Use of terpenoids in the treatment or prevention of fibrotic diseases

Abstract

The present invention relates to a method for preventing or treating a fibrotic condition, comprising administering to a subject in need thereof an effective amount of a composition comprising triterpenes extracted from Antrodia camphorate or Anisomeles indica.

Description

[Technical Field]

[0001] The present invention relates to herbal terpenoids derived from Antrodia camphorata and Anisomeles indica extracts, particularly to medicinal and dietary formulations for the effective alleviation of fibrotic diseases. [Background technology]

[0002] Fibroproliferative disorders are a burden to an increasing number of individuals and are a common pathological consequence of many persistent inflammatory diseases such as pulmonary fibrosis, progressive kidney disease, liver cirrhosis, atherosclerosis and benign prostatic hyperplasia.

[0003] Impaired renal repair after acute kidney injury can induce fibrosis and ultimately lead to the development of chronic kidney disease. Kidney injury activates multipotent progenitor cells to repair tissue. However, as injury supports the development of renal fibrosis, these multipotent progenitor cells become dysfunctional, inducing fibrogenic repair. The pathogenesis of renal fibrosis is a progressive process that ultimately leads to end-stage renal failure, a devastating disorder requiring dialysis or kidney transplantation.

[0004] Nonalcoholic fatty liver disease (NAFLD) is a major form of chronic liver disease with significant unmet medical needs. Nonalcoholic steatohepatitis (NASH), a progressive variant of NAFLD, can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. NAFLD and NASH have become a focus of attention across the medical community, particularly due to the increasing prevalence of diabetes and obesity in the global population. Nonalcoholic fatty liver disease and its spectrum should always be considered during the clinical evaluation of patients with abnormal aminotransferase levels, especially if they are obese or diabetic. While the prognosis of uncomplicated NAFLD is generally benign, the presence of fibrosis, hepatocyte hypertrophy, inflammation, and Mallory bodies increases the risk of progression to cirrhosis.

[0005] Autoimmune hepatitis (AIH) is a chronic liver disease characterized by hepatocellular inflammation without a clear etiology. Severe AIH can progress to cirrhosis, hepatocellular carcinoma, or death. Cirrhosis occurs in approximately 40% of treated patients with autoimmune hepatitis, depending on the duration of follow-up. Antifibrotic therapies can complement the anti-inflammatory and immunosuppressive effects of current regimens, and these regimens have emerged as promising treatments for reorienting the treatment of autoimmune hepatitis toward preventing, stabilizing, and reversing liver fibrosis.

[0006] Atherosclerosis, one of the major causes of cardiovascular disease progression, involves vascular fibrosis. Vascular fibrosis involves the accumulation of extracellular matrix (ECM) proteins, particularly collagen and fibronectin, in the vascular media, contributing to structural remodeling and scar formation. A lack of elastin or an excess of collagen in the vascular wall leads to vascular fibrosis and increased stiffness.

[0007] In benign prostatic hyperplasia, the deposition of collagen fibers in the prostate compensates for broken muscle fibers, resulting in stiffness and weakness of the muscle tissue and the deposition of prostatic fluid in the glandular ducts. Prostatic fibrosis plays a central role in the development of bladder outlet obstruction in aging men.

[0008] The medicinal fungus Antrodia camphorata (AC) is a well-known Chinese folk medicine known for its numerous biological activities, particularly antitumor effects in vitro in cancer cells and in vivo in animal models. Given its diverse bioactive compounds, it is considered an effective alternative phytotherapeutic agent or adjuvant for cancer treatment and immune-related diseases. To date, a total of 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), terpenoids (lanostane triterpenes, ergostane triterpenes, diterpenes, monoterpenes, and steroids), nucleotides (nucleobases and nucleosides), fatty acids, and fatty acid esters.

[0009] Accumulative in vitro and in vivo studies have revealed its antidiabetic, antihyperlipidemic, antihypertensive, anti-inflammatory, antioxidant, antibacterial, cardiovascular disease preventive, immunomodulatory, hepatoprotective, and neuroprotective effects. However, the efficacy of Antrodia camphorata and its components in the treatment of fibrosis has not been evaluated.

[0010] Anisomeles indica, commonly known as "Indian catmint," is a source of medicinally active compounds and possesses various pharmacological properties. This plant has traditionally been used as an analgesic, anti-inflammatory, and for skin problems. Medicinally, it has been shown to possess various pharmacological activities, including antioxidant, antibacterial, anti-HIV, anti-Helicobacter pylori, and anti-cancer activities. It has also been used for chronic rheumatism. Further research has revealed the presence of various phytochemical constituents, primarily triterpenes, beta-sitosterol, stigmasterol, flavones, apigenin, and ovatodiolides. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the separation of anthocine K, dehydrosulfurenic acid / sulfurenic acid, versisponic acid D, and dehydroebriochoic acid from Antrodia camphorata extract.

[0012] [Figure 2] Figure 2: Protective effects of Antrodia camphorata extract and compounds against cisplatin-induced renal injury in AKI mice. To analyze the effects of Antrodia camphorata extract and compounds, mice were administered cisplatin daily for 7 days starting 3 weeks after the initial administration and sacrificed at week 4. Renal morphological changes (A). Blood urea nitrogen (BUN) levels (B). Serum creatinine (CRE) levels (C). Data are shown as mean ± SEM (n = 5). ### indicates p < 0.001 compared to the control group. ** p < 0.01, *** p < 0.001 compared to the cisplatin group.

[0013] [Figure 3] Figure 3. Protective effects of Antrodia camphorata extract and compounds against cisplatin-induced renal injury in AKI mice. To analyze the effects of Antrodia camphorata extract and compounds, mice were administered cisplatin daily for 7 days starting 3 weeks after the initial administration and sacrificed at week 4. Kidneys were stained with H&E. After cisplatin challenge, kidneys from each group were prepared for histological evaluation. Representative histological sections of the kidneys were stained with H&E at 400x magnification. Data are shown as mean ± SEM (n = 5). ### indicates p < 0.001 compared to control samples. ** p < 0.01, *** p < 0.001 compared to cisplatin group. Tubular cell necrosis is indicated by arrows, and the bar indicates 50 μm.

[0014] [Figure 4]Figure 4: Serum levels of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) TGF-β, and (E) albumin were modulated by Antrodia camphorata extract and compounds. Serum levels of TNF-α, IL-1β, IL-6, TGF-β, and albumin were measured using commercially available ELISA kits. Data are shown as mean ± SEM (n=5). ### indicates p < 0.01 compared to the control group sample. ** p < 0.01, *** p < 0.001 compared to the cisplatin alone group.

[0015] [Figure 5] Figure 5: Effects of ARH005-EA (A) and ARH (B) on cisplatin-induced TWEAK, α-SMA, P53, and P21 signaling expression in the kidney. Protein levels of TWEAK, α-SMA, P53, and P21 protein expression in kidney homogenates are assessed by Western blot analysis after cisplatin challenge.

[0016] [Figure 6] FIG. 6 shows the course of the CCl4-induced fibrosis model.

[0017] [Figure 7] FIG. 7 shows (A) delta weight, (B) liver weight, and (C) liver weight / body weight ratio, respectively.

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

[0019] [Figure 9] FIG. 9 shows the liver (A) inflammation, (B) vacuolization, (C) necrosis, (D) fibrosis, and (E) total histological scores.

[0020] [Figure 10] Figure 10: Representative histological section of liver stained with H&E stain.

[0021] [Figure 11] FIG. 11 shows the course of Con A (concanavalin A)-induced acute hepatitis model.

[0022] [Figure 12] Figure 12 shows the course of obatodiolide (AR100-DS1) on GOT, GPT, and body weight. (A) Serum GOT and (B) serum GPT 24 hours after 15 mg / kg Con A challenge. (C) Body weight before and after 15 mg / kg Con A challenge. Data are shown as mean ± SEM (n=9). *p<0.05 vs. Veh by t-test. Veh, vehicle; Dex, dexamethasone.

[0023] [Figure 13] Figure 13: Effect of obatodiolide (AR100-DS1) on liver injury. Liver histology of A) Naive, (B) 15 mg / kg Con A (Veh), (C) 2019-0321-1, and (D) dexamethasone, and (E) histopathological scores of necrosis. Data are shown as mean ± SEM (n=9). ***p < 0.001 vs. Veh by Student's t-test. Veh, vehicle; Dex, dexamethasone.

[0024] [Figure 14] FIG. 14 shows the course of the rabbit atherosclerosis model.

[0025] [Figure 15] Figure 15 shows the initial and final mean body weights of the rabbits. † and * indicate P < 0.05 compared with the control and HF groups, respectively.

[0026] [Figure 16] FIG. 16 shows the changes in AST, ALT, and BUN between WO groups in each group of rabbits, where † and * indicate P<0.05 compared with the control group and the HF group, respectively.

[0027] [Figure 17] Figure 17 shows the changes in TG, TC, HDL-C, and LDL-C between WO groups in each group of rabbits, where † and * indicate P < 0.05 compared with the control and HF groups, respectively.

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

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

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

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

[0032] [Figure 22] Figure 22 shows the changes in AST, ALT, and BUN between W12 groups in each group of rabbits, where † and * indicate P < 0.05 compared with the control and HF groups, respectively.

[0033] [Figure 23]Figure 23 shows the changes in TG, TC, HDL-C, and LDL-C between W12 groups in each group of rabbits. † and * indicate P < 0.05 compared with the control and HF groups, respectively.

[0034] [Figure 24] FIG. 24 shows histopathological examination of aortic fatty streak lesions in a hypercholesterolemic rabbit model after a 12-week study.

[0035] [Figure 25] FIG. 25 shows HE staining of coronary artery sections after sacrifice in each group of rabbits.

[0036] [Figure 26] 26 shows HE staining of coronary arteries in each group of rabbits after sacrifice: N, neointima layer; M, media layer.

[0037] [Figure 27] Figure 27 shows the onset of vascular restenosis expressed as the ratio of neointima to media area (N / M ratio), N, neointimal layer; M, medial layer. *p < 0.05, **p < 0.01, ***p < 0.001, respectively, compared with the HFD group.

[0038] [Figure 28] FIG. 28 shows histopathological examination of cardiac tissue in a hypercholesterolemic rabbit model after a 12-week study.

[0039] [Figure 29] FIG. 29 shows photographs of the appearance of the liver in a hypercholesterolemic rabbit model after a 12-week study.

[0040] [Figure 30] FIG. 30 shows histopathological examination of liver tissues from a hypercholesterolemic rabbit model after a 12-week study.

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

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

[0043] [Figure 33] FIG. 33 shows Masson's trichrome staining of lungs in mice with bleomycin-induced pulmonary fibrosis.

[0044] [Figure 34] FIG. 34 shows the effects of A. camphorata extracts and compounds on hydroxyproline content in bleomycin-induced lung injury in mice.

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

[0046] [Figure 36] FIG. 36 shows the modulatory effects of A. camphorata extracts and compounds on pulmonary MPO activity in mice induced by BLM.

[0047] Detailed Description of the Invention For the convenience of explaining the present invention, the central idea expressed in the above summary of the present invention will be expressed by specific examples. Various items in the embodiments are shown in proportions, dimensions, deformations, or displacements suitable for the explanation, but are not shown in the proportions of the actual elements as described above.

[0048] The term "terpene" refers to a large and diverse class of organic compounds whose basic structure follows a general principle: a 2-methylbutane residue, usually, but not exactly, an isoprene unit, (C5) n These carbon atoms, also known as 2-methylbutane (isoprene), make up the carbon skeleton of terpenes. Currently, approximately 30,000 terpenes are known in the literature. Depending on the number of 2-methylbutane (isoprene) subunits, they are classified as hemi- (C5), mono- (C 10 ), sesquioxane (C 15 ), Ji-(C 20 ), Cester (C 25 ), Tri-(C 30 ) and tetraterpenes (C 40 ) are distinguished. [ka]

[0049] The terms "subject," "individual," "host," and "patient" are used interchangeably herein to refer to living animals, including humans and non-human animals. A subject can be, for example, an organism that has immune cells capable of responding to antigenic stimulation and stimulatory and inhibitory signaling through cell surface receptor binding. A subject can be a mammal, such as a human or 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 disease, or who are normal in all respects.

[0050] The term "treatment" refers to a therapeutic or prophylactic measure. Treatment may be applied to a subject having a medical disorder, or a subject who may ultimately acquire the disorder, to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrence of the disorder, or to prolong the subject's survival beyond that expected in the absence of such treatment.

[0051] The term "therapeutically effective amount" refers to an amount of a subject compound that is capable of inducing a desired response, e.g., a biological or medical response, in a tissue, system, animal, or human, as desired, e.g., by a researcher, veterinarian, medical doctor, or other clinician.

[0052] Measurement of biochemical parameters: Serum creatinine and serum urea were assessed using colorimetric kits according to the manufacturer's instructions. The kit for the former marker was purchased from HUMAN Diagnostics Worldwide, Magdeburg, Germany, and from a chemistry analyzer (Roche Diagnostics, Cobas Mira Plus, Rotkreuz, Switzerland).

[0053] Renal histopathology: The anterior portion of the left liver lobe from each mouse was fixed in 10% formaldehyde phosphate buffer, embedded in paraffin, sectioned at 5 μm, and then stained with hematoxylin and eosin (H&E) for histological examination under a light microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images were taken at an original magnification of 400x using a digital camera (NIS-Elements D 2.30, SP4, Build 387).

[0054] Serum TNF-α, IL-6, and IL-1β cytokines: The concentrations of pro-inflammatory cytokines (i.e., tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β) in serum will be assessed using the relevant enzyme-linked immunosorbent assay (ELISA) kits (Biosource International Inc., Sunnyvale, CA, USA) according to the manufacturer's instructions.

[0055] Western blot analysis of kidney tissue: A lysis buffer consisting of 0.6% NP-40, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, and 0.5 mM PMSF was used to homogenize liver tissue at 4°C. The homogenized sample was then centrifuged at 3,000 revolutions per minute (rpm) for 10 minutes at 4°C to obtain the supernatant. Equal total cellular protein content of the supernatant was measured using a bovine serum albumin (BSA) protein standard. Protein samples (50 μg) were resolved by denaturing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods, then transferred to a PVDF membrane (Immobilon, Millipore, Bedford, MA, USA) for electroblotting and blocking with 10% skim milk. The membranes were incubated with the appropriate dilution of specific primary antibodies at 4°C, washed three times with TBS / Tween (TBST) buffer, and then incubated with horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 hour (or overnight). The membranes were washed three times and subjected to immunoreactive protein testing using enhanced chemiluminescence (ECL) reagents (Thermo Scientific, Hudson, NH, USA). Band intensities on the scanned films were quantified compared to control groups using Image J software (NIH, Bethesda, MD, USA) and are presented as relative intensities.

[0056] Statistical analysis: Data obtained from animal experiments are presented as the mean and standard error of the mean (±SEM). Student's t-test is used to examine differences between multiple groups or between two groups. Statistical significance is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001.

[0057] Example 1. Preparation of Antrodia camphorata extract

[0058] 100 g of the fruiting body of Antrodia camphorata is refluxed in methanol for 6 hours, and the extract is collected and dried to obtain a total of 15 g of methanol extract of Antrodia camphorata.

[0059] Example 2. Preparation of active ingredients: anthocin K, dehydrosulfurenic acid / sulfurenic acid, versisponic acid D, and dehydroebric acid

[0060] The methanol extract of Antrodia camphorata is further separated by silica column chromatography using n-hexane / ethyl acetate / methanol as an eluent to obtain fractions (shown in Figure 1). ARH101-DS1 (RS-Antocin K), ARH101-DS2 (dehydrosulfurenic acid / sulfurenic acid), ARH101-DS3 (versisponate D) and ARH101-DS4 (dehydroebulic acid) [ka]

[0061] Example 3. Preparation of AR003 extract

[0062] 100 g of Antrodia camphorata (Petri dish culture) was refluxed with methanol for 6 hours, and the extract was collected and dried under reduced pressure to obtain 15 g of ARH003 extract of Antrodia camphorata.

[0063] Example 4. Preparation of AR003-E extract

[0064] 200 g of Antrodia camphorata (Petri dish culture) was refluxed with ethanol for 6 hours, and the extract was collected and dried to obtain a total of 18 g of AR003-E Antrodia camphorata extract.

[0065] Example 5. Preparation of AR004 extract

[0066] 100 g of Antrodia camphorata (tree culture) is refluxed with methanol for 6 hours, and the extract is collected and dried under reduced pressure to obtain Antrodia camphorate ARH004 extract.

[0067] Example 6. Preparation of AR005-EA extract

[0068] 100 g of Antrodia camphorata (solid culture) was refluxed with ethyl acetate for 6 hours, and the extract was collected and dried to obtain a total of 12 g of EA extract of Antrodia camphorata.

[0069] Example 7. Preparation of Anisomeles indica extract

[0070] The Anisomeles indica extract is prepared by the following process: (1) collecting the ethanol extract of Anisomeles indica and loading it onto a chromatographic column packed with silica, followed by gradient elution with the eluents "n-hexane / ethyl acetate", "hexane / ethyl acetate / methanol", and "methanol" to obtain an Anisomeles indica isolate; (2) separating the Anisomeles indica isolate using a chromatographic column packed with silica, followed by gradient elution with the eluents "dichloromethane", "dichloromethane / methanol", and "methanol" to obtain an isolated concentrate; (3) recrystallizing the isolated concentrate with the solvent "n-hexane / ethyl acetate" to obtain Anisomeles indica microcrystals.

[0071] Example 8. Preparation of the active ingredient: Obatodiolide (AR100-DS1)

[0072] 200 g of ethanol extract of Anisomeles indica was taken and placed in a silica-packed chromatography column (10 x 15 cm). Gradient elution was performed with 1200 ml of each of the following eluents: n-hexane / ethyl acetate (ratios 10 / 1, 5 / 1, 3 / 1, 1 / 1), hexane / ethyl acetate / methanol (ratios 6 / 4 / 1, 3 / 2 / 1), and methanol, to obtain 140 g of initial isolated solution.

[0073] 140 g of the initial solution was separated using a silica-packed chromatographic column (10 x 15 cm). Gradient elution was performed using 1000 ml of dichloromethane, dichloromethane / methanol (10 / 1, 5 / 1, 7 / 3 ratios), and methanol to obtain a concentrated product. The concentrated product was further recrystallized using n-hexane / ethyl acetate to obtain crystals. Nuclear magnetic resonance spectroscopy (H-NMR) of the crystals confirmed that the diterpenoid compound was obatodiolide. High-performance liquid chromatography (HPLC) analysis of the crystals confirmed that they contained the obatodiolide compound. [ka]

[0074] Metabolites from obatdiolide (AR100-DS1): +O, +Cysteine: m / z:466, M2, M3, M4 +Glutathione: m / z:636, M6, M7 +O: m / z:345, M8, M9

[0075] [Table 1-1] [Table 1-2] [Table 1-3]

[0076] Example 9. Mouse model of cisplatin-induced kidney injury

[0077] Male C57BL / 6 mice, 7–8 weeks old, were obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). Animals were housed in Plexiglas cages at a constant temperature of 22 ± 1°C and a relative humidity of 55 ± 5% with a 12-hour dark-light cycle for at least 2 weeks prior to the experiment. Animals were allowed free access to food and water. All experimental procedures were performed in accordance with the guidelines of the Institutional Animal Ethics Committee, and the protocol was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals.

[0078] Renal fibrosis was induced via multiple injections of low-dose cisplatin. Cisplatin (5 mg / kg / injection; P4394, Sigma-Aldrich, St. Louis, MO) was injected intraperitoneally at 0, 1, and 3 weeks for a total of three injections. Mice were sacrificed 6 weeks after the first cisplatin administration (n=6). To analyze the effects of the samples, mice were injected intraperitoneally daily for 7 days from 4 weeks after the first cisplatin administration and sacrificed at 4 weeks (n=6).

[0079] Example 10. Antrodia camphorata extracts and compounds reduced cisplatin-induced renal dysfunction and histopathological changes in mice.

[0080] Renal morphological changes are shown in Figure 2A. CRE and BUN are characteristic of renal function. Figures 2B and 2C show that, compared with the control group, cisplatin injection at three 10 mg / kg CP doses (at weeks 0, 1, and 3) significantly increased serum CRE and BUN levels (p < 0.001), indicating the occurrence of nephrotoxicity in cisplatin-treated mice. Treatment with ARH005-EA and ARH003-E at 1000 mg / kg and with the compounds AR101-DS4 and AR100-DS1 exerted significant renal protective effects in a dose-dependent manner, as indicated by the normalization of CRE and BUN (p < 0.001) compared with the cisplatin-stimulated group.

[0081] Example 11. Antrodia camphorata extracts and compounds attenuate renal dysfunction and injury induced by multiple doses of cisplatin.

[0082] Histopathological changes were analyzed to determine whether Antrodia camphorata extract and compounds affected renal failure in cisplatin-induced mice. Kidney tissue in the control group was completely normal, characterized by clear tubular and glomerular structures with clear, normal nuclei. In cisplatin-induced mice, the kidneys exhibited severe renal damage, including tubular epithelial damage, inflammatory cell infiltration, tubular cell swelling, intratubular mold formation, and tubular dilation. However, treatment with Antrodia camphorata extract (AR005-EA) (at a dose of 1000 mg / kg) and compound (AR100-DS1) significantly improved necrosis and inflammatory infiltrates in the kidney tissue (see Figure 3).

[0083] Example 12. Antrodia camphorata extract and compounds prevented cisplatin-induced changes in inflammatory cytokines and albumin.

[0084] Serum levels of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β were assessed by ELISA. Cisplatin-treated mice with kidney injury had significantly elevated serum NO, TNF-α, IL-1β, and IL-6 levels compared with the control group (Figures 4A-4E, respectively). Treatment with Antrodia camphorata extract (AR005-EA) (at a dose of 1000 mg / kg) and compound (AR100-DS1) significantly improved necrosis and inflammatory infiltrates in kidney tissue and improved the production of NO, TNF-α, IL-1β, and IL-6 after cisplatin challenge.

[0085] Example 13. Cisplatin-induced renal injury. Inhibition of TWEAK, α-SMA, P53 and P21 protein expression.

[0086] We investigated whether pretreatment with Antrodia camphorata extract (ARH005-EA) and compound (AR100-DS1) inhibited cisplatin-induced TWEAK, α-SMA, p53, and p21 protein expression in kidney tissues after cisplatin challenge. Treatment with ARH005-EA and ARH inhibited TWEAK, α-SMA, p53, and p21 protein expression in kidney tissues after cisplatin challenge (Figures 5A and 5B).

[0087] Example 14. CCl4-induced chronic liver fibrosis in rats

[0088] As shown in Figure 6, 8-week-old male SD rats were administered CCl4 at 0.4 mg / kg twice weekly for 8 weeks. Blood samples were collected at weeks 0, 2, 4, 6, and 8. At the end of the 8-week period, the animals were sacrificed for histopathological examination. Figures 7A, 7B, and 7C show the delta weight, liver weight, and liver / body weight ratio, respectively. The liver weight of the naive group was not significantly different from that of the vehicle group, but the liver / body weight ratio of the naive group was significantly lower than that of the vehicle group. The 50 mg / kg AR100-DS1-treated group had significantly higher liver weight and liver / body weight ratio than the vehicle and naive groups.

[0089] Example 15. Serum liver enzyme profile

[0090] Clinical biochemistry levels, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were evaluated to determine liver enzyme activity in the control and experimental groups (shown in Figures 8A-8C). The AST, ALT, and AST / ALT ratio levels in the naive group showed no significant changes during the experiment. While serum AST and ALT levels in animals from each experimental group significantly increased as the experiment progressed, a smaller increase in AST and ALT was observed in the 50 mg / kg AR100-DS1 group at W6 and W8 compared to the vehicle group.

[0091] Example 16. Liver histological evaluation

[0092] Eight weeks after CCl4 induction, the vehicle group exhibited significant liver injury, including increased AST and ALT, decreased AST / ALT ratios, inflammation, fibrosis, vacuolization, and necrosis. As shown in Figures 9A-9E and 10, the livers of the 50 mg / kg AR100-DS1 group had smooth surfaces without atrophy or hardening, and the liver weight and liver / body weight ratio were significantly greater than those of the vehicle and naive groups. AR100-DS1 demonstrated the potential to partially repair CCl4-induced liver injury.

[0093] Example 17. Effect of obatodiolide (AR100-DS1) on Con A (concanavalin A)-induced acute hepatitis in BALB / c mice

[0094] Intravenous injection of concanavalin A (Con A) is a widely used strategy to study T cell-mediated hepatitis. Con A inhibits CD4 + Dexamethasone (Dex) is a lectin that can activate T cells, produce cytokines, and cause liver cell damage. Dexamethasone (Dex) is a long-acting synthetic corticosteroid that has been used as an anti-inflammatory and immunosuppressant. The effects of obatdiolide (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.

[0095] Con A and Dex were purchased from Sigma Aldrich (USA). TM The immunoassay kit was purchased from Corning Inc. (USA). GOP and GPT Fuji Dri-Chem slides were purchased from Winning Medical Inc. (Taiwan).

[0096] Male BALB / c mice (7–9 weeks old) were purchased from BioLASCO Taiwan Co., Ltd. or the National Laboratory Animal Center (NLAC, Taiwan). Animals were housed five per cage and provided with free access to food and water throughout the experiment. The room temperature was maintained at 23 ± 2°C with a 12-hour light / dark cycle. Animals were allowed to acclimate for one week before the experiment to minimize the effects of stress. All experimental protocols involving animals and their care were approved by ITRI's Institutional Animal Care and Use Committee (IACUC) (ITRI-IACUC-2018-041 and ITRI-IACUC-2018-050; approved by AAALAC) and were conducted in accordance with the regulations of the Council of Agriculture, Taiwan.

[0097] Con A was dissolved in pyrogen-free saline at a concentration of 3 mg / mL and injected intravenously at 15 or 20 mg / kg body weight to induce hepatitis. Obatodiolide (AR100-DS1) and Dex were orally administered 30 minutes before, 4 hours after, and 8 hours after Con A treatment. Blood and liver tissue samples were collected 24 hours after Con A treatment (Figure 11). Serum was stored at 80°C until analysis.

[0098] To assess the level of hepatocellular injury after Con A treatment, serum GPT and GOT levels were measured using Fuji Dri-Chem slides (Fuji, Japan). Serum from the same group was pooled and used for cytokine assays. Cytokine levels were measured using ProcartaPlex TMMeasurements were performed using an immunoassay kit according to the manufacturer's instructions. Data are presented as mean ± SEM. A T test was used to analyze differences between drug-treated and vehicle-treated groups. Differences were considered statistically significant when p values ​​were less than 0.05. 50 mg / kg obatodiolide (AR100-DS1) significantly reduced the Con A-induced elevated GPT level (109 ± 25 vs 368 ± 107 U / L, p < 0.05) and slightly improved the elevated GOT (261 ± 45 vs 410 ± 56 U / L) (Figure 12).

[0099] Liver tissues were fixed in 10% phosphate-buffered formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E) to identify histopathological lesions. Histopathological lesions were examined microscopically by veterinary pathologists at BioLASCO Taiwan Co., Ltd. A severity grading system for all microscopic lesions was used, scoring from 0 to 4: 0 = none; 1 = individual cell necrosis; 2 = ≤30% lobular necrosis; 3 = ≤60% lobular necrosis; and 4 = >60% lobular necrosis. Histopathological analysis showed that obatodiolide (AR100-DS1) improved liver necrosis (score 0.2 ± 0.2 vs. 1.4 ± 0.2, p < 0.05) (Figure 13). The results indicate that obatodiolide (AR100-DS1) reduces serum GOP and GPT and attenuates Con A-induced liver necrosis.

[0100] Example 18. Evaluation of the efficacy of Antrodia camphorata extract and AR101-DS2 in preventing atherosclerosis and liver fibrosis

[0101] Experimental model

[0102] Male New Zealand White rabbits weighing 2–3 kg were individually caged and housed in a temperature- and humidity-controlled room with a 12-hour light / dark cycle. After several days of acclimation, the animals were sequentially assigned to six feeding groups: standard rabbit chow, standard rabbit chow containing 0.5% cholesterol, standard rabbit chow containing both 0.5% cholesterol and 10 mg / kg lovastatin, standard rabbit chow containing both 0.5% cholesterol and 1% ARH003, standard rabbit chow containing both 0.5% cholesterol and 1% ARH004, and standard rabbit chow containing both 0.5% cholesterol and 10 mg / kg AR101-DS2. All other groups, except the standard rabbit chow group, were fed standard rabbit chow containing 0.5% cholesterol for four weeks (see Figures 14–15). Each rabbit was fed 50 g / kg body weight per day. The diet was continued for eight weeks after the animals had adapted to their new environment. 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, the rabbits were sacrificed, and the aorta (from the aortic arch to the bifurcation of the iliac arteries) and whole liver were harvested for further histopathological analysis.

[0103] Male New Zealand White rabbits (n=30) weighing 2-3 kg were divided into the following groups: (ND) standard rabbit chow, n = 5; (HF) standard rabbit chow containing 0.5% cholesterol, n = 6; (L) standard rabbit chow containing both 0.5% cholesterol and 10 mg / kg lovastatin, n = 4; (AR003) standard rabbit chow containing both 0.5% cholesterol and 1% ARH003, n = 5; (AR004) standard rabbit chow containing both 0.5% cholesterol and 1% ARH004, n = 5; (AR101-DS2) standard rabbit chow containing both 0.5% cholesterol and 10 mg / kg AR101-DS2, n = 5; Each rabbit is fed 50g / kg body weight per day.

[0104] blood chemistry analysis

[0105] Animals are fasted overnight before blood collection. Blood is collected from the rabbit marginal ear vein into BD Vacutainer EDTA Blood Collection Tubes. Plasma is separated by centrifugation at 3,000 rpm for 10 minutes at 4°C. Figures 16-23 show the results of measurements of 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).

[0106] Aortic Fat Streak Staining

[0107] The aorta was opened longitudinally to expose the intimal surface and gently rinsed with normal saline (see Figures 24-26). The aorta was incubated in 2% (w / v) Sudan IV, then flushed with several concentrations of ethanol (100%, 90%, 80%, 70%, and 60%) for 1 minute, and then rinsed with pure water. Photographs shown in Figure 28 were taken using a digital camera (Nikon D80, Japan) and quantified using an Alpha Imager 2200 documentation system (Alpha Innotech, USA). The progression of fatty streak lesions is shown as the percentage of stained area relative to the total area (Figure 27).

[0108] method 1. Hydrate cells or tissues: i. Use microscope slides with frozen or rehydrated tissue sections fixed in alcohol or aldehyde-based fixatives (see step 12 in Cutting Sections of Paraffin Embedded Tissues) (Fischer et al. 2008). ii. Immerse slides in H2O with manual agitation for 30 seconds. The HO rinse is important; hematoxylin precipitates with salts and buffers. Staining can be performed using immunohistochemistry or hybridization reactions followed by non-fluorescent detection systems. 2. Immerse the slides in a Coplin jar containing Mayer's hematoxylin and agitate for 30 seconds. 3. Rinse slides in H2O for 1 minute. At this point, estimate the staining intensity and repeat steps 2 and 3 if necessary. 4. Stain with 1% eosin Y solution for 10 to 30 seconds with agitation. 5. The sections were dehydrated twice in 95% alcohol and twice in 100% alcohol for 30 seconds each. 6. Extract the alcohol twice with 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. 7. Add 1-2 drops of mounting medium and cover with a coverslip. If alcohols are not available, mount the coverslips with glycerol or other aqueous mounting medium.

[0109] reagent Cells or tissues of interest on a microscope slide (see step 1.i) Eosin Y (1% aqueous solution; EM diagnostic system) Ethanol (95%, 100%) Methanol or Flex alcohols (Richard-Allan Scientific) can be used in place of ethanol (see step 5). Hematoxylin, Mayer's (Sigma) Mayer's hematoxylin is the easiest to use and is compatible with most colorimetric substrates. Mounting medium (Canada Balsam, Sigma C1795) If alcohols are not available, use glycerol or other aqueous mounting media (see step 7). xylene

[0110] Cryosectioning of liver tissue

[0111] Rabbit liver tissue (shown in Figure 29) was perfused with normal saline and fixed in 10% (v / v) formalin neutralizing solution (JT Baker, Inc., USA) for 24 hours. The tissue was then embedded in Tissue Tek OCT Compound (#4583; Sakura Finetek Inc., USA). The embedded tissue was cut into 10-μm-thick slices and stained with Sudan IV and hematoxylin (Merck, USA). Briefly, the sections were washed in pure water for 1 minute to remove the OCT compound, then washed in 50% (v / v) ethanol for 30 seconds, and then stained with 2% (w / v) Sudan IV for 1 hour. After a further 2-minute wash in 50% (v / v) ethanol and pure water, the sections were counterstained with hematoxylin. The photographs shown in Figure 30 were taken using a microscope equipped with a 10x magnification objective lens and quantified using an Alpha Imager 2200 documentation system (Alpha Innotech, USA). The signs of fatty liver progression are shown as the ratio of the area of ​​oil droplets to the total liver tissue (cells).

[0112] [Table 2]

[0113] Example 19. Protective effects of Antrodia camphorata extracts and compounds against bleomycin-induced pulmonary fibrosis in mice.

[0114] Animals and treatments

[0115] Specific pathogen-free ICR mice (male, weighing 18–22 g) were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). Animals were housed in Plexiglas cages at a constant temperature of 22 ± 1°C and a relative humidity of 55 ± 5% with a 12-h dark-light cycle for at least 2 weeks prior to the experiment. Animals were provided with food and water ad libitum. All experimental procedures were performed in accordance with the guidelines of the Institutional Animal Ethics Committee, and the protocol was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals.

[0116] BLM-induced PF in mice

[0117] Mice were divided into five groups based on body weight, with five mice per group: control, BLM, BLM+DEX (7.5 mg / kg), BLM+ACH (50 mg / kg), and BLM+ACM (25 mg / kg), BLM+ACH (50 mg / kg), and BLM+ACM (25 mg / kg), BLM+AH (50 mg / kg), and BLM+AM (25 mg / kg), BLM+BH (50 mg / kg), and BLM+BM (25 mg / kg), BLM+CH (50 mg / kg), and BLM+CM (25 mg / kg), BLM+DH (50 mg / kg), and BLM+DM (25 mg / kg). Mice were treated with BLM+EH (50 mg / kg) and BLM+EM (25 mg / kg) BLM. Pulmonary fibrosis (PF) was established in mice by a single intratracheal administration of BLM at 7.5 mg / kg body weight. Various doses of BLM were administered intragastrically daily for 21 days after BLM injury, with DEX used as a positive control. The control and model groups received an equal volume of vehicle (0.9% NaCl) using the same schedule and route of administration.

[0118] Mouse weights were recorded daily. Mice were sacrificed on day 21 using an overdose of chloral hydrate hydrochloride anesthesia. Blood was collected for ELISA analysis, and whole lungs were removed and weighed. The right lung was fixed in 10% formalin, dehydrated, and embedded in paraffin. The left lung was used for hydroxyproline measurements. Lung index was calculated as lung weight / body weight × 100%.

[0119] Experimental design

[0120] Male C57BL / 6 mice were randomly divided into the following 8 groups (n=6): 1. Group I: control; 2. Group II: Mice received a single intraperitoneal injection of BLM (7.5 mg / kg BW). 3. Group III: Single dose (ACH, 0.5g / kg) 4. IV group: single dose (ACM, 1.0g / kg) 5. Group V: Purified AR101-DS1 (50mg / kg) 6. Group VI: Purified AR101-DS1 (25mg / kg) 7. Group VII: Purified AR101-DS2 (50mg / kg) 8. Group VIII: Purified AR101-DS2 (25mg / kg) 7. Group VII: Purified AR101-DS4 (50mg / kg) 8. Group VIII: Purified AR101-DS4 (25mg / kg) 7. Group VII: Purified AR100-DS1 (50mg / kg) 8. Group VIII: Purified AR100-DS1 (25mg / kg) 7. Group VII: Purified ARH013-RA1 (50mg / kg) 8. Group VIII: Purified ARH013-RA1 (25mg / kg)

[0121] BALF sampling

[0122] Under anesthesia, BALF was collected four times via a tracheal cannula using 0.7 mL of saline. Approximately 2.5 mL (90%) of BALF fluid was collected from each mouse. The BALF supernatant was stored at -80°C until use.

[0123] lung histopathology

[0124] The anterior portion of the right lung of each mouse was fixed in 10% formaldehyde phosphate buffer, embedded in paraffin, and sectioned at 5 μm. Hematoxylin and eosin (H&E) staining was then performed for histological examination under a light microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images were taken at an original magnification of 400x using a digital camera (NIS-Elements D 2.30, SP4, Build 387).

[0125] Hydroxyproline assay

[0126] Hydroxyproline content in lung tissue was analyzed according to the manufacturer's instructions for a hydroxyproline assay kit (Biosource International Inc., Sunnyvale, CA, USA). Mouse lung tissue was pulverized, homogenized in 1 ml of 6 mol / L potassium chloride solution, and hydrolyzed at 95°C for 5 hours, with the pH adjusted to 6.0–6.8. According to the manufacturer's instructions, the corresponding reagent was added to the reaction system, mixed thoroughly, and then incubated at 60°C for 15 minutes. After cooling, the mixture was centrifuged at 3500 rpm for 10 minutes and the supernatant was collected. The absorbance of the supernatant at 550 nm was measured spectrophotometrically, and the hydroxyproline content of each group was calculated.

[0127] Serum TNF-α, IL-6, and IL-1β cytokines

[0128] Serum concentrations of pro-inflammatory cytokines (i.e., tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β) in serum were assessed using the relevant enzyme-linked immunosorbent assay (ELISA) kits (Biosource International Inc., Sunnyvale, CA, USA) according to the manufacturer's instructions.

[0129] Myeloid peroxidase (MPO) assay

[0130] Pulmonary MPO activity was a reliable indicator for estimating inflammatory cell infiltration in the lung. Lung tissue was homogenized and MPO levels were detected using a kit according to the manufacturer's instructions.

[0131] Histopathological analysis

[0132] The right lung was embedded in paraffin wax, fixed in 10% formalin, and sectioned, and the sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome.

[0133] statistical analysis

[0134] Data from animal experiments are presented as the mean and standard error of the mean (±SEM). Student's t-test was used to examine differences between multiple groups or between two groups. Statistical significance is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001.

[0135] At the end of all experiments, the body weight and lung weight of the animals were recorded. Compared with the control animals, the body weight change of the bleomycin (BLM)-treated animals was significantly reduced. Compared with the other experimental groups, the lung index [(lung weight / body weight) × 100] showed a significant increase in the bleomycin-treated animals (Table 1 and Figure 31). The lung index of ACH, BH, and DH was significantly reduced.

[0136] [Table 3]

[0137] Example 20. Antrodia camphorata extracts and compounds reduced pulmonary dysfunction and histopathological changes in BLM-induced mice.

[0138] To explore the therapeutic effects of A. camphorata extract and compounds, histopathological changes in the lungs of mice were evaluated. Inflammatory infiltration and tissue structural integrity were observed using H&E staining (Figure 32); the degree of fibrosis in lung tissue was assessed using Masson staining (Figure 33). Histological findings in the control group included thin alveolar walls, intact alveolar structure, normal alveolar septa, and minimal inflammatory cell infiltration into the pulmonary mesenchyme. After 21 days of BLM administration, significant increases in alveolar edema, septal width, and inflammatory cell infiltration were observed. Administration of A. camphorata extract and compounds improved inflammatory infiltration and damaged structure in lung tissue compared to the BLM group.

[0139] Twenty-one days after BLM administration, lung tissue and septa were stained blue extensively by Masson staining, suggesting a higher degree of pulmonary fibrosis in the BLM group compared with the control group. Treatment with A. camphorata extract and compounds reduced the blue area and the degree of fibrosis. Twenty-one days after BLM formation, the alveolitis and fibrosis scores were significantly reduced after A. camphorata extract and compound treatment. These results suggest that A. camphorata extract and compounds reduce lung inflammation and fibrosis in mice with pulmonary fibrosis.

[0140] Example 21 Pulmonary fibrosis markers

[0141] Hydroxyproline content is an important indicator of collagen deposition in lung tissue. To quantify the degree of pulmonary fibrosis, the hydroxyproline content in lung tissue was measured in each group, and the results are shown in Figure 34. BLM significantly increased HP content compared with the control group (p<0.001). A. camphorata extract (1.0 g / kg) as well as AH, BH, and DH significantly restored pulmonary HP (p<0.001).

[0142] Example 22. Antrodia camphorata extracts and compounds evoked bleomycin-induced changes in pro-inflammatory cytokines.

[0143] Serum levels of inflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β were assessed by ELISA. BLM-treated mice with renal injury had significantly increased serum NO, TNF-α, IL-1β, and IL-6 levels compared with the control group (Figures 35A-35E, respectively). Treatment with A. camphorata extract and compounds (BH and DH) at a dose of 1.0 g / kg significantly improved lung tissue necrosis and inflammatory infiltrates, and improved the production of TNF-α, IL-1β, IL-6, and TGF-β after BLM challenge (p<0.001).

[0144] Example 23 Effects of Antrodia camphorata extract and compounds on pulmonary MPO activity

[0145] As shown in Figure 36, in response to BLM challenge, MPO levels were significantly increased compared to the control group (p<0.01). In contrast, administration of A. camphorata extract, AH, BH, DH, and Dex all significantly suppressed MPO activity compared to the BLM group (p<0.001), and exerted a stronger effect than the A. camphorata extract and compound groups (p<0.05) (Figure 36). Furthermore, the present invention includes the following aspects. [Aspect 1] A method for preventing or treating a fibrotic condition, comprising administering to a subject in need thereof an effective amount of a composition comprising a 4-fused ring triterpene extracted from Antrodia camphorate. [Aspect 2] 2. The method of claim 1, wherein the 4-fused ring triterpene is obtained from an ethanol extract of Antrodia camphorate. [Aspect 3] 2. The method of embodiment 1, wherein the 4-fused ring triterpene is obtained from an ethyl acetate extract of Antrodia camphorate. [Aspect 4] 2. The method of claim 1, wherein the 4-fused ring triterpene is obtained from a methanol extract of Antrodia camphorate. [Aspect 5] 2. The method of claim 1, wherein the 4-fused ring triterpenes are obtained from an organic eluate by loading a methanol extract of Antrodia camphorate onto a normal phase chromatography column and eluting the column with hexane / ethyl acetate / methanol. [Aspect 6] 4. The organic eluate contains a compound of the formula:

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Claims

1. The following formula: 【Chemistry 1】 and combinations thereof, The composition, wherein the fibrotic condition is renal fibrosis, vascular fibrosis or pulmonary fibrosis.

2. 10. The composition of claim 1, wherein the composition further reduces renal dysfunction and damage.

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