8 weeks were selected and acclimatized to SPF for one week. Mice were weighed, their toes clipped, and numbered. They were then randomly divided into 6 groups of 8 mice each using a random number table. The normal control group (CON) was fed a standard diet, while the model group (HFD) and all treatment groups were fed a high-fat, high-fructose, high-cholesterol diet (Xyz Biotech, XT310). Mice were allowed free access to food for 16 weeks. From the start of modeling, the treatment groups were administered longevity glycosides (5, 10, 20 mg / kg / day) via gavage as low, medium, and high doses (LROS, MROS, HROS), respectively. Polyene phosphatidylcholine (PPC, 178 mg / kg / day, human equivalent dose) was administered as a positive control.

[0039] 1.2 TG, TC, ALT, AST detection

[0040] Detected using a fully automated biochemical analyzer.

[0041] 1.3 Methods for detecting reactive oxygen species levels

[0042] Fresh mouse liver tissue was collected, washed thoroughly with PBS, and 450 μL of homogenate was added to 50 mg of tissue. The tissue was homogenized thoroughly. Centrifuged at 10000 g, 4 °C for 10 min, the precipitate was discarded, and the supernatant was collected. 90 μL of homogenate buffer, 10 μL of tissue homogenate supernatant, and 1 μL of 100×DHE staining solution were added to a black 96-well plate. The plate was incubated at 37 °C in the dark for 20 min. The absorbance was measured using a microplate reader with an excitation wavelength of 520 nm and an emission wavelength of 605 nm. The absorbance value represents the level of reactive oxygen species (ROS).

[0043] 1.4 Protein content determination method

[0044] Mouse livers were harvested and homogenized using RIPA lysis buffer supplemented with protease and phosphatase inhibitors, then centrifuged at 14000g for 15 minutes. The supernatant was collected, and protein concentration was determined using a BCA protein assay kit. Equal volumes of protein from each sample were mixed with loading buffer and boiled for 5 minutes to denature the protein. Then, based on the molecular weight of the target protein, samples were subjected to SDS-PAGE gel electrophoresis on gels of appropriate concentrations, and the separated proteins were transferred to PVDF membranes. The membranes were blocked and incubated overnight at 4°C with a target protein-specific antibody. After washing with TBST, the membranes were incubated with the corresponding secondary antibody at room temperature, followed by detection of protein bands using ECL chemiluminescence. The relative content of SIRT1 protein was calculated based on the gray values ​​of the protein bands. Beta-actin was used as an internal control. SIRT1 (DF6033, Affinity) and Beta-actin (abs171598, Absin) were both commercially available products.

[0045] 1.5 HE staining method

[0046] Liver tissue samples were removed from formalin fixative and rinsed continuously with running water for 4 hours to remove residual fixative and impurities. The tissue samples were then dehydrated in an automated dehydrator. After dehydration, the samples were embedded in paraffin to form paraffin blocks. The paraffin blocks were sliced ​​into 5 μm thick sections using a microtome and attached to glass slides. The dried sections were then immersed in xylene for 10 minutes, repeated once, to completely remove the paraffin. Next, they were immersed in anhydrous ethanol for 5 minutes, then again for 2 minutes, followed by immersion in 95%, 90%, 80%, and 70% ethanol for 2 minutes each. Finally, the sections were rinsed with distilled water and then washed three times with PBS solution for 5 minutes each time to ensure adequate tissue hydration and removal of impurities. The sections were then stained with hematoxylin for 1 minute to stain the cell nuclei. The sections were rinsed with distilled water to remove excess stain. Next, the sections were immersed in 0.4% hydrochloric acid ethanol for 4 seconds for differentiation, rinsed again with distilled water, and then immersed in 0.6% ammonia water for bluing to make the cell nuclei more clearly stained. Finally, the sections were rinsed with running water. The sections were then stained with eosin for 2 minutes to stain the cytoplasm. The stained sections were then immersed in 95% ethanol for 5 minutes, then in anhydrous ethanol for 5 minutes, and finally in xylene for 5 minutes to completely dehydrate the sections. After the sections were air-dried, they were mounted with neutral resin and photographed.

[0047] 1.6 Oil Red O Staining Method

[0048] Pre-cool the cryostat to ensure the machine temperature is stable at a suitable low temperature. Remove the frozen tissue sample and, after the sample temperature has equilibrated, thaw it on ice in preparation for subsequent sectioning. Attach the thawed tissue sample to the cryostat holder using OCT adhesive, ensuring the sample is firmly fixed. Place the specimen holder on the pre-cooled cryostat and, after the OCT adhesive has solidified, section it to a thickness of 8 μm. Gently attach the section to a pre-labeled glass slide, ensuring the section adheres firmly. Remove the frozen section and rinse it in a container of 60% isopropanol for 20 seconds to remove impurities and residual OCT adhesive from the section surface. Mix Oil Red O dye and diluent at a ratio of 5:2 and filter twice with filter paper to ensure the staining solution is homogeneous and free of impurities. Immerse the section in the Oil Red O staining solution for 10 minutes to ensure the lipid droplets are fully stained. After staining, rinse the sections with 60% isopropanol for 20 seconds to remove excess Oil Red O staining solution, then wash the sections with preheated double-distilled water at 37°C to ensure the section surface is clean. Counterstain the sections with hematoxylin (counterstain) for 4 minutes to stain the cell nuclei. Mount the sections with a water-based mounting medium preheated to liquid state at 60°C and photograph them.

[0049] 1.7 Data Analysis

[0050] Data were processed using GraphPadPrism 7.0 software, and are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and repeated measures ANOVA was used for comparisons of repeated measures data. P < 0.05 was considered statistically significant.

[0051] 2 Experimental Results

[0052] 2.1 Effects of Kalanchoe glycosides on body weight and liver weight in HFD-induced MASLD mice

[0053] The body weight and liver weight of the mice showed that at the beginning of the experiment, there was no significant difference in body weight among the groups (P>0.05). During the modeling treatment, the mice fed a high-fat, high-fructose, and high-cholesterol diet gained weight rapidly, with the HFD group showing the fastest weight gain. By the end of the experiment, the body weight of the CON mice was basically stable, while the body weight of the HFD group continued to increase, with a statistically significant difference compared to the CON group (P<0.05). The body weight of the mice in each treatment group also continued to increase, but the rate of increase was significantly lower than that in the HFD group. Figure 1 A). At the end of the experiment, the livers of the mice were weighed. Compared with the CON group, the liver weight of the HFD group was significantly increased (P < 0.05); while compared with the HFD group, the liver weight of the mice in each treatment group was significantly decreased, and the differences were statistically significant (P < 0.05). Among them, the specific treatment effects were HROS > MROS > LROS > PPC ( Figure 1 B). This result indicates that longevity glycosides can inhibit body weight gain and reduce liver weight in HFD mice.

[0054] 2.2 Effects of Kalanchoe glycosides on blood lipids in HFD-induced MASLD mice

[0055] Detection of TC and TG in the blood lipids of mice showed that, compared with the CON group, the serum TC and TG levels in the HFD group were significantly increased (P < 0.05); while compared with the HFD group, the TC and TG levels in each treatment group were significantly decreased, and the differences were statistically significant (P < 0.05). Figure 2 Among them, the specific treatment effects are HROS > MROS > PPC > LROS. Figure 2 This result indicates that longevity glycosides can reduce the levels of TC and TG in mouse serum and lower blood lipids in mice.

[0056] 2.3 Effects of Kalanchoe glycosides on liver function in HFD-induced MASLD mice

[0057] Detection of ALT and AST in the liver of mice showed that, compared with the CON group, the serum ALT and AST levels in the HFD group were significantly increased (P < 0.05); while compared with the HFD group, the ALT and AST levels in all treatment groups were significantly decreased, and the differences were statistically significant (P < 0.05). Figure 3 Among them, the specific therapeutic effects of ALT are HROS > MROS > LROS > PPC ( Figure 3 A), the specific therapeutic effects of AST are: HROS > MROS > PPC > LROS ( Figure 3 B). This result indicates that longevity flower glycosides can reduce the levels of ALT and AST in the liver and promote the recovery of liver function.

[0058] 2.4 Effects of Kalanchoe glycosides on liver histopathology in HFD-induced MASLD mice

[0059] After HE and Oil Red O staining, observation of mouse liver cells revealed that, compared with the CON group, the HFD group showed significant fatty degeneration, fatty vacuoles, and inflammatory cell infiltration in the liver tissue; while compared with the HFD group, the liver inflammation and fatty degeneration of mice in all treatment groups were improved; among them, the most significant improvement was observed in the HROS group. Figure 4 This result indicates that longevity glycosides can reduce liver inflammation and improve hepatic steatosis in mice.

[0060] 2.5 Effects of Kalanchoe glycosides on oxidative stress levels in liver tissue of HFD-induced MASLD mice

[0061] ROS (Reactive Oxidant Scale) is an important indicator of hepatic oxidative stress. Analysis of ROS levels in mouse liver tissue showed that, compared to the CON group, the HFD group had significantly higher levels of oxidative stress in liver tissue (P < 0.05); while compared to the HFD group, all treatment groups showed significantly lower levels of oxidative stress in liver tissue, with statistically significant differences (P < 0.05). Specifically, the treatment efficacy was HROS > MROS > LROS > PPC (Reactive Oxidant Scale). Figure 5 This result indicates that Kalanchoe glycosides can regulate the level of oxidative stress in liver tissue.

[0062] 2.6 Effect of Kalanchoe glycosides on SIRT1 protein expression in liver tissue of HFD-induced MASLD mice

[0063] Detection of SIRT1 protein in mouse liver tissue showed that all groups expressed SIRT1 protein ( Figure 6A). Compared with the CON group, the expression level of SIRT1 in the liver tissue of mice in the HFD group was significantly decreased (P < 0.05); while compared with the HFD group, the SIRT1 content in mice in each treatment group was restored, and the difference was statistically significant (P < 0.05). Figure 6 B). This result indicates that Kalanchoe glycosides can restore SIRT1 protein expression in liver tissue, further demonstrating that the pharmacodynamic mechanism of Kalanchoe glycosides is related to SIRT1-mediated oxidative stress.

[0064] In summary, Kalanchoe glycosides can alleviate HFD-induced increases in body weight and liver weight in mice, reduce the levels of blood lipids and liver function indicators such as TG, TC, ALT, and AST, and improve hepatic steatosis and inflammatory response. Its mechanism of action is related to SIRT1-mediated oxidative stress. Kalanchoe glycosides can be used as the sole active ingredient or in combination with other drugs to treat metabolic-related fatty liver disease.

[0065] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Use of a longevous flower glycoside in the preparation of a drug for treating metabolic-related fatty liver disease.

2. Use according to claim 1, characterized in that, The longevous flower glycoside is used as the only active ingredient or in combination with other drugs to treat metabolic-related fatty liver disease.

3. Use according to claim 2, characterized in that, The drug is used to reduce the content of TC and TG in serum to reduce blood lipids.

4. Use according to claim 2, characterized in that, The drug is used to reduce the content of ALT and AST in liver tissue to promote liver function recovery.

5. Use according to claim 2, characterized in that, The drug is used to improve liver inflammation and fatty change.

6. Use according to claim 2, characterized in that, The drug is used to reduce the level of oxidative stress in liver tissue.

7. Use according to claim 2, characterized in that, The drug is used to restore the expression amount of SIRT1 protein in liver tissue.

8. Use according to claim 2, characterized in that, The drug comprises the longevous flower glycoside and a medically permissible excipient.

9. Use according to claim 2, characterized in that, The drug comprises various acceptable dosage forms.

10. Use according to claim 9, characterized in that, The preparation is an injection, a pill, a capsule, a granule, a tablet or an oral liquid.

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