Uses of terpenoids in the treatment or prevention of fibrotic diseases

Abstract

This invention relates to a method for preventing or treating fibrotic conditions, comprising administering an effective amount of a composition to a subject in need; wherein the composition comprises a triterpenoid compound extracted from Antrodia camphorata or Gynostemma pentaphyllum.

Description

Technical Field

[0001] This invention relates to plant-derived terpenoids derived from extracts of Antrodia camphorata and Anisomeles indica, particularly a medicinal and dietary formulation that can alleviate fibrosis. Background Technology

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

[0003] Kidney repair following acute kidney injury can induce fibrosis, potentially leading to chronic kidney disease. Initially, kidney injury activates multipotent progenitor cells to repair tissue; however, as the injury persists and renal fibrosis develops, these cells become dysfunctional and induce fibrotic repair. The pathogenesis of renal fibrosis is a progressive process, ultimately leading to end-stage renal failure, a serious condition requiring dialysis or kidney transplantation.

[0004] Non-alcoholic fatty liver disease (NAFLD) is a major form of chronic liver disease with urgent medical need. Non-alcoholic steatohepatitis (NASH) is a progressive variant of NAFLD that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. NAFLD and NASH have become a topic of widespread concern in the medical community, especially due to the increasing prevalence of diabetes and obesity in the world's population. Clinical evaluation of every patient with abnormal aminotransferase levels should consider non-alcoholic fatty liver disease and its spectrum, especially in obese or diabetic patients. The prognosis of isolated NAFLD is usually benign, but if fibrosis, hepatocellular ballooning, inflammation, and Mallory bodies are present, there is a risk of progression to cirrhosis.

[0005] Autoimmune hepatitis (AIH) is a chronic liver disease with no clear cause, characterized by inflammation of hepatocellular cells. Severe AIH can progress to cirrhosis, hepatocellular carcinoma, and even death. Depending on the duration of observation, up to 40% of AIH patients develop cirrhosis. Therefore, in addition to current anti-inflammatory and immunosuppressive therapies, emerging anti-fibrotic therapies can be added, which promise to redefine the treatment goals of AIH as preventing, stabilizing, and reversing liver fibrosis.

[0006] Atherosclerosis is a major cause of cardiovascular disease and is associated with vascular fibrosis. Vascular fibrosis involves the accumulation of extracellular matrix (ECM) proteins, especially collagen and fibronectin in the vascular mediator, and promotes structural remodeling and scar formation. A lack of elastin or an excess of collagen in the blood vessel wall leads to vascular fibrosis and increased stiffness.

[0007] In benign prostatic hyperplasia (BPH), the deposition of collagen fibers in the prostate gland is intended to replace broken muscle fibers, but this can lead to stiffness and weakness of the muscle tissue, causing prostatic fluid to accumulate in the glandular ducts. Prostatic fibrosis is a major factor in bladder outlet obstruction in older men.

[0008] Antrodia camphorata, a medicinal fungus, is a well-known traditional Chinese medicine known for its diverse biological activities, particularly its anti-tumor effects in in vitro cancer cells and in vivo animal models. Given its diverse range of bioactive compounds, it is considered an effective alternative herbal therapy or an adjuvant for cancer treatment and immune-related diseases. To date, 225 compounds have been isolated, identified, and elucidated, including macromolecules (nucleic acids, proteins, and polysaccharides), small molecules (benzenoids, lignans, benzoquinones, and maleic acid / succinic acid derivatives), terpenes (lanostane triterpenes, ergostane triterpenes, diterpenes, monoterpenes, and steroids), nucleotides (nucleobases and nucleosides), fatty acids, and fatty acid esters.

[0009] Cumulative in vitro and in vivo experiments have shown that it has anti-diabetic, anti-hyperlipidemic, anti-hypertensive, anti-inflammatory, antioxidant, antibacterial, cardiovascular disease prevention, immunomodulatory, hepatoprotective, and neuroprotective effects. However, the efficacy of Antrodia camphorata and its components in treating fibrosis has not yet been evaluated.

[0010] Fish needle grass (Anisomeles indica), known as "Indian catmint," is a source of medicinally active compounds with a variety of pharmacological effects. This plant has traditionally been used as an analgesic, anti-inflammatory agent, and for treating skin problems. Medically proven pharmacological activities include antioxidant, antibacterial, anti-HIV, anti-Helicobacter pylori, and anticancer activities. Further research has revealed the presence of various phytochemicals, primarily triterpenes, β-sitosterol, stigmasterol, flavones, apigenin, and ovatodiolides. Attached Figure Description

[0011] Figure 1 Antcin K, dehydrosulphurenic acid / sulphurenic acid, versisponic acid D, and dehydroeburicoic acid were isolated from Antrodia camphorata extract.

[0012] 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 cisplatin daily for 7 days starting 3 weeks after the first dose, and sacrificed at week 4. (A) Morphological changes in the kidneys; (B) Blood urea nitrogen (BUN) levels; (C) Serum creatinine (CRE) levels. Data are expressed as mean ± SEM (n = 5). ### indicates p < 0.001 compared to the control group. ** indicates p < 0.01 compared to the cisplatin group, and *** indicates p < 0.001.

[0013] Figure 3Protective effect 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 cisplatin daily for 7 days starting 3 weeks after the first dose and sacrificed at 4 weeks. Kidneys were stained with H&E. After cisplatin induction, kidneys from each group were prepared for histological evaluation. Representative histological sections of the kidneys were stained with H&E and magnified 400 times. Data are presented as mean ± SEM (n = 5). ### indicates p < 0.001 compared with the control group. ** indicates p < 0.01 compared with the cisplatin group, and *** indicates p < 0.001. Tubular cell necrosis is marked with arrows; line scale bar is 50 μm.

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

[0015] Figure 5A Effects of RH005-EA (A) and ARH (B) on cisplatin-induced renal TWEAK, α-SMA, P53, and P21 protein expression. Following cisplatin stimulation, the expression levels of TWEAK, α-SMA, P53, and P21 proteins in renal homogenates were analyzed using the Western ink dot method.

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

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

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

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

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

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

[0022] Figure 12 illustrates the effects of scutellarin (AR100-DS1) on GOT, GPT, and body weight. (A) Serum GOT and (B) Serum GPT 24 hours after Con A induction. (C) Body weight before and after Con A induction. Data are presented as mean ± SEM (n = 9).

[0023] Figure 13 Effects of strychnine lactone (AR100-DS1) on liver injury. (A) (B) Liver histopathology of 15mg / kg Con A (Veh), (C) AR100-DS1 and (D) dexamethasone, and (E) histopathological score of necrosis.

[0024] Figure 14 The process of establishing a rabbit model of atherosclerosis is described.

[0025] Figure 15 The initial and final average weights of the rabbits are depicted. * indicates that P < 0.05 compared with the control group and HF group, respectively.

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

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

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

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

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

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

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

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

[0034] Figure 24 Histopathological and chemical examination of aortic atherosclerotic plaque lesions in a rabbit model of hypercholesterolemia after a 12-week study was described.

[0035] Figure 25 H&E staining of coronary artery sections after each group of rabbits was depicted.

[0036] Figure 26 H&E staining of coronary artery sections after rabbit sacrifice was depicted in each group. N, neointima layer; M, media layer.

[0037] Figure 27 The manifestations of vascular stenosis were described, expressed as the ratio of the area of ​​the neointima layer to the medial layer (N / M ratio), where N is the neointima layer and M is the medial layer. Compared with the HFD group, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0038] Figure 28 Histopathological and chemical examination of cardiac tissue in a rabbit model with hypercholesterolemia after 12 weeks of study was described.

[0039] Figure 29 The appearance of the liver in a rabbit model with hypercholesterolemia was depicted after a 12-week study.

[0040] Figure 30 The histochemical examination of liver tissue in a rabbit model with hypercholesterolemia after 12 weeks of study was described.

[0041] Figure 31 The animal's body weight and lung weight are described.

[0042] Figure 32 The histopathological changes in the lungs during bleomycin-induced pulmonary fibrosis in mice were described.

[0043] Figure 33 Masson's trichrome staining of the lungs in bleomycin-induced pulmonary fibrosis in mice was depicted.

[0044] Figure 34 The effects of Antrodia camphorata extract and its compounds on hydroxyproline content in bleomycin-induced lung injury in mice were described.

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

[0046] Figure 36 The regulation of MPO activity in mouse lungs by Antrodia camphorata extract and its compounds was described. Detailed Implementation

[0047] To facilitate the explanation of this invention, the central ideas expressed in the above-described inventive summary are illustrated through specific examples. The various items in the embodiments are depicted according to scale, dimensions, deformations, or displacements suitable for illustration, rather than being drawn to the scale of the actual elements described above.

[0048] The term "terpenes" refers to a large and diverse class of organic compounds whose basic structures follow a general principle: they begin with 2-methylbutane residues (often also with isoprene units or C5). n The carbon skeleton of terpenes is composed of 2-methylbutane (C5, C6). Currently, approximately 30,000 terpenes are known in the literature. Based on the number of 2-methylbutane residues, they can be classified as hemi- (C5, C6) and mono- (C5, C6) terpenes. 10 ), sesqui-(C 15 ), double (di-,C 20 ), sester-(C 25 ), three (tri-,C 30 ) and four (tetra-,C 40 Terpenoids.

[0049]

[0050] The terms “subject,” “individual,” “host,” and “patient” are used interchangeably in this specification to refer to a living animal, including humans and non-human animals. For example, a subject can be an organism possessing immune cells capable of responding to antigen stimulation and transducing stimulatory and inhibitory signals through binding to cell surface receptors. A subject can be a mammal, such as a human or non-human mammal, including dogs, cats, pigs, cattle, sheep, goats, horses, rats, and mice. The term “subject” does not exclude individuals who are completely disease-free or normal in all respects.

[0051] The term "treatment" can be applied to a subject who has a medical condition or who may eventually develop it, in order to prevent, cure, delay, reduce the severity of one or more symptoms of the condition or recurrence of the condition, or prolong the subject's survival.

[0052] The term "medically effective amount" refers to the amount of a subject compound that can elicit a desired response, including biological or medical responses in tissues, systems, animals, or humans sought by researchers, veterinarians, physicians, or other clinicians.

[0053] Biochemical parameters were determined according to the manufacturer's instructions, using colorimetric kits to assess serum creatinine and serum urea. The biomarker kit for creatinine was purchased from HUMAN Diagnostics Worldwide, Magdeburg, Germany, and the chemical analyzer was from Roche Diagnostics, Cobas Mira Plus, Rotkreuz, Switzerland.

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

[0055] Serum cytokines TNF-α, IL-6, and IL-1β: Serum concentrations of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) were assessed using an enzyme-linked immunosorbent assay (ELISA) kit (Biosource International Inc., Sunnyvale, CA, USA) according to the manufacturer's instructions.

[0056] The lysis buffer for kidney tissue analysis using the Western ink dot method consisted of 0.6% NP-40, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, and 0.5 mM PMSF. Liver tissue was homogenized at 4°C. The homogenized sample was centrifuged at 3000 rpm for 10 minutes at 4°C to obtain the supernatant. The total cellular protein content in the supernatant was determined by bovine serum albumin (BSA). Protein samples (50 μg) were analyzed using standard methods by denaturing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Immobilon, Millipore, Bedford, MA, USA) with 10% skim milk as a barrier. The PVDF membrane was reacted with an appropriately diluted primary antibody at 4°C, washed three times with TBST buffer, and then reacted with a horseradish peroxidase-conjugated secondary antibody at 37°C for 1 hour. The PVDF membrane was washed three times, and immunoreactive proteins were detected using ECL reagent (Thermo Scientific, Hudson, NH, USA). The results were compared with a control group using ImageJ software (NIH, Bethesda, MD, USA), and the band brightness on the film was quantified and expressed as relative intensity.

[0057] Statistical analysis of data obtained from animal experiments is expressed as the mean and the standard error of the mean (±SEM). The t-test is used to examine differences between multiple groups or between two groups. Statistical significance is expressed as *p<0.05, **p<0.01, and ***p<0.001.

[0058] Example 1: Preparation of Antrodia camphorata extract

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

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

[0061] The methanol extract of Antrodia camphorata was further separated by silicone column chromatography using n-hexane / ethyl acetate / methanol as the chromatographic solvent, yielding the following fractions (e.g.) Figure 1 As shown):

[0062] AR101-DS1(RS-Antcin K)

[0063] AR101-DS2 (Dehydrothiochrome polyporic acid / Thiochrome polyporic acid)

[0064] AR101-DS3 (Versisponic acid D)

[0065] AR101-DS4 (Dehydrodentinoic acid)

[0066]

[0067] Example 3: Preparation of ARH003 extract

[0068] 100g of Antrodia camphorata (pectomy) was refluxed with methanol for 6 hours, the extract was collected and dried, and a total of 15g of Antrodia camphorata ARH003 extract was obtained.

[0069] Example 4: Preparation of ARH003-E extract

[0070] 200g of Antrodia camphorata (pectomy) was refluxed with ethanol for 6 hours, the extract was collected and dried, and a total of 18g of Antrodia camphorata ARH003-E extract was obtained.

[0071] Example 5: Preparation of ARH004 Extract

[0072] 100g of Antrodia camphorata (wood-grown) was refluxed with methanol for 6 hours, the extract was collected and dried, and a total of 18g of Antrodia camphorata ARH004 extract was obtained.

[0073] Example 6: Preparation of ARH005-EA Extract

[0074] 100g of Antrodia camphorata (solid culture) was refluxed with ethyl acetate for 6 hours, the extract was collected and dried, and a total of 12g of Antrodia camphorata EA extract was obtained.

[0075] Example 7: Preparation of Fish Needle Grass Extract

[0076] The preparation process of the fish needle grass extract is as follows: (1) Take the fish needle grass ethanol extract and add it to a silicone-packed column. Then, perform gradient extraction with the extraction solution "n-hexane / ethyl acetate", "n-hexane / ethyl acetate / methanol" and "methanol" to obtain a fish needle grass separation solution; (2) Further process the above fish needle grass separation solution with a silicone-packed column. Then, perform gradient extraction with the extraction solution "dichloromethane", "dichloromethane / methanol" and "methanol" to obtain a separated concentrate; (3) Co-crystallize the separated concentrate with "n-hexane / ethyl acetate" solvent to obtain fish needle grass microcrystals.

[0077] Example 8: Preparation of the active ingredient: ovatodiolide (AR100-DS1)

[0078] 200 g of ethanol extract of *Needlea natans* was added to a silicone-packed column (10 x 15 cm) and subjected to gradient extraction with the following eluents: hexane / ethyl acetate (ratios of 10:1, 5:1, 3:1, 1:1), hexane / ethyl acetate / methanol (ratios of 6:4:1, 3:2:1), and methanol, to obtain 140 g of initial separation solution.

[0079] 140 g of the initial separated liquid was added to a silicone-packed column (10 x 15 cm) for further separation. Gradient extraction was performed using dichloromethane, dichloromethane / methanol (ratios of 10:1, 5:1, and 7:3), and methanol to obtain a concentrated extract. This concentrated extract was further recrystallized in n-hexane / ethyl acetate to obtain a crystal. The crystal was identified by 1H NMR spectroscopy as a diterpenoid compound with the chemical structure of sedge lactone. High-performance liquid chromatography (HPLC) analysis, comparing the crystal with sedge lactone standards, confirmed its identity as sedge lactone.

[0080]

[0081] Metabolites of strychnine lactone (AR100-DS1):

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

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

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

[0085]

[0086]

[0087]

[0088]

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

[0090] Male C57BL / 6 mice, aged seven to eight weeks, were obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan, China). Prior to the experiment, the animals were housed in acrylic cages at a temperature of 22±1℃ and a relative humidity of 55±5%, under a 12-hour dark-light cycle for at least two weeks, with free access to food and water. All experimental procedures were conducted in accordance with the guidelines of the relevant authorities and were approved by them.

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

[0092] Example 10: Antrodia camphorata extract and compounds reduced cisplatin-induced renal dysfunction and histopathological changes in mice.

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

[0094] Example 11: Antrodia camphorata extract and compounds alleviate renal dysfunction and kidney damage induced by repeated cisplatin treatment.

[0095] Histopathological changes were analyzed to determine whether Antrodia camphorata extract and its compounds affected cisplatin-induced renal failure in mice. The control group showed completely normal kidney tissue with hyaline tubular and glomerular structures and clear, normal cell nuclei. In cisplatin-stimulated mice, severe kidney damage was observed, leading to tubular epithelial damage, inflammatory cell infiltration, tubular cell swelling, intratubular cast formation, and tubular dilation. However, administration of 1000 mg / kg of Antrodia camphorata extract (AR005-EA) and its compound (AR100-DS1) significantly improved necrosis and inflammatory cell infiltration in the kidney tissue (see [link to study]). Figure 3 ).

[0096] Example 12: Antrodia camphorata extract and compounds reduce cisplatin-induced changes in pro-inflammatory cytokines and albumin.

[0097] Serum levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β were assessed using ELISA. Compared to the control group, serum levels of NO, TNF-α, IL-1β, and IL-6 were significantly increased in cisplatin-treated mice with kidney injury (respectively, respectively). Figures 4A-4E Treatment with Antrodia camphorata extract (AR005-EA) and compound (AR100-DS1) at a dose of 1000 mg / kg significantly improved necrotic and inflammatory infiltrating cells in renal tissue and also reduced the production of NO, TNF-α, IL-1β and IL-6 after cisplatin stimulation.

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

[0099] This study examined the effects of pretreatment with Antrodia camphorata extract (ARH005-EA) and compound (AR100-DS1) on the inhibition of cisplatin-induced TWEAK, α-SMA, P53, and P21 protein expression. The results showed that pretreatment with ARH005-EA and ARH inhibited the expression of TWEAK, α-SMA, P53, and P21 proteins in kidney tissue after cisplatin stimulation. Figure 5A and 5B ).

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

[0101] like Figure 6 As shown, eight-week-old male SD rats were administered carbon tetrachloride at a dose of 0.4 mg / kg weekly for eight weeks. Blood samples were collected at weeks 0, 2, 4, 6, and 8, and the animals were sacrificed at the end of week 8 for histopathological examination. Figure 7A , 7B 7C depicted weight changes, liver weight, and liver / body weight ratio, respectively. (Control group) Liver weight was not significantly different from the vehicle group; however, the liver / body weight ratio in the control group was significantly smaller than that in the vehicle group. The liver weight and liver / body weight ratio in the 50 mg / kg AR100-DS1 group were significantly greater than those in the vehicle and control groups.

[0102] Example 15 Serum Liver Enzyme Analysis

[0103] Assess clinical biochemical levels, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT), to determine the enzyme activity in the livers of the control and experimental groups (e.g., Figures 8A-8C(As shown). AST, ALT, and the AST / ALT ratio in the blank control group showed no significant changes during the experiment. Serum AST and ALT levels in all experimental groups increased significantly as the experiment progressed; however, compared to the vector group, less increase in AST and ALT was observed in the 50 mg / kg AR100-DS1 group at weeks 6 and 8.

[0104] Example 16 Liver Histological Assessment

[0105] After 8 weeks of carbon tetrachloride induction, the vector group showed significant liver damage, including elevated AST and ALT levels, decreased AST / ALT ratio, inflammation, fibrosis, vacuolation, and necrosis. Figures 9A-9E As shown in Figure 10, the liver surface of the 50 mg / kg AR100-DS1 group was smooth, without atrophy or cirrhosis, and the liver weight and liver / body weight ratio were significantly greater than those of the carrier and blank groups. Overall, AR100-DS1 demonstrates the potential to partially repair carbon tetrachloride-induced liver injury.

[0106] Example 17 Effect of scutellarin (AR100-DS1) on Con A protein-induced acute hepatitis in BALB / c mice

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

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

[0109] Male BALB / c mice (7-9 weeks old) were purchased from BioLASCO Taiwan Co., Ltd. or related institutions (Taiwan, China). Throughout the experiment, five animals were housed per cage, with free access to food and water. The room temperature was maintained at 23±2℃, with alternating 12-hour light-dark cycles. Animals were allowed one week of acclimatization before the experiment to minimize the effects of stress. All experimental protocols involving animals and their care were approved by the relevant departments of ITRI (ITRI-IACUC-2018-041 and ITRI-IACUC-2018-050; accredited by AAALAC) and conducted in accordance with the relevant departmental regulations.

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

[0111] To assess the extent of hepatocellular damage following Con A administration, serum GPT and GOT levels were measured using the Fuji Dri-Chem Slide kit. Serum samples from the same group were pooled for cytokine assay. Cytokine levels were measured using the ProcartaPlex™ immunoassay kit according to the manufacturer's instructions. Data are presented as mean ± SEM. A t-test was used to analyze differences between the drug treatment group and the carrier group. A p-value less than 0.05 was considered statistically significant. 50 mg / kg of scutellarin (AR100-DS1) significantly reduced the increase in GPT levels induced by Con A (109 ± 25 vs. 368 ± 107 U / L, p < 0.05) and slightly improved the increase in GOT (261 ± 45 vs. 410 ± 56 U / L) (Figure 12).

[0112] 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 veterinary pathologists at BioLASCO Taiwan Co., Ltd. The severity of all microscopic lesions was graded using a standardized scale from 0 to 4 as follows: 0 = none; 1 = single-cell necrosis; 2 = ≤30% lobular necrosis; 3 = ≤60% lobular necrosis; 4 => 60% lobular necrosis. Histopathological analysis showed that strychnine lactone (AR100-DS1) improved liver necrosis (score 0.2±0.2 vs 1.4±0.2, p<0.05). Figure 13The above results indicate that strychnine lactone (AR100-DS1) can reduce serum GOP and GPT and alleviate Con A-induced liver necrosis.

[0113] Example 18: Evaluation of the efficacy of Antrodia camphorata extract and AR101-DS2 in preventing atherosclerosis and liver fibrosis.

[0114] Experimental model

[0115] Male New Zealand White rabbits weighing 2-3 kg were kept individually in cages in a room with controlled temperature and humidity, with a 12-hour light-dark cycle. After several days of acclimatization, the animals were sequentially assigned to six feeding groups: standard rabbit pellets, standard rabbit pellets containing 0.5% cholesterol, standard rabbit pellets containing 0.5% cholesterol and 10 mg / kg lovastatin, standard rabbit pellets containing 0.5% cholesterol and 1% ARH003, standard rabbit pellets containing 0.5% cholesterol and 1% ARH004, and standard rabbit pellets containing 0.5% cholesterol and 10 mg / kg AR101-DS2. Except for the standard rabbit pellet group, the other groups were given standard rabbit pellets containing 0.5% cholesterol for 4 weeks (see [link to feeding instructions]). Figure 14-15 Each rabbit was fed 50 grams of food per kilogram of body weight per day. After the animals acclimatized to their new environment, they were fed this diet for 8 weeks. At the beginning and end of the 12-week study, rabbits were anesthetized by intramuscular injection of Zoltil 50 (1 mL / kg) (Virbac Ltd., France), and blood samples were collected. Finally, after sacrificing the rabbits, the aorta (from the aortic arch to the bifurcation of the iliac artery) and the entire liver were collected for further histopathological analysis.

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

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

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

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

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

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

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

[0123] Each rabbit should be fed 50 grams of food per kilogram of body weight per day.

[0124] Blood chemistry analysis

[0125] 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 3000 rpm for 10 minutes at 4°C. Figures 16-23 depict the measurements of changes in blood chemical parameters, including serum levels of low-density lipoprotein (LDL), cholesterol (Chol), triglycerides (TG), glutamate-oxaloacetate transaminase (GOT), and glutamate-pyruvate transaminase (GPT).

[0126] Aortic Fatty Streak Staining

[0127] The aorta was opened longitudinally to expose the intima and gently flushed with saline (see below). Figure 24-26 The aorta was cultured in 2% (w / v) Sudan IV, rinsed for 1 minute with several concentrations of ethanol (100%, 90%, 80%, 70%, and 60%), and then rinsed with pure water. Figure 28 The images shown were taken using a digital camera (Nikon D80, Japan) and quantified using Alpha Imager 2200 (Alpha Innotech, USA). Progression of sclerotic plaque lesions is expressed as a percentage of the total stained area. Figure 27 ).

[0128] method

[0129] 1. Hydrated cells or tissues:

[0130] i. Fix the tissue with alcohol or aldehyde fixative using a microscope slide with frozen or rehydrated tissue sections (see step 12 in Cutting Sections of Paraffin-Embedded Tissue) (Fischer et al., 2008).

[0131] ii. Immerse the slide in H2O for 30 seconds while stirring by hand. Rinsing in H2O is important; hematoxylin precipitates along with the salt and buffer solution. Staining can be performed using a non-fluorescent detection system after immunohistochemistry or hybridization.

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

[0133] 3. Rinse the slide in H2O for 1 minute. Estimate the staining intensity at this point, and repeat steps 2 and 3 if necessary.

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

[0135] 5. Dehydrate the sections by applying 95% alcohol twice and 100% alcohol twice, for 30 seconds each time.

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

[0137] 7. Add one or two drops of sealing agent and cover with a coverslip. If alcohol cannot be used, use glycerin or other water-based sealing agents to seal the coverslip.

[0138] reagents

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

[0140] Eosin Y (1% aqueous solution; EM diagnosis is systematic)

[0141] Ethanol (95%, 100%)

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

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

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

[0145] If alcohol cannot be used, please use glycerin or other water-based sealant (see step 7).

[0146] Xylene

[0147] Liver tissue frozen sections

[0148] Rabbit liver tissue (e.g.) Figure 29(As shown) The tissue was perfused with physiological saline and fixed in 10% (v / v) formalin neutralization solution (JTBaker, Inc., USA) for 24 hours, followed by embedding in Tissue Tek OCT Compound (#4583; Sakura Finetek Inc., USA). The embedded tissue was cut into 10 μm thick sections and stained with Sudan IV and hematoxylin (Merck, USA). Briefly, the sections were washed with pure water for 1 minute to remove the OCT compound, washed with 50% (v / v) ethanol for 30 seconds, and then stained with 2% (w / v) Sudan IV for 1 hour. After further washing with 50% (v / v) ethanol and pure water for 2 minutes, the sections were counterstained with hematoxylin. Figure 30 The images shown were obtained using a microscope equipped with a 10x magnification objective and quantified using Alpha Imager 2200 (Alpha Innotech, USA). The progression of fatty liver is expressed as the percentage of oil droplet area to total liver tissue (cells).

[0149]

[0150] Fatty liver score

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

[0152] 1:5-33%

[0153] 2:33-66%

[0154] 3: >66%

[0155] Location

[0156] 0: Region 3, leaflet center

[0157] Zone 1:2, Central Zone

[0158] Zones 2 and 3, around the mouth

[0159] 3: panacinar

[0160] Fibrosis score

[0161] 0: None

[0162] 1: Mild perisinusitis or periportal vein

[0163] 2: Perisinus and portal vein / periportal area

[0164] 3: Bridging Fiberization

[0165] 4: Cirrhosis

[0166] Inflammation score

[0167] 0: No lesions

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

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

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

[0171] Example 19: Protective effect of Antrodia camphorata extract and compounds against bleomycin-induced pulmonary fibrosis in mice.

[0172] Animals and treatment

[0173] Male ICR mice (weighing 18-22g) free of specific pathogens were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan, China). Prior to the experiment, the animals were housed in resin glass cages at a constant temperature of 22±1℃ and relative humidity of 55±5%, with a 12-hour dark-light cycle for at least two weeks. The animals were provided with free access to food and water. All experimental procedures were conducted in accordance with the relevant department's guidelines, and this protocol was approved by the relevant department for the control and supervision of animal experiments.

[0174] Bleomycin (BLM)-induced pulmonary fibrosis in mice

[0175] Mice were divided into the following groups according to body weight, with 5 mice in each group: control group, BLM group, BLM+DEX group (7.5 mg / kg), BLM+ACH dose group (ARH003 ext. 1.0 g / kg), BLM+ACM dose group (ARH003 ext. 0.5 g / kg), BLM+AH dose group (AR101-DS1 50 mg / kg), BLM+AM dose group (AR101-DS1 25 mg / kg), BLM+BH dose group (AR101-DS2 50 mg / kg), BLM+BM dose group (AR101-DS2 25 mg / kg), BLM+CH dose group (AR101-DS4 50 mg / kg), BLM+CM dose group (AR101-DS4 25 mg / kg), BLM+DH dose group (AR100-DS1 50 mg / kg), and BLM+DM dose group (AR100-DS1 50 mg / kg). The mice were divided into two groups: a 25 mg / kg BLM+EH dose group (ARH013-RA1 50 mg / kg) and a BLM+EM dose group (ARH013-RA1 25 mg / kg). Pulmonary fibrosis (PF) was established in mice by a single intratracheal administration of BLM at a dose of 7.5 mg / kg body weight. Different doses of BLM were administered daily by gavage for 21 days post-BLM injury, with DEX serving as a positive control. The control and experimental groups received the same volume of carrier (0.9% NaCl) via the same schedule and route of administration.

[0176] Mice body weight was recorded daily. On day 21, mice were sacrificed using an overdose of chloral hydrate anesthetic, and blood was collected for 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 to determine hydroxyproline. Lung specific gravity was calculated using the following formula: Lung weight / Body weight × 100%.

[0177] Experimental Design

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

[0179] (n=6):

[0180] 1. Group 1: Control group;

[0181] 2. Group 2: Mice received a single intraperitoneal injection of BLM (7.5 mg / kg).

[0182] 3. Group 3: Single dose (ACH, ARH003 ext. 1.0 g / kg)

[0183] 4. Group 4: Single dose (ACM, ARH003 ext. 0.5g / kg)

[0184] 5. Group 5: Purified AR101-DS1 (50 mg / kg)

[0185] 6. Group Six: Purified AR101-DS1 (25 mg / kg)

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

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

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

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

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

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

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

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

[0194] BALF sampling

[0195] Under anesthesia, four BALF (Basal Acid Fluid) treatments were performed via endotracheal intubation with 0.7 mL of physiological saline. Approximately 2.5 mL (90%) of BAL (Basal Acid Fluid) was recovered from each mouse examined. The supernatant of the BALF was stored at -80°C for later use.

[0196] Lung histopathology

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

[0198] Determination of hydroxyproline

[0199] The hydroxyproline content in lung tissue was analyzed according to the instructions of the hydroxyproline assay kit (Biosource International Inc., Sunnyvale, CA, USA). Mouse lung tissue was ground, 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. The appropriate reagents were added to the reaction mixture according to the instructions and thoroughly mixed, then incubated at 60°C for 15 minutes. After cooling, the supernatant was collected after centrifugation at 3500 rpm for 10 minutes. The absorbance of the sample supernatant was measured at 550 nm using a spectrophotometer, and the hydroxyproline content of each group was calculated.

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

[0201] Serum concentrations of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) were assessed using an enzyme-linked immunosorbent assay (ELISA) kit (Biosource International Inc., Sunnyvale, CA, USA) in accordance with the manufacturer’s instructions.

[0202] Myeloperoxidase (MPO) detection

[0203] MPO activity in the lungs is a reliable indicator for assessing inflammatory cell infiltration in the lungs. Lung tissue was homogenized, and MPO levels were measured using a kit according to the manufacturer's instructions.

[0204] Lung histopathological analysis

[0205] The right lung was embedded in paraffin, fixed with 10% formalin, and prepared into sections. The sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome stain.

[0206] Statistical analysis

[0207] Data obtained from animal experiments are expressed as mean and standard error of mean (±SEM). The t-test is used to examine differences between multiple groups or between two groups. Statistical significance is expressed as *p<0.05, **p<0.01, and ***p<0.001.

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

[0209] Effects of Antrodia cinnamomea extracts and compounds on lung index in bleomycin-induced pulmonary fibrosis

[0210]

[0211] Example 20: Antrodia camphorata extract and compounds reduced BLM-induced pulmonary dysfunction and histopathological changes in mice.

[0212] To assess histopathological lung changes in mice and explore the therapeutic effects of Antrodia camphorata extract and compounds. Inflammatory infiltration and integrity of tissue structures were observed using H&E staining. Figure 32 The degree of fibrosis in lung tissue was determined by Masson staining. Figure 33 The control group exhibited several histological changes, such as thin alveolar walls, intact alveolar structure, normal alveolar septa, and less inflammatory cell infiltration in the pulmonary mesenchyme. After 21 days of BLM administration, alveolar edema, significantly increased septal width, and increased inflammatory cell infiltration were observed. Compared to the BLM group, administration of Antrodia camphorata extract and compounds improved inflammatory infiltration and damaged structures in the lung tissue.

[0213] Twenty-one days after BLM administration, Masson staining showed extensive blue staining in the lung tissue and septum, indicating that the degree of pulmonary fibrosis in the BLM group was more severe than in the normal group. Treatment with Antrodia camphorata extract and compounds reduced the blue area and alleviated the degree of fibrosis. Twenty-one days after BLM induction, the scores for alveolitis and fibrosis were significantly reduced after treatment with Antrodia camphorata extract and compounds. These results indicate that Antrodia camphorata extract and compounds alleviated the degree of inflammation and fibrosis in the lungs of mice with pulmonary fibrosis.

[0214] Example 21 Pulmonary fibrosis markers

[0215] Hydroxyproline (HP) 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 displayed. Figure 34 In the control group, BLM significantly increased HP content (p<0.001). Antrodia camphorata extract (1.0 g / kg) and AH, BH, and DH significantly reduced the recovery of HP in the lungs (p<0.001).

[0216] Example 22: Antrodia camphorata extract and compounds remind us of changes in bleomycin-induced pro-inflammatory cytokines.

[0217] Serum levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and TGF-β were assessed using ELISA. Compared with the control group, serum levels of NO, TNF-α, IL-1β, and IL-6 were significantly increased in BLM-treated mice with kidney injury (respectively, respectively). Figures 35A-35DTreatment with 1.0 g / kg of Antrodia camphorata extract and compounds (BH and DH) significantly improved necrotic and inflammatory infiltrating cells in lung tissue and improved the production of TNF-α, IL-1β, IL-6 and TGF-β after BLM induction (p<0.001).

[0218] Example 23 Effects of Antrodia camphorata extract and compounds on lung MPO activity

[0219] like Figure 36 As shown, compared with the control group, the MPO level induced by BLM was significantly increased (p<0.01). Conversely, the AH, BH, DH, and Dex extracts of Antrodia camphorata significantly inhibited MPO activity compared with the BLM group (p<0.001), and their effects were stronger than those of the Antrodia camphorata extract and compound groups (p<0.05). Figure 36 ).

Claims

1. The use of a strychnine lactone having the following formula in the preparation of a medicament for the prevention or treatment of pulmonary fibrosis:

Images