Pharmaceutical application of harpagide

Abstract

The invention discloses application of harpagide in preparation of a medicine for preventing and / or treating a non-alcoholic fatty liver disease. The harpagide can obviously reduce the increased contents of triglyceride, free fatty acid and hydroxyproline in the liver tissue of a mouse with the non-alcoholic fatty liver disease, and reduce the increased myeloperoxidase activity in the liver tissue; meanwhile, the harpagide can also reduce the increased NAS score and reduce lipid accumulation, inflammatory cell infiltration and fibrosis in the liver of the NAFLD mouse.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to the pharmaceutical use of harbazoside, specifically to the use of harbazoside in the preparation of drugs for the prevention and / or treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD), also suggested by some studies to be renamed metabolic associated fatty liver disease (MAFLD), is a spectrum of liver diseases characterized by ectopic fat deposition and hepatic steatosis. It ranges from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH), including varying degrees of liver fibrosis, progressing to cirrhosis and end-stage liver disease, and involving related complications such as hepatocellular carcinoma. NAFLD affects 25% of adults worldwide and is the most common chronic liver disease globally. With the increasing prevalence of obesity and diabetes, NAFLD has become the leading cause of chronic liver disease and abnormal liver function tests in developed Western countries, and it shows a global and younger age of onset trend. Therefore, NAFLD is gradually becoming a major threat to public health.

[0003] Harpagide (HAR) is an iridoid glycoside extracted from plants such as Scrophularia ningpoensis Hemsl. It is an indicator component for evaluating the quality of Scrophularia ningpoensis in the 2020 edition of the Chinese Pharmacopoeia. Modern research shows that harpagide has antioxidant, antiviral, anti-inflammatory, anti-osteoporosis, and neuroprotective effects. Summary of the Invention

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

[0005] Use of harbazoside in the preparation of medicines for the prevention and / or treatment of non-alcoholic fatty liver disease.

[0006] Furthermore, the aforementioned non-alcoholic fatty liver disease includes non-alcoholic fatty liver, non-alcoholic steatohepatitis, fatty liver fibrosis, and / or cirrhosis.

[0007] Furthermore, the stated use is the use of harbazoside in the preparation of a medicament for the prevention and / or treatment of liver fibrosis by exerting at least one of the following effects: reducing triglyceride levels in the liver, reducing free fatty acid levels in the liver, reducing hydroxyproline levels in the liver, reducing myeloperoxidase activity in the liver, reducing lipid accumulation in the liver, reducing hepatocyte necrosis, reducing inflammatory cell infiltration, and reducing fibrosis. Furthermore, the dosage form of the medicament is a solid or liquid formulation.

[0008] Furthermore, the solid dosage form includes tablets, sustained-release tablets, dispersible tablets, capsules, sustained-release capsules, granules, pills, and powder for injection. The liquid dosage form includes oral liquid preparations or injectable liquid preparations. The oral liquid preparation includes oral liquids, suspensions, and drop pills.

[0009] The beneficial effects of this invention are:

[0010] Harbaguline significantly reduced elevated triglyceride (TG), free fatty acid (NEFA), and hydroxyproline (HYP) levels in the liver tissue of mice with non-alcoholic fatty liver disease induced by methionine-choline deficient diets (MCDD) or high-fat diets (HFD), and decreased elevated myeloperoxidase (MPO) activity in the liver tissue. Simultaneously, harbaguline also reduced elevated NAS scores and decreased lipid accumulation, inflammatory cell infiltration, and fibrosis in the livers of NAFLD mice. These results indicate that harbaguline can significantly improve NAFLD. Attached Figure Description

[0011] Figure 1 The results of H&E and Oil Red-O staining of liver tissue from the MCDD model.

[0012] Figure 2 The NAS score results for the MCDD model.

[0013] Figure 3 The results of F4 / 80 and Ly6G staining of liver tissue from the MCDD model.

[0014] Figure 4 H&E and Oil Red-O staining results of liver tissue from an HFD model

[0015] Figure 5 The NAS score results for the HFD model.

[0016] Figure 6 The results of F4 / 80 and Ly6G staining of liver tissue from the HFD model. Detailed Implementation

[0017] The technical solutions of the present invention are described in detail through examples. Through the description of the examples, the features, objectives, and advantages of the present invention will become more obvious.

[0018] Example 1

[0019] Feeding mice with methionine-choline deficient diet (MCDD) to induce NAFLD is a commonly used animal model for NAFLD. In this experiment, the MCDD-induced NAFLD animal model was used to evaluate the therapeutic effect of harpagide on NAFLD.

[0020] 1.1 Experimental materials

[0021] 1.1.1 Experimental animals

[0022] Male clean-grade C57BL / 6J mice, weighing 18 - 20 g, were purchased from Shanghai Slack Experimental 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 feeding temperature of the mice was (22 ± 1) °C, the relative humidity was (65 ± 10)%, and the light was cycled for 12 h / 12 h.

[0023] 1.1.2 Drugs

[0024] Harpagide was purchased from Chengdu Efa Biotechnology Co., Ltd.

[0025] 1.1.3 Reagents

[0026] Triglyceride (TG) test kit, nonesterified fatty acid (NEFA) test kit, hydroxyproline (HYP) assay kit, and myeloperoxidase (MPO) test kit were all purchased from Nanjing Jiancheng Bioengineering Institute.

[0027] 1.2 Experimental methods

[0028] 1.2.1 Animal experiment protocol

[0029] C57BL / 6J mice were randomly divided into 4 groups: (1) methionine-and-choline supplement diet (MCSD) group (blank control group); (2) MCDD group (model group); (3) MCDD+HAR30 group (single dose 30 mg / kg, dissolved in physiological saline and administered by gavage, low dose of harbazoside); (4) MCDD+HAR60 group (single dose 60 mg / kg, dissolved in physiological saline and administered by gavage, high dose of harbazoside). Except for the MCSD group (7 mice), the other three groups had 8 mice each. After one week of acclimatization, the MCSD group was fed MCS diet daily, while the other three groups were fed MCD diet for 6 consecutive weeks to induce NAFLD model. After successful establishment of NAFLD model, the MCDD+HAR30 and MCDD+HAR60 groups were administered harbazoside by gavage for 4 consecutive weeks, while the MCSD and MCDD groups were given the same dose of physiological saline. Four weeks after treatment, blood and liver tissue were collected for drug efficacy testing and liver tissue section staining.

[0030] 1.2.2 Observation of liver tissue morphology through staining

[0031] Liver tissue from the same location (liver lobule) of mice was fixed with 4% paraformaldehyde solution, embedded in paraffin, and sectioned. Some paraffin sections were stained with hematoxylin and eosin (H&E), while others were stained with Oil Red O dye. The stained sections were observed under an optical microscope to assess the morphological changes and lipid accumulation of the liver tissue.

[0032] 1.2.3 NAS Rating

[0033] The NAS score is a semi-quantitative scoring system. Based on the results of H&E-stained pathological sections, it scores the hepatocyte steatosis, lobular inflammation and necrosis, and hepatocyte ballooning. The scores are then summed to obtain the NAS score. A NAS score ≥ 5 can diagnose NASH, while a NAS score < 3 can exclude NASH. The scoring criteria are shown in Table 1.

[0034] Table 1. NAS Scoring Details

[0035]

[0036] 1.2.4 Detection of Triglyceride (TG) Content in Liver Tissue

[0037] Weigh 25 mg of liver tissue and add 9 times the volume of anhydrous ethanol homogenizing medium (g:mL) at a ratio of 1:9. Homogenize mechanically under ice-water bath conditions, centrifuge at 2500 rpm for 10 minutes, and collect the supernatant for analysis. In a 96-well plate, select one blank well, one standard well, and the rest as sample wells. Add 2.5 μL of distilled water, standard, and sample to the blank well, standard well, and sample well, respectively. Add 250 μL of working solution to each well, mix well, and incubate at 37°C for 10 minutes. Measure the absorbance at 510 nm using a microplate reader. Calculate the triglyceride content in the tissue sample.

[0038] Triglyceride content = (Sample OD value - Blank OD value) ÷ (Calibration OD value - Blank OD value) × Calibrator concentration (mmol / L) ÷ Protein concentration of the sample to be tested (gprot / L)

[0039] 1.2.5 Detection of Free Fatty Acids (NEFA) Content in Liver Tissue

[0040] Accurately weigh 20 mg of tissue. Add 9 times the volume of physiological saline according to the ratio of liver tissue to anhydrous ethanol (g:mL) = 1:9. Homogenize mechanically under ice-water bath conditions to prepare a 10% homogenate. Centrifuge at 2500 rpm for 10 minutes and use the supernatant for analysis. In a 96-well plate, select one blank well and add 4 μL of double-distilled water, select one calibration well and add 4 μL of 1.00 mmol / L calibrator, and add 4 μL of sample to the remaining sample wells. Add 200 μL of reagent one to each well, mix well, incubate at 37°C for 5 min, and read the absorbance value A1 at 546 nm. Add 50 μL of reagent two to each well, mix well, incubate at 37°C for 5 min, and read the absorbance value A2. Calculate ΔA = A2 - A1.

[0041] NEFA content in tissue sample = (ΔA sample - ΔA blank) ÷ (ΔA standard - ΔA blank) × calibrator concentration (mmol / L) ÷ protein concentration of sample to be tested (gprot / L)

[0042] 1.2.6 Determination of myeloperoxidase (MPO) content in liver tissue

[0043] Accurately weigh 10 mg of tissue and homogenize it mechanically under ice-water bath conditions at a ratio of liver tissue weight (g):volume (mL) = 1:19 to prepare a 5% tissue homogenate. Mix the 5% tissue homogenate with reagent No. 3 at a volume ratio of 9:1 and incubate at 37°C for 15 minutes. Set up test tubes and control tubes. Add 3 mL of double-distilled water to the control tube and 3 mL of colorimetric reagent to the test tube. Add 0.2 mL of sample and 0.2 mL of reagent No. 4 to each tube, mix well, and incubate at 37°C for 30 minutes. Then add 0.05 mL of reagent No. 7, mix well, and incubate at 60°C for 10 minutes. Immediately after incubation, measure the absorbance of each tube at 460 nm. Calculate the MPO content in the tissue sample.

[0044] MPO activity (U / g tissue wet weight) = (Measured OD value - Control OD value) ÷ (11.3 × Sample weight (g))

[0045] 1.2.7 Determination of hydroxyproline (HYP) content in liver tissue

[0046] Accurately weigh 50 mg of tissue wet weight and place it in a test tube. Accurately add 1 mL of hydrolysis solution and mix well. Hydrolyze at 95℃ or in a boiling water bath for 20 minutes (mixing once every 10 minutes of hydrolysis). After cooling each test tube, add 10 μL of indicator to each tube and shake well. Accurately add 1.0 mL of pH adjustment solution A to each tube and shake well. Using a 200 μL pipette, carefully add pH adjustment solution B dropwise to each tube until the indicator in the liquid turns yellow-green. Then add double-distilled water to 10 mL and mix well. Take 3–4 mL of the diluted hydrolysis solution, add an appropriate amount of activated carbon (approximately 20–30 mg, until the supernatant is clear and colorless after centrifugation), mix well, and centrifuge at 3500 rpm for 10 minutes. Carefully take 1 mL of the supernatant for testing. Add 1.0 mL of double-distilled water, 5 μg / mL of standard working solution, and test solution to the blank tube, standard tube, and test tube, respectively. Add 0.5 mL of reagent I to each tube, mix well, and let stand for 10 minutes. Add 5 mL of reagent II to each tube, mix well, and let stand for 5 minutes. Add 0.5 mL of reagent III to each tube, mix well, incubate in a 60℃ water bath for 15 minutes, cool, centrifuge at 3500 rpm for 10 minutes, and measure the absorbance of each tube at a wavelength of 550 nm. Calculate the HYP content of the tissue sample.

[0047] HYP content in tissue sample (μg / mg wet weight) = (Measured OD value - Blank OD value) ÷ (Standard OD value - Blank OD value) × Calibrator content (5μg / ml) × Total volume of hydrolysate (10ml) ÷ Tissue wet weight (mg)

[0048] 1.2.8 Immunohistochemical staining analysis

[0049] Liver tissue from the same location (liver lobule) of mice was used for pathological examination. The tissue was fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned, dewaxed, hydrated, and subjected to antigen retrieval. The sections were then incubated with F4 / 80 and Ly6G antibodies, developed with a chromogenic agent, and observed under a light microscope. F4 / 80 is a surface marker of mouse macrophages, and a positive staining is brown; Ly6G is a surface marker of neutrophils, and a positive staining is brown. Immunohistochemical staining was used to reflect the infiltration of inflammatory macrophages and neutrophils.

[0050] 1.2.9 Statistical Analysis of Experimental Data

[0051] Experimental data are expressed as mean ± standard error (x ± SEM). Statistical analysis was performed using SPSS 16.0 software, and one-way ANOVA was used for analysis of variance between groups. 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.

[0052] 1.3 Experimental Results

[0053] 1.3.1 Results of H&E and Oil Red O staining of liver tissue and its NAS score

[0054] like Figure 1 As shown, H&E staining of liver tissue revealed significant hepatocyte necrosis and inflammatory cell infiltration in the livers of MCDD-treated mice, while different doses of HAR inhibited hepatocyte necrosis and inflammatory cell infiltration. Oil Red O staining of liver tissue showed significant lipid accumulation in the livers of MCDD-treated mice, while different doses of HAR reduced the elevated lipid accumulation in the livers. These results indicate that HAR can improve hepatocyte necrosis and inflammatory damage induced by MCDD in the livers of NAFLD mice and reduce lipid accumulation induced by MCDD in the livers of NAFLD mice. NAS scores were calculated for H&E pathological sections, and the results are as follows. Figure 2 As shown, the NAS scores of MCDD mice were above 5, indicating that MCDD mice had progressed to the severe NASH stage of the late NAFLD stage. After administration of different doses of HAR, the scores were significantly reduced (P<0.05), indicating that HAR can significantly inhibit the NASH process.

[0055] 1.3.2 Results of TG, NEFA, HYP content and MPO activity in liver tissue

[0056] The experimental results are shown in Table 2. Compared to the MCSD group, the levels of TG and NEFA in the liver tissue of mice in the MCDD group were significantly increased. However, different doses of HAR significantly reduced this increase, suggesting that HAR can alleviate lipid accumulation in the liver tissue of MCDD-induced NAFLD mice. Compared to the MCSD group, the levels of HYP and MPO enzyme activity in the liver tissue of mice in the MCDD group were significantly increased. However, different doses of HAR reduced this increase, suggesting that HAR can improve the inflammatory response and fibrosis in the liver of MCDD-induced NAFLD mice.

[0057] Table 2. HAR reduces elevated TG, NEFA, HYP levels and MPO activity in liver tissue of NAFLD mice.

[0058]

[0059] Note: Compared to the MCSD group, ** P<0.01, *** P<0.001; compared with the MCDD group, # P<0.05, ## P<0.01, ### P<0.001; n=5~8.

[0060] 1.3.3 Immunohistochemical staining results of liver tissue

[0061] The results of immunohistochemical staining analysis of liver tissue are shown below. Figure 3 The results showed that the infiltration of macrophages (F4 / 80+) and neutrophils (Ly6G+) in the liver of NAFLD mice fed with MCDD was significantly increased. Administration of HAR (30, 60 mg / kg) could reduce the inflammatory infiltration of macrophages and neutrophils in the liver of NAFLD mice, suggesting that HAR can inhibit the inflammatory response in the liver of NAFLD lesions.

[0062] Example 2

[0063] High-fat diet (HFD) feeding in mice to induce NAFLD is another commonly used classic animal model of NAFLD. This experiment used an HFD-induced NAFLD animal model to further confirm the therapeutic effect of harbazoside on NAFLD.

[0064] 2.1 Experimental Materials

[0065] 2.1.1 Laboratory Animals

[0066] Male clean-grade C57BL / 6J mice, weighing 18 - 20 g, were purchased from Shanghai Slack Experimental Animal Co., Ltd., with the certificate number: SYXK(Shanghai)2020 - 0009, and were raised in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. The feeding temperature of the mice was (22 ± 1)°C, the relative humidity was (65 ± 10)%, and the light was cycled for 12 h / 12 h.

[0067] 2.1.2 Drugs

[0068] Harpagoside was purchased from Chengdu Efar Biotechnology Co., Ltd.

[0069] 2.1.3 Reagents

[0070] The triglyceride (TG) test kit was purchased from Nanjing Jiancheng Bioengineering Institute.

[0071] 2.2 Experimental Methods

[0072] 2.2.1 Animal Experiment Protocol

[0073] C57BL / 6J mice were randomly divided into 4 groups. Grouping: (1) Normal chow diet (NCD) group (blank control group); (2) HFD group (model group); (3) HFD + HAR30 group (single-dose administration of 30 mg / kg, dissolved in normal saline and administered by gavage, low-dose harpagoside group); (4) HFD + HAR60 group (single-dose administration of 60 mg / kg, dissolved in normal saline and administered by gavage, high-dose harpagoside group); 6 mice in each group. After one week of adaptive feeding of the mice, the NCD group was fed with normal chow diet every day, and the remaining 3 groups were each fed with HFD for continuous 8 weeks to induce the NAFLD model. After successfully establishing the NAFLD model, the mice in the HFD + HAR30 group and HFD + HAR60 group were continuously administered harpagoside by gavage for 4 weeks, and the mice in the NCD group and HFD group were given an equal dose of normal saline. The body weight changes of the mice were monitored during the experiment. After 4 weeks of treatment, blood and liver tissues were taken for the detection of efficacy indexes and the preparation of liver tissue sections.

[0074] 2.2.2 Observation of Liver Tissue Morphology by Staining

[0075] Same as 1.2.2.

[0076] 2.2.3 NAS Scoring

[0077] Same as 1.2.3.

[0078] 2.2.4 Detection of Triglyceride (TG) Content in Liver Tissue

[0079] Same as 1.2.4.

[0080] 2.2.5 Detection of free fatty acid (NEFA) content in liver tissue

[0081] Same as 1.2.5.

[0082] 2.2.6 Immunohistochemical staining analysis

[0083] Same as 1.2.8.

[0084] 2.2.7 Statistical Analysis of Experimental Data

[0085] Same as 1.2.9.

[0086] 2.3 Experimental Results

[0087] 2.3.1 Results of H&E and Oil Red O staining of liver tissue and its NAS score

[0088] The results of H&E staining observation of liver tissue pathology are as follows: Figure 4 As shown, the livers of HFD-induced mice exhibited significant hepatocyte necrosis and inflammatory cell infiltration, and different doses of HAR could improve hepatocyte necrosis and inflammatory cell infiltration in the liver. Oil Red O staining results showed lipid accumulation in the livers of HFD-induced mice, and different doses of HAR could reduce the elevated lipid accumulation in the liver. These results indicate that HAR can improve hepatocyte necrosis and inflammatory damage in the livers of HFD-induced NAFLD mice and reduce lipid accumulation in the livers of HFD-induced NAFLD mice. NAS scoring of H&E pathological sections yielded the following results: Figure 5 As shown, the NAS scores of mice in the HFD group were all above 5, indicating that the mice in the HFD group had progressed to the severe NASH stage of late NAFLD. The HAR score decreased significantly after administration (P<0.05), further demonstrating that HAR can significantly inhibit the NASH process.

[0089] 2.3.2 TG content in blood and liver tissue

[0090] The experimental results are shown in Table 3. Compared to the NCD group, the levels of TG and NEFA in the liver tissue of mice in the HFD group were significantly increased. Different doses of HAR significantly reduced the elevated TG level in the liver, and high-dose HAR significantly reduced the elevated NEFA level in the liver. This suggests that HAR has significant lipid-lowering activity.

[0091] Table 3. HAR reduces elevated TG and NEFA levels in liver tissue of NAFLD mice.

[0092]

[0093] Note: Compared to the NCD group ** P<0.01, *** P<0.001; compared with the HFD group,# P < 0.05. (n = 5 - 6)

[0094] 2.3.3 Immunohistochemical staining results of liver tissue

[0095] The results of immunohistochemical staining analysis of liver tissue are shown below. Figure 6 The results showed that the infiltration of macrophages (F4 / 80+) and neutrophils (Ly6G+) in the liver of NAFLD mice fed with HFD was significantly increased. Administration of HAR (30, 60 mg / kg) could reduce the inflammatory infiltration of macrophages and neutrophils in the liver of NAFLD mice, suggesting that HAR can inhibit the inflammatory response in the liver of NAFLD lesions.

[0096] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this specification can be used in any compositional form, and each feature disclosed in the specification can 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.

[0097] 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 present 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 harbazoside in the preparation of drugs for the prevention and / or treatment of non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: The aforementioned non-alcoholic fatty liver disease includes non-alcoholic fatty liver, non-alcoholic steatohepatitis, fatty liver fibrosis, and / or cirrhosis.

3. The use according to claim 1, characterized in that: The dosage form of the drug is a solid dosage form or a liquid dosage form.

4. The use according to claim 3, characterized in that: The solid dosage forms mentioned are tablets, sustained-release tablets, dispersible tablets, capsules, sustained-release capsules, granules, pills, and powder injections.

5. The use according to claim 3, characterized in that: The liquid formulation is an oral liquid formulation or an injectable liquid formulation.

6. The use according to claim 5, characterized in that: The oral liquid preparations mentioned are oral liquids, suspensions, and droplets.

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