Pharmaceutical application of harpagoside

Abstract

The invention discloses an application of harpagoside in preparation of a medicine for preventing and / or treating hepatic fibrosis. The invention discloses an application of harpagoside in preparation of a health care product with an auxiliary protection effect on chemical liver injury. The animal level proves that the harpagoside can reduce hepatic fibrosis such as chemical hepatic fibrosis induced by carbon tetrachloride, reduce liver collagen deposition, reduce hydroxyproline content in hepatic tissue, reduce increased HA and Col IV content in serum, and reduce expression of fibrosis and collagen deposition related genes, so that the harpagoside can be used for treating hepatic fibrosis. The traditional Chinese medicine composition has a good effect of treating or preventing hepatic fibrosis.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to the pharmaceutical use of harpagosine, specifically to the use of harpagosine in the preparation of medicaments for the prevention and / or treatment of liver fibrosis, and particularly to the use of harpagosine in the preparation of medicaments for the prevention and / or treatment of chemical liver fibrosis. Background Technology

[0002] Liver fibrosis is a persistent process of liver damage and repair following various forms of chronic liver injury. This repeated process leads to excessive and abnormal deposition of extracellular matrix (ECM) in the liver. In the early stages of liver fibrosis, the symptoms can be reversed if the underlying cause of liver damage is eliminated. However, without effective treatment, chronic liver damage cannot be improved, and the process of liver damage and repair continues, eventually leading to cirrhosis and even liver cancer. The pathogenesis of liver fibrosis is complex, involving different cell types and signaling pathways in the liver, and the mechanisms are not yet fully understood. Therefore, its treatment remains a major challenge in international medicine. Although the pathogenesis of liver fibrosis is complex, excessive activation of hepatic stellate cells (HSCs) is recognized as a core event in the development and progression of liver fibrosis. It has been reported that the incidence of liver fibrosis in people with different types of chronic liver disease is 18%–27%, and approximately 1.2 million people die from cirrhosis and 810,000 from liver cancer worldwide each year. Currently, there are no universally recognized, specific, and effective chemical drugs or biological agents for the prevention and treatment of liver fibrosis. Liver transplantation is the only treatment option for decompensated liver disease. Therefore, finding drugs that can effectively inhibit or improve liver fibrosis is of great significance.

[0003] Harpagoside (HPG) is an iridoid glycoside extracted from plants such as Scrophularia ningpoensis Hemsl. It is an indicator chemical component for evaluating the quality of Scrophularia ningpoensis medicinal materials in the 2020 edition of the Chinese Pharmacopoeia. Modern research shows that harpagoside has effects such as lowering blood sugar, protecting the myocardium, improving Alzheimer's disease, and protecting the nervous system. Summary of the Invention

[0004] The purpose of this invention is to provide new pharmaceutical uses for harpagoside.

[0005] Use of harpagoside in the preparation of medicines for the prevention and / or treatment of liver fibrosis.

[0006] The liver fibrosis described herein is characterized by at least one of the following: increased collagen deposition in the liver, elevated hydroxyproline content in liver tissue, elevated serum hyaluronic acid, elevated serum type IV collagen, and increased expression of fibrosis and collagen deposition-related genes.

[0007] Preferably, the use is the use of harpagoside in the preparation of a medicament for the prevention and / or treatment of liver fibrosis by inhibiting liver collagen deposition, reducing hydroxyproline content in liver tissue, reducing serum hyaluronic acid content, reducing serum type IV collagen content, and inhibiting the expression of fibrosis and collagen deposition-related genes.

[0008] The dosage form of the drug is a pharmaceutically acceptable oral dosage form or injectable formulation.

[0009] The oral preparations are tablets, capsules, granules, suspensions, drops, pills, or oral liquid preparations; the injectable preparations are injection solutions or powder injections.

[0010] Preferably, the liver fibrosis is chemical liver fibrosis.

[0011] Specifically, the aforementioned chemical liver injury refers to carbon tetrachloride-induced liver fibrosis.

[0012] Use of harpagoside in the preparation of health products that have an adjunctive protective effect against chemically induced liver damage.

[0013] The aforementioned chemical liver injury is characterized by at least one of the following: increased liver collagen deposition, elevated hydroxyproline content in liver tissue, elevated serum hyaluronic acid, elevated serum type IV collagen, and increased expression of fibrosis and collagen deposition-related genes.

[0014] The health supplement is in a pharmaceutically acceptable oral dosage form.

[0015] The oral preparations mentioned are tablets, capsules, granules, suspensions, drops, pills, and oral liquids.

[0016] The genes related to fibrosis and collagen deposition are Acta2, Col1a1, and Col3a1.

[0017] The beneficial effects of this invention are:

[0018] This invention demonstrates in animal studies that harpagoside can reduce liver fibrosis, such as carbon tetrachloride-induced chemical liver fibrosis, reduce liver collagen deposition, decrease hydroxyproline content in liver tissue, reduce elevated HA and Col IV levels in serum, and decrease the expression of genes related to fibrosis and collagen deposition, thus exhibiting good therapeutic or preventive effects on liver fibrosis. Attached Figure Description

[0019] Figure 1Results of Masson and Sirius Red staining of liver tissue.

[0020] Figure 2 Results of Real-Time PCR. Detailed implementation

[0021] The technical solution of the present invention is described in detail in specific implementation manners. Through the description of specific implementation manners, the features, purposes and advantages of the present invention will become more obvious.

[0022] Example 1

[0023] The carbon tetrachloride (CCl4)-induced mouse liver fibrosis model is an animal experimental model widely used in liver fibrosis research. In this invention, the CCl4-induced mouse liver fibrosis animal model is used to evaluate the therapeutic effect of harpagoside on liver fibrosis.

[0024] 1.1 Experimental materials

[0025] [[ID=二十一]]1.1.1 Experimental animals

[0026] Male SPF-grade C57BL / 6J mice, weighing 18 - 20 g, were purchased from Shanghai Slac Laboratory Animal Co., Ltd. [Certificate number: SYXK (Shanghai) 2020 - 0009], and were raised in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. The temperature for raising mice was (22 ± 1) °C, the relative humidity was (65 ± 10)%, and the light was cycled for 12 h / 12 h.

[0027] 1.1.2 Drugs

[0028] Harpagoside (HPG) was purchased from Chengdu Efa Biotechnology, and the positive drug silymarin (SIL) was purchased from Shanghai Yuanye Biotechnology.

[0029] 1.2 Experimental methods

[0030] 1.2.1 Animal experiment protocol

[0031] Male C57BL / 6 mice were randomly divided into 5 groups, with 5 mice in each group. The groups were: Control group (blank control); CCl4 group (model group); CCl4+SIL group (positive control group, silymarin administered at a dose of 200 mg / kg, prepared as a suspension using double-distilled water, and administered by gavage); CCl4+HPG low-dose group (harpagosine administered at a dose of 30 mg / kg, prepared as a solution using double-distilled water, and administered by gavage); and CCl4+HPG high-dose group (harpagosine administered at a dose of 60 mg / kg, prepared as a solution using double-distilled water, and administered by gavage). After one week of acclimatization, except for the Control group mice, the other mice were intraperitoneally injected with a CCl4-ol-ol mixture (CCl4 dissolved in olive oil at a volume ratio of 1:3), at a dose of 2 mL / kg. One week later, mice injected intraperitoneally with CCl4 were randomly and evenly divided into four groups: a CCl4 group, a CCl4+SIL group, a low-dose CCl4+HPG group, and a high-dose CCl4+HPG group. All four groups received daily drug intervention, while the control group received an equal volume of double-distilled water. This treatment continued for three weeks. After four weeks of intraperitoneal injection of a CCl4-ol-marine mixture (equivalent to three weeks of HPG administration), the mice were anesthetized, their eyeballs were enucleated for blood collection, and the animals were euthanized. Liver tissue was then harvested, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0032] 1.2.2 Observation of Masson and Sirius Red staining in liver tissue

[0033] Liver tissues from the same location (liver lobule) of mice were taken for pathological examination. The tissues were fixed with 4% paraformaldehyde solution, embedded in paraffin, sectioned, and stained with Masson and Sirius Red. The liver morphological changes in each group were evaluated under an optical microscope.

[0034] 1.2.3 Detection of hydroxyproline (HYP) content in liver tissue

[0035] Accurately weigh 50 mg of liver tissue sample and add 0.5 mL of hydrolysis buffer, then mix well. Cap the sample and hydrolyze at 95°C for 20 min. Add 0.5 mL of pH adjustment solution A to each tube, mix well, then adjust the pH to 6.0–6.8 with pH adjustment solution B, add double-distilled water to 5 mL, and mix well. Take 0.5 mL of the diluted hydrolysis buffer, mix with activated charcoal, centrifuge at 1250 × g for 10 min, and collect 0.25 mL of the supernatant. Add reagents I, II, and III respectively according to the instructions of the hydroxyproline (HYP) assay kit (alkaline hydrolysis method) from Nanjing Jiancheng Biotechnology Co., Ltd., mix well, incubate at 60°C for 15 min, cool to room temperature, centrifuge at 1250 × g for 10 min, collect the supernatant, and measure the absorbance at 550 nm. Calculate the hydroxyproline content in the liver tissue.

[0036] Hydroxyproline content = (Measured OD value - Blank OD value) ÷ (Standard OD value - Blank OD value) × Standard concentration (5 μg / mL) × (Total volume of hydrolysate ÷ Tissue wet weight);

[0037] The blank OD value is the absorbance of double-distilled water measured at 550 nm after the experiment was conducted according to the kit requirements. The standard OD value is the absorbance of 5 μg / mL hydroxyproline standard provided by the kit measured at 550 nm after the experiment was conducted according to the kit requirements.

[0038] 1.2.4 Detection of serum hyaluronic acid (HA) and collagen type IV (Col IV) levels

[0039] Mouse serum was collected, and the levels of HA and Col IV in mouse serum were detected using the Shanghai Weiao Biotechnology ELISA kit according to the kit instructions, employing an antibody sandwich method. The procedure is summarized as follows: Samples and standards were added to a pre-coated ELISA plate, incubated at 37°C for 50 minutes, and washed three times; biotinylated detection antibody was added, incubated at 37°C for 50 minutes, and washed three times; SABC complex was added, incubated at 37°C for 30 minutes, and washed three times; TMB chromogenic solution was added, incubated at 37°C for 10-20 minutes, and stop solution was added. The ELISA plate was placed in an ELISA reader, and the absorbance was measured at 450 nm. A standard curve was plotted with standard concentration on the x-axis and OD value on the y-axis. The levels of HA and Col IV in the sample were calculated based on the sample OD value and the standard curve. 1.2.5 Detection of mRNA expression in liver tissue

[0040] Total RNA extraction: 15 mg of liver tissue was cut and placed in a 1.5 mL centrifuge tube. 1 mL of Trizol homogenate was added and the mixture was incubated on ice for 5 min. 200 μL of chloroform was added and shaken for 15 s. The mixture was centrifuged at 12000 × g for 15 min at 4 °C. The colorless aqueous layer was transferred to a new tube and 250 μL of isopropanol was added. The mixture was shaken thoroughly and incubated for 10 min. The mixture was centrifuged at 12000 × g for 10 min at 4 °C to obtain RNA precipitate. The supernatant was discarded. 1 mL of pre-cooled 75% ethanol was added and the mixture was centrifuged at 7500 × g for 5 min at 4 °C. After centrifugation, the ethanol was discarded. After the precipitate was dried, 200 μL of DEPC water was added to dissolve it completely. The resulting RNA solution was used to determine the RNA concentration in each sample. The solution was stored at -80 °C for later use.

[0041] Reverse transcription: Prepare the reverse transcription system by adding the appropriate reagents according to the kit instructions and mixing thoroughly. Incubate at 37℃ for 30 min, then at 85℃ for 1 min. Perform reverse transcription according to this temperature program. After reverse transcription, store the cDNA sample at -80℃ for later use.

[0042] Real-time PCR: Prepare the reaction mixture according to the kit instructions, with a loading volume of 10 μL. Perform real-time PCR amplification experiments according to the instrument instructions. 2 -ΔΔ Ct method to calculate gene expression differences between two groups.

[0043] Table 1. Primer sequences

[0044]

[0045] 1.2.6 Data Statistics

[0046] Experimental data are expressed as mean ± standard deviation (Mean ± SD). SPSS 16.0 software was used for statistical analysis, and one-way ANOVA was performed to compare the variances between groups. All data are expressed as mean ± standard deviation (Mean ± SD). Compared with the blank control group, * P<0.05, ** P<0.01, *** P<0.001; compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0047] 1.3 Experimental Results

[0048] 1.3.1 Effects of HPG on collagen deposition in liver tissue of CCl4-induced liver fibrosis mice

[0049] Masson staining and Serius Red staining results of liver tissue are shown in the figure. Figure 1 CCl4 can cause significant collagen deposition in the liver of mice, starting in the portal area and spreading outward, bridging with collagen in adjacent portal areas to form obvious fibrous septa that completely isolate the liver lobules. SIL (200 mg / kg) can reduce CCl4-induced collagen deposition, but the liver lobules are not completely isolated, and some "portal-portal" bridging fibrous septa still exist. Low-dose harpagoside (30 mg / kg) has comparable efficacy to SIL, while high-dose harpagoside (60 mg / kg) has significantly better efficacy than SIL, with collagen deposition only appearing around the portal area, and no bridging fibrous septa forming in adjacent areas.

[0050] 1.3.2 HPG reduces elevated HYP content in liver tissue of mice with CCl4-induced liver fibrosis.

[0051] The experimental results are shown in Table 2. Compared with the blank control group, the HYP content in the liver of mice in the CCl4 group was significantly increased (P<0.01), and high-dose harpagosine (60mg / kg) could reduce the CCl4-induced increase in HYP content in the liver of mice (P<0.01).

[0052] Table 2. Effects of HPG on HYP content in liver tissue of mice with CCl4-induced liver fibrosis

[0053]

[0054] Note: Compared with the blank control group ** P<0.01; compared with the CCl4 group, # P<0.05, ## P<0.01; n=5.

[0055] 1.3.3 HPG reduces elevated HA and Col IV levels in the serum of CCl4-induced liver fibrosis mice.

[0056] The ELISA results are shown in Table 3. Compared to the blank control group, the serum HA content in the CCl4 group was significantly increased (P<0.01), while different doses of harpagosine (30, 60 mg / kg) significantly reduced the CCl4-induced increase in HA content (P<0.01). Similarly, compared to the blank control group, the serum Col IV content in the CCl4 group was significantly increased (P<0.01), while different doses of harpagosine (30, 60 mg / kg) significantly reduced the CCl4-induced increase in Col IV content (P<0.01).

[0057] Table 3. Effects of HPG on serum HA and Col IV levels in mice with CCl4-induced liver fibrosis

[0058]

[0059] Note: Compared with the blank control group ** P<0.01; compared with the CCl4 group, ## P<0.01; n=5.

[0060] 1.3.4 HPG reduces the expression of genes related to fibrosis and collagen deposition in CCl4-induced liver fibrosis mice.

[0061] Acta2 encodes smooth muscle actin (α-SMA), and increased expression of Acta2 represents activation of hepatic stellate cells and is considered a typical marker of liver fibrosis. Col3a1 mainly encodes type III collagen, an important component of the extracellular matrix and a hallmark molecule of fibrosis.

[0062] The results of the real-time PCR experiment are shown below. Figure 2 The results showed that different doses of HPG (30, 60 mg / kg) could significantly reduce the expression of genes such as Acta2 and Col3a1 induced by CCl4 (P<0.05), inhibit the activation of hepatic stellate cells and reduce collagen deposition in the liver, thereby playing a role in improving liver fibrosis.

[0063] In summary, harpagoside can reduce hepatic collagen deposition, decrease hepatic and serum fibrosis markers (HYP, HA, and Col IV), inhibit hepatic stellate cell activation, and reduce the expression of genes related to fibrosis and collagen deposition, thereby improving liver fibrosis.

[0064] All features disclosed in this specification may be used in any compositional form, and each feature disclosed in the specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0065] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness or practicality of the invention. The scope of protection claimed by the present invention should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims. In conclusion, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. Use of harpagoside in the preparation of drugs for the prevention and / or treatment of liver fibrosis.

2. The use according to claim 1, characterized in that: The liver fibrosis described herein is characterized by at least one of the following: increased collagen deposition in the liver, elevated hydroxyproline content in liver tissue, elevated serum hyaluronic acid, elevated serum type IV collagen, and increased expression of fibrosis and collagen deposition-related genes.

3. The use according to claim 1, characterized in that: The dosage form of the drug is a pharmaceutically acceptable oral dosage form or injectable formulation.

4. The use according to claim 3, characterized in that: The oral preparations are tablets, capsules, granules, suspensions, drops, pills, or oral liquid preparations; the injectable preparations are injection solutions or powder injections.

5. Use of harpagoside in the preparation of health products that have an auxiliary protective effect against chemically induced liver damage.

6. The use according to claim 5, characterized in that: The aforementioned chemical liver injury is characterized by at least one of the following: increased liver collagen deposition, elevated hydroxyproline content in liver tissue, elevated serum hyaluronic acid, elevated serum type IV collagen, and increased expression of fibrosis and collagen deposition-related genes.

7. The use according to claim 5, characterized in that: The health supplement is in a pharmaceutically acceptable oral dosage form.

8. The use according to claim 7, characterized in that: The oral preparations mentioned are tablets, capsules, granules, suspensions, drops, pills, and oral liquids.

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