Use of terpenoids in the treatment or prevention of fibrotic diseases

Abstract

The present invention relates to a method for preventing or treating fibrotic diseases, comprising administering an effective amount of a composition to a subject in need thereof; wherein the composition comprises triterpenoid compounds extracted from Antrodia camphorata or Vernonia cinerea.

Description

Technical Field

[0001] The present invention relates to plant terpenoids derived from extracts of Antrodia camphorata and Anisomeles indica (also known as Acne Plant, Anisomeles indica), and particularly to a medicinal and dietary formulation capable of alleviating fibrotic diseases. Background Art

[0002] For an increasing number of individuals, fibrotic diseases are a troublesome problem and a common pathological sequela of many persistent inflammatory diseases, such as pulmonary fibrosis, progressive kidney disease, liver cirrhosis, atherosclerosis, and benign prostatic hyperplasia.

[0003] Renal repair after acute kidney injury induces fibrosis, which may ultimately deteriorate into chronic kidney disease. First, kidney injury activates multipotent progenitor cells to repair tissues. However, as the injury continuously triggers renal fibrosis, these cells become dysfunctional and induce fibrotic repair. The pathogenesis of renal fibrosis is a progressive process that ultimately leads to end-stage renal failure, a major disease that requires dialysis or kidney transplantation.

[0004] Non-alcoholic fatty liver disease (NAFLD) is the main form of chronic liver disease with urgent medical needs. 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 common topic of concern in the medical community, especially because of the increasing prevalence of diabetes and obesity in the world's population. The clinical evaluation of each patient with abnormal aminotransferase levels should take into account non-alcoholic fatty liver 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.

[0005] Autoimmune hepatitis (AIH) is a chronic liver disease without a clear etiology and is characterized by hepatocyte inflammation. Severe AIH may progress to cirrhosis, hepatocellular carcinoma, and even death. Up to 40% of patients with autoimmune hepatitis develop cirrhosis depending on the length of observation time. Therefore, in addition to current anti-inflammatory and immunosuppressive therapies, emerging antifibrotic therapies can be added, which are expected to reposition the treatment goals of autoimmune hepatitis to the prevention, stabilization, and reversal of liver fibrosis.

[0006] Atherosclerosis is one of the main causes of the development of cardiovascular diseases and is related to vascular fibrosis. Vascular fibrosis involves the accumulation of extracellular matrix (ECM) proteins, especially collagen and fibronectin in the vascular medium, and contributes to the remodeling of the structure and the formation of scars. The lack of elastin or excessive collagen in the blood vessel wall leads to vascular fibrosis and increased stiffness.

[0007] In benign prostatic hyperplasia, the deposition of collagen fibers in the prostate replaces the broken muscle fibers, but it causes the muscle tissue to become stiff and weak, making the prostatic fluid accumulate in the glandular ducts. Prostatic fibrosis is the main factor for bladder outlet obstruction in elderly men.

[0008] The medicinal fungus Antrodia camphorata is a well-known folk medicine and is known to have various biological activities, especially antitumor effects in vitro cancer cells and in vivo animal models. Given its diverse bioactive compounds, it is considered an effective alternative plant therapeutic agent or an adjuvant for cancer treatment and immune-related diseases. So far, 225 compounds have been isolated, identified, and their structures elucidated, including macromolecules (nucleic acids, proteins, and polysaccharides), small molecules (benzenoids, lignans, benzoquinones, and maleic acid / succinic acid derivatives), terpenoids (lanostane triterpenes, ergostane triterpenes, diterpenes, monoterpenes, and steroids), nucleotides (nucleobases and nucleosides), fatty acids, and fatty acid esters.

[0009] Accumulated in vitro and in vivo experiments have shown that it has effects such as anti-diabetes, anti-hyperlipidemia, anti-hypertension, anti-inflammatory, antioxidant, antibacterial, prevention of cardiovascular diseases, immunomodulation, liver protection and neuroprotection. However, the efficacy of Antrodia camphorata and its components in the treatment of fibrosis has not been evaluated yet.

[0010] Anisomeles indica, known as "Indian catmint", is a source of medicinally active compounds with various pharmacological effects. The plant has traditionally been used as an analgesic, anti-inflammatory agent and for treating skin problems. Pharmacological activities proven by medicine include antioxidant, antibacterial, anti-human immunodeficiency virus, anti-Helicobacter pylori and anti-cancer activities, etc. Further research has revealed the presence of various phytochemical components, mainly triterpenes, β-sitosterol, stigmasterol, flavones, apigenin and ovatodiolides, etc. Description of the Drawings

[0011] Figure 1 Described are antcin K, dehydrosulphurenic acid / sulphurenic acid, versisponic acid D and dehydroeburicoic acid isolated from Antrodia camphorata extracts.

[0012] Figure 2 Protective effects of Antrodia camphorata extracts and compounds on cisplatin-induced kidney injury in AKI mice. To analyze the effects of Antrodia camphorata extracts and compounds, mice were given daily for 7 days starting 3 weeks after the first dose of cisplatin and sacrificed at the 4th week. (A) Morphological changes of 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 samples. Compared with the cisplatin group, ** indicates p < 0.01 and *** indicates p < 0.001.

[0013] Figure 3Protective effects of Antrodia camphorata extracts and compounds against cisplatin-induced kidney injury in AKI mice. 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. 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 400×. Data are expressed as mean ± SEM (n = 5). indicates p < 0.001 compared with the control group samples. Compared with the cisplatin group, ** indicates p < 0.01 and *** indicates p < 0.001. Tubular cell necrosis was marked with arrows; the scale bar is 50 μm.

[0014] Figure 4 Antrodia camphorata extracts and compounds regulate (A) TNF-α, (B) IL-1β, (C) IL-6, (D) TGF-β and (E) albumin in serum. Serum levels of TNF-α, IL-1β, IL-6, TGF-β and albumin were measured using commercially available ELISA kits. Data are expressed as mean ± S.E.M. (n = 5). indicates p < 0.001 compared with the control group samples. Compared with the cisplatin-only group, ** indicates p < 0.01 and *** indicates p < 0.001.

[0015] Figure 5A Effects of RH005-EA (A) and ARH (B) on the expression of TWEAK, α-SMA, P53 and P21 in cisplatin-induced kidneys. After cisplatin challenge, the levels of TWEAK, α-SMA, P53 and P21 protein expression in renal homogenates were analyzed by Western blotting.

[0016] Figure 6 The process of establishing a CCl4-induced fibrosis model is depicted.

[0017] Figure 7 depicts (A) the weight difference, (B) the liver weight, and (C) the liver / body weight ratio.

[0018] Figure 8 depicts the serum levels of (A) AST, (B) ALT, and (C) AST / ALT in rats after CCl4-induced liver injury.

[0019] Figure 9 depicts (A) inflammation, (B) vacuolization, (C) necrosis, (D) fibrosis, (E) the total histological score in the liver.

[0020] Figure 10 Representative tissue sections of the liver were stained with H&E.

[0021] Figure 11 The process of establishing a Con A-induced acute hepatitis model is depicted.

[0022] Figure 12 depicts the effects of arnicolide (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 expressed as mean ± SEM (n = 9).

[0023] Figure 13 Effect of arnicolide (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) histopathological score of necrosis.

[0024] Figure 14 Depicts the process of establishing an atherosclerotic rabbit model.

[0025] Figure 15 Depicts the initial and final average body weights of the rabbits. Depicts the initial and final average body weights of the rabbits. and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0026] Figure 16 depicts the changes in AST, ALT, and BUN in the W0 group of each group of rabbits, and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0027] Figure 17 depicts the changes in TG, TC, HDL-C, and LDL-C in the W0 group of each group of rabbits, and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0028] Figure 18 depicts the changes in AST, ALT, and BUN in the W4 group of each group of rabbits, and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0029] Figure 19 depicts the changes in TG, TC, HDL-C, and LDL-C in the W4 group of each group of rabbits, and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0030] Figure 20 depicts the changes in AST, ALT, and BUN in the W8 group of each group of rabbits, and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0031] Figure 21 depicts the changes in TG, TC, HDL-C, and LDL-C in the W8 group of each group of rabbits, and * indicate P < 0.05 compared with the control group and HF group, respectively.

[0032] Figure 22 depicts the changes in AST, ALT, and BUN among the W12 group in each set of rabbits. And * represent P < 0.05 compared with the control group and the HF group, respectively.

[0033] Figure 23 depicts the changes in TG, TC, HDL-C, and LDL-C among the W12 group in each group of rabbits. And * represent P < 0.05 compared with the control group and the HF group, respectively.

[0034] Figure 24 Depicts the histochemical examination of atherosclerotic plaque lesions in the aorta of the hypercholesterolemic rabbit model after a 12-week study.

[0035] Figure 25 Depicts the H&E staining of coronary artery sections after sacrificing each group of rabbits.

[0036] Figure 26 Depicts the H&E staining of coronary artery sections after sacrificing each group of rabbits. N, neointima layer; M, medial layer.

[0037] Figure 27 Depicts the manifestation of vascular stenosis, expressed as the ratio of the area of the neointima layer to the media layer (N / M ratio). N, neointima layer; M, media layer. Compared with the HFD group, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0038] Figure 28 Depicts the histochemical examination of the heart tissue of the hypercholesterolemic rabbit model after a 12-week study.

[0039] Figure 29 Depicts the appearance of the liver of the hypercholesterolemic rabbit model after a 12-week study.

[0040] Figure 30 Depicts the histochemical examination of the liver tissue of the hypercholesterolemic rabbit model after a 12-week study.

[0041] Figure 31 Depicts the body weight and lung weight of the animals.

[0042] Figure 32 Depicts the histopathological changes in the lungs of mice with bleomycin-induced pulmonary fibrosis.

[0043] Figure 33 Depicts the Masson trichrome staining of the lungs of mice with bleomycin-induced pulmonary fibrosis.

[0044] Figure 34 Depicts the effects of Antrodia camphorata extracts and compounds on the hydroxyproline content in bleomycin-induced mouse lung injury.

[0045] Figure 35 depicts that Antrodia camphorata extracts and compounds in BALF regulated (A) TNF-α, (B) IL-1β, (C) IL-6, (D) TGF-β.

[0046] Figure 36 Depicts the regulation of Antrodia camphorata extracts and compounds on the MPO activity in the lungs of BLM-induced mice. Detailed implementation modes

[0047] For the convenience of explaining the present invention, the central idea expressed in the above-mentioned invention content of the present invention is expressed through specific examples. Each item in the examples is depicted according to a proportion, size, deformation amount or displacement amount suitable for illustration, rather than drawn according to the proportion of the above actual components.

[0048] The term "terpene" refers to a large and diverse class of organic compounds whose basic structure follows a general principle: the carbon skeleton of terpenes is composed of 2-methylbutane residues (2-Methylbutane residues, usually also referred to as isoprene units or (C5) n Called). There are approximately 30,000 known terpenes in the current literature. According to the number of 2-methylbutane residues, they can be classified into hemi- (C5), mono- (C 10 ), sesqui- (C 15 ), di- (C 20 ), sester- (C 25 ), tri- (C 30 ), and tetra- (C 40 ) terpene compounds.

[0049]

[0050] The terms "subject", "individual", "host", and "patient" are used interchangeably in this specification to refer to living animals, including humans and non-human animals. For example, a subject can be an organism with immune cells capable of responding to antigen stimulation and transducing stimulation and inhibition signals through cell surface receptor binding. The subject can be a mammal. For example, a human or a non-human mammal, such as a dog, a cat, a pig, a cow, a sheep, a goat, a horse, a rat, and a mouse. The term "subject" does not exclude an individual who is completely normal in terms of disease or normal in all aspects.

[0051] The term "treatment" can be applied to a subject having or ultimately at risk of having a medical condition to prevent, cure, delay, reduce the severity of one or more symptoms of the condition or recurrence of the condition, or to prolong the survival time of the subject.

[0052] The term "medically effective amount" refers to the amount of a subject compound that elicits a desired response, which response includes a biological or medical response of tissue, system, animal or human that a researcher, veterinarian, physician or other clinician is seeking.

[0053] Determination of biochemical parameters Serum creatinine and serum urea were evaluated using a colorimetric kit 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 (Roche Diagnostics, Cobas Mira Plus, Rotkreuz, Switzerland).

[0054] Renal histopathology The anterior part of the left lateral hepatic lobe of each mouse was fixed in 10% buffered formalin, embedded in paraffin, cut into 5-μm sections, stained with hematoxylin and eosin (H&E), and examined histologically under an optical microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images were taken with a digital camera (NIS-Elements D 2.30, SP4, Build 387) at an original magnification of 400×.

[0055] Proinflammatory cytokine concentrations (TNF-α, IL-6 and IL-1β) in serum The concentrations of proinflammatory cytokines TNF-α, IL-6 and IL-1β in serum were evaluated using an Enzyme-linked immunosorbent assay (ELISA) kit (Biosource International Inc., Sunnyvale, CA, USA) according to the manufacturer's instructions.

[0056] The lysis buffer for Western blot analysis of kidney tissues consisted of 0.6% NP-40, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, and 0.5 mM PMSF. Liver tissues were homogenized at 4°C. The homogenized samples were centrifuged at 3000 revolutions per minute (rpm) for 10 minutes at 4°C to obtain the supernatant. The total cellular protein amount in the supernatant was calibrated with bovine serum albumin (BSA). Protein samples (50 μg) were resolved by denaturing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods and transferred to a PVDF membrane (Immobilon, Millipore, Bedford, MA, USA), which was blocked with 10% non-fat milk. The PVDF membrane was reacted with appropriately diluted specific primary antibodies at 4°C, washed 3 times with TBST buffer, and then reacted with horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 hour. The PVDF membrane was washed 3 times, and immunoreactive proteins were detected with ECL reagent (Thermo Scientific, Hudson, NH, USA). Band brightness on the film was quantified and expressed as relative intensity by comparison with the control group using Image J software (NIH, Bethesda, MD, USA).

[0057] Data obtained from animal experiments in statistical analysis were expressed as the mean and standard error of the mean (±S.E.M.). The t-test was used to examine differences between multiple groups or between two groups. Statistical significance was expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0058] Example 1 Preparation of Antrodia camphorata extract

[0059] 100 g of Antrodia camphorata fruiting bodies were refluxed with methanol for 6 hours. The extract was collected and dried, yielding 15 g of Antrodia camphorata methanol extract.

[0060] Example 2 Preparation of active ingredients: Antcin K, dehydrosulphurenic acid / sulphurenic acid, Versisponic acid D, and dehydroeburicoic acid

[0061] The Antrodia camphorata methanol extract was further separated by silica gel column chromatography using n-hexane / ethyl acetate / methanol as the eluent, yielding the following fractions (as Figure 1 shown):

[0062] ARH101-DS1 (RS-Antcin K)

[0063] ARH101-DS2 (Dehydrothioabietic acid / Thioabietic acid)

[0064] ARH101-DS3 (Versisponic acid D)

[0065] ARH101-DS4 (Dehydroeburicoic acid)

[0066]

[0067] Preparation of Extract AR003 in Example 3

[0068] 100 grams of Ganoderma tsugae (plate culture) was refluxed with methanol for 6 hours, and the extract was collected and dried, obtaining 15 grams of Ganoderma tsugae ARH003 extract in total.

[0069] Preparation of Extract AR003-E in Example 4

[0070] 200 grams of Ganoderma tsugae (plate culture) was refluxed with ethanol for 6 hours, and the extract was collected and dried, obtaining 18 grams of Ganoderma tsugae ARH003-E extract in total.

[0071] Preparation of Extract AR004 in Example 5

[0072] 100 grams of Ganoderma tsugae (wood culture) was refluxed with methanol for 6 hours, and the extract was collected and dried, obtaining 18 grams of Ganoderma tsugae ARH004 extract in total.

[0073] Preparation of Extract AR005-EA in Example 6

[0074] 100 grams of Ganoderma tsugae (solid culture) was refluxed with ethyl acetate for 6 hours, and the extract was collected and dried, obtaining 12 grams of Ganoderma tsugae EA extract in total.

[0075] Preparation of Extract of Herba Stachydis Nees in Example 7

[0076] The preparation process of the extract of Herba Stachydis Nees is as follows: (1) Take the ethanol extract of Herba Stachydis Nees and add it to a silica gel-packed column, and perform gradient elution with eluents "n-hexane / ethyl acetate", "n-hexane / ethyl acetate / methanol", and "methanol" to obtain a separated solution of Herba Stachydis Nees; (2) Further process the above-mentioned separated solution of Herba Stachydis Nees with a silica gel-packed column, and perform gradient elution with eluents "dichloromethane", "dichloromethane / methanol", and "methanol" to obtain a separated concentrate; (3) Co-crystallize the separated concentrate with the solvent "n-hexane / ethyl acetate" to obtain microcrystals of Herba Stachydis Nees.

[0077] Preparation of Active Ingredient: Ovatodiolide (AR100-DS1)

[0078] 200 g of the ethanol extract of *Veronicastrum villosulum* was added to a silica gel-packed column (10 x 15 cm), and gradient elution was performed with eluents "n-hexane / ethyl acetate (ratios of 10:1, 5:1, 3:1, 1:1)", "n-hexane / ethyl acetate / methanol (ratios of 6:4:1, 3:2:1)", and "methanol" to obtain 140 g of a preliminary separation solution.

[0079] 140 g of the above-mentioned preliminary separation solution was taken and added to a silica gel-packed column (10 x 15 cm) for further separation. Gradient elution was performed with eluents "dichloromethane", "dichloromethane / methanol (ratios of 10:1, 5:1, 7:3)", and "methanol" to obtain a separated concentrate. The separated concentrate was further recrystallized with the solvent "n-hexane / ethyl acetate" to obtain a crystal. The chemical structure of the crystal was identified by 1H-NMR as a diterpene compound of *Veronicastrum villosulum* lactone. By HPLC analysis, the crystal was compared with the *Veronicastrum villosulum* lactone standard product and confirmed to be the *Veronicastrum villosulum* lactone compound.

[0080]

[0081] Metabolites of *Veronicastrum villosulum* lactone (AR100-DS1):

[0082] +O, +Cysteine: m / z: 466, M2, M3, M4

[0083] +Glutathione: m / z: 636, M6, M7

[0084] +O: m / z: 345, M8, M9

[0085]

[0086]

[0087]

[0088]

[0089] Example 9 Cisplatin-induced mouse model of kidney injury

[0090] Male C57BL / 6 mice, 7 to 8 weeks old, were obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan, China). Before the experiment, the animals were housed in acrylic cages at a temperature of 22 ± 1 °C and a relative humidity of 55 ± 5%, and were maintained in a 12-hour light-dark cycle for at least 2 weeks. The animals had free access to food and water. All experimental procedures were carried out in accordance with the guidelines of the relevant institutions and were approved by the relevant institutions.

[0091] Renal fibrosis was induced by multiple injections of low-dose cisplatin. Intraperitoneal injection of cisplatin (5 mg / kg per injection; P4394, Sigma-Aldrich, St Louis, MO) was performed at weeks 0, 1, and 3 for a total of 3 times. Mice were sacrificed 6 weeks after the first dose of cisplatin (n = 6). To analyze the effect of the sample, starting 4 weeks after the first dose of cisplatin, mice were intraperitoneally injected with the sample daily for 7 days and sacrificed at 4 weeks (n = 6).

[0092] Example 10 Antrodia camphorata extracts and compounds reduce cisplatin-induced renal dysfunction and histopathological changes in mice

[0093] The morphological changes of the kidneys are as Figure 2A shown. CRE and BUN are indicators of renal function, Figure 2B and 2C show that compared with the control group, mice injected with 3 doses of cisplatin at 10 mg / kg (at weeks 0, 1, and 3) had significantly increased serum CRE and BUN levels (p < 0.001), indicating that cisplatin-treated mice developed nephrotoxicity. Normalized CRE and BUN compared with the cisplatin-stimulated group demonstrated that mice treated with the 1000 mg / kg doses of ARH005-EA and ARH003-E, as well as the compounds AR101-DS4 and AR100-DS1, exerted significant renal protection in a dose-dependent manner (p < 0.001).

[0094] Example 11 Antrodia camphorata extracts and compounds alleviate cisplatin-induced renal dysfunction and kidney injury

[0095] Histopathological changes were analyzed to determine whether Antrodia camphorata extracts and compounds affected cisplatin-induced renal failure in mice. The renal tissue of the control group was completely normal, showing clear tubular and glomerular structures with normal nuclei. In cisplatin-stimulated mice, the kidneys had severe damage, resulting in damaged renal tubular epithelium, infiltration of inflammatory cells, swelling of renal tubular cells, formation of intratubular casts, and dilation of renal tubules. However, administration of the 1000 mg / kg doses of Antrodia camphorata extract (AR005-EA) and compound (AR100-DS1) significantly improved necrosis and inflammatory infiltrating cells in the renal tissue (see Figure 3 ).

[0096] Example 12 Antrodia camphorata extracts and compounds alleviate changes in cisplatin-induced pro-inflammatory cytokines and albumin

[0097] The levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and TGF-β in serum were evaluated by ELISA. Compared with the control group, the levels of NO, TNF-α, IL-1β and IL-6 in the serum of cisplatin-induced kidney injury mice were significantly increased (respectively Figures 4A - 4E ). Treatment with 1000 mg / kg dose of Ganoderma lucidum extract (AR005-EA) and compound (AR100-DS1) significantly improved necrosis and inflammatory infiltrating cells in kidney tissues, and also reduced the production of NO, TNF-α, IL-1β and IL-6 after being stimulated by cisplatin.

[0098] Example 13 Inhibition of TWEAK, α-SMA, P53 and P21 protein expressions in cisplatin-induced kidney injury

[0099] Examine the effects of pretreatment with Ganoderma lucidum extract (ARH005-EA) and compound (AR100-DS1) on inhibiting cisplatin-induced TWEAK, α-SMA, P53 and P21 protein expressions. The experimental results showed that pretreatment with ARH005-EA and ARH inhibited the protein expressions of TWEAK, α-SMA, P53 and P21 in kidney tissues after being stimulated by cisplatin ( Figure 5A and 5B ).

[0100] Example 14 Carbon tetrachloride (CCl4)-induced chronic liver fibrosis in rats

[0101] As Figure 6 shown, male SD rats at 8 weeks old were administered 0.4 mg / kg of carbon tetrachloride weekly for 8 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 respectively depict the weight change, liver weight and liver / body weight ratio. The liver weight of the blank group had no significant difference from that of the vehicle group, however, 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.

[0102] Example 15 Serum liver enzyme analysis

[0103] Evaluate clinical biochemical levels, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), to determine the enzyme activities of the livers in the control group and the experimental groups (as Figures 8A - 8CAs shown, the AST, ALT, and AST / ALT ratio in the blank group did not change significantly during the experiment. The serum AST and ALT levels in each experimental group of animals increased significantly as the experiment progressed. However, compared with the vehicle group, less increase in AST and ALT was observed in the 50 mg / kg AR100-DS1 group at week 6 and week 8.

[0104] Example 16 Histological Evaluation of Liver

[0105] After 8 weeks of carbon tetrachloride induction, the vehicle group showed obvious liver injuries such as increased AST and ALT, decreased AST / ALT ratio, inflammation, fibrosis, vacuolization, and necrosis. As Figures 9A - 9E shown in Figures 9 and 10, the liver surface of the 50 mg / kg AR100-DS1 group was smooth, without atrophy and 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 injury.

[0106] Example 17 Effect of Achyranthone (AR100-DS1) on Concanavalin A (Con A)-induced Acute Hepatitis in BALB / c Mice

[0107] Intravenous injection of Con A is one of the widely used strategies for studying T cell-mediated hepatitis. Con A is a lectin that can activate CD4 + T cells, produce cytokines, and cause hepatocyte injury. Dexamethasone (Dex) is a long-acting synthetic corticosteroid used as an anti-inflammatory and immunosuppressive drug. The effects of achyranthone (AR100-DS1) on serum glutamic-pyruvic transaminase (GOT), glutamic-oxaloacetic transaminase (GPT), circulating cytokines, and liver histopathology in Con A-induced acute hepatitis were evaluated in BALB / c mice.

[0108] Con A and Dex were purchased from Sigma Aldrich (USA). The ProcartaPlex TM immunoassay kit was purchased from Corning Inc. (USA). The Fuji Dri-Chem Slide GOP / GPT serum test kit was purchased from Winning Medical Inc. (Taiwan, China).

[0109] 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 and allowed free access to food and water. The room temperature was maintained at 23 ± 2 °C with a 12-hour light / dark cycle. 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 Committee of ITRI (ITRI-IACUC-2018-041 and ITRI-IACUC-2018-050; accredited by AAALAC) and conducted in accordance with the regulations of the relevant institution.

[0110] Con A was dissolved in pyrogen-free saline at a concentration of 3 mg / mL and injected intravenously at a dose of 15 mg / kg or 20 mg / kg body weight to induce hepatitis. Arnebinol (AR100-DS1) and Dex were orally administered 30 minutes before, 4 hours after, and 8 hours after the administration of Con A. Blood and liver tissues were collected 24 hours after the administration of Con A ( Figure 11 ). Serum was stored at -80 °C until removed for analysis.

[0111] To evaluate the degree of hepatocyte damage after the administration of Con A, serum GPT and GOT levels were measured using a Fuji Dri-Chem Slide kit. Serum from the same group was pooled for cytokine determination. According to the manufacturer's instructions, cytokine levels were measured using a ProcartaPlex TM immunoassay kit. Data are presented as mean ± SEM. The t-test was used to analyze the differences between the drug treatment group and the vehicle group. A difference was considered statistically significant when the p-value was less than 0.05. 50 mg / kg of arnebinol (AR100-DS1) significantly reduced the GPT level increased by Con A (109 ± 25 vs 368 ± 107 U / L, p < 0.05) and slightly improved the elevation of GOT (261 ± 45 vs 410 ± 56 U / L) (Figure 12).

[0112] 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 a veterinary pathologist from BioLASCO Taiwan Co., Ltd. The severity grading of all microscopic lesions was unified and graded from 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 arnicolide (AR100-DS1) improved liver necrosis (score 0.2 ± 0.2 vs 1.4 ± 0.2, p<0.05)( Figure 13 ). The above results showed that arnicolide (AR100-DS1) could reduce serum GOP and GPT and alleviate Con A-induced liver necrosis.

[0113] Example 18 Evaluation of the Efficacy of Antrodia camphorata Extract and AR101-DS2 in Preventing Atherosclerosis and Liver Fibrosis

[0114] Experimental Model

[0115] Male New Zealand white rabbits weighing 2 to 3 kg were individually caged and housed in a room with controlled temperature and humidity, with a 12-hour light-dark cycle. After several days of adaptation, the animals were sequentially assigned to six feeding groups: standard rabbit chow, standard rabbit chow containing 0.5% cholesterol, standard rabbit chow containing 0.5% cholesterol and 10 mg / kg lovastatin, standard rabbit chow containing 0.5% cholesterol and 1% ARH003, standard rabbit chow containing 0.5% cholesterol and 1% ARH004, and standard rabbit chow containing 0.5% cholesterol and 10 mg / kg AR101-DS2. Except for the standard rabbit chow group, the other groups were given standard rabbit chow containing 0.5% cholesterol for 4 weeks (see Figures 14 - 15 ). The daily feeding amount for each rabbit was 50 g / kg body weight per day. After the animals adapted to the new environment, the diet was continued for 8 weeks. At the beginning and end of the 12-week study, the rabbits were anesthetized by intramuscular injection of Zoletil50 (1 mL / kg) (Virbac Ltd., France), and blood samples were collected. Finally, the aorta (from the aortic arch to the iliac artery bifurcation) and the entire liver were collected after sacrificing the rabbits for further histopathological analysis.

[0116] Male New Zealand white rabbits (n = 30) weighing 2 to 3 kg were divided into the following groups:

[0117] (ND) Standard rabbit chow, n = 5;

[0118] (HF) Standard rabbit chow containing 0.5% cholesterol, n = 6;

[0119] (L) Standard rabbit diet containing 0.5% cholesterol and 10 mg / kg lovastatin, n = 4;

[0120] (AR003) Standard rabbit diet containing 0.5% cholesterol and 1% ARH003, n = 5;

[0121] (AR004) Standard rabbit diet containing 0.5% cholesterol and 1% ARH004, n = 5;

[0122] (AR101-DS2) Standard rabbit diet containing 0.5% cholesterol and 10 mg / kg AR101-DS2, n = 5;

[0123] The daily feeding amount for each rabbit was 50 grams per kilogram of body weight per day.

[0124] Blood chemistry analysis

[0125] Animals were fasted overnight before blood sampling. Blood was collected from the marginal ear vein of rabbits 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 the measured values of blood chemistry parameter changes, including serum levels of low-density lipoprotein (LDL), cholesterol (Chol), triglycerides (TG), glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT).

[0126] Aortic fatty streak staining

[0127] The aorta was opened longitudinally to expose the intimal surface and gently rinsed with saline (see Figures 24 - 26 ). The aorta was incubated in 2% (w / v) Sudan IV, rinsed with several concentrations (100%, 90%, 80%, 70%, and 60%) of ethanol for 1 minute, and then rinsed with pure water. Figure 28 The photos shown in Figure 27 were taken using a digital camera (Nikon D80, Japan) and quantified on an Alpha Imager 2200 document system (Alpha Innotech, USA). The progression of atherosclerotic plaque lesions was expressed as the percentage of the stained area to the total area (

[0128] Method

[0129] 1. Hydrate cells or tissues:

[0130] i. Use microscope slides with frozen sections or rehydrated tissue sections (see Step 12 in Cutting Sections of Paraffin-Embedded Tissues) (Fischer et al., 2008), fixed with alcohol or aldehyde fixatives.

[0131] ii. Immerse the slides in H2O by hand stirring for 30 seconds. Rinsing in H2O is important; hematoxylin precipitates with salts and buffers. Staining can be carried out with non-fluorescent detection systems after immunohistochemistry or hybridization reactions.

[0132] 2. Immerse the slides in a Coplin staining jar containing Mayer's hematoxylin and stir for 30 seconds.

[0133] 3. Rinse the slides in H2O for 1 minute. Estimate the staining intensity at this time and repeat Steps 2 and 3 if necessary.

[0134] 4. Stain the slides with 1% eosin Y solution for 10 - 30 seconds and stir.

[0135] 5. Dehydrate the sections with two changes of 95% alcohol and two changes of 100% alcohol, 30 seconds each time.

[0136] 6. Extract the alcohol with two changes of xylene. Do not use xylene or xylene-based mounting media if using plastic slides or staining in plastic petri dishes as they will dissolve the plastic.

[0137] 7. Add one or two drops of mounting medium and cover with a coverslip. If alcohol cannot be used, use glycerol or other aqueous mounting media to cover the coverslip.

[0138] Reagents

[0139] Cells or tissues of interest on microscope slides (see Method 1.i)

[0140] Eosin Y (1% aqueous solution; EM Diagnostic Systems)

[0141] Ethanol (95%, 100%)

[0142] Methanol or Flex alcohols (Richard-Allan Scientific) can be used instead of ethanol (see Step 5).

[0143] Mayer's hematoxylin is the easiest to use and is compatible with most colorimetric substrates.

[0144] Mounting medium(Canada Balsam,Sigma C1795)

[0145] If alcohol cannot be used, use glycerol or other aqueous mounting media (see Step 7).

[0146] Xylene

[0147] Frozen liver sections

[0148] Rabbit liver tissues (as Figure 29 shown) were perfused with saline and fixed in 10% (v / v) formalin solution (J.T. Baker, Inc., USA) for 24 hours, and then the tissues were 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 pure 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 pure water for 2 minutes, the sections were counterstained with hematoxylin. Figure 30 The photographs shown were obtained using a microscope equipped with a 10× magnification objective lens and quantified on an Alpha Imager 2200 document system (Alpha Innotech, USA). The manifestation of fatty liver progression was expressed as the percentage of the oil droplet area in the total liver tissue (cells).

[0149]

[0150] Fatty liver score

[0151] 0: low-to medium-power evaluation of parenchymal involvement <5%

[0152] 1: 5 - 33%

[0153] 2: 33 - 66%

[0154] 3: >66%

[0155] Location

[0156] 0: Zone 3, centrilobular

[0157] 1: Zone 2, midzone

[0158] 2: Zone 3, periportal

[0159] 3: panacinar

[0160] Fibrosis score

[0161] 0: None

[0162] 1: Mild perisinusoidal or periportal

[0163] 2: Perisinusoidal and portal / peri-portal

[0164] 3: Bridging fibrosis

[0165] 4: Liver cirrhosis

[0166] Inflammation score

[0167] 0: No lesions

[0168] 1: Mild, 2 lesions per 200 fields

[0169] 2: Moderate, 2 - 4 lesions per 200 fields

[0170] 3: Severe, 4 lesions per 200 fields

[0171] Example 19 Protective Effects of Antrodia camphorata Extracts and Compounds on Bleomycin-Induced Pulmonary Fibrosis in Mice

[0172] Animals and Treatments

[0173] Male ICR mice (weight 18 - 22 g) specific pathogen free were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan, China). Before the experiment, the animals were housed in plexiglass cages at a constant temperature of 22 ± 1°C, relative humidity of 55 ± 5%, and a 12-hour light-dark cycle for at least 2 weeks. The animals had free access to food and water. All experimental procedures were conducted in accordance with the guidelines of the relevant institution, and the protocol was approved by the relevant institution for the control and supervision of animal experiments.

[0174] Bleomycin (BLM)-Induced Pulmonary Fibrosis in Mice

[0175] Mice were divided into the following groups according to 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 the sample were administered by gavage daily for 21 days after BLM injury, with DEX as the positive control. The control group and the experimental groups received an equal volume of vehicle (0.9% NaCl) using the same schedule and route of administration.

[0176] The body weight of the mice was recorded daily. On day 21, the mice were sacrificed with an overdose of chloral hydrate anesthetic, blood was collected for ELISA analysis, and the whole lungs were 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 calculation formula is as follows: lung weight / body weight × 100%

[0177] Experimental design

[0178] Male C57BL / 6 mice were randomly divided into the following 8 groups:

[0179] (n = 6):

[0180] 1. First group: control group;

[0181] 2. Second group: mice received a single intraperitoneal injection of BLM (7.5 mg / kg)

[0182] 3. Third group: single dose (ACH, 0.5 g / kg)

[0183] 4. Fourth group: single dose (ACM, 1.0 g / kg)

[0184] 5. Fifth group: purified AR101-DS1 (50 mg / kg)

[0185] 6. Sixth group: purified AR101-DS1 (25 mg / kg)

[0186] 7. Group VII: Purified AR101-DS2 (50 mg / kg)

[0187] 8. Group VIII: Purified AR101-DS2 (25 mg / kg)

[0188] 7. Group VII: Purified AR101-DS4 (50 mg / kg)

[0189] 8. Group VIII: Purified AR101-DS4 (25 mg / kg)

[0190] 7. Group VII: Purified AR100-DS1 (50 mg / kg)

[0191] 8. Group VIII: Purified AR100-DS1 (25 mg / kg)

[0192] 7. Group VII: Purified ARH013-RA1 (50 mg / kg)

[0193] 8. Group VIII: Purified ARH013-RA1 (25 mg / kg)

[0194] BALF Sampling

[0195] Under anesthesia, four BALFs were performed with 0.7 mL of normal saline through tracheal intubation. In each mouse examined, approximately 2.5 mL (90%) of BAL fluid (BALF) was recovered. The supernatant of the BALF was stored at -80 °C for later use.

[0196] Lung Histopathology

[0197] The anterior part of the right lung of each mouse was fixed in 10% buffered formalin, paraffin-embedded, cut into 5-μm sections, and then stained with hematoxylin and eosin (H&E) and examined histologically under an optical microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images were taken at an original magnification of 400× using a digital camera (NIS-Elements D 2.30, SP4, Build 387).

[0198] Determination of Hydroxyproline

[0199] The content of hydroxyproline in lung tissues was analyzed according to the instructions of a hydroxyproline assay kit (Biosource International Inc., Sunnyvale, CA, USA). The mouse lung tissues were ground and homogenized with 1 ml of 6 mol / L potassium chloride solution, hydrolyzed at 95 °C for 5 hours, and the pH value was adjusted to 6.0 - 6.8. According to the instructions, the corresponding reagents were added to the reaction system and mixed well, and then incubated at 60 °C for 15 minutes. After cooling, the supernatant was collected after centrifugation at 3500 rpm for 10 minutes. The absorbance value of the sample supernatant was measured at 550 nm using a spectrophotometer, and the content of hydroxyproline in each group was calculated.

[0200] TNF-α, IL-6 and IL-1β cytokines in serum

[0201] According to the manufacturer's instructions, an enzyme-linked immunosorbent assay (ELISA) kit (Biosource International Inc., Sunnyvale, CA, USA) was used to evaluate the serum concentrations of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) in serum.

[0202] Myeloperoxidase (MPO) detection

[0203] The MPO activity in the lungs is a reliable indicator for evaluating pulmonary inflammatory cell infiltration. The lung tissues were homogenized, and the MPO level was detected using a kit according to the manufacturer's instructions.

[0204] Pathological analysis of lung tissues

[0205] 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 stain.

[0206] Statistical analysis

[0207] The data obtained from the animal experiments were expressed as the mean and the standard error of the mean (±S.E.M.). 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.

[0208] At the end of the whole experiment, the body weight and lung weight of the animals were recorded. Compared with the control animals, the change in body weight of the animals administered with bleomycin (BLM) was significantly reduced. Compared with other experimental groups, the lung index [(lung weight / body weight) × 100] showed a significant increase in the animals administered with bleomycin (the following table and Figure 31 ). The lung indices of ACH, BH and DH were significantly reduced.

[0209] Lung Index of Antrodia camphorata Extracts and Compounds Against Bleomycin-Induced Pulmonary Fibrosis

[0210]

[0211]

[0212] Example 20 Antrodia camphorata Extracts and Compounds Reduce Lung Dysfunction and Histopathological Changes in BLM-Induced Mice

[0213] The histopathological lung changes in mice were evaluated to explore the therapeutic effects of Antrodia camphorata extracts and compounds. Inflammatory infiltration and integrity of tissue structure were observed by H&E staining ( Figure 32 ); the degree of pulmonary fibrosis was determined by Masson staining in the lung tissue ( Figure 33 ). The control group showed some histological phenomena such as thin alveolar walls, intact alveolar structures, normal alveolar septa, and less inflammatory cell infiltration in the lung mesenchyme. After 21 days of BLM administration, alveolar edema, a significant increase in septal width, and increased inflammatory cell infiltration were observed. Compared with the BLM group, administration of Antrodia camphorata extracts and compounds improved inflammatory infiltration and damaged structures in the lung tissue.

[0214] Twenty-one days after BLM administration, Masson staining showed extensive blue staining in the lung tissue and septa, indicating that the degree of pulmonary fibrosis in the BLM group was more severe than that in the normal group. After treatment with Antrodia camphorata extracts and compounds, the blue area decreased and the degree of fibrosis was alleviated. Twenty-one days after BLM induction, the scores of alveolitis and fibrosis were significantly reduced after treatment with Antrodia camphorata extracts and compounds. The above results indicate that Antrodia camphorata extracts and compounds alleviate inflammation and the degree of fibrosis in the lungs of mice with pulmonary fibrosis.

[0215] Example 21 Pulmonary Fibrosis Markers

[0216] The content of hydroxyproline (HP) is an important indicator of collagen deposition in lung tissue. To quantify the degree of pulmonary fibrosis, the HP content in lung tissue was measured in each group and shown in Figure 34 . Compared with the control group, BLM significantly increased the HP content (p < 0.001). Antrodia camphorata extract (1.0 g / kg) and AH, BH, and DH significantly reduced the HP recovery in the lung (p < 0.001).

[0217] Example 22 Antrodia camphorata Extracts and Compounds Alleviate Changes in Proinflammatory Cytokines Induced by Bleomycin

[0218] The levels of proinflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β in serum were evaluated by ELISA. Compared with the control group, the levels of NO, TNF-α, IL-1β, and IL-6 in serum of mice with kidney injury treated with BLM were significantly increased (respectivelyFigures 35A - 35D ). Treatment with 1.0 g / kg of Antrodia camphorata extract and compounds (BH and DH) significantly improved necrosis and inflammatory infiltrating cells in the lung tissue and also improved the production of TNF-α, IL-1β, IL-6 and TGF-β after BLM induction (p < 0.001).

[0219] Example 23 Effects of Antrodia camphorata extract and compounds on pulmonary MPO activity

[0220] As Figure 36 shown, compared with the control group, the MPO level in response to BLM induction was significantly increased (p < 0.01). Conversely, Antrodia camphorata extracts AH, BH, DH and Dex significantly inhibited MPO activity compared with the BLM group (p < 0.001), and their effects were stronger than those of the Antrodia camphorata extract and compound group (p < 0.05)( Figure 36 ).

Claims

1. Use of a compound for the preparation of a medicament for preventing or treating pulmonary fibrosis disorders, wherein the compound is a compound of the following formula:

Images