Use of terpenoids in the treatment or prevention of fibrous 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 Anisomeles indica.

Description

[Technical Field]

[0001] This invention relates to herbal terpenoids derived from extracts of Antrodia camphorata and Anisomeles indica, particularly to medicinal and edible formulations for effectively alleviating fibrotic diseases. [Background technology]

[0002] Fibroproliferative disorders are a growing problem for individuals and are a common pathological consequence of many persistent inflammatory diseases, including pulmonary fibrosis, progressive kidney disease, cirrhosis, atherosclerosis, and benign prostatic hyperplasia.

[0003] Impaired renal repair after acute kidney injury can induce fibrosis, ultimately leading to the development of chronic kidney disease. Kidney injury activates pluripotent progenitor cells to repair tissue. However, these pluripotent progenitor cells become dysfunctional, inducing fibrinolytic repair, as the injury supports the development of renal fibrosis. The development of renal fibrosis is a progressive process that ultimately leads to end-stage renal failure, a catastrophic impairment requiring dialysis or a kidney transplant.

[0004] Non-alcoholic fatty liver disease (NAFLD) is a major form of chronic liver disease with a significant unmet need. Non-alcoholic steatohepatitis (NASH), a progressive variant of NAFLD, can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. NAFLD and NASH are attracting attention throughout the medical community, particularly due to the increasing prevalence of diabetes and obesity in the global population. When clinically evaluating patients with abnormal aminotransferase levels, non-alcoholic fatty liver disease and its spectrum should always be considered, especially if the patient is obese or diabetic. The prognosis for simple NAFLD is generally benign, but there is a risk of progression to cirrhosis if fibrosis, hepatocyte hypertrophy, inflammation, and Mallory bodies are present.

[0005] Autoimmune hepatitis (AIH) is a chronic liver disease characterized by inflammation of liver cells, although it does not have a clear etiology. Severe AIH can progress to cirrhosis, hepatocellular carcinoma, or death. Cirrhosis occurs in approximately 40% of patients treated for autoimmune hepatitis, depending on the observation period. Antifibrotic therapy can complement the anti-inflammatory and immunosuppressive effects of current regimens, and these regimens are emerging as treatments that are expected to shift the focus of autoimmune hepatitis treatment to the prevention, stabilization, and recovery of 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 deficiency of elastin or an excess of collagen in the vascular wall leads to vascular fibrosis and increased stiffness.

[0007] In benign prostatic hyperplasia (BPH), the deposition of collagen fibers in the prostate gland serves to replace damaged muscle fibers, but it results in stiffness and weakness of the muscle tissue, as well as 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] Antrodia camphorata (AC), a medicinal fungus, is a well-known Chinese folk medicine with numerous biological activities, particularly antitumor effects in in vitro cancer cells and in vivo animal models. Its diverse bioactive compounds have led to its consideration as an efficient alternative plant-based therapeutic agent or adjuvant for cancer treatment and immune-related diseases. To date, a total of 225 compounds, including macromolecules (nucleic acids, proteins, and polysaccharides), small molecules (benzenoids, lignans, benzoquinones, and malein / succinic acid derivatives), terpenoids (lanostan triterpenes, ergostan triterpenes, diterpenes, monoterpenes, and steroids), nucleotides (nucleic acid bases and nucleosides), fatty acids, and fatty acid esters, have been isolated, identified, and structurally elucidated.

[0009] Cumulative in vitro and in vivo studies have revealed its anti-diabetic and anti-hyperlipidemia, antihypertensive, anti-inflammatory, antioxidant, antibacterial, cardiovascular disease prevention, 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 medicinal compounds and possesses a variety of pharmacological effects. This plant has traditionally been used as an analgesic, anti-inflammatory, and for treating skin problems. Medicinally, it has been proven to have various pharmacological activities, including antioxidant, antibacterial, anti-HIV, anti-Helicobacter pylori, and anti-cancer activity. It is also used for chronic rheumatism. Further research has revealed the presence of various phytochemicals, primarily triterpenes, β-sitosterol, stigmasterol, flavones, apigenin, and ovatodiolides. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the separation of anthocinic acid K, dehydrosulfurenic acid / sulfurenic acid, pericisponate D, and dehydroeblichoic acid from an extract of Antrodia camphorata.

[0012] [Figure 2] Figure 2: Protective effects of Antrodia camphorata extract and compounds against cisplatin-induced kidney injury in AKI mice. To analyze the effects of Antrodia camphorata extract and compounds, mice were administered daily for 7 days starting 3 weeks after the first dose of cisplatin, and sacrificed at 4 weeks. Morphological changes in the kidney (A). Blood urea nitrogen (BUN) levels (B). Serum creatinine (CRE) levels (C). Data are shown mean ± SEM (n=5). ### indicates p < 0.001 compared to the control group sample. ** 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 kidney injury in AKI mice. To analyze the effects of Antrodia camphorata extract and compounds, mice were administered daily for 7 days from 3 weeks after the first dose of cisplatin, and sacrificed at 4 weeks. Kidneys were stained with H&E. After cisplatin loading, kidneys from each group were prepared and used for histological evaluation. Representative histological sections of kidneys were stained with H&E at a magnification of (400×). Data are shown as mean ± SEM (n=5). ### indicates p < 0.001 compared to the control group sample. Compared to the cisplatin group, ** p < 0.01 and *** p < 0.001. Tubular cell necrosis is indicated by arrows, and bars represent 50 μm.

[0014] [Figure 4]Figure 4: Serum (A) TNF-α, (B) IL-1β, (C) IL-6, (D) TGF-β, and (E) albumin levels modulated by Antrodia camphorata extracts 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 ± S.E.M. (n = 5). indicates p < 0.01 compared to samples from the control group. ** 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 were evaluated by Western blot analysis after cisplatin challenge.

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

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

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

[0019] [Figure 9] Figure 9 is a diagram showing (A) inflammation, (B) vacuolization, (C) necrosis, (D) fibrosis, and (E) total histological score of the liver.

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

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

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

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

[0024] [Figure 14] Figure 14 shows the process of the atherosclerotic model in rabbits.

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

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

[0027] [Figure 17] Figure 17 shows the changes in TG, TC, HDL-C, and LDL-C between the W0 groups in each rabbit group, with † and * indicating P < 0.05 compared to the control group and HF group, respectively.

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

[0029] [Figure 19] Figure 19 shows the changes in TG, TC, HDL-C, and LDL-C among the W4 groups in each rabbit group, with † and * indicating P < 0.05 compared to the control group and HF group, respectively.

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

[0031] [Figure 21] Figure 21 shows the changes in TG, TC, HDL-C, and LDL-C among the W8 groups in each rabbit group, with † and * indicating P < 0.05 compared to the control group and HF group, respectively.

[0032] [Figure 22] Figure 22 shows the changes in AST, ALT, and BUN among the W12 groups in each rabbit group, with † and * indicating P < 0.05 compared to the control group and HF group, respectively.

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

[0034] [Figure 24] Figure 24 shows the histopathological examination of aortic fatty stripe lesions in a rabbit model of hypercholesterolemia after a 12-week trial.

[0035] [Figure 25] Figure 25 shows HE staining of post-sacrificial coronary artery sections from each group of rabbits.

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

[0037] [Figure 27] Figure 27 shows the occurrence of vascular restenosis, expressed as the ratio of new intima to medial area (N / M ratio) N, new intima layer; M, and medial layer. Compared to the HFD group, *p < 0.05, **p < 0.01, and ***p < 0.001, respectively.

[0038] [Figure 28] Figure 28 shows the histopathological examination of cardiac tissue in a rabbit model of hypercholesterolemia after a 12-week trial.

[0039] [Figure 29] Figure 29 shows a photograph of the liver appearance in a rabbit model of hypercholesterolemia after a 12-week trial.

[0040] [Figure 30] Figure 30 shows the histopathological examination of liver tissue from a rabbit model with hypercholesterolemia after a 12-week trial.

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

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

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

[0044] [Figure 34] Figure 34 shows the effect of Antrodia camphorata extract and compounds on hydroxyproline content in bleomycin-induced lung injury in mice.

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

[0046] [Figure 36] Figure 36 shows the modulating effects of Antrodia camphorata extract and compounds on pulmonary MPO activity in BLM-induced mice.

[0047] Detailed description of the present invention For the sake of clarity in describing the present invention, the central ideas expressed in the above summary of the invention will be illustrated with specific examples. Various items in the embodiments are shown in proportions, dimensions, deformations, or displacements suitable for explanation, and not in proportions of actual elements as described above.

[0048] The term "terpene" refers to a large and diverse class of organic compounds whose basic structure follows general principles: a 2-methylbutane residue, usually an isoprene unit (C5), though not precisely. n Also known as 2-methylbutane (isoprene), it forms the carbon skeleton of terpenes. Currently, approximately 30,000 types of terpenes are known in the literature. Depending on the number of 2-methylbutane (isoprene) subunits, they are classified as hemi-(C5) or mono-(C) 10 ), sesqui-(C 15 ), G-(C 20 ), Sester-(C 25 ), Tri-(C 30 ) and tetraterpene (C 40 They are distinguished as follows: [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 may be, for example, immune cells capable of responding to antigenic stimulation, and organisms having stimulative and inhibitory signaling through cell surface receptor binding. A subject may be a mammal, such as human or non-human mammals, including dogs, cats, pigs, cattle, sheep, goats, horses, rats, and mice. The term “subject” does not exclude individuals that are completely normal with respect to disease, or normal in all respects.

[0050] The term “treatment” means therapeutic or preventive measures. Treatment may be applied to subjects with a medical disability, or subjects who are likely to acquire a disability, to prevent, cure, delay, reduce the severity of, or improve one or more symptoms of the disability or recurrent disability, or to extend the subject’s survival beyond what would be expected in the absence of such treatment.

[0051] The term "therapeutic dose" means the amount of a target compound that can induce a desired response in a tissue, system, animal, or human, such as a biological or medical response, as sought by a researcher, veterinarian, medical doctor, or other clinician.

[0052] Measurement of biochemical parameters: Serum creatinine and serum urea are evaluated using colorimetric kits according to the manufacturer's instructions for use. Kits for the former marker are purchased from HUMAN Diagnostics Worldwide, Magdeburg, Germany, and chemical analyzers from Roche Diagnostics, Cobas Mira Plus, Rotkreuz, Switzerland.

[0053] Kidney histopathology: The anterior portion of the left hepatic lobe of each mouse was fixed in 10% formaldehyde phosphate buffer, embedded in paraffin, and prepared as 5 μm sections. These sections were then treated with hematoxylin and eosin (H&E) staining and examined histologically using a light microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images were taken using a digital camera (NIS-Elements D 2.30, SP4, Build 387) at 400x magnification.

[0054] Serum TNF-α, IL-6, and IL-1β cytokines: Serum concentrations of pro-inflammatory cytokines (i.e., tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β) are assessed using the relevant enzyme-linked immunosorbent assay (ELISA) kit (Biosource International Inc., Sunnyvale, CA, USA) as per the manufacturer's instructions for use.

[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 is used to homogenize liver tissue at 4°C. The homogenized sample is then centrifuged at 3000 rpm for 10 minutes at 4°C to obtain the supernatant. The equal total cellular protein content of the supernatant is measured using a bovine serum albumin (BSA) protein standard. Protein samples (50 μg) are degraded by denatured 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a standard method, then transferred to a PVDF membrane (Immobilon, Millipore, Bedford, MA, USA) for electroblotting and blocking with 10% skim milk. The membrane was incubated at 4°C with the appropriate dilution of the specific primary antibody, washed three times with TBS / Tween (TBST) buffer, and then incubated at 37°C for 1 hour (overnight) with the horseradish peroxidase-conjugated secondary antibody. The membrane was washed three times, and immunoreactive protein assays were performed using enhanced chemiluminescence (ECL) reagent (Thermo Scientific, Hudson, NH, USA). Band intensities on the scanned film were quantified using ImageJ software (NIH, Bethesda, MD, USA) in comparison to the control group and expressed as relative intensities.

[0056] Statistical Analysis: Data obtained from animal experiments are presented as the mean and the 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 Antrodia camphorata fruiting bodies were refluxed with methanol for 6 hours, and the extract was collected and dried to obtain a total of 15 g of methanol extract from Antrodia camphorata.

[0059] Example 2. Preparation of active ingredients: Anthocinic acid K, dehydrosulfurenic acid / sulfurenic acid, pericisponate D, and dehydroeblichoic acid

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

[0061] Example 3. Preparation of AR003 extract

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

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

[0064] 200g 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 18g of AR003-E Antrodia camphorata extract.

[0065] Example 5. Preparation of AR004 extract

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

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

[0068] 100g 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 12g of Antrodia camphorata EA extract.

[0069] Example 7. Preparation of Anisomeles indica Extract

[0070] An extract of Anisomeles indica is prepared by the following process: (1) An ethanol extract of Anisomeles indica is collected and added to a silica-packed chromatographic column, and gradient elution is performed with the eluents "n-hexane / ethyl acetate", "hexane / ethyl acetate / methanol", and "methanol" to obtain an Anisomeles indica separator; (2) The Anisomeles indica separator is separated using a silica-packed chromatographic column, and gradient elution is performed with the eluents "dichloromethane", "dichloromethane / methanol", and "methanol" to obtain a separated concentrate; (3) The separated concentrate is recrystallized with the solvent "n-hexane / ethyl acetate" to obtain Anisomeles indica microcrystals.

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

[0072] 200 g of ethanol extract of Anisomeles indica was placed on a silica-packed chromatography column (10 x 15 cm) and gradient elution was performed using 1200 ml 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 separation.

[0073] 140 g of the initial separation solution was separated using a silica-packed chromatography column (10 x 15 cm), and gradient elution was performed with 1000 ml each of dichloromethane, dichloromethane / methanol (in 10 / 1, 5 / 1, and 7 / 3 ratios), and methanol to obtain the separated concentrate. The separated concentrate was further recrystallized in the solvent n-hexane / ethyl acetate to obtain crystals. Nuclear magnetic resonance spectroscopy (H1-NMR) was used to confirm that these crystals are diterpenoid compounds with the chemical structure of ovatodiolide. High-performance liquid chromatography (HPLC) analysis of these crystals was performed to compare them with ovatodiolide standards and to confirm the presence of an ovatodiolide compound. [ka]

[0074] Metabolites from ovatodiolide (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). The animals were housed in plexiglass cages for at least two weeks prior to the experiment at a constant temperature of 22±1°C and a relative humidity of 55±5% with a 12-hour dark-light cycle. Food and water were provided ad libitum. All experimental procedures were carried out 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 is induced via multiple injections of low-dose cisplatin. Intraperitoneal injections of cisplatin (5 mg / kg / injection; P4394, Sigma-Aldrich, St Louis, MO) are administered a total of three times, at weeks 0, 1, and 3. Mice are sacrificed 6 weeks after the first dose of cisplatin (n=6). To analyze the effects of the samples, mice are given daily intraperitoneal injections of cisplatin for 7 days starting 4 weeks after the first dose, and then sacrificed at 4 weeks (n=6).

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

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

[0081] Example 11. Antrodia camphorata extract and compounds reduce renal failure and kidney damage induced by multiple doses of cisplatin.

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

[0083] Example 12. Antrodia camphorata extract and compounds were observed to cause changes in cisplatin-induced inflammatory cytokines and albumin.

[0084] Serum levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β were evaluated by ELISA. Cisplatin-treated renal injury mice showed significantly elevated serum NO, TNF-α, IL-1β, and IL-6 levels compared to 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 necrotic and inflammatory infiltrating cells in renal tissue treatment and improved the production of NO, TNF-α, IL-1β, and IL-6 after cisplatin challenge.

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

[0086] We investigated whether pretreatment with Antrodia camphorata extract (ARH005-EA) and compound (AR100-DS1) inhibits cisplatin-induced TWEAK, α-SMA, P53, and P21 protein expression. The results showed that treatment with ARH005-EA and ARH inhibited TWEAK, α-SMA, P53, and P21 protein expression in kidney tissue 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 a dose of 0.4 mg / kg twice a week for 8 weeks. Blood samples were collected at weeks 0, 2, 4, 6, and 8. At the end of the 8 weeks, the animals were sacrificed for histopathological examination. Figures 7A, 7B, and 7C show δ weight, liver weight, and liver / body weight ratio, respectively. There was no significant difference in liver weight between the naive group and the vehicle group, but the liver / body weight ratio in the naive group was significantly smaller than in the vehicle group. The AR100-DS1 administration group at 50 mg / kg had significantly larger liver weight and liver / body weight ratio compared to both the vehicle and naive groups.

[0089] Example 15. Serum liver enzyme profile

[0090] The levels of clinical biochemistry such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were evaluated to determine the hepatic enzyme activity of the control and experimental groups (shown in Figures 8A-8C). The levels of AST, ALT, and AST / ALT ratio in the naive group did not show significant changes during the experiment. While serum AST and ALT levels in animals in each experimental group increased significantly with the progress of the experiment, the increase in AST and ALT was less pronounced in the 50 mg / kg AR100-DS1 group at W6 and W8 compared to the vehicle group.

[0091] Example 16. Histological evaluation of the liver

[0092] Eight weeks after CCl4 induction, the vehicle group showed significant liver injury, including increased AST and ALT, decreased AST / ALT ratio, inflammation, fibrosis, vacuolation, and necrosis. As shown in Figures 9A-9E and 10, the livers of the 50 mg / kg AR100-DS1 group had a smooth surface without atrophy or hardening, and liver weight and liver / body weight ratio were significantly larger than those of the vehicle and naive groups. This suggests that AR100-DS1 may partially repair CCl4-induced liver injury.

[0093] Effect of Obatoclide (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 for studying T cell-mediated hepatitis. Con A is a lectin that can activate CD4 + T cells, produce cytokines, and cause damage to liver cells. Dexamethasone (Dex) is a long-acting synthetic corticosteroid that has been used as an anti-inflammatory and immunosuppressive drug. In BALB / c mice, the effect of obatoclide (AR100-DS1) on serum glutamate pyruvate transaminase (GOT), glutamate oxaloacetate transaminase (GPT), the effect of circulating cytokines on Con A-induced acute hepatitis, and the effect on liver histopathology were evaluated.

[0095] Con A and Dex were purchased from Sigma Aldrich (USA). ProcartaPlex TM Immunoassay kits were 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 in cages of five and given free access to food and water throughout the experiment. Room temperature was maintained at 23±2°C with a 12-hour light-dark cycle. Animals were acclimatized for one week prior to the experiment to minimize stress. All experimental protocols and care, including the animals, were approved by the ITRI's Institutional Animal Care and Use Committee (IACUC) (ITRI-IACUC-2018-041 and ITRI-IACUC-2018-050; approved by AAALAC) and implemented in accordance with the regulations of the Council of Agriculture, Taiwan.

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

[0098] To assess the level of hepatocyte damage 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 shown as mean ± SEM. The T test was used to analyze the difference between the drug-treated group and the vehicle-treated group. A difference was considered statistically significant if the p-value was less than 0.05. 50 mg / kg of ovatodiolide (AR100-DS1) significantly reduced GPT levels elevated 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).

[0099] Liver tissue was fixed in 10% phosphate-buffered formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E) to confirm histological lesions. Histological lesions were examined under a microscope by a veterinary pathologist at BioLASCO Taiwan Co., Ltd. The severity of all microscopic lesions was assessed on a scale of 0 to 4 as follows: 0 = none; 1 = individual cell necrosis; 2 = ≤30% lobular necrosis; 3 = ≤60% lobular necrosis; 4 = >60% lobular necrosis. Histopathological analysis showed that ovatodiolide (AR100-DS1) improved hepatic necrosis (score 0.2±0.2 vs 1.4±0.2, p < 0.05) (Figure 13). These results indicate that ovatodiolide (AR100-DS1) reduces serum GOP and GPT levels and attenuates Con A-induced hepatic necrosis.

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

[0101] Experimental model

[0102] Male New Zealand white rabbits weighing 2-3 kg are placed individually in cages in a room with controlled temperature and humidity. The light-dark cycle is 12 hours each. After several days of acclimatization, the animals are sequentially assigned to six feeding groups: standard rabbit solid feed, standard rabbit solid feed containing 0.5% cholesterol, standard rabbit solid feed containing both 0.5% cholesterol and 10 mg / kg lovastatin, standard rabbit solid feed containing both 0.5% cholesterol and 1% ARH003, standard rabbit solid feed containing both 0.5% cholesterol and 1% ARH004, and standard rabbit solid feed containing both 0.5% cholesterol and 10 mg / kg AR101-DS2. The groups other than the standard rabbit solid feed group are fed standard rabbit solid feed containing 0.5% cholesterol for 4 weeks (see Figures 14-15). The daily feeding amount for each rabbit is 50 g / kg body weight. The feed is provided for 8 weeks after the animals have adapted to the new environment. At the beginning and end of the 12-week study, rabbits are anesthetized by intramuscular injection of Zoletil 50 (1 mL / kg) (Virbac Ltd., France), and blood samples are collected. Finally, after the rabbits are sacrificed, the aorta (from the aortic arch to the bifurcation of the iliac arteries) and the entire liver are collected for further histopathological analysis.

[0103] Male New Zealand white rabbits weighing 2-3 kg (n=30) are divided into the following groups: (ND) Standard rabbit solid feed, n=5; (HF) Standard rabbit solid feed containing 0.5% cholesterol, n=6; (L) Standard rabbit solid feed containing both 0.5% cholesterol and 10 mg / kg of lovastatin, n=4; Standard rabbit solid feed containing both (AR003) 0.5% cholesterol and 1% ARH003, n=5; (AR004) Standard rabbit solid feed containing both 0.5% cholesterol and 1% ARH004, n=5; Standard rabbit solid feed containing both (AR101-DS2) 0.5% cholesterol and 10 mg / kg of AR101-DS2, n=5; The daily feeding amount for each rabbit should be 50g / kg body weight.

[0104] blood chemistry analysis

[0105] Animals were fasted overnight before blood collection. Blood was collected from the marginal ear vein of rabbits into BD Vacutainer EDTA Blood Collection Tubes. Plasma was separated by centrifugation at 3,000 rpm and 4°C for 10 minutes. Figures 16-23 show the measurement results regarding changes in blood chemistry parameters, including serum levels of low-density lipoprotein (LDL), cholesterol (Chol), triglycerides (TG), glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT).

[0106] Aortic fat streak staining

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

[0108] method 1. Hydrate cells or tissues: i. Use microscope slides containing frozen sections or rehydrated tissue sections fixed with alcohol or aldehyde-based fixatives (see step 12 of Cutting Sections of Paraffin Embedded Tissues) (Fischer et al. 2008). ii. Immerse the slide in H2O for 30 seconds while stirring by hand. Washing with H2O is important; hematoxylin precipitates with salts and buffers. Staining can be performed using a non-fluorescent detection system after immunohistochemical or hybridization reactions. 2. Immerse the slide in a Coplin jar containing Mayer hematoxylin and stir for 30 seconds. 3. Rinse the slide in H2O for 1 minute. At this point, estimate the staining intensity and repeat steps 2 and 3 as needed. 4. Stain with 1% eosin Y solution while stirring for 10-30 seconds. 5. The sections were dehydrated twice with 95% alcohol and twice with 100% alcohol for 30 seconds each time. 6. Extract the alcohol twice using xylene. When 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 cannot be used, the coverslip should be sealed with glycerol or other aqueous mounting agent.

[0109] reagent Target cells or tissue 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 instead of ethanol (see step 5). Hematoxylin, Mayer brand (Sigma) Mayer's hematoxylin is the easiest to use and works well with most colorimetric substrates. Mounting medium (Canada Balsam, Sigma C1795) If alcohols cannot be used, use glycerol or other aqueous mounting agent (see step 7). xylene

[0110] Frozen sectioning of liver tissue

[0111] Rabbit liver tissue (shown in Figure 29) was perfused with normal saline and fixed in 10% (v / v) formalin neutralization 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 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. Further washing with 50% (v / v) ethanol and pure water for 2 minutes followed by counterstaining with hematoxylin. The images shown in Figure 30 were acquired using a microscope equipped with a 10x objective lens and quantified using the Alpha Imager 2200 documentation system (Alpha Innotech, USA). Signs of fatty liver progression are indicated as the percentage of oil droplet area relative to the total liver tissue (cells).

[0112] [Table 2]

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

[0114] Animals and treatments

[0115] ICR mice (male) (body weight 18-22g) free of specific pathogens were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). The animals were housed in plexiglass cages for at least two weeks prior to the experiment, at a constant temperature of 22±1°C and a relative humidity of 55±5% with a 12-hour dark-light cycle. Food and water were provided ad libitum. All experimental procedures followed the guidelines of the facility's 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 of five based on body weight: control group, BLM group, BLM+DEX group (7.5 mg / kg), BLM+ACH dose group (50 mg / kg), and BLM+ACM dose group (25 mg / kg), BLM+ACH dose group (50 mg / kg), and BLM+ACM dose group (25 mg / kg), BLM+AH dose group (50 mg / kg), and 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) were used. Pulmonary fibrosis (PF) was established in mice by a single intratracheal administration of BLM at a dose of 7.5 mg / kg mg / kg body weight. For 21 days after BLM injury, various doses of samples were administered intragastricly daily, and DEX was used as a positive control. The control group and model group were administered an equal volume of vehicle (0.9% NaCl) using the same schedule and route of administration.

[0118] The weight of the mice was recorded daily. The mice were sacrificed on day 21 using an excess of chloral hydrate hydrochloride anesthesia. Blood was collected and performed ELISA analysis. The entire lung was removed and weighed. The right lung was fixed in 10% formalin, dehydrated, and embedded in paraffin. The left lung was used for hydroxyproline measurement. The lung coefficient was calculated as lung weight / body weight × 100%.

[0119] Experimental Design

[0120] Male C57BL / 6 mice were randomly divided into the following eight groups (n=6): 1. Group I: Control; 2. Group II: Mice were given a single intraperitoneal injection of BLM (7.5 mg / kg BW). 3. Group III: Single dose (ACH, 0.5 g / kg) 4. Group IV: Single dose (ACM, 1.0 g / 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 sample collection

[0122] Under anesthesia, four BALF (Body-Absorbed Lung Focus) procedures were performed via tracheal cannula using 0.7 mL of physiological saline. Approximately 2.5 mL (90%) of BALF solution was collected from each mouse examined. The supernatant of the BALF solution 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 into 5 μm sections. These sections were then stained with hematoxylin and eosin (H&E), and histological examination was performed using a light microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images were captured using a digital camera (NIS-Elements D 2.30, SP4, Build 387) at 400x magnification.

[0125] Hydroxyproline assay

[0126] The hydroxyproline content in lung tissue was analyzed according to the instructions for use of the Hydroxyproline Assay Kit (Biosource International Inc., Sunnyvale, CA, USA). Mouse lung tissue was pulverized, homogenized with 1 ml of 6 mol / L potassium chloride solution, hydrolyzed at 95°C for 5 hours, and the pH was adjusted to 6.0-6.8. Following the instructions, the corresponding reagents were added to the reaction system, thoroughly mixed, 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 was measured at 550 nm using a spectrophotometer, 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) were assessed using the relevant enzyme-linked immunosorbent assay (ELISA) kit (Biosource International Inc., Sunnyvale, CA, USA) according to the manufacturer's instructions for use.

[0129] Bone marrow peroxidase (MPO) assay

[0130] Pulmonary MPO activity was a reliable indicator for estimating inflammatory cell infiltration in the lungs. 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. The sections were stained with hematoxylin and eosin (H&E) or Masson's tricolor staining.

[0133] statistical analysis

[0134] Data obtained from animal experiments are presented as the mean and the standard error of the mean (±SEM). Student's t-tests were 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 to control animals, the change in body weight was significantly reduced in animals treated with bleomycin (BLM). Compared to other experimental groups, the lung index [(lung weight / body weight) × 100] showed a significant increase in animals treated with bleomycin (Table 1 and Figure 31). The lung index was significantly reduced in ACH, BH, and DH.

[0136] [Table 3]

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

[0138] To explore the therapeutic effects of Antrodia camphorata extract and compounds, histopathological changes in the lungs of mice were evaluated. Inflammatory infiltration and structural integrity were observed by H&E staining (Figure 32); the degree of fibrosis in lung tissue was assessed by Masson staining (Figure 33). In the control group, histological findings included thin alveolar walls, complete alveolar structure, normal alveolar septa, and minimal infiltration of inflammatory cells into the mesenchymal tissue. Twenty-one days after BLM administration, alveolar edema, a significant increase in septal width, and increased inflammatory cell infiltration were observed. Administration of Antrodia 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 stained extensively blue by Masson staining, suggesting a higher degree of pulmonary fibrosis in the BLM group compared to the normal group. After treatment with Antrodia camphorata extract and compounds, the blue area decreased, indicating a reduction in the degree of fibrosis. On day 21 after BLM fabrication, the scores for alveolitis and fibrosis were significantly reduced after Antrodia camphorata extract and compound therapy. These results suggest that Antrodia camphorata extract and compounds reduce the degree of 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, hydroxyproline content in lung tissue was measured in each group and is shown in Figure 34. BLM clearly increased HP content compared to the control group (p<0.001). Antrodia camphorata extract (1.0 g / kg), as well as AH, BH, and DH, significantly restored lung HP (p<0.001).

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

[0143] Serum levels of inflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β were evaluated by ELISA. BLM-treated renal injury mice showed significantly increased serum levels of NO, TNF-α, IL-1β, and IL-6 compared to the control group (Figures 35A-34E, respectively). Treatment with 1.0 g / kg of Antrodia camphorata extract and compounds (BH and DH) significantly improved necrosis and inflammatory infiltrates in lung tissue 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, a significant improvement in MPO levels was observed in response to the BLM challenge compared to the control group (p<0.01). In contrast, administration of both Antrodia camphorata extract, AH, BH, DH, and Dex resulted in a clear suppression of MPO activity compared to the BLM group (p<0.001), and exerted a stronger effect than Antrodia camphorata extract and the compound groups (p<0.05) (Figure 36). Furthermore, the present invention includes the following embodiments. [Aspect 1] A method for preventing or treating a fibrous condition, comprising administering an effective amount of a composition containing a triterpene extracted from anisomeles indica to a subject in need thereof. [Aspect 2] The method according to Embodiment 1, wherein the triterpene is obtained from an organic eluate based on introducing an ethanol extract of anisomeleth indica into a normal-phase chromatography column and eluting the column with hexane / ethyl acetate / methanol. [Aspect 3] The organic eluate is given by the following formula:

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Claims

1. A composition comprising an anisomeles indica extract for the prevention or treatment of a fibrous condition, wherein the fibrous condition is selected from renal fibrosis, vascular fibrosis, and pulmonary fibrosis.

2. Anisomeles indica extract is given by the following formula: 【Chemistry 1】 The composition according to claim 1, comprising at least one compound selected from the group consisting of compounds represented by and combinations thereof.

3. The following formulas are used to prevent or treat fibrous conditions: 【Chemistry 2】 A composition comprising an effective amount of a compound selected from the group consisting of the compounds shown and combinations thereof, A composition in which the fibrotic condition is selected from renal fibrosis, vascular fibrosis, and pulmonary fibrosis.

4. The composition according to claim 1 or 3, wherein the composition further reduces renal insufficiency and renal damage.

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