Use of a renormycin type diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating liver disease

Abstract

This invention discloses the application of a renolane-type diterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention or treatment of liver disease; the renolane-type diterpenoid compound has at least one of the structural formulas shown in formulas (I), (II), (III), (IV), (V), and (VI); the renolane-type diterpenoid compounds of formulas (I) to (VI) can significantly alleviate alcohol-induced hepatocellular damage, and the renolane-type diterpenoid compounds of formulas (III) to (VI) can significantly alleviate palmitic acid-induced hepatocellular damage, indicating that the renolane-type diterpenoids of this invention have hepatoprotective activity and can be used to prepare hepatoprotective drugs, showing good research and development prospects. This invention belongs to the field of natural product chemistry.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry, specifically relating to the application of renin-type diterpenoid compounds in the preparation of drugs for the prevention or treatment of liver diseases. Background Technology

[0002] The liver, a vital solid organ in the human body, plays an irreplaceable role in maintaining the body's physiological balance through its normal functioning. However, under the combined influence of numerous internal and external pathogenic factors, the liver is susceptible to damage, leading to a series of complex liver diseases. Common factors causing liver damage include viruses, alcohol, high-fat and high-sugar diets, and medications. Long-term excessive alcohol consumption is a well-established and common cause of liver damage; a long-term high-fat and high-sugar diet and a sedentary lifestyle are the main culprits leading to metabolic liver diseases. Currently, there are few drugs for preventing and treating liver damage, and most play an adjunctive role in treatment. Therefore, finding effective and safe drugs to combat liver damage is imperative.

[0003] Ryanodane diterpenoids are a limited class of pentacyclic natural products characterized by a complex 6 / 5 / 5 / 6 / 6 fused ring skeleton. They can be divided into nine subclasses, and to date, fewer than 140 ryanodane diterpenoids have been reported in nature. Approximately 40% of ryanodane diterpenoids are isolated from plants in the genus *Cinnamomum*. Therefore, ryanodane diterpenoids are considered characteristic components of *Cinnamomum*. Ryanodane diterpenoids possess various biological activities, including anti-inflammatory, antiviral, antifeedant, and insecticidal activities. Due to their complex skeletal structure and significant biological activities, ryanodane diterpenoids have attracted widespread attention from chemists in recent years. Summary of the Invention

[0004] The purpose of this invention is to provide the application of reninose diterpenoid compounds in the preparation of medicaments for the prevention or treatment of liver diseases.

[0005] To solve the above problems, the technical solution provided by the present invention is as follows:

[0006] The use of a reninokine-type diterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of liver disease, wherein the reninokine-type diterpenoid compound has a structural formula of at least one of the following: formula (I), formula (II), formula (III), formula (IV), formula (V), and formula (VI):

[0007] ;

[0008] Preferably, the use of the above-mentioned renin-type diterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of liver diseases, including liver injury, fatty liver, hepatitis, liver fibrosis, and cirrhosis.

[0009] More preferably, the use of the above-mentioned renin-type diterpenoid compound or its pharmaceutically acceptable salt in the preparation of a medicament for the prevention or treatment of liver disease, wherein the liver injury is alcoholic liver injury, metabolic liver injury, chemical liver injury, drug-induced liver injury, or viral liver injury.

[0010] More preferably, the use of the above-mentioned renin-type diterpenoid compound or its pharmaceutically acceptable salt in the preparation of a medicament for the prevention or treatment of liver disease, wherein the fatty liver is alcoholic fatty liver or metabolic fatty liver.

[0011] More preferably, the use of the above-mentioned renin-type diterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of liver disease, wherein the hepatitis is alcoholic hepatitis, metabolic hepatitis, chemical hepatitis, drug-induced hepatitis, or viral hepatitis.

[0012] More preferably, the use of the above-described renin-type diterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of liver disease, wherein the medicament further comprises a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, medium, or combination thereof.

[0013] More preferably, the use of the above-described renin-type diterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of liver disease, wherein the medicament further comprises other medicaments for the prevention or treatment of liver disease.

[0014] In this invention, the pharmaceutically acceptable salt refers to a salt that is substantially non-toxic and provides the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism, or excretion.

[0015] Specifically, the term "pharmaceutically acceptable salt" includes both inorganic and organic salts of the compound.

[0016] The inorganic salts mentioned include: hydrobromide, hydroiodide, phosphate, sulfate, perchlorate, etc.

[0017] The organic acid salts mentioned include: aspartate, acetate, oxalate, benzenesulfonate, maleate, formate, tartrate, adipate, succinate, malonate, citrate, alginate, benzoate, ascorbate, cyclopentylpropionate, ethanesulfonate, heptaate, hexanoate, lacturonate, lactate, malate, laurate, etc.

[0018] Furthermore, the drug comprises one or a combination of pharmaceutically acceptable carriers, excipients, diluents, and adjuvants.

[0019] The aforementioned pharmaceutically acceptable carriers, excipients, diluents, and excipients can ensure the stability, efficacy, and safety of the drug.

[0020] When preparing drugs for the prevention or treatment of liver diseases, appropriate combinations of carriers, excipients, diluents, and adjuvants are selected according to different dosage forms and methods of administration.

[0021] For example, for oral preparations, starch or sucrose may be used as excipients, and water or ethanol as diluents to ensure that the drug can be absorbed smoothly by the body. For injectable preparations, a carrier such as physiological saline is used to ensure medication safety.

[0022] The drug of the present invention may also contain other drugs for the prevention or treatment of liver disease, such as silymarin, to achieve synergistic effects.

[0023] When used in combination with other drugs for the prevention or treatment of liver disease, reninolane diterpenoids can exert a synergistic effect, enhancing therapeutic efficacy. For example, in the treatment of viral hepatitis, they can be used in conjunction with antiviral drugs to reduce viral damage to the liver and promote the repair and regeneration of damaged liver cells through the hepatoprotective effect of reninolane diterpenoids.

[0024] Further in-depth research can explore the optimal combination ratio of renin-type diterpenoids with other drugs and the timing of administration, in order to achieve more precise and efficient treatment of liver diseases.

[0025] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0026] The technical solution provided by this invention uses a hepatoprotective activity evaluation model induced by alcohol, palmitic acid, etc., and found that the renoline diterpenoid compound of this invention can effectively alleviate hepatocellular damage induced by alcohol, palmitic acid, etc., and reduce lipid accumulation, indicating that the renoline diterpenoid compound of this invention has hepatoprotective activity and can be used in the preparation of hepatoprotective drugs, and has good research and development prospects. Attached Figure Description

[0027] Figure 1 This is the 400 MHz 1H NMR spectrum of compound (I) in deuterated methanol;

[0028] Figure 2 This is the 100 MHz carbon NMR spectrum of compound (I) in deuterated methanol;

[0029] Figure 3 This is the 400 MHz 1H NMR spectrum of compound (ⅠⅠ) in deuterated methanol;

[0030] Figure 4 This is the 100 MHz carbon NMR spectrum of compound (ⅠⅠ) in deuterated methanol;

[0031] Figure 5This is the 400 MHz 1H NMR spectrum of compound (IIII) in deuterated methanol;

[0032] Figure 6 This is the 100 MHz carbon NMR spectrum of compound (IⅠⅠ) in deuterated methanol;

[0033] Figure 7 This is the 400 MHz 1H NMR spectrum of compound (IV) in deuterated methanol;

[0034] Figure 8 This is the 100 MHz carbon NMR spectrum of compound (IV) in deuterated methanol;

[0035] Figure 9 This is the 400 MHz 1H NMR spectrum of compound (V) in deuterated dimethyl sulfoxide;

[0036] Figure 10 This is the 100 MHz carbon NMR spectrum of compound (V) in deuterated dimethyl sulfoxide;

[0037] Figure 11 This is the 400 MHz 1H NMR spectrum of compound (VI) in deuterated dimethyl sulfoxide;

[0038] Figure 12 This is the 100 MHz carbon NMR spectrum of compound (VI) in deuterated dimethyl sulfoxide;

[0039] Figure 13 The protective effects of compounds of formulas (I) to (VI) on an alcohol-induced hepatocellular injury model;

[0040] Figure 14 The protective effects of compounds of formulas (I) to (VI) on palmitic acid-induced hepatocyte injury models are shown. Detailed Implementation

[0041] The present invention will be further described below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0042] All the devices in the following embodiments are as follows:

[0043] The mass spectrometer was a Bruker Amazon SL mass spectrometer manufactured by Burker Daltonics, USA. The superconducting nuclear magnetic resonance spectrometer was a Bruker AV-400. The X-ray single-crystal diffractometer was a Bruker D8 Venture. The silica gel GF254 for thin-layer chromatography and the silica gel (200-300 mesh) for column chromatography were both products of Qingdao Ocean Chemical Plant. The reversed-phase ODS packing material (50 μm) was a product of YMC Corporation, Japan. The medium- and low-pressure liquid chromatograph was a product of Shanghai Lishui Electronic Technology Co., Ltd. The preparative-grade column used in the liquid phase separation was a Cosmosil Packed C1000. 18 Column (20.0 × 250 mm, 5 μm). Acetonitrile or methanol was of chromatographic grade for liquid chromatography, water was double-distilled water, and all other reagents were of analytical grade. A multi-functional microplate reader (MEMB) was used: the Tecan Infinite F50 MEB reader (Switzerland). Normal mouse hepatocytes were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0044] The compounds of formulas (I) to (VI) in this application are prepared by the following methods:

[0045] 20.0 kg of dried cinnamon bark was extracted three times by reflux in 200 L of ethanol-water (60:40, v / v), each time for 2 hours. After filtration, the filtrate was concentrated under reduced pressure to obtain a concentrate. The concentrate was then extracted successively with dichloromethane and ethyl acetate, and then concentrated to obtain dichloromethane extract and ethyl acetate extract.

[0046] 255.0 g of ethyl acetate extract was subjected to silica gel column chromatography at atmospheric pressure. The extract was eluted sequentially with dichloromethane-methanol-water eluents at volume ratios of 99:1:0.1, 98:2:0.2, 97:3:0.3, 95:5:0.5, 93:7:0.7, 90:10:1, 85:15:1.5, 80:20:2, 70:30:3, 60:40:4, and 0:100:0, yielding 18 fractions: E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13, E14, E15, E16, E17, and E18. (See Table 1 for the corresponding fractions obtained from elution with different volume ratios of dichloromethane-methanol-water).

[0047] Table 1. Correlation table of fractions obtained with different volume ratios of dichloromethane-methanol-water eluent

[0048]

[0049] The fraction E4 obtained by elution with dichloromethane-methanol-water at a volume ratio of 98:2:0.2 was subjected to medium-low pressure reversed-phase column chromatography, successively eluted with methanol-water at volume ratios of 20:80:0.1, 25:75:0.1, 30:70:0.1, 35:65:0.1, 40:60:0.1, 45:55:0.1, 50:50:0.1, 60:40:0.1, 70:30:0.1, and 100:0:0.0. The water-formic acid eluent was used for elution to obtain 19 fractions: E4.1, E4.2, E4.3, E4.4, E4.5, E4.6, E4.7, E4.8, E4.9, E4.10, E4.11, E4.12, E4.13, E4.14, E4.15, E4.16, E4.17, E4.18, and E4.19 (see Table 2 for the fractions corresponding to methanol-water-formic acid elution with different volume ratios).

[0050] Table 2. Correlation table of fractions obtained with methanol-water-formic acid eluent at different volume ratios.

[0051]

[0052] The fraction E4.11 (83.2 mg) obtained by elution with methanol-water-formic acid at a volume ratio of 35:65:0.1 was prepared by reversed-phase preparative high-performance liquid chromatography (HPLC) using a methanol-water-formic acid eluent at a flow rate of 8 mL / min and a volume ratio of 55:45:0.1 to obtain compound (t) of formula (V). R (41.4 min, 38.8 mg, purity 95%).

[0053]

[0054] Fraction E6, obtained by elution with dichloromethane-methanol-water at a volume ratio of 95:5:0.5, was subjected to medium-low pressure reversed-phase column chromatography, successively eluted with methanol-water-formic acid eluents at volume ratios of 10:90:0.1, 15:85:0.1, 20:80:0.1, 25:75:0.1, 30:70:0.1, 40:60:0.1, 50:50:0.1, and 100:0:0.0, to obtain fractions E6.1, E6.2, E6.3, and E6.4. 6.4, E6.5, E6.6, E6.7, E6.8, E6.9, E6.10, E6.11, E6.12, E6.13, E6.14, E6.15, E6.16, E6.17, E6.18, E6.19, E6.20, E6.21, E6.22, E6.23, E6.24, E6.25, and E6.26, a total of 26 fractions (see Table 3 for the fractions corresponding to methanol-water-formic acid elution with different volume ratios).

[0055] Table 3. Correlation table of fractions obtained with methanol-water-formic acid eluent at different volume ratios.

[0056]

[0057] The fraction E6.8 (500.9 mg) obtained by elution with methanol-water-formic acid at a volume ratio of 15:85:0.1 was prepared by reversed-phase preparative high-performance liquid chromatography using acetonitrile-water-formic acid eluent at a flow rate of 8 mL / min to obtain compound (t) of formula (II). R : 28.2 min, 10.1 mg, purity 95%) and compound of formula (III) (t R : 29.9 min, 10.4 mg, purity 95%. The fraction E6.18 (325.8 mg) obtained by elution with methanol-water-formic acid at a volume ratio of 30:70:0.1 was prepared by reversed-phase preparative high-performance liquid chromatography using acetonitrile-water-formic acid at a volume ratio of 23:77:0.1 at a flow rate of 8 mL / min to obtain compound (t). R (23.7 min, 128.5 mg, purity 95%).

[0058]

[0059] The fraction E8 obtained by elution with dichloromethane-methanol-water at a volume ratio of 90:10:1 was subjected to medium-low pressure reversed-phase column chromatography, successively eluted with methanol-water-formic acid eluates at volume ratios of 10:90:0.1, 15:85:0.1, 20:80:0.1, 25:75:0.1, 30:70:0.1, 40:60:0.1, 50:50:0.1, and 100:0:0.0. After elution, 19 fractions were obtained: E8.1, E8.2, E8.3, E8.4, E8.5, E8.6, E8.7, E8.8, E8.9, E8.10, E8.11, E8.12, E8.13, E8.14, E8.15, E8.16, E8.17, E8.18, and E8.19 (see Table 4 for the fractions corresponding to different volume ratios of methanol-water-formic acid elution).

[0060] Table 4. Correlation table of fractions obtained with methanol-water-formic acid eluent at different volume ratios.

[0061]

[0062] The fraction E8.8 (325.2 mg) obtained by elution with methanol-water-formic acid at a volume ratio of 15:85:0.1 was prepared by reversed-phase preparative high-performance liquid chromatography using acetonitrile-water-formic acid eluent at a flow rate of 8 mL / min to obtain compound (t) of formula (IV). R : 25.0 min, 21.6 mg, purity 95%). The fraction E8.11 (506.4 mg) obtained by elution with methanol-water-formic acid at a volume ratio of 25:75:0.1 was prepared by reversed-phase preparative high-performance liquid chromatography using acetonitrile-water-formic acid at a volume ratio of 12:88:0.1 at a flow rate of 8 mL / min to obtain compound (t) of formula (I). R (46.5 min, 20.3 mg, purity 95%).

[0063]

[0064] The physicochemical constants are as follows:

[0065] Compound of formula (I): white amorphous powder; [α]27D +12.5 (c 0.1, methanol); UV (methanol) λ max (log ε) 219 (3.00) nm; ECD (c 6.54 ×10 −3 M, methanol) λ max (Δε) 194 (−60.70), 200(+56.86); IR (KBr) v max 3445, 2944, 1723, 1596, 1029 cm –1 ESIMS (positive ion) m / z 383.23 [M + H] + ESIMS (negative ions) m / z 381.32 [M − H] - HRESIMS (positive ion) m / z 383.2072 [M + H] + (Calculated value [C]) 20 H 31 O7] + (383.2070), the molecular formula of the compound was determined to be C 20 H 30 O7; see the hydrogen spectrum. Figure 1 See the carbon spectrum. Figure 2 The data is shown in Table 5.

[0066] Compound of formula (II): white amorphous powder; ESIMS (positive ion) m / z 399.02 [M + H] + The molecular formula of the compound was determined to be C. 20 H 30 O8; see the hydrogen spectrum. Figure 3 See the carbon spectrum. Figure 4 The data is shown in Table 5.

[0067] Compounds of formula (ⅠII): white amorphous powder; ESIMS (positive ion) m / z 407.06 [M + Na] + The molecular formula of the compound was determined to be C. 20 H 32 O7; see the hydrogen spectrum. Figure 5 See the carbon spectrum. Figure 6 The data is shown in Table 5.

[0068] Compound (IV): White amorphous powder; ESIMS (positive ion) m / z 423.04 [M + Na] + The molecular formula of the compound was determined to be C. 20 H 32 O8; see the hydrogen spectrum. Figure 7 See the carbon spectrum. Figure 8 The data is shown in Table 6.

[0069] Compound (V): White amorphous powder; ESIMS (positive ion) m / z 431.20 [M + Na] + The molecular formula of the compound was determined to be C. 22 H 32 O7; see the hydrogen spectrum. Figure 9 See the carbon spectrum. Figure 10 The data is shown in Table 6.

[0070] Compound of formula (VI): white, massive crystals; ESIMS (positive ion) m / z 733.12 [2M + H] + The molecular formula of the compound was determined to be C. 20 H 30 O6; see the hydrogen spectrum. Figure 11 See the carbon spectrum. Figure 12 The data is shown in Table 6.

[0071] Table 5. Carbon and hydrogen spectral data of compounds of formulas (I) to (III) and their attribution.

[0072]

[0073] aThe test solvent was deuterated methanol (H1N1 spectrum: 400 MHz, C1N1 spectrum: 100 MHz).

[0074] b Signals were not labeled due to overlap or complex splitting.

[0075] Table 6. Carbon and proton spectral data of compounds of formulas (IV) to (VI) and their attribution

[0076]

[0077] a The test solvent was deuterated methanol (H1N1 spectrum: 400 MHz, C1N1 spectrum: 100 MHz).

[0078] b The test solvent was deuterated dimethyl sulfoxide (H1N M: 400 MHz, C1N M: 100 MHz).

[0079] c Signals were not labeled due to overlap or complex splitting.

[0080] Crystal diffraction data of compounds of formula (VI) in Table 7

[0081]

[0082] Example 1: Hepatoprotective Activity Experiment of Rhenorane-type Diterpenoid Compounds

[0083] The following provides the hepatoprotective activity test methods and results of the renoside-type diterpenoid compounds (I)-(VI) described in this application and the positive control drug silymarin.

[0084] Experiment 1: Alcohol-induced hepatocellular injury model

[0085] Establishment of an alcoholic hepatocyte injury model: Normal mouse hepatocytes were suspended in DMEM / F-12 complete culture medium and inoculated with 1×10⁻⁶ cells / mL. 4 pcs / hole and 3×10 4 The culture medium was seeded at a density of 100 μL / well into 96-well and 24-well plates (500 μL / well). After incubation for 24 hours, the culture medium was aspirated from the wells.

[0086] The wells of the drug group were filled with DMEM / F-12 medium containing 10 μmol / L of the test monomer (the test monomer is one of the renoside diterpenoid compounds with structural formulas as shown in formulas (I) to (VI) or the positive control drug silymarin) and 1000 mmol / L anhydrous ethanol; the wells of the model group were filled with DMEM / F-12 medium containing 1000 mmol / L anhydrous ethanol; and the wells of the control group were filled with DMEM / F-12 medium. The cells were cultured for another 24 hours. Cell viability was assessed using the CCK-8 assay in 96-well plates, and Nile Red staining was used in 24-well plates for qualitative imaging and quantitative analysis.

[0087] Quantitative detection of cell viability: Cell viability was detected in 96-well plates using the CCK-8 assay. After 24 hours of cell culture following modeling and drug administration, the culture medium in each well was aspirated, and 100 μL of DMEM / F-12 medium containing 10 wt% CCK-8 reagent was added to each well. The cells were incubated at 37 ℃ in the dark for 2.5 hours. Subsequently, the absorbance values ​​of the treatment group and the control group were detected at 450 nm using a microplate reader. The cell viability (%) was calculated as A(treatment group) / A(control group) × 100%, and the results are shown in Table 8 below.

[0088] Table 8 Cell viability results

[0089]

[0090] ** p < 0.01, *** p < 0.001 vs. model group.

[0091] Nile Red Staining and Quantitative Detection: After the cells in the 24-well plates had been cultured for the appropriate time, the liquid in the wells was discarded. The cells were washed twice with PBS (500 μL per well), and 300 μL of 4wt% paraformaldehyde solution was added to each well for fixation in the dark for 30 minutes. Following the instructions of the Nile Red Staining Kit from Beijing Solarbio Science & Technology Co., Ltd., solutions A and B in the kit were diluted 1:400 (volume:volume) and mixed to obtain the staining working solution. The fixative in the wells was discarded, and 300 μL of the staining working solution was added for staining in the dark for 10 minutes, after which the staining solution was discarded. Subsequently, 500 μL of PBS was added to each well for washing twice. After the final wash, the original 500 μL of PBS in the wells was discarded, and 200 μL of PBS was added to each well. The staining was observed under a fluorescence microscope at a wavelength of 598 nm. The lipid droplet staining area was quantified using ImageJ software. The calculation formula was: Nile Red lipid quantification = (average gray value of the treatment group / average gray value of the control group) × 100%. The results are shown in Table 9.

[0092] Table 9 Quantitative results of Nile Red staining

[0093]

[0094] *** p < 0.001 vs. model group.

[0095] The results are shown in Table 8 and Figure 13 As shown, alcohol stimulation significantly reduced the survival rate of normal hepatocytes in mice to 50.4%. Administration of renin-type diterpenoid compounds (I) to (VI) restored the cell survival rate to 65.8%, 59.4%, 62.7%, 70.4%, 71.8%, and 66.7%, respectively (the positive control drug silymarin restored the cell survival rate to 64.5%), all of which significantly improved the cell survival rate. Among them, compounds (IV) and (V) showed relatively better improvement effects.

[0096] The results are shown in Table 9 and Figure 13 As shown, alcohol increased the lipid accumulation rate of normal mouse hepatocytes to 375.2%. Treatment with renin-type diterpenoid (I)-(VI) compounds reduced the lipid accumulation rate to 194.5%, 177.6%, 164.9%, 172.7%, 168.1%, and 149.5%, respectively (the positive control drug silymarin reduced the lipid accumulation rate to 213.7%), all of which significantly reduced lipid accumulation. The ratio of Nile Red staining quantification to cell viability was compared. Alcohol stimulation increased the ratio to 7.44, while treatment with renin-type diterpenoid (I)-(VI) compounds reduced the ratio to 2.96, 2.99, 2.63, 2.45, 2.34, and 2.24, respectively (the positive control drug silymarin reduced the ratio to 3.31), all demonstrating good hepatoprotective activity. The above experimental results show that the renin-type diterpenoid (I)-(VI) compounds of the present invention can significantly increase hepatocyte survival rate and reduce fat accumulation, exhibiting good hepatoprotective activity. Therefore, the renin-type diterpenoid (I)-(VI) compounds of the present invention can be used to prepare hepatoprotective drugs.

[0097] Experiment 2: Palmitic acid-induced hepatocyte injury model

[0098] Establishment of a palmitic acid-induced hepatocyte injury model:

[0099] To prepare a 100 mM palmitic acid stock solution: Weigh 0.1 g of palmitic acid and dissolve it in 3.9 mL of anhydrous ethanol. After dissolving by heating in a metal bath at 65 °C, store at 4 °C.

[0100] To prepare 10% BSA: Weigh 1 g of BSA and dissolve it in 10 mL of PBS. Dissolve the BSA by heating in a metal bath at 65 °C. Filter the solution through a 0.22 μM filter and aliquot the solution for storage at 4 °C.

[0101] To prepare a 10 mM palmitic acid-BSA modeling solution: Immediately before use, after preheating the above two solutions in a 65 ℃ metal bath, take 150 μL of the mother solution and add it to 1.35 mL of 10 wt% BSA. Heat at 65 ℃ for a short time until clear and transparent, and then filter through a 0.22 μM sterile filter membrane.

[0102] Normal mouse hepatocytes were suspended in DMEM / F-12 complete medium and cultured at 8 × 10⁸ cells per well. 3 Each hole is 12×10 4 Cells were seeded at a density of 100 μL / well in 96-well and 500 μL / well in 24-well plates. After 24 hours of adherent growth, when the cell density of normal mouse hepatocytes in the 96-well plates reached 30% and that in the 24-well plates reached 60%, the original complete culture medium was discarded, and DMEM / F-12 medium containing 2 wt% fetal bovine serum was added for starvation culture for 12 hours to synchronize cell growth. The original culture medium was then discarded. The model group received DMEM / F-12 medium containing 0.4 mM palmitic acid-BSA per well; the drug group received DMEM / F-12 medium containing 0.4 mM palmitic acid-BSA and 1 μmol / L of the test monomer (the test monomer is one of the renin-type diterpenoid compounds with structural formulas as shown in formulas (I) to (VI)) per well; and the control group received DMEM / F-12 medium per well. After culturing for another 24 hours, CCK-8 cell viability was detected and Oil Red O staining was performed in 96-well and 24-well plates, respectively.

[0103] Quantitative detection of cell viability: Cell viability was detected in 96-well plates using the CCK-8 assay. After 24 hours of cell culture following modeling and drug administration, the culture medium in each well was aspirated, and 100 μL of DMEM / F-12 medium containing 10 wt% CCK-8 reagent was added to each well. The cells were then incubated in a cell culture incubator in the dark for 2 hours. Subsequently, the absorbance values ​​of the treatment group and the control group were detected at 450 nm using a microplate reader. The calculation formula was: Cell viability (%) = A(treatment group) / A(control group) × 100%. The results are shown in Table 10 below.

[0104] Table 10 Cell viability results

[0105]

[0106] * p < 0.05, ** p < 0.01, *** p < 0.001 vs. model group.

[0107] Oil Red O staining for cellular lipid detection:

[0108] Preparation of Oil Red O stock solution: Weigh 150 mg of Oil Red O solid in the dark, add 25 mL of isopropanol, and dissolve by sonication.

[0109] Preparation of Oil Red O working solution: Under light-protected conditions, mix Oil Red O stock solution and ultrapure water in a ratio of 3:2 (volume:volume), filter with a 0.22μm microporous membrane, and use immediately after preparation.

[0110] Oil Red O staining and detection: After 24 hours of culture in 24-well plates following modeling and drug administration, the culture medium was aspirated from each well, and 500 μL of PBS was added to each well for washing twice. Subsequently, 4 wt% paraformaldehyde solution was added for fixation at room temperature in the dark for 30 minutes, followed by rinsing twice with 60% isopropanol-water solution (300 μL / well). Then, 300 μL of Oil Red O working solution was added to each well for staining in the dark for 30 minutes. After staining, the 24-well plates were washed with PBS until no excess red was visible. 200 μL of PBS was added to each well, and images were taken using a 40× inverted microscope. After imaging, the PBS in the 24-well plates was discarded, and 250 μL of isopropanol was added to each well and gently pipetted in. 200 μL of the solution was transferred from each well to a 96-well plate, and the absorbance was measured at 492 nm using a microplate reader. The lipid accumulation rate was calculated as (absorbance of the treatment group / absorbance of the control group) × 100%, and the results are shown in Table 11.

[0111] Table 11 Quantitative results of Oil Red O staining

[0112]

[0113] * p < 0.05, ** p < 0.01, *** p < 0.001 vs. model group. ns No significant difference vs. model group.

[0114] The results are shown in Table 10 and Figure 14 As shown, palmitic acid stimulation significantly reduced the survival rate of normal mouse hepatocytes to 63.3%. Administration of renin-type diterpenoid (I)-(VI) compounds restored the cell survival rate to 69.6%, 68.3%, 71.5%, 67.8%, 71.3% and 71.3%, respectively, all of which significantly improved the cell survival rate.

[0115] The results are shown in Table 11 and Figure 14As shown, palmitic acid increased the lipid accumulation rate of normal mouse hepatocytes to 140.6%. Treatment with renin-type diterpenoids (IV) and (VI) reduced the lipid accumulation rate to 109.2% and 123.8%, respectively, significantly reducing lipid accumulation. The ratio of quantitative results obtained by Oil Red O staining to cell viability was compared. Palmitic acid stimulation increased the ratio to 2.22, while treatment with renin-type diterpenoids (III) to (VI) reduced the ratio to 1.97, 1.61, 1.95, and 1.74, respectively. These experimental results demonstrate that the renin-type diterpenoids (III) to (VI) of this invention possess good hepatoprotective activity.

[0116] In summary, the renoline-type diterpenoids (I) to (VI) prepared in this invention can all significantly alleviate alcohol-induced hepatocellular damage, and the renoline-type diterpenoids (III) to (VI) can all significantly alleviate palmitic acid-induced hepatocellular damage. This indicates that the renoline-type diterpenoids of this invention have hepatoprotective activity and can be used to prepare hepatoprotective drugs, showing good research and development prospects.

[0117] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of a reninolane-type diterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of liver disease, wherein the reninolane-type diterpenoid compound has at least one of the structural formulas shown in formula (I), formula (II), formula (III), formula (IV), formula (V), and formula (VI): 。 2. The application according to claim 1, characterized in that, The pharmaceutically acceptable salt of the renolan-type diterpenoid compound is an inorganic salt containing the renolan-type diterpenoid compound of claim 1.

3. The application according to claim 1, characterized in that, The pharmaceutically acceptable salts of the renin-type diterpenoid compounds are organic salts.

4. The application according to claim 1, characterized in that, The liver diseases mentioned include liver damage, fatty liver, hepatitis, liver fibrosis, and cirrhosis.

5. The application according to claim 4, characterized in that, The liver injuries mentioned include alcoholic liver injury, metabolic liver injury, chemical liver injury, drug-induced liver injury, and viral liver injury.

6. The application according to claim 4, characterized in that, The fatty liver mentioned refers to alcoholic fatty liver and metabolic fatty liver.

7. The application according to claim 4, characterized in that, The hepatitis mentioned includes alcoholic hepatitis, metabolic hepatitis, chemical hepatitis, drug-induced hepatitis, and viral hepatitis.

8. The application according to claim 1, characterized in that, The drug also includes one or a combination of pharmaceutically acceptable carriers, excipients, diluents, and adjuvants.

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