Application of terpenoids in treatment or prevention of fibrotic diseases

Abstract

The invention relates to application of terpenoids in treating or preventing fibrotic diseases. The application comprises the step of applying an effective amount of a composition to a subject in need of the terpenoids, wherein the composition comprises a triterpenoid compound extracted from antrodia camphorata or needle herb.

Description

[0001] This application is a divisional application of the PCT patent application entered China with the international application number PCT / CN2021 / 096593, the title of invention is “Use of terpenoids in treating or preventing fibrotic diseases”, and the international filing date is May 19, 2021. TECHNICAL FIELD

[0002] The present invention relates to plant terpenoids derived from Antrodia camphorata and Anisomeles indica extracts, in particular, a pharmaceutical and dietary formulation for alleviating fibrotic diseases. BACKGROUND

[0003] Fibroproliferative diseases are a growing concern for an increasing number of individuals and are a common pathological sequelae of many persistent inflammatory diseases, such as pulmonary fibrosis, progressive kidney disease, liver cirrhosis, atherosclerosis, and benign prostatic hyperplasia.

[0004] Renal repair after acute kidney injury induces fibrosis, which can eventually worsen into chronic kidney disease. First, kidney injury activates multipotent progenitor cells to repair the tissue, however, as the injury persists, these cells become dysfunctional and induce fibrotic repair, leading to kidney fibrosis. The pathogenesis of kidney fibrosis is a progressive process that eventually leads to end-stage renal function failure, a major disease that requires dialysis or kidney transplantation.

[0005] Non-alcoholic fatty liver disease (NAFLD) is a major form of chronic liver disease with unmet medical need. Non-alcoholic steatohepatitis (NASH) is a progressive variant of NAFLD that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. NAFLD and NASH have become a subject of widespread interest in the medical community, especially as the prevalence of diabetes and obesity in the world population increases. The clinical evaluation of every patient with abnormal aminotransferase levels should consider non-alcoholic fatty liver disease and its spectrum, especially in obese or diabetic patients. The prognosis of simple NAFLD is usually benign, but if fibrosis, hepatocellular ballooning, inflammation, and Mallory bodies are present, there is a risk of progression to cirrhosis.

[0006] Autoimmune hepatitis (AIH) is a chronic liver disease of unknown etiology characterized by inflammation of hepatocytes. Severe AIH can progress to cirrhosis, hepatocellular carcinoma, and even death. Up to 40% of patients with autoimmune hepatitis develop cirrhosis depending on the length of observation. Therefore, in addition to current anti-inflammatory and immunosuppressive therapies, emerging anti-fibrotic therapies can be added, which can be expected to reposition the treatment goal of autoimmune hepatitis to prevent, stabilize, and reverse liver fibrosis.

[0007] Atherosclerosis is a major cause of the development of cardiovascular disease, which is associated with vascular fibrosis. Vascular fibrosis involves the accumulation of extracellular matrix (ECM) proteins, especially collagen and fibronectin in the vascular media, and contributes to remodeling structures and scar formation. A lack of elastin or excess collagen in the vessel wall leads to increased vascular fibrosis and stiffness.

[0008] In benign prostatic hyperplasia, the deposition of collagen fibers in the prostate is to replace the broken muscle fibers, but it causes the muscle tissue to be stiff and weak, so that the prostatic fluid is deposited in the ducts. Prostatic fibrosis is a major factor for bladder outlet obstruction in older men.

[0009] The medicinal fungus Antrodia camphorata is a well-known traditional Chinese medicine with diverse biological activities, particularly antitumor effects in vitro against cancer cells and in vivo in animal models. Given its diverse bioactive compounds, it is considered an effective alternative phytotherapy agent or adjuvant for cancer treatment and immune-related diseases. To date, 225 compounds have been isolated, identified, and structurally elucidated, including macromolecules (nucleic acids, proteins, and polysaccharides), small molecules (benzenoids, lignans, benzoquinones, and maleic / succinic acid derivatives), terpenes (lanostane triterpenes, ergostane triterpenes, diterpenes, monoterpenes, and steroids), nucleotides (nucleobases and nucleosides), fatty acids, and fatty acid esters.

[0010] Cumulative in vitro and in vivo studies have demonstrated anti-diabetic, anti-hyperlipidemic, anti-hypertensive, anti-inflammatory, antioxidant, antibacterial, cardiovascular disease prevention, immunomodulatory, hepatoprotective, and neuroprotective effects. However, the efficacy of Antrodia cinnamomea and its components in the treatment of fibrosis has not yet been evaluated.

[0011] Anisomeles indica, commonly known as "Indian catmint," is a source of medicinally active compounds with diverse pharmacological properties. The plant has traditionally been used as an analgesic, anti-inflammatory agent, and for skin problems. Its pharmacological activities have been shown to include antioxidant, antibacterial, anti-HIV, anti-Helicobacter pylori, and anticancer activities. Further research has revealed the presence of various phytochemicals, primarily triterpenes, β-sitosterol, stigmasterol, flavones, apigenin, and ovatodiolides. Summary of the Invention

[0012] The present invention provides a method for preventing or treating fibrotic diseases, comprising administering a medically effective amount of a composition to a subject in need thereof; wherein the composition comprises 4-fused-rings triterpenes extracted from Antrodia camphorate.

[0013] In some embodiments, the tetra-fused-ring triterpenoid compound is obtained from the ethanol extract of Antrodia cinnamomea.

[0014] In some embodiments, the tetra-fused-ring triterpenoid compound is obtained from the ethyl acetate extract of Antrodia cinnamomea.

[0015] In some embodiments, the tetra-fused-ring triterpenoid compound is obtained from the methanol extract of Antrodia cinnamomea.

[0016] In some embodiments, the tetra-fused-ring triterpenoid compound is obtained from an organic eluent by introducing a methanol extract of Antrodia cinnamomea into a normal phase chromatography column and eluting with hexane / ethyl acetate / methanol.

[0017] In some embodiments, the organic eluent comprises at least one compound selected from the following formulae or a combination thereof:

[0018]

[0019]

[0020] In another aspect, the present invention also provides a method for preventing or treating a fibrotic disorder, comprising administering to a subject in need thereof a medically effective amount of a composition comprising a compound selected from the following formulae or a combination thereof:

[0021]

[0022] In some embodiments, the fibrotic disorder comprises liver fibrosis, kidney fibrosis, vascular fibrosis, pulmonary fibrosis, and benign prostatic hyperplasia.

[0023] In some embodiments, the composition further reduces renal dysfunction and renal injury.

[0024] In some embodiments, the composition further reduces non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), and inflammation, vacuolation, and necrosis of the liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Described are antcin K, dehydrosulphurenic acid / sulphurenic acid, versisponic acid D, and dehydroeburicoic acid isolated from an extract of Antrodia cinnamomea.

[0026] Figure 2A-2C Protective effects of Antrodia camphorata extracts and compounds against cisplatin-induced renal injury in mice with AKI. To analyze the effects of Antrodia camphorata extracts and compounds, mice were administered daily for 7 days, starting 3 weeks after the first dose of cisplatin, and sacrificed at 4 weeks. (A) Morphological changes in the kidneys; (B) Blood urea nitrogen (BUN) levels; (C) Serum creatinine (CRE) levels. Data are expressed as mean ± SEM (n = 5). ### indicates p < 0.001 compared with the control group. ** indicates p < 0.01, and *** indicates p < 0.001 compared with the cisplatin group.

[0027] Figure 3 Protective effects of Antrodia cinnamomea extracts and compounds on cisplatin-induced renal injury in AKI mice. To analyze the effects of Antrodia cinnamomea extracts and compounds, mice were given daily for 7 days starting 3 weeks after the first dose of cisplatin and sacrificed at 4 weeks. Kidneys were stained with H&E. After cisplatin induction, kidneys in each group were prepared for histological evaluation. Representative tissue sections of the kidneys were stained with H&E at a magnification of 400x. Data are expressed as mean ± SEM (n = 5). ### indicates p < 0.001 compared with the control group sample. ** indicates p < 0.01, *** indicates p < 0.001 compared with the cisplatin group. Tubular cell necrosis is marked with arrows; the bar is 50 μm.

[0028] Figures 4A-4E Antrodia camphorata extracts and compounds regulate serum levels of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) TGF-β, and (E) albumin. Serum levels of TNF-α, IL-1β, IL-6, TGF-β, and albumin were determined using commercially available ELISA kits. Data are expressed as mean ± SEM (n = 5). ### indicates p < 0.001 compared with the control group. ** indicates p < 0.01, and *** indicates p < 0.001 compared with the cisplatin-only group.

[0029] Figure 5A-5BEffect of ARH005-EA (A) and ARH (B) on cisplatin-induced renal TWEAK, a-SMA, P53, and P21 mRNA expression. Western blot analysis of TWEAK, a-SMA, P53, and P21 protein expression levels in renal homogenates after cisplatin challenge.

[0030] Figure 6 The process of establishing CCl4-induced fibrosis model is depicted

[0031] Figures 7A-7C The (A) weight difference, (B) liver weight, and (C) liver / body weight ratio are depicted.

[0032] Figures 8A-8C The serum levels of (A) AST, (B) ALT, and (C) AST / ALT of rats after CCl4-induced liver injury are depicted.

[0033] Figures 9A-9E The (A) inflammation, (B) vacuolization, (C) necrosis, (D) fibrosis, and (E) total histological score in the liver are depicted.

[0034] Figure 10 Representative histological sections of the liver were stained with H&E.

[0035] Figure 11 The process of establishing Con A-induced acute hepatitis model is depicted.

[0036] Figures 12A-12C The effect of ardisia crispae lactone (AR100-DS1) on GOT, GPT, and body weight. (A) Serum GOT and (B) serum GPT 24 hours after Con A induction. (C) Body weight before and after Con A induction. Data are presented as mean ± SEM (n = 9).

[0037] Figure 13 The effect of ardisia crispae lactone (AR100-DS1) on liver injury. (A) (B) Liver histopathology of 15 mg / kg Con A (Veh), (C) 2019-0321-1, and (D) dexamethasone, and (E) necrosis histopathology score.

[0038] Figure 14 The process of establishing atherosclerotic rabbit model is depicted.

[0039] Figure 15 The initial and final average body weights of rabbits are depicted. The initial and final average body weights of rabbits are depicted. * and ** represent P < 0.05 compared to the control and HF groups, respectively.

[0040] Figures 16A-16CThe changes of AST, ALT and BUN between the W0 group and the rabbits in each group are depicted. and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0041] Figures 17A-17D The changes of TG, TC, HDL-C and LDL-C in the W0 group of rabbits in each group are depicted. and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0042] Figures 18A-18C The changes of AST, ALT and BUN between W4 groups in each group of rabbits are depicted. and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0043] Figures 19A-19D The changes of TG, TC, HDL-C and LDL-C in the W4 group of rabbits were depicted. and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0044] Figures 20A-20C The changes of AST, ALT and BUN between W8 groups in each group of rabbits are depicted. and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0045] Figures 21A-21D The changes of TG, TC, HDL-C and LDL-C in the W8 group of rabbits in each group were depicted. and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0046] Figures 22A-22C The changes of AST, ALT and BUN between W12 groups in each group of rabbits are depicted. and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0047] Figures 23A-23D The changes of TG, TC, HDL-C and LDL-C in the W12 group of rabbits in each group were depicted. and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0048] Figure 24 Depicted are histopathological examinations of aortic sclerotic plaque lesions in a hypercholesterolemic rabbit model after a 12-week study.

[0049] Figure 25 Depicted are H&E staining of coronary artery sections after sacrifice of rabbits in each group.

[0050] Figure 26Depicted are H&E-stained sections of coronary arteries from rabbits sacrificed in each group. N, neointima layer; M, medialayer.

[0051] Figure 27 The appearance of vascular stenosis is depicted as the ratio of the neointima layer to the medial layer area (N / M ratio). N, neointima layer; M, media layer. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001, respectively, compared with the HFD group.

[0052] Figure 28 Depicted are histopathological examinations of cardiac tissue in a hypercholesterolemic rabbit model after a 12-week study.

[0053] Figure 29 Depicts the appearance of the liver in a rabbit model of hypercholesterolemia after a 12-week study.

[0054] Figure 30 Depicted are liver histopathological examinations of a hypercholesterolemic rabbit model after a 12-week study.

[0055] Figure 31 The body and lung weight of the animals are depicted.

[0056] Figure 32 Depicts the histopathological changes in the lungs of mice with bleomycin-induced pulmonary fibrosis.

[0057] Figure 33 Depicted is Masson's trichrome staining of the lung in mice with bleomycin-induced pulmonary fibrosis.

[0058] Figure 34 The effects of Antrodia cinnamomea extracts and compounds on hydroxyproline content in bleomycin-induced lung injury in mice were characterized.

[0059] Figures 35A-35D Depicts the regulation of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) TGF-β, and (E) by Antrodia camphorata extracts and compounds in BALF.

[0060] Figure 36 Describe the regulation of BLM-induced MPO activity in mouse lungs by Antrodia cinnamomea extracts and compounds. DETAILED DESCRIPTION

[0061] To facilitate the description of the present invention, the central idea of ​​the present invention is expressed through specific examples. Each item in the embodiment is depicted according to the proportion, size, deformation or displacement suitable for description, rather than being drawn according to the proportion of the actual elements.

[0062] The term "terpenes" refers to a large and diverse class of organic compounds whose basic structure follows a general principle: 2-Methylbutane residues (usually also isoprene units or (C5) n There are about 30,000 known terpenes in the literature, which can be divided into hemi- (hemi-, C5), mono- (mono-, C 10 )、sesqui-(C 15 ), double (di-, C 20 )、sester-(C 25 ), three (tri-, C 30 ) and four (tetra-,C 40 ) terpenoid compounds.

[0063]

[0064] The terms "subject," "individual," "host," and "patient" are used interchangeably herein to refer to living animals, including humans and non-human animals. For example, a subject can be an organism that has immune cells capable of responding to antigenic stimulation and stimulating and inhibiting signal transduction through cell surface receptor binding. The subject can be a mammal, for example, a human or a non-human mammal, such as a dog, cat, pig, cow, sheep, goat, horse, rat, and mouse. The term "subject" does not exclude individuals who are completely normal with respect to the disease or who are normal in all respects.

[0065] The term "treatment" may be administered to a subject having a medical condition or who may eventually acquire the condition to prevent, cure, delay, reduce the severity of one or more symptoms of the condition or a recurring condition, or prolong the subject's survival.

[0066] The term "therapeutically effective amount" refers to that amount of the subject compound that will elicit a desired response, including the biological or medical response in a tissue, system, animal or human, that is being sought by a researcher, veterinarian, medical doctor or other clinician.

[0067] Biochemical parameters were determined using colorimetric kits for the assessment of serum creatinine and serum urea according to the manufacturer's instructions. The kit for the former biomarker was purchased from HUMAN Diagnostics Worldwide, Magdeburg, Germany, and the chemical analyzer was purchased from Roche Diagnostics, Cobas Mira Plus, Rotkreuz, Switzerland.

[0068] Renal histopathology. The left anterior lobe of each mouse kidney was fixed in 10% phosphate-buffered formalin, paraffin-embedded, sectioned at 5 pm, processed with hematoxylin and eosin (H&E) staining, and examined histologically under a light microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images were taken at 400x original magnification using a digital camera (NIS-Elements D 2.30, SP4, Build 387).

[0069] Cytokines. The concentrations of proinflammatory cytokines (TNF-a, IL-6, and IL-1 b) in serum were evaluated by enzyme-linked immunosorbent assay (ELISA) kits (Biosource International Inc., Sunnyvale, CA, USA) according to the manufacturer’s instructions.

[0070] Western blot analysis of kidney tissue. The lysis buffer for Western blot analysis of kidney tissue consisted of 0.6% NP-40, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, and 0.5 mM PMSF, and liver tissue was homogenized at 4°C. The homogenized samples were centrifuged at 3000 revolutions per minute (rpm) for 10 min at 4°C to obtain the supernatant. The total cellular protein amount of the supernatant was calibrated with bovine serum albumin (BSA). Protein samples (50 pg) were resolved using standard methods with denaturing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Immobilon, Millipore, Bedford, MA, USA) blocked with 10% nonfat milk. The PVDF membranes were reacted with the appropriate dilution of specific primary antibodies at 4°C, washed three times with TBST buffer, and then reacted with horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 h. The PVDF membranes were washed three times, and the immunoreactive proteins were detected with ECL reagent (Thermo Scientific, Hudson, NH, USA), and the band intensities on the film were quantified by comparison with the control group using Image J software (NIH, Bethesda, MD, USA) and expressed as relative intensity.

[0071] Statistical Analysis Data obtained from animal experiments are expressed as mean ± standard error of the mean (± SEM). t-test was used to examine differences between multiple groups or between two groups. Statistical significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0072] Example 1 Preparation of Antrodia cinnamomea extract

[0073] 100 g of Antrodia cinnamomea fruiting bodies were refluxed with methanol for 6 hours, and the extract was collected and dried to obtain 15 g of Antrodia cinnamomea methanol extract.

[0074] Example 2 Preparation of active ingredients: Antcin K, dehydrosulphurenic acid / sulphurenic acid, Versisponic acid D and dehydroeburicoic acid

[0075] The methanol extract of Antrodia cinnamomea was further separated by silica gel column chromatography using n-hexane / ethyl acetate / methanol as the chromatography solvent to obtain the following fractions (eg Figure 1 (shown): ARH101-DS1 (RS-Antcin K)

[0076] ARH101-DS2 (dehydrothiochromic acid / thiochromic acid)

[0077] ARH101-DS3(Versisponic acid D)

[0078] ARH101-DS4 (dehydroporous acid)

[0079]

[0080] Example 3 Preparation of AR003 Extract

[0081] 100 g of Antrodia cinnamomea (cultivated in a dish) was refluxed with methanol for 6 hours, and the extract was collected and dried to obtain 15 g of Antrodia cinnamomea ARH003 extract.

[0082] Example 4 Preparation of AR003-E Extract

[0083] 200 g of Antrodia cinnamomea (cultivated in a dish) was refluxed with ethanol for 6 hours, and the extract was collected and dried to obtain 18 g of Antrodia cinnamomea ARH003-E extract.

[0084] Example 5 Preparation of AR004 Extract

[0085] 100 g of Antrodia cinnamomea (Cultivars antrodiae) was refluxed with methanol for 6 hours, and the extract was collected and dried to obtain 18 g of Antrodia cinnamomea ARH004 extract.

[0086] Example 6 Preparation of AR005-EA Extract

[0087] 100 g of Antrodia cinnamomea (solid culture) was refluxed with ethyl acetate for 6 hours, and the extract was collected and dried to obtain 12 g of Antrodia cinnamomea EA extract.

[0088] Example 7 Preparation of Dictyophora japonica Extract

[0089] The preparation process of the nematode extract is as follows: (1) the nematode ethanol extract is added to a silica gel-filled column, and gradient elution is performed with an eluent of "n-hexane / ethyl acetate", "n-hexane / ethyl acetate / methanol" and "methanol" to obtain a nematode separated liquid; (2) the nematode separated liquid is further treated with a silica gel-filled column, and gradient elution is performed with an eluent of "dichloromethane", "dichloromethane / methanol" and "methanol" to obtain a separated concentrate; (3) the separated concentrate is co-crystallized with a "n-hexane / ethyl acetate" solvent to obtain nematode microcrystals.

[0090] Example 8 Preparation of active ingredient: ovatodiolide (AR100-DS1)

[0091] 200 g of the ethanol extract of D. nepalensis was added to a silica gel-packed column (10 x 15 cm) and gradient eluted with eluents consisting of n-hexane / ethyl acetate (ratios of 10:1, 5:1, 3:1, and 1:1), n-hexane / ethyl acetate / methanol (ratios of 6:4:1 and 3:2:1), and methanol to obtain 140 g of a primary fraction.

[0092] 140 g of the initial separation solution was added to a silica gel-packed column (10 x 15 cm) for further separation. Gradient elution was performed using eluents such as dichloromethane, dichloromethane / methanol (ratios of 10:1, 5:1, and 7:3), and methanol, yielding a separated concentrate. This separated concentrate was further recrystallized with the solvent n-hexane / ethyl acetate to yield a crystal. Proton nuclear magnetic resonance (H1-NMR) analysis of the crystal confirmed the chemical structure of the diterpene compound, nephrolactone. Analysis by high-performance liquid chromatography (HPLC) confirmed the crystal to be a nephrolactone compound by comparison with a nephrolactone standard.

[0093]

[0094] Metabolites of sylvatolide (AR100-DS1):

[0095] +O,+Cysteine:m / z:466,M2,M3,M4

[0096] +Glutathione:m / z:636,M6,M7

[0097] +O:m / z:345,M8,M9

[0098]

[0099]

[0100]

[0101]

[0102] Example 9 Cisplatin-induced renal injury mouse model

[0103] Seven- to eight-week-old male C57BL / 6 mice were obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan, China). Animals were housed in acrylic cages at a temperature of 22 ± 1°C and a relative humidity of 55 ± 5% for at least 2 weeks prior to the experiment. Animals had free access to food and water. All experimental procedures were performed in accordance with the guidelines of the relevant institutions and were approved by the relevant institutions.

[0104] Renal fibrosis was induced by multiple low-dose cisplatin injections. Cisplatin (5 mg / kg / injection; P4394, Sigma-Aldrich, St Louis, MO) was injected intraperitoneally three times at weeks 0, 1, and 3. Mice were sacrificed 6 weeks after the first dose of cisplatin (n=6). To analyze the effects of the samples, mice were injected intraperitoneally daily for 7 days, starting 4 weeks after the first dose of cisplatin, and sacrificed at 4 weeks (n=6).

[0105] Example 10 Antrodia camphorata extracts and compounds reduce cisplatin-induced renal dysfunction and histopathological changes in mice

[0106] Changes in kidney morphology Figure 2A As shown. CRE and BUN are indicators of renal function. Figure 2B and 2CThe results showed that compared with the control group, mice injected with cisplatin at 10 mg / kg for three times (at weeks 0, 1, and 3) had significantly increased serum CRE and BUN levels (p<0.001), indicating that cisplatin-treated mice developed nephrotoxicity. Compared with the cisplatin-stimulated group, normalized CRE and BUN demonstrated that mice treated with 1000 mg / kg doses of ARH005-EA and ARH003-E, as well as compounds AR101-DS4 and AR100-DS1, exerted significant renal protective effects in a dose-dependent manner (p<0.001).

[0107] Example 11 Antrodia camphorata extracts and compounds alleviate renal dysfunction and renal injury induced by multiple cisplatin treatments

[0108] The histopathological changes were analyzed to determine whether the extracts and compounds of Antrodia cinnamomea affect the renal failure of mice stimulated by cisplatin. The renal tissue of the control group was completely normal, showing transparent tubular and glomerular structures, and the cell nuclei were clear and normal. In mice stimulated by cisplatin, the kidneys had severe damage, resulting in damage to the renal tubular epithelium, inflammatory cell infiltration, swelling of renal tubular cells, formation of intratubular casts, and dilation of renal tubules. However, the administration of 1000 mg / kg dose of Antrodia cinnamomea extract (AR005-EA) and compound (AR100-DS1) significantly improved the necrosis and inflammatory infiltrating cells in the renal tissue (see Figure 3 ).

[0109] Example 12 Antrodia camphorata extracts and compounds alleviate changes in proinflammatory cytokines and albumin induced by cisplatin

[0110] The levels of proinflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β in serum were assessed by ELISA. Compared with the control group, the levels of NO, TNF-α, IL-1β, and IL-6 in the serum of mice with renal injury treated with cisplatin were significantly increased ( Figures 4A-4E Treatment with 1000 mg / kg of antrodia cinnamomea extract (AR005-EA) and compound (AR100-DS1) significantly improved necrosis and inflammatory infiltrating cells in renal tissue, and also reduced the production of NO, TNF-α, IL-1β, and IL-6 after stimulation with cisplatin.

[0111] Example 13 Inhibition of TWEAK, α-SMA, P53 and P21 Protein Expression in Cisplatin-Induced Renal Injury

[0112] The effect of pretreatment with Antrodia camphorata extract (ARH005-EA) and compound (AR100-DS1) on the inhibition of cisplatin-induced TWEAK, α-SMA, P53 and P21 protein expression was examined. The experimental results showed that the pretreatment with ARH005-EA and ARH inhibited the protein expression of TWEAK, α-SMA, P53 and P21 in renal tissue after cisplatin stimulation ( Figure 5A and 5B ).

[0113] Example 14 Carbon tetrachloride (CCl4)-induced chronic liver fibrosis in rats

[0114] like Figure 6 As shown, eight-week-old male Sprague-Dawley rats were administered 0.4 mg / kg of carbon tetrachloride weekly for eight weeks. Blood samples were collected at weeks 0, 2, 4, 6, and 8, and the animals were sacrificed at the end of week 8 for histopathological examination. Figure 7A 、 7B 7C depict weight change, liver weight and liver / body weight ratio respectively. The liver weight was not significantly different from that of the vehicle group, but the liver / body weight ratio of the blank group was significantly smaller than that of the vehicle group. The liver weight and liver / body weight ratio of the 50 mg / kg AR100-DS1 group were significantly greater than those of the vehicle and blank groups.

[0115] Example 15 Serum liver enzyme analysis

[0116] Evaluate clinical biochemical levels, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), to determine the enzyme activities in the liver of the control and experimental groups (e.g. Figures 8A-8C AST, ALT, and the AST / ALT ratio in the blank group did not change significantly during the experiment. Serum AST and ALT levels in all experimental groups increased significantly over the course of the experiment; however, compared with the vehicle group, smaller increases in AST and ALT were observed in the 50 mg / kg AR100-DS1 group at weeks 6 and 8.

[0117] Example 16 Histological evaluation of liver

[0118] After 8 weeks of carbon tetrachloride induction, the vehicle group showed obvious liver damage such as increased AST and ALT, decreased AST / ALT ratio, inflammation, fibrosis, vacuolization and necrosis. Figures 9A-9EAs shown in Figure 10, the liver surface of the 50 mg / kg AR100-DS1 group was smooth, without atrophy or sclerosis, and the liver weight and liver / body weight ratio were significantly greater than those of the vehicle and blank groups. Overall, AR100-DS1 demonstrated the potential to partially repair carbon tetrachloride-induced liver damage.

[0119] Example 17 Effect of aralia lactone (AR100-DS1) on Con A protein (concanavalin A)-induced acute hepatitis in BALB / c mice

[0120] Intravenous injection of Con A protein is a widely used strategy to study T cell-mediated hepatitis. Con A protein is a lectin that can activate CD4 + T cells, producing cytokines and leading to hepatocellular damage. Dexamethasone (Dex) is a long-acting synthetic corticosteroid used as an anti-inflammatory and immunosuppressive drug. The effects of scutellaria lactone (AR100-DS1) on serum glutamic-pyruvic transaminase (GOT), glutamic-oxaloacetic transaminase (GPT), circulating cytokines, and liver histopathology in BALB / c mice induced by Con A acute hepatitis were evaluated.

[0121] Con A and Dex were purchased from Sigma Aldrich (USA). TM Immunoassay kits were purchased from Corning Inc. (USA), and Fuji Dri-Chem Slide GOP / GPT serum test kits were purchased from Winning Medical Inc. (Taiwan, China).

[0122] Male BALB / c mice (7-9 weeks old) were purchased from BioLASCO Taiwan Co., Ltd. or related institutions (Taiwan, China). Throughout the experiment, five animals were housed per cage with free access to food and water. The room temperature was maintained at 23 ± 2°C with a 12-h light-dark cycle. The animals were acclimated for one week before the experiment to minimize the effects of stress. All experimental protocols involving animals and their care were approved by the Institutional Animal Care and Use of ITRI (ITRI-IACUC-2018-041 and ITRI-IACUC-2018-050; accredited by AAALAC) and performed in accordance with the regulations of the relevant institutions.

[0123] Con A was dissolved in pyrogen-free saline at a concentration of 3 mg / mL and intravenously injected at a dose of 15 mg / kg or 20 mg / kg body weight to induce hepatitis. Ichthyolide (AR100-DS1) and Dex were orally administered 30 minutes before, 4 hours, and 8 hours after Con A administration. Blood and liver tissue were collected 24 hours after Con A administration ( Figure 11 The serum was stored at -80°C until analysis.

[0124] To assess the extent of hepatocellular damage after administration of Con A, serum GPT and GOT levels were measured using a Fuji Dri-Chem Slide kit. Sera from the same group were pooled and used for cytokine determination. TM Cytokine levels were measured using immunoassay kits. Data are expressed as mean ± SEM. Student's t-test was used to analyze differences between the drug-treated and vehicle groups. Differences were considered statistically significant when the p value was less than 0.05. 50 mg / kg of aralia lactone (AR100-DS1) significantly reduced Con A-induced increases in GPT levels (109 ± 25 vs. 368 ± 107 U / L, p < 0.05) and slightly improved the elevated GOT levels (261 ± 45 vs. 410 ± 56 U / L) ( Figure 12 ).

[0125] Liver tissues were fixed in 10% phosphate-buffered formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E) to confirm tissue lesions. Tissue lesions were examined microscopically by veterinary pathologists from BioLASCO Taiwan Co., Ltd. The severity of all microscopic lesions was graded on a scale of 0 to 4 as follows: 0 = none; 1 = single cell necrosis; 2 = ≤30% lobular necrosis; 3 = ≤60% lobular necrosis; 4 = >60% lobular necrosis. Histopathological analysis showed that nebulosa lactone (AR100-DS1) improved liver necrosis (score 0.2±0.2 vs 1.4±0.2, p<0.05) ( Figure 13 The above results show that nematolide (AR100-DS1) can reduce serum GOP and GPT, and alleviate Con A-induced liver necrosis.

[0126] Example 18 Evaluation of the efficacy of Antrodia camphorata extract and AR101-DS2 in preventing atherosclerosis and liver fibrosis

[0127] Experimental model

[0128] Male New Zealand white rabbits weighing 2 to 3 kg were individually housed in cages in a temperature- and humidity-controlled room with a 12-hour light-dark cycle. After a few days of acclimatization, the animals were sequentially assigned to six feeding groups: standard rabbit diet, standard rabbit diet containing 0.5% cholesterol, standard rabbit diet containing 0.5% cholesterol and 10 mg / kg lovastatin, standard rabbit diet containing 0.5% cholesterol and 1% ARH003, standard rabbit diet containing 0.5% cholesterol and 1% ARH004, and standard rabbit diet containing 0.5% cholesterol and 10 mg / kg AR101-DS2. Except for the standard rabbit diet group, the remaining groups were fed a standard rabbit diet containing 0.5% cholesterol for 4 weeks (see Figure 14-15 Each rabbit was fed 50 g / kg of body weight per day. After the animals were acclimated to the new environment, the diet was maintained for 8 weeks. At the beginning and end of the 12-week study, rabbits were anesthetized with an intramuscular injection of Zoletil 50 (1 mL / kg) (Virbac Ltd., France), and blood samples were collected. Finally, after sacrificing the rabbits, the aorta (from the aortic arch to the iliac artery bifurcation) and the entire liver were collected for further histopathological analysis.

[0129] Male New Zealand White rabbits weighing 2 to 3 kg (n=30) were divided into the following groups:

[0130] (ND) standard rabbit diet, n = 5;

[0131] (HF) standard rabbit diet containing 0.5% cholesterol, n = 6;

[0132] (L) Standard rabbit diet containing 0.5% cholesterol and 10 mg / kg lovastatin, n = 4;

[0133] (AR003) standard rabbit diet containing 0.5% cholesterol and 1% ARH003, n = 5;

[0134] (AR004) standard rabbit diet containing 0.5% cholesterol and 1% ARH004, n = 5;

[0135] (AR101-DS2) standard rabbit diet containing 0.5% cholesterol and 10 mg / kg AR101-DS2, n = 5;

[0136] The daily feeding amount for each rabbit was 50 g / kg body weight per day.

[0137] Blood chemistry analysis

[0138] Animals were fasted overnight before blood draw. Blood was collected from the rabbits' ear veins into BD Vacutainer EDTA blood collection tubes. Plasma was separated by centrifugation at 3000 rpm for 10 minutes at 4°C. Figures 16-23 depict measured changes in blood chemistry parameters, including serum levels of low-density lipoprotein (LDL), cholesterol (Chol), triglycerides (TG), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT).

[0139] Aortic Fatty Streak Staining

[0140] The aorta was opened longitudinally to expose the intimal surface and gently flushed with saline (see Figures 24-26 The aorta was incubated in 2% (w / v) Sudan IV, rinsed with ethanol at various concentrations (100%, 90%, 80%, 70%, and 60%) for 1 minute, and then rinsed with pure water. Figure 28 The images shown in the figure were taken with a digital camera (Nikon D80, Japan) and quantified on an Alpha Imager 2200 imaging system (Alpha Innotech, USA). The progression of sclerotic plaque lesions was expressed as the percentage of stained area to total area ( Figure 27 ).

[0141] method

[0142] 1. Hydrate cells or tissues:

[0143] i. Using microscope slides with frozen or rehydrated tissue sections (see step 12 in Sectioning of Paraffin-Embedded Tissue) (Fischer et al., 2008), fix with alcohol- or aldehyde-based fixative.

[0144] ii. Immerse the slide in H2O for 30 seconds, stirring manually. Rinsing in H2O is important; hematoxylin precipitates with the salts and buffer. Staining can be performed using a nonfluorescent detection system after immunohistochemistry or hybridization.

[0145] 2. Immerse the slide in a Coplin jar containing Mayer's hematoxylin and agitate for 30 seconds.

[0146] 3. Rinse the slides in H2O for 1 minute. Estimate the staining intensity at this point and repeat steps 2 and 3 if necessary.

[0147] 4. Stain the slides with 1% eosin Y solution for 10-30 seconds with agitation.

[0148] 5. Dehydrate the sections with two washes of 95% alcohol and two washes of 100% alcohol for 30 seconds each.

[0149] 6. Extract the alcohol with two changes of xylene. If using plastic slides or staining in plastic culture dishes, do not use xylene or xylene-based mounting media as they will dissolve the plastic. If using plastic slides or staining in plastic culture dishes, do not use xylene or xylene-based mounting media as they will dissolve the plastic.

[0150] 7. Add one or two drops of mounting medium and cover with a coverslip. If alcohol cannot be used, use glycerol or another aqueous mounting medium to mount the coverslip.

[0151] Reagents

[0152] Cells or tissues of interest on a microscope slide (see Method 1.i)

[0153] Eosin Y (1% aqueous solution; EM diagnostic system)

[0154] Ethanol (95%, 100%)

[0155] Methanol or Flex alcohols (Richard-Allan Scientific) can be used instead of ethanol (see step 5).

[0156] Mayer's hematoxylin is the easiest to use and is compatible with most colorimetric substrates.

[0157] Mounting medium(Canada Balsam,Sigma C1795)

[0158] If alcohol cannot be used, use glycerol or another aqueous mounting medium (see step 7).

[0159] Xylene

[0160] Liver tissue frozen sections

[0161] Rabbit liver tissue (such as Figure 29The tissues were perfused with saline and fixed in 10% (v / v) formalin neutralization solution (JTBaker, Inc., USA) for 24 hours. The tissues were then embedded in Tissue Tek OCT Compound (#4583; Sakura Finetek Inc., USA). The embedded tissues were cut into 10 μm thick sections and stained with Sudan IV and hematoxylin (Merck, USA). Briefly, the sections were washed with purified water for 1 minute to remove the OCT compound, washed with 50% (v / v) ethanol for 30 seconds, and then stained with 2% (w / v) Sudan IV for 1 hour. After further washing with 50% (v / v) ethanol and purified water for 2 minutes, the sections were counterstained with hematoxylin. Figure 30 The images shown were obtained using a microscope equipped with a 10x magnification objective and quantified on an Alpha Imager 2200 imaging system (Alpha Innotech, USA). The progression of fatty liver disease was expressed as the percentage of oil droplet area to total liver tissue (cells).

[0162]

[0163] Fatty liver score

[0164] 0:low-to medium-power evaluation of parenchymal involvement<5%

[0165] 1:5-33%

[0166] 2: 33-66%

[0167] 3: >66%

[0168] Location

[0169] 0: Region 3, center of lobule

[0170] 1:2 Zone, Central Zone

[0171] Zone 2:3, perioral

[0172] 3: panacinar

[0173] Fibrosis score

[0174] 0: None

[0175] 1: Mild perisinusoidal or periportal

[0176] 2: Peri-sinusoidal and portal / peri-portal

[0177] 3: Bridging fibrosis

[0178] 4: Cirrhosis

[0179] Inflammation score

[0180] 0: No lesion

[0181] 1: Mild, 2 lesions per 200 visual fields

[0182] 2: Moderate, 2-4 lesions per 200 fields

[0183] 3: Severe, 4 lesions per 200 fields of view

[0184] Example 19 Protective Effects of Antrodia Camphorata Extracts and Compounds on Bleomycin-Induced Pulmonary Fibrosis in Mice

[0185] Animals and treatment

[0186] Specific pathogen-free male ICR mice (weighing 18–22 g) were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan, China). Animals were housed in Plexiglas cages at a constant temperature of 22 ± 1°C, a relative humidity of 55 ± 5%, and a 12-h light-dark cycle for at least 2 weeks prior to the experiment. Animals had free access to food and water. All experimental procedures were performed in accordance with the guidelines of the relevant institutions, and this protocol was approved by the relevant institutions for the control and supervision of animal experiments.

[0187] Bleomycin (BLM)-induced pulmonary fibrosis in mice

[0188] The mice were divided into the following groups according to their body weight, with 5 mice in each group: control group, BLM group, BLM+DEX group (7.5 mg / kg), BLM+ACH dose group (50 mg / kg), BLM+ACM dose group, BLM+ACH dose group (25 mg / kg), BLM+ACH dose group (50 mg / kg), BLM+ACM dose group (25 mg / kg), BLM+AH dose group (50 mg / kg), BLM+AM dose group (25 mg / kg), BLM+BH dose group (50 mg / kg) and BLM+BM dose group (25 mg / kg), BLM+CH dose group (50 mg / kg) and BLM+CM dose group (25 mg / kg), BLM+DH dose group (50 mg / kg) and BLM+DM dose group (25 mg / kg), BLM+EH dose group (50 mg / kg) and BLM+EM dose group (25 mg / kg), BLM. Pulmonary fibrosis (PF) was established in mice by intratracheal administration of BLM at a single dose of 7.5 mg / kg body weight. Different doses of samples were administered orally daily for 21 days after BLM injury, with DEX serving as a positive control. The control and experimental groups received an equal volume of vehicle (0.9% NaCl) using the same schedule and route of administration.

[0189] The weight of the mice was recorded daily. On day 21, the mice were sacrificed using an overdose of chloral hydrate anesthesia. Blood was collected for ELISA analysis, and the whole lung was removed and weighed. The right lung was fixed with 10% formalin, dehydrated, and embedded in paraffin. The left lung was used for the determination of hydroxyproline. The lung specific gravity was calculated as follows: lung weight / body weight × 100%

[0190] Experimental design

[0191] Male C57BL / 6 mice were randomly divided into the following 8 groups:

[0192] (n=6):

[0193] 1. Group 1: control group;

[0194] 2. Group 2: Mice received a single intraperitoneal injection of BLM (7.5 mg / kg)

[0195] 3. Group 3: Single dose (ACH, 0.5g / kg)

[0196] 4. Group 4: single dose (ACM, 1.0 g / kg)

[0197] 5. Group 5: Purified AR101-DS1 (50 mg / kg)

[0198] 6. Group 6: Purified AR101-DS1 (25 mg / kg)

[0199] 7. Group 7: Purified AR101-DS2 (50 mg / kg)

[0200] 8. Group 8: Purified AR101-DS2 (25 mg / kg)

[0201] 7. Group 7: Purified AR101-DS4 (50 mg / kg)

[0202] 8. Group 8: Purified AR101-DS4 (25 mg / kg)

[0203] 7. Group 7: Purified AR100-DS1 (50 mg / kg)

[0204] 8. Group 8: Purified AR100-DS1 (25 mg / kg)

[0205] 7. Group 7: Purified ARH013-RA1 (50 mg / kg)

[0206] 8. Group 8: Purified ARH013-RA1 (25 mg / kg)

[0207] BALF sampling

[0208] Under anesthesia, BALF was collected four times with 0.7 mL of normal saline via endotracheal intubation. Approximately 2.5 mL (90%) of BAL fluid (BALF) was recovered from each mouse examined. The supernatant of BALF was stored at -80°C until use.

[0209] Lung histopathology

[0210] The anterior portion of the right lung of each mouse was fixed in 10% phosphate-formaldehyde buffer, embedded in paraffin, cut into 5 μm sections, and then stained with hematoxylin and eosin (H&E). Histological examination was performed under a light microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images were captured using a digital camera (NIS-Elements D 2.30, SP4, Build 387) at an original magnification of 400 times.

[0211] Determination of hydroxyproline

[0212] Hydroxyproline content in lung tissue was analyzed according to the instructions of the Hydroxyproline Assay Kit (Biosource International Inc., Sunnyvale, CA, USA). Mouse lung tissue was ground and homogenized with 1 ml of 6 mol / L potassium chloride solution. Hydrolysis was performed at 95°C for 5 hours, and the pH was adjusted to 6.0-6.8. According to the instructions, the corresponding reagents were added to the reaction mixture and mixed thoroughly. The mixture was then incubated at 60°C for 15 minutes. After cooling, the supernatant was collected by centrifugation at 3500 rpm for 10 minutes. The absorbance of the sample supernatant was measured at 550 nm using a spectrophotometer, and the hydroxyproline content of each group was calculated.

[0213] TNF-α, IL-6, and IL-1β cytokines in serum

[0214] The serum concentrations of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in serum were assessed using enzyme-linked immunosorbent assay (ELISA) kits (Biosource International Inc., Sunnyvale, CA, USA) according to the manufacturer's instructions.

[0215] Myeloperoxidase (MPO) detection

[0216] Lung MPO activity is a reliable indicator for assessing lung inflammatory cell infiltration. Lung tissue was homogenized and MPO levels were measured using a kit according to the manufacturer's instructions.

[0217] Lung histopathological analysis

[0218] The right lung was embedded in paraffin, fixed with 10% formalin, and processed into sections. The sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome.

[0219] Statistical analysis

[0220] The data obtained from the animal experiments are expressed as the mean and standard error of the mean (±SEM). The t-test was used to examine the differences between multiple groups or between two groups. Statistical significance was expressed as *p<0.05, **p<0.01, and ***p<0.001.

[0221] At the end of the experiment, the body weight and lung weight of the animals were recorded. Compared with the control animals, the weight change of animals treated with bleomycin (BLM) was significantly reduced. Compared with the other experimental groups, the lung index [(lung weight / body weight) × 100] showed that the animals treated with bleomycin had a significant increase (Table 2 and Table 3). Figure 31 ). The lung index of ACH, BH and DH was significantly reduced.

[0222] Effects of Antrodia cinnamomea extracts and compounds on lung index in bleomycin-induced pulmonary fibrosis

[0223]

[0224] Example 20 Antrodia camphorata extracts and compounds reduce BLM-induced lung dysfunction and histopathological changes in mice

[0225] The histopathological changes in the lungs of mice were evaluated to explore the therapeutic effects of Antrodia cinnamomea extracts and compounds. The inflammatory infiltration and integrity of the tissue structure were observed by H&E staining ( Figure 32 ); the degree of lung fibrosis was assessed by Masson staining ( Figure 33 The control group exhibited some histological findings, including thin alveolar walls, intact alveolar structure, normal alveolar septa, and reduced mesenchymal inflammatory cell infiltration. After 21 days of BLM administration, alveolar edema, a significant increase in septal width, and increased inflammatory cell infiltration were observed. Compared to the BLM group, administration of the Antrodia cinnamomea extract and compound improved inflammatory infiltration and damaged lung tissue.

[0226] Twenty-one days after BLM administration, Masson staining revealed widespread blue staining in the lung tissue and diaphragm, indicating more severe pulmonary fibrosis in the BLM group than in the control group. Treatment with the Antrodia cinnamomea extract and compound reduced the blue area and alleviated the degree of fibrosis. Twenty-one days after BLM induction, treatment with the Antrodia cinnamomea extract and compound significantly reduced alveolitis and fibrosis scores. These results demonstrate that the Antrodia cinnamomea extract and compound alleviate inflammation and fibrosis in the lungs of mice with pulmonary fibrosis.

[0227] Example 21 Pulmonary Fibrosis Markers

[0228] Hydroxyproline (HP) content is an important indicator of collagen deposition in lung tissue. To quantify the extent of pulmonary fibrosis, the hydroxyproline content in lung tissue was measured in each group and displayed in Figure 34 Compared with the control group, BLM significantly increased HP content (p < 0.001). Antrodia cinnamomea extract (1.0 g / kg) and AH, BH, and DH significantly reduced HP recovery in the lungs (p < 0.001).

[0229] Example 22: Antrodia camphorata extracts and compounds induce changes in pro-inflammatory cytokines induced by bleomycin

[0230] The levels of proinflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β in serum were assessed by ELISA. Compared with the control group, the levels of NO, TNF-α, IL-1β, and IL-6 in the serum of BLM-treated mice with renal injury were significantly increased (P<0.05). Figures 35A-35D Treatment with 1.0 g / kg of Antrodia camphorata extract and compounds (BH and DH) significantly improved necrosis and inflammatory infiltration in lung tissue, and also improved the production of TNF-α, IL-1β, IL-6, and TGF-β after BLM induction (p<0.001).

[0231] Example 23 Effects of Antrodia cinnamomea extracts and compounds on lung MPO activity

[0232] like Figure 36 As shown in Figure 2, compared with the control group, the MPO level induced by BLM was significantly increased (p<0.01). In contrast, the extracts of Antrodia cinnamomea AH, BH, DH and Dex significantly inhibited the MPO activity compared with the BLM group (p<0.001), and their effects were stronger than those of the extracts of Antrodia cinnamomea and the compound group (p<0.05) ( Figure 36 ).

Claims

1. Use of a compound for preparing a drug for preventing or treating atherosclerosis, wherein the compound is a compound of the following formula:

Images