6-methoxybenzoxazolinone for prevention and / or treatment of liver fibrosis and use of coix lacryma-jobi var. ma-yuen extract containing 6-methoxybenzoxazolinone

Abstract

To provide a novel composition for the prevention and / or treatment of liver fibrosis.SOLUTION: The present invention provides 6-methoxybenzoxazolinone for the prevention and / or treatment of liver fibrosis and the use of Coix lacryma-jobi var. ma-yuen extract containing 6-methoxybenzoxazolinone. According to the results of multiple efficacy experiments, the 6-methoxybenzoxazolinone and the Coix lacryma-jobi var. ma-yuen extract containing 6-methoxybenzoxazolinone exhibit effects in preventing and / or treating liver fibrosis.SELECTED DRAWING: Figure 13J

Description

[Technical field]

[0001] The present invention relates to the use of 6-methoxybenzoxazolinone (6-MBOA) and an extract of Job's tears (Coix lachryma-jobi L.) containing 6-methoxybenzoxazolinone for preventing and / or treating liver fibrosis. [Background technology]

[0002] The liver is an important organ of the human body and is composed of parenchymal and non-parenchymal cells. Parenchymal cells, also called hepatocytes, account for 60% of the total liver cells and 80% of the liver volume. Non-parenchymal cells are composed of sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells (HSCs), and other biologically important cells such as hepatic natural killer cells. Each of the above cells accounts for 3-20% of the liver volume.

[0003] Liver fibrosis is the result of excessive repair caused by viral infection, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), autoimmune liver disease (AILD), and cholestatic liver disease. As excess extracellular matrix (ECM) accumulates, the structure of the liver changes, further damaging liver function. Activation of hepatic stellate cells appears to be a serious condition, and when the liver is damaged, activated hepatic stellate cells become the main source of extracellular matrix production.

[0004] In current clinical practice, the effectiveness of drug treatment for liver fibrosis is limited and serious side effects occur, so many patients cannot continue to receive treatment. More importantly, drugs only alleviate symptoms and cannot fundamentally solve the problem. Therefore, the development of new drugs that can effectively treat and / or prevent liver fibrosis is the problem that the present invention aims to solve. Summary of the Invention [Problem to be solved by the invention]

[0005] To solve the above problems, those skilled in the art focus on developing novel compositions for preventing and / or treating liver fibrosis so as to help the many people in need. [Means for solving the problem]

[0006] In view of this, an object of the present invention is to provide a use of 6-methoxybenzoxazolinone (6-MBOA) for the manufacture of a composition for preventing and / or treating liver fibrosis.

[0007] In one embodiment of the present invention, the 6-MBOA reduces inflammatory infiltration, liver structure deformation, collagen deposition, α-SMA expression levels, hepatic hydroxyproline content, upregulation of serum transforming growth factor beta 1 (TGF-β1), liver weight, liver function parameters, TGF-β1-induced reactive oxygen species (ROS) generation, and TGF-β1-induced fibronectin expression levels in individuals who are preventing and / or treating liver fibrosis.

[0008] In one embodiment of the present invention, the 6-MBOA down-regulates the expression levels of phosphorylated SMAD2 (p-SMAD2) and phosphorylated SMAD3 (p-SMAD3) in an individual for preventing and / or treating liver fibrosis.

[0009] In one embodiment of the present invention, the 6-MBOA enhances the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) content in an individual for preventing and / or treating liver fibrosis, and also reduces the expression level of NADPH oxidase 4 (NOX4), and reduces the content of malondialdehyde (MDA) caused by oxidative stress.

[0010] In one embodiment of the present invention, 6-MBOA inhibits hepatic stellate cell (HSC) activation and epithelial-mesenchymal transition (EMT), including suppressing fibronectin expression and maintaining E-cadherin expression levels.

[0011] In one embodiment of the present invention, the negative regulation is achieved by 6-MBOA decreasing the expression levels of zinc finger proteins SNAIL1 / 2, TWIST1 / 2, and zinc finger E-box-binding homeobox 1 (ZEB1).

[0012] Another object of the present invention provides use of an extract of Job's Tears (Coix lachryma-jobi L.) containing 6-methoxybenzoxazolinone (6-MBOA) for the manufacture of a composition for preventing and / or treating liver fibrosis.

[0013] In one embodiment of the present invention, the Job's Tears extract is obtained by extracting Job's Tears with a solvent, and the solvent is water, alcohol, an alcohol-water mixture, or a combination thereof.

[0014] In one embodiment of the present invention, the Job's Tears extract down-regulates serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum total bilirubin (TBIL), serum total glyceride (TG), and serum total cholesterol (TC) levels in an individual for preventing and / or treating liver fibrosis, and up-regulates serum glucose levels in the individual for preventing and / or treating liver fibrosis.

[0015] In one embodiment of the present invention, the Job's Tears extract reduces the levels of hepatic α-smooth muscle actin (α-SMA) gene, Collagen type I alpha 1 (COL1A1) gene, tumor necrosis factor-α (TNF-α) gene, and interleukin-6 (IL-6) gene in an individual for preventing and / or treating liver fibrosis.

[0016] In one embodiment of the present invention, the Job's Tears is Job's Tears root, leaf or seed.

[0017] In one embodiment of the present invention, the composition is a pharmaceutical composition, a food composition, or a topical composition.

[0018] In one embodiment of the invention, the pharmaceutical composition is in a dosage form for oral administration.

[0019] In one embodiment of the invention, the pharmaceutical composition is in a dosage form for parenteral administration.

[0020] In one embodiment of the present invention, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient, vector, adjuvant, and / or food additive.

[0021] In one embodiment of the present invention, the composition is in the form of a powder, granules, solution, gel, or paste. Effect of the Invention

[0022] In summary, the effect of the Job's Tears extract containing 6-methoxybenzoxazolinone and 6-methoxybenzoxazolinone according to the present invention improves liver fibrosis and reduces fibrotic and inflammatory factors, and 6-MBOA improves oxidative stress in the liver and reduces the production of biomarkers of liver fibrosis by regulating the TGF-β / SMAD signaling pathway, and inhibits the progression of hepatic stellate cells to mesenchymal transition, indicating the potential of the Job's Tears extract containing 6-methoxybenzoxazolinone and 6-methoxybenzoxazolinone to treat liver fibrosis.

[0023] The following provides a further description of the embodiments of the present invention. The examples listed below are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Since a person skilled in the art may make some changes and modifications without departing from the spirit and scope of the present invention, the scope of protection of the present invention is deemed to be defined by the scope of the patent application attached later. [Brief description of the drawings]

[0024] [Figure 1]Schematic diagram of animal experimental design for Job's tears group. In this experiment, C57BL / 6J mice were intraperitoneally injected with thioacetamide (TAA) to induce liver fibrosis. Six-week-old mice were randomly divided into seven groups and fed different diets (NT, TAA, 10% ASP, 1% ALP, 1% ASPS, 0.025% ASE, and 0.1% ASE, n=6). After co-rearing for 6 weeks, TAA was injected intraperitoneally three times a week, and the mice were euthanized at 12 weeks of age. NT indicates no treatment, ASP indicates Job's tears powder, ALP indicates Job's tears leaf powder, ASPS indicates Job's tears polysaccharide, and ASE indicates ethanol extract of Job's tears seeds. [Fig. 2A-2F] The effect of Job's tears extract on the pathological and physiological values ​​of liver tissue in TAA-induced liver fibrosis C57BL / 6J mice is shown. Figure 2A shows the results of observing the pathological changes of liver tissue by hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining (original magnification ×200, scale bar 100 μm). Figure 2B shows the results of quantification of IHC staining of α-smooth muscle actin (α-SMA) by Image J software. Figure 2C shows the changes in liver hydroxyproline content. Figures 2D and 2E show the changes in liver weight and body weight. Figure 2F shows the ratio of liver weight change divided by body weight, i.e., liver-body weight ratio. Data are shown as mean ± standard deviation (SD) (n=6 in each group). Regarding the English letters on the bar graphs, the same letters indicate that no significant difference was observed between the two groups (p>0.05), and different letters indicate that a significant difference was observed between the two groups (p<0.05). [Figure 3A-3F]The effect of Job's tears extract on liver function damage and nutrition metabolism in TAA-induced liver fibrosis C57BL / 6J mice is shown. Figures 3A to 3C show serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL), which are indicators of liver function damage. Figures 3D to 3F show serum levels of total glyceride (TG), total cholesterol (TC), and glucose to observe nutrition metabolism. Data are shown as mean ± standard deviation (SD) (n=6 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p>0.05), and different English letters indicate that there was a significant difference between the two groups (p<0.05). [Figure 4A-4E] The effect of Job's tears extract on fibrosis and inflammatory gene levels in TAA-induced hepatic fibrosis C57BL / 6J mice is shown. Figures 4A and 4B show the mRNA levels of fibrosis markers α-smooth muscle actin (α-SMA) and collagen type I, alpha 1 (COL1A1). Figures 4C to 4E show the mRNA levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-10. Data are shown as mean ± standard deviation (SD) (n=6 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p>0.05), and different English letters indicate that there was a significant difference between the two groups (p<0.05). [Figure 5A-5H]Figure 5 shows the effect of Job's tears extract on HSC-T6 cell viability, fibrotic protein expression, and inflammatory gene levels. Figure 5A is a schematic diagram showing drug treatment of HSC-T6 cells. Figure 5B shows cell viability of HSC-T6 cells treated with different concentrations of Job's tears leaf, root, and seed extracts. Figures 5C-5E show the results of Western blotting of α-SMA and COL1A1 in HSC-T6 cells treated with TGF-β1 and Job's tears extract, and the results of quantification by Image J software. Figures 5F-5H show the mRNA levels of inflammatory factors in HSC-T6 cells treated with TGF-β1 and Job's tears extract. Data are shown as mean ± standard deviation (SD) (n=3 in each group). * in the bar graph of cell viability indicates that the concentration is 0 compared to other concentrations, ** indicates p<0.01, and *** indicates p<0.001. For the English letters in the bar graphs of Western blotting and real-time PCR, the same English letter indicates that there was no significant difference between the two groups (p>0.05), and different English letters indicate that there was a significant difference between the two groups (p<0.05). In Figures 5C and 5F to 5H, L indicates leaf, in Figures 5D and 5F to 5H, R indicates root, and in Figures 5E to 5H, S indicates seed. [Figure 6] A schematic diagram showing the animal experimental design of the 6-methoxybenzoxazolinone (6-MBOA) (CX) group. In the animal experiment, 6-week-old C57BL / 6J mice were randomly divided into NT group, TAA group, CX20 group (TAA+20mg / kg CX), and CX50 group (TAA+50mg / kg CX) (n=8 in each group). TAA was injected intraperitoneally into the mice three times a week to induce liver fibrosis. CX or vehicle was also administered once a day via a feeding tube. After the animal experiment was completed, the mice were euthanized at 12 weeks of age. [Figures 7A-7K]The effect of CX on the pathological and physiological values ​​of liver tissue in TAA-induced liver fibrosis C57BL / 6J mice is shown. Figure 7 shows the chemical structure of CX (C8H7NO3, MW=165.15). Figure 7B shows the results of observing the pathological changes of liver tissue with H&E, picrosirius red and IHC staining (original magnification ×200, scale bar 100 μm). Figure 7C shows the results of quantifying collagen picrosirius red staining. Figures 7D-7F show the results of quantifying IHC staining of α-SMA, phosphorylated SMAD homolog 2 (phosphor-Mothers against decapentaplegic homolog 2, p-SMAD2), and phosphorylated SMAD homolog 3 (phosphor-Mothers against decapentaplegic homolog 3, p-SMAD3). Figure 7G shows the change in the content of hepatic hydroxyproline. Figure 7H shows the expression of TGF-β1 in serum. Figures 7I and 7J show liver and body weight changes. Figure 7K shows the ratio of liver weight change divided by body weight, i.e., liver-body weight ratio. Image J was used to quantify picrosirius red and IHC staining. Data are shown as mean ± standard deviation (SD) (n=8 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p>0.05), and different English letters indicate that there was a significant difference between the two groups (p<0.05). [Figure 8A-8D] The effect of CX on antioxidant enzymes and lipid peroxidation in TAA-induced hepatic fibrosis C57BL / 6J mice is shown. Figures 8A and 8B show the changes in superoxide dismutase (SOD) and catalase (CAT) activities. Figure 8C shows the changes in glutathione (GSH) content. Figure 8D shows the changes in malondialdehyde (MDA) content. Data are shown as mean ± standard deviation (SD) (n = 8 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p > 0.05), and different English letters indicate that there was a significant difference between the two groups (p < 0.05). [Figures 9A-9G] The effect of CX on liver function damage and nutritional metabolism in TAA-induced liver fibrosis C57BL / 6J mice is shown. Figures 9A to 9D show serum levels of ALT, AST, TBIL, and ALP, which are indicators of liver function damage. Figures 9E to 9G show serum levels of TG, TC, and glucose to observe nutritional metabolism. Data are shown as mean ± standard deviation (SD) (n = 8 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p > 0.05), and different English letters indicate that there was a significant difference between the two groups (p < 0.05). ALP indicates powder of Job's tears leaves. [Figure 10A-10E] Figure 10 shows the effect of CX on fibrosis and inflammatory gene levels in TAA-induced liver fibrosis C57BL / 6J mice. Figures 10A and 10B show hepatic mRNA levels of α-SMA and COL1A1 detected by real-time PCR. Figures 10C to 10E show hepatic mRNA levels of inflammatory factors TNF-α, IL-6, and IL-10. Data are shown as mean ± standard deviation (SD) (n=8 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p>0.05), and different English letters indicate that there was a significant difference between the two groups (p<0.05). [Figures 11A-11F]The effect of CX on cell viability, inflammatory gene levels, and reactive oxygen species (ROS) of TGF-β1-activated HSC-T6 is shown. FIG. 11A is a schematic diagram of HSC-T6 cells treated with TGF-β1 and CX. FIG. 11B shows cell viability of HSC-T6 cells treated with different concentrations of CX, and 6-MBOA stands for 6-methoxybenzoxazolinone. FIG. 11C-FIG. 11E show mRNA levels of inflammatory factors in HSC-T6 cells treated with TGF-β1 and CX. FIG. 11F shows the results of detecting ROS levels of HSC-T6 by DCFDA and normalizing with protein content. Data are shown as mean ± standard deviation (SD) (n=3 in each group). * in the bar graph of cell viability indicates that the concentration is 0 compared to other concentrations, and *** indicates p<0.001. Regarding the English letters in the bar graphs of Western blotting and real-time PCR, the same letters indicate that no significant difference was observed between the two groups (p>0.05), and different letters indicate that a significant difference was observed between the two groups (p<0.05). [Figures 12A-12G]Figure 12 shows the effect of CX on HSC activation and epithelial-mesenchymal transition (EMT) in TGF-β1-activated HSC-T6 cells. Figure 12A shows the results of Western blotting analysis of E-cadherin, fibronectin, α-SMA and COL1A1. Figures 12B-12E show the results of quantifying Western blotting of E-cadherin, fibronectin, α-SMA and COL1A1 by Image J software. Figures 12F and 12G show the results of quantifying wound healing test results (magnification ×200, scale bar 100 μm) at 0 h, 24 h and 48 h by Image J software. Wound healing was defined as (wound area at 0 h-wound area at 24 h or 48 h) / wound area at 0 h×100%. Data are shown as mean ± standard deviation (SD) (n = 3 in each group). The same alphabetical letter in the bar graph indicates that there was no significant difference between the two groups (p > 0.05), and different alphabetical letters indicate that there was a significant difference between the two groups (p < 0.05). [Figures 13A-13J]Figure 13 shows the effect of CX on the TGF-β / SMAD signaling pathway in TGF-β1-activated HSC-T6 cells. Figure 13A shows a schematic diagram of the stimulation of the TGF-β1 pathway in HSC-T6 cells and the results of CX treatment. Figure 13B shows the results of Western blotting of phosphorylated SMAD homolog 2 (p-SMAD2), phosphorylated SMAD homolog 3 (p-SMAD3), SMAD2 / 3, zinc finger proteins SNAIL1 / 2, TWIST1 / 2, zinc finger E-box-binding homeobox 1 (ZEB1), and NADPH oxidase 4 (NOX4) normalized to α-tubulin. Figure 13C-13I show the results of Western blotting of p-SMAD2, p-SMAD3, SMAD2 / 3, SNAIL1 / 2, TWIST1 / 2, ZEB1 and NOX4 quantified by Image J software. Data are shown as mean ± standard deviation (SD) (n=3 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p>0.05), and different English letters indicate that there was a significant difference between the two groups (p<0.05). Figure 13J illustrates the process of the present invention on liver fibrosis message transfer, gene transcription, translation, and protein expression, and the process by which CX completely suppresses liver fibrosis from TGF-β1 message transfer. [Figures 14A-14C]Figure 14 shows the effect of CX on the signaling pathways of phosphorylated phosphoinositide 3-kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (ERK) in TGF-β1-activated HSC-T6 cells. Figure 14A shows the results of Western blotting in which p-PI3K and p-ERK1 / 2 were normalized with α-tubulin. Figures 14B and 14C show the results of quantifying p-PI3K and p-ERK1 / 2 by Western blotting using Image J. Data are shown as mean ± standard deviation (SD) (n = 3 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p > 0.05), and different English letters indicate that there was a significant difference between the two groups (p < 0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] definition The numerical values ​​given herein are approximate values, all experimental data are shown to be within a range of ±20%, preferably within a range of ±10%, more preferably within a range of ±5%.

[0026] In this specification (particularly in the claims), unless specifically stated otherwise, the terms "a," "an," "the," and similar terms include both the singular and the plural.

[0027] The Job's tears (Coix lachryma-jobi L., Adlay) (e.g., Coix lachryma-jobi L. var. ma-yuen Stapf) according to the present invention is also called Job's tears, Job's tears, or Job's tears, and is an annual or perennial herbaceous plant of the Poaceae family, a common herbal medicine and dietary supplement in Asia, used to improve inflammatory diseases, and has a height of about 1 to 1.8 m and a cultivation period of about 5 months. Job's tears is composed of four parts from the outside to the inside, namely, bran, husk, seed coat, and endosperm (white Job's tears), and each of the four parts has its own biological activity.

[0028] The 6-methoxybenzoxazolinone (6-MBOA) (CX) (chemical formula C8H7NO3) according to the present invention is a compound found in grasses and has anti-hepatic fibrosis and hormone regulation activities.

[0029] The pharmaceutical composition according to the present invention may be prepared into a dosage form for parenteral or oral administration by techniques well known to those skilled in the art, including, but not limited to, an injection (e.g., a sterile aqueous solution or dispersion), a sterile powder, a tablet, a troche, a lozenge, a pill, a capsule, a dispersible powder or granule, a solution, a suspension, an emulsion, a syrup, an elixir, a slurry, and the like.

[0030] The pharmaceutical composition of the present invention may be administered by one parenteral route selected from the group consisting of intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, intralesional injection, sublingual administration, and transdermal administration.

[0031] The pharmaceutical composition of the present invention may contain one of the vectors widely used in pharmaceutical manufacturing technology and pharma- ceutical acceptable. The pharma-ceutical acceptable vector may contain one or more agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption delaying agents, liposomes, and the like. The choice and quantity of these agents are within the expertise and routine skill of the person skilled in the art.

[0032] The pharma- ceutically acceptable vector of the present invention may comprise a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), sugar solution, aqueous solution containing alcohol, and combinations thereof.

[0033] The chemicals and reagents used in the following examples of the present invention are described below: 6-methoxybenzoxazolinone (6-MBOA) (CX) (purity ≧96.5%), thioacetamide (TAA) (purity ≧99.0%), and 2',7'-dichlorofluorescin diacetate (DCFDA, purity ≧97.0%) are purchased from Sigma-Aldrich (USA).

[0034] Recombinant human transforming growth factor beta 1 (TGF-β1) is purchased from PeproTech (PeproTech, USA). TBARS and catalase (CAT) detection kits are purchased from Cayman Chemical (USA). Reduced glutathione (GSH) and superoxide dismutase (SOD) detection kits are purchased from Abbkine (Abbkine Scientific, China).

[0035] Hydroxyproline detection kit is purchased from Elabscience (Elabscience Biotechnology, China). Mouse TGF-β1 ELISA kit is purchased from Invitrogen (Thermo Fisher Scientific, USA).

[0036] α-smooth muscle actin (α-SMA) (GTX100458), collagen, type I, alpha 1, COL1A1 (GTX112731), fibronectin (GTX112794), E-cadherin (GTX629691), NADPH oxidase 4 (NOX4) (GTX121929), TWIST1 / 2 (GTX127310), zinc finger E-box-binding homeobox 1 (Zinc finger E-box-binding homeobox 1) (GTX127310), and β-glucose-binding ... 1, ZEB1 (GTX105278), β-actin (GTX109639), α-Tubulin (GTX628802), phosphorylation of SMAD homolog 2 (phosphor-Mothers against decapentaplegic homolog 2, p-SMAD2) (GTX133614), phosphorylation of SMAD homolog 3 (phosphor-Mothers against decapentaplegic homolog 3, p-SMAD3) (GTX129841), phosphorylation of extracellular signal-regulated kinase 1 / 2 (ERK1 / 2) (GTX635617), phosphorylation of phosphoinositide 3-kinase (phosphoinositide Antibodies against PI3K (GTX132597), SMAD2 / 3 (GTX111123), and horseradish peroxidase (HRP) were purchased from GeneTex (USA).

[0037] The zinc finger protein SNAIL1 / 2 (ab180714) was purchased from Abcam (UK).

[0038] The animals used in the following examples of the present invention are described below. Male C57BL / 6J mice (5 weeks old) are obtained from the National Laboratory Animal Center (Taipei, Taiwan). Mice are kept in a temperature-controlled room with a 12-hour light / dark cycle and are allowed to access food and water ad libitum (National Yang Ming Chiao Tung University Animal Center, Taipei, Taiwan). All experiments are approved by the National Yang Ming Chiao Tung University IACUC.

[0039] For the Job's tears extract test, mice were randomly divided into the following seven groups (n=6 per group): (1) No treatment group (NT, normal diet), (2) Thioacetamide group (TAA, 200mg / kg, normal diet), (3) 10% Job's tears powder (ASP) group, (4) 1% Job's tears leaf powder (ALP) group, (5) 1% Job's tears polysaccharide (ASPS) group, (6) 0.025% Job's tears seed ethanol extract (ASE) group, and (7) 0.1% ASE group. The NT group was given pure saline, and the other groups were given intraperitoneal injections of saline containing TAA dissolved therein three times a week.

[0040] For the CX study, mice were randomly divided into four groups (n=8 per group): (1) NT group, (2) TAA group (200mg / kg), (3) CX group (CX20, 20mg / kg), and (4) CX group (CX50, 50mg / kg). The NT group was given pure saline, and the other groups were given intraperitoneal injections of saline containing TAA three times a week. In addition, the NT and TAA groups were given 5% propylene glycol once a day via a feeding tube, and the CX group was given 5% propylene glycol (prepared in 0.9% saline).

[0041] At the end of the experiment, the mice are euthanized. Serum is obtained by centrifugation of mouse whole blood and stored at -80°C, and liver tissue is collected for histological staining.

[0042] The statistical analysis procedure according to the present invention is described below. Statistical analysis was performed using one-way ANOVA, followed by Tukey's multiple comparison test using GraphPad Prism 7. Cell viability data was analyzed using two-way ANOVA, followed by Dunnett's multiple comparison test. All data are shown as mean ± standard deviation (SD). Differences of P<0.05 were considered to be statistically significant.

[0043] The present invention will be further described below by the following examples. These examples are provided for illustrative purposes only and do not limit the protection scope of the present invention. The protection scope of the present invention is as set forth in the scope of the patent application.

[0044] Example 1. Preparation of pearl barley (Coix lachryma-jobi L.) extract For cell experiments, Job's tears leaves (L), roots (R) and seeds (S) are used. Dry and ground Job's tears are extracted with water, alcohol, alcohol-water mixture or a combination thereof (preferably 70% ethanol), concentrated and dried under reduced pressure. Meanwhile, for animal experiments, Job's tears powder (ASP, containing 10% ASP in normal diet), Job's tears leaf powder (ALP, containing 1% ALP in normal diet), Job's tears polysaccharide (ASPS, containing 1% ASPS in normal diet), and Job's tears seed ethanol extract (ASE, containing 0.025% and 0.1% ASE in normal diet) are used. All Job's tears extracts are provided by Dr. Yu-Hsin Chen (Taiwan Agricultural Improvement Farm).

[0045] The flow of the schematic diagram of the animal experiment design of the Job's tears group is described below. In order to verify the effect of Job's tears on liver fibrosis, in this example, a thioacetamide (TAA)-induced liver fibrosis C57BL / 6J mouse model is used. Figure 1 is a schematic diagram of the animal experiment design of the Job's tears group. In this experiment, TAA is injected intraperitoneally into C57BL / 6J mice to induce liver fibrosis. Six-week-old mice are randomly divided into seven groups and given different diets (NT, TAA, 10% ASP, 1% ALP, 1% ASPS, 0.025% ASE, and 0.1% ASE, n=6).

[0046] After 6 weeks of co-rearing, TAA was injected intraperitoneally three times a week, and finally, at 12 weeks of age, the mice were euthanized. NT indicates no treatment, ASP indicates Job's tears powder, ALP indicates Job's tears leaf powder, ASPS indicates Job's tears polysaccharide, and ASE indicates ethanol extract of Job's tears seeds. Six-week-old mice were randomly divided into seven groups: NT, TAA, 10% ASP, 1% ALP, 1% ASPS, 0.025% ASE, and 0.1% ASE (see FIG. 1). The NT and TAA groups were fed a normal diet, and the Job's tears group was fed a normal diet with the addition of Job's tears extract. The mice were intraperitoneally injected with TAA or saline three times a week for six weeks. After six weeks, the mice were euthanized, and liver tissues and serum were collected for later analysis.

[0047] Example 2. Effects of Job's Tears extract on the pathological and physiological values ​​of liver tissue in TAA-induced liver fibrosis mice After 6 weeks of exposure, pathological changes in liver tissue sections stained with TAA-induced hematoxylin and eosin (H&E) and immunohistochemistry (IHC) will be observed.

[0048] The flow of pathological observation of liver tissue is described below. Liver tissue is fixed with 10% neutral buffered formalin solution, embedded in paraffin, and cut into 4 μm thick sections. H&E staining kit (CIS-Biotechnology, Taiwan) is used to examine the pathological sections and evaluate the changes in tissue structure. Picrosirius red staining kit (CIS-Biotechnology, Taiwan) is used to evaluate the status of collagen deposition. Immunohistochemistry kit (Agilent, USA), phosphorylation of SMAD2 (1:100, GeneTex, USA), phosphorylation of SMAD3 (1:100, GeneTex, USA), and α-smooth muscle actin (α-SMA) (1:100, GeneTex, USA) antibodies are used to evaluate the expression of specific proteins in the tissue. All staining is observed using an optical microscope (BEL Photonics, Italy).

[0049] The procedure for measuring hydroxyproline is described below. Hydroxyproline is an indirect indicator of collagen content in the liver and is measured by using a hydroxyproline detection kit according to the manufacturer's guidelines. That is, liver tissue samples are hydrolyzed with HCl at 95°C for 6 hours, the pH value is adjusted to 7, impurities are adsorbed with carbon powder, and centrifuged, after which the supernatant is collected and used for analysis. Hydroxyproline content at a wavelength of 558 nm is measured using an ELISA reader (TECAN, Switzerland).

[0050] Figures 2A to 2F show the effects of Job's tears extract on the pathological and physiological values ​​of liver tissue in TAA-induced liver fibrosis C57BL / 6J mice. Figure 2A shows the results of observing the pathological changes of liver tissue by H&E and IHC staining (original magnification: ×200, scale bar: 100 μm). Figure 2B shows the results of quantifying IHC staining of α-smooth muscle actin (α-SMA) by Image J software. Figure 2C shows the changes in liver hydroxyproline content. Figures 2D and 2E show the changes in liver weight and body weight. Figure 2F shows the ratio of liver weight change divided by body weight, i.e., liver-body weight ratio.

[0051] Data are shown as mean ± standard deviation (SD) (n = 6 in each group). The same alphabetical letter in the bar graph indicates that there was no significant difference between the two groups (p > 0.05), and different alphabetical letters indicate that there was a significant difference between the two groups (p < 0.05).

[0052] As can be seen from Figures 2A and 2B, compared with the NT group, the TAA group showed inflammatory infiltration, liver structure deformation, and overexpression of α-SMA, while the Job's tears group showed a significant reduction in inflammatory infiltration, improved liver deformation, and reduced expression of α-SMA. Hydroxyproline (a collagen component) was measured to detect changes in collagen content in the liver. As can be seen from Figure 2C, the hydroxyproline content of the TAA group was higher than that of the NT group, indicating an increase in collagen. Meanwhile, the hydroxyproline content of all Job's tears groups was significantly reduced, indicating that Job's tears can reduce TAA-induced liver collagen deposition.

[0053] As can be seen from Figures 2D and 2E, compared with the NT group, the TAA group lost weight and increased liver tissue. In the Job's tears group, TAA-induced hepatomegaly was significantly reduced in the 10% ASP, 1% ALP, 1% ASPS, and 0.1% ASE groups, but no significant difference was observed in body weight. In Figure 2F, the changes in liver weight and body weight were corrected by the liver-to-body weight ratio to avoid individual differences. Compared with the TAA group, only the 0.025% ASE group showed no obvious changes, and the liver function values ​​of the other groups were significantly reduced.

[0054] Example 3. Effects of Job's Tears extract on the biochemical characteristics of the liver in mice with TAA-induced liver fibrosis The flow of biochemical analysis is explained below. A biochemical analyzer is used to measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), total cholesterol (TC), total glyceride (TG), and glucose levels. That is, after centrifuging whole blood from mice, serum is collected and dropped onto a test chip, and analyzed by an automatic clinical chemistry analyzer (Fuji, Japan).

[0055] Figures 3A to 3F show the effects of Job's tears extract on liver function damage and nutritional metabolism in TAA-induced liver fibrosis C57BL / 6J mice. Figures 3A to 3C show serum ALT, AST, and TBIL levels, which are indicators of liver function damage. Figures 3D to 3F show serum TG, TC, and glucose levels to observe nutritional metabolism.

[0056] Data are shown as mean ± standard deviation (SD) (n = 6 in each group). The same alphabetical letter in the bar graph indicates that there was no significant difference between the two groups (p > 0.05), and different alphabetical letters indicate that there was a significant difference between the two groups (p < 0.05).

[0057] Serum AST, ALT, and TBIL are indicators of liver function damage, and serum TG, TC, and glucose are related to liver nutrition metabolism. As shown in Figures 3A to 3C, compared with the NT group, only the TAA group has improved AST, ALT, and TBIL levels. In comparison, the upregulation of indicators of liver function damage due to TAA downregulation in the Job's tears group is observed, especially in the 10% ASP, 1% ALP, 1% ASPS, and 0.1% ASE groups. Meanwhile, as shown in Figures 3D to 3F, compared with the NT group, the TG and TC levels of the TAA group are significantly improved and the glucose level is reduced. Treatment with Job's tears extract significantly improves the TAA-induced TG and TC levels of all Job's tears groups, and the glucose levels of the 10% ASP and 1% ASPS groups are maintained at the same level as the control group.

[0058] Example 4. Effect of Job's Tears extract on liver fibrosis and mRNA levels of inflammatory genes in TAA-induced liver fibrosis mice The flow of real-time PCR analysis is described below. Total RNA is extracted with Trizol reagent (Ambion, USA) and reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). PCR is amplified using SYBR Green PCR Master Mix (Applied Biosystems, USA), and gene mRNA expression is standardized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All primers are obtained from Genomics (Taiwan Genomics).

[0059] The primer sequences used in this example are shown in Table 1. The rat primer sequences were used for real-time PCR in HSC-T6 cells, and the mouse primer sequences were used for real-time PCR in C57BL / 6J mice.

[0060] [Table 1]

[0061] To confirm the changes in gene levels in mice with liver fibrosis, real-time PCR was used to observe the mRNA levels of fibrosis genes. Figures 4A to 4E show the effects of Job's tears extract on fibrosis and inflammatory gene levels in TAA-induced liver fibrosis C57BL / 6J mice. Figures 4A and 4B show the mRNA levels of fibrosis markers α-smooth muscle actin (α-SMA) and collagen, type I, alpha 1 (COL1A1). Figures 4C to 4E show the mRNA levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10.

[0062] Data are shown as mean ± standard deviation (SD) (n = 6 in each group). The same alphabetical letter in the bar graph indicates that there was no significant difference between the two groups (p > 0.05), and different alphabetical letters indicate that there was a significant difference between the two groups (p < 0.05).

[0063] As shown in Figures 4A and 4B, the levels of fibrosis markers α-SMA and COL1A1 are significantly increased in the TAA group compared with the NT group. Conversely, the levels of α-SMA and COL1A1 are significantly decreased in all of the Job's Tears groups. This result shows that Job's Tears extract has the effect of reducing the expression of fibrosis markers. Since inflammation is related to fibrosis, the mRNA levels of inflammatory genes are also detected. Tumor necrosis factor-α (TNF-α) and IL-6 are pro-inflammatory cytokines, and IL-10 is an anti-inflammatory cytokine.

[0064] As can be seen from the real-time PCR results shown in Figures 4C to 4E, TNF-α and IL-6 are up-regulated and IL-10 is down-regulated in the TAA group compared with the NT group. Although no significant difference was found between the 1% ALP, 0.025% ASE, and 0.1% ASE groups and the TAA group, all of the Job's tears extract groups reduced the TAA-induced TNF-α and IL-6 mRNA levels, and significantly up-regulated the IL-10 mRNA level.

[0065] Example 5. Effects of Job's Tears extract on HSC-T6 cell viability, fibrotic protein expression and inflammatory gene levels The cell culture procedure is described below. HSC-T6 cells are immortalized cell lines isolated from primary astrocytes transformed with SV40 large T antigen from male Sprague-Dawley rats. HSC-T6 cells are maintained in DMEM (Dulbecco's Modified Eagle Medium, Sigma-Aldrich, USA) containing 8% FBS (FBS, HyClone, USA), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, USA).

[0066] To activate hepatic stellate cells (HSCs), cells were cultured for 24 hours, then replaced with serum-free medium for 24 hours, and then treated with transforming growth factor beta 1 (TGF-β1) (10 ng / mL) for 24 hours in the presence or absence of CX (10-100 μM) or extracts of Job's tears leaves, roots, and seeds (25 and 50 μg / mL), or intracellular signaling pathways for 30 minutes.

[0067] The procedure for cell viability analysis is explained below. 4HSC-T6 cells were seeded in a 96-well plate and treated with CX (0-800 μM) or Job's tears extract (0-800 μg / mL) for 24 hours. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA) was used to measure cell viability. That is, MTT was added to each well to a final concentration of 0.5 μg / mL and incubated at 37°C for 1 hour. Then, MTT was removed and dimethyl sulfoxide (DMSO) was added to dissolve the MTT-formazan crystals formed from metabolic live cells. Finally, the absorbance of each well at a wavelength of 570 nm was detected using a spectrophotometer.

[0068] The flow of Western blotting analysis is explained below. Cells are dissolved in RIPA buffer containing protease inhibitors and centrifuged at 4°C and 12000 rpm for 10 minutes. The supernatant is collected, the total protein concentration is quantified to 50 μg, and after heating at 95°C for 10 minutes, the protein sample is separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane and blocked with 6% milk, reacted with the primary antibody overnight at 4°C, and reacted with the secondary antibody at room temperature for 1 hour. Images are displayed using ECL reagent (GeneDireX, USA) and Amersham Imager 680 (Cytiva, USA).

[0069] HSC activation is considered to be a major factor in liver fibrosis. In this example, HSC-T6 cells were used as a cell model, and TGF-β1 was added as an HSC activation inducer.

[0070] 5A-5H show the effects of Job's tears extract on HSC-T6 cell viability, fibrotic protein expression and inflammatory gene levels. FIG. 5A is a schematic diagram showing drug treatment of HSC-T6 cells. FIG. 5B shows cell viability of HSC-T6 cells treated with Job's tears leaf, root and seed extracts. FIG. 5C-5E show the results of Western blotting of α-SMA and COL1A1 in HSC-T6 cells treated with TGF-β1 and Job's tears extract, and the results quantified by Image J software.

[0071] Figures 5F to 5H show the mRNA levels of inflammatory factors in HSC-T6 treated with TGF-β1 and Job's tears extract. Data are shown as mean ± standard deviation (SD) (n=3 for each group). * in the bar graph of cell viability indicates that the concentration is 0 compared to other concentrations, ** indicates p<0.01, and *** indicates p<0.001.

[0072] For the English letters in the bar graphs of Western blotting and real-time PCR, the same English letter indicates that there was no significant difference between the two groups (p>0.05), and different English letters indicate that there was a significant difference between the two groups (p<0.05). In Figures 5C and 5F to 5H, L indicates leaf, in Figures 5D and 5F to 5H, R indicates root, and in Figures 5E to 5H, S indicates seed.

[0073] FIG. 5A shows the results of determining the amount of Job's tears extract (leaves, roots and seeds) suitable for cell experiments and examining the effect of Job's tears extract on cell viability. As can be seen from the cell viability shown in FIG. 5B, when the amount of Job's tears leaf and root extract is less than 100 μg / mL and the amount of Job's tears seed extract is less than 200 μg / mL, there is no significant effect on cell viability. Therefore, in the following experiments, 20 and 50 μg / mL of Job's tears extract are used. The expression of fibrosis markers in HSC-T6 cells treated with TGF-β1 is detected by Western blotting.

[0074] As a result, as shown in Figures 5C to 5E, all of the Job's Tears extracts dose-dependently reduce the expression of TGF-β1-induced α-SMA and COL1A. For inflammation, real-time PCR is used to evaluate the mRNA levels of TNF-α, IL-6, and IL-10. As a result, as shown in Figures 5F to 5H, all of the Job's Tears extracts dose-dependently down-regulate the mRNA levels of TGF-β1-induced TNF-α and IL-6, and up-regulate the mRNA level of IL-10. As shown by the changes in fibrotic proteins and inflammatory genes, Job's Tears extracts improve inflammation and HSC activation in HSC-T6 cells treated with TGF-β1.

[0075] Example 6. Schematic diagram of animal experimental design for 6-methoxybenzoxazolinone (6-MBOA) (CX) group To verify the effect of CX on liver fibrosis, this example utilizes a TAA-induced liver fibrosis C57BL / 6J mouse model. Figure 6 is a schematic diagram of the animal experiment design of the CX group. For the animal experiment, 6-week-old C57BL / 6J mice are randomly divided into NT group, TAA group, CX20 group (TAA+20mg / kg CX), and CX50 group (TAA+50mg / kg CX) (n=8 in each group). TAA is injected intraperitoneally into the mice three times a week to induce liver fibrosis. In addition, CX or vehicle is administered via a feeding tube once a day. After the animal experiment is completed, the mice are euthanized at 12 weeks of age.

[0076] In Figure 6, 6-week-old mice are randomly divided into four groups: NT, TAA, CX20, and CX50. The NT group receives vehicle via a feeding tube. The TAA group receives vehicle and TAA via a feeding tube. The CX group receives CX and TAA via a feeding tube. The mice are fed once a day for 6 weeks, and intraperitoneally injected three times a week. The mice are then euthanized, and liver tissue and serum are collected for further analysis.

[0077] Example 7. Effects of 6-methoxybenzoxazolinone (6-MBOA) (CX) on the pathological and physiological values ​​of liver tissue in TAA-induced liver fibrosis mice To observe the effect of CX on the pathology of liver tissue, mice with liver fibrosis were treated with CX, and the liver tissue was stained with H&E and picrosirius red, and then IHC staining was performed to detect fibrosis markers α-SMA, and TGF-β / SMAD pathway-related proteins p-SMAD2 and p-SMAD3 in liver sections.

[0078] The flow of the enzyme-linked immunosorbent assay (ELISA) for TGF-β1 is described below. A commercially available mouse ELISA kit (Invitrogen, USA) is used to detect TGF-β1 levels in serum. The procedure is performed according to sandwich ELISA. That is, serum is added to wells coated with TGF-β1 antibody, and reacted with biotin conjugate, streptavidin-HRP, substrate solution, and stop solution in order. Immediately after reacting with the stop solution, the color intensity at 450 nm is measured by an ELISA reader (TECAN, Switzerland).

[0079] Figure 7A-7K show the effects of CX on the pathological and physiological values ​​of liver tissue in TAA-induced liver fibrosis C57BL / 6J mice. Figure 7A shows the chemical structure of CX (C8H7NO3, MW=165.15). Figure 7B shows the results of observing the histopathological changes of the liver with H&E, picrosirius red and IHC staining (original magnification ×200, scale bar 100 μm). Figure 7C shows the results of quantifying collagen picrosirius red staining. Figure 7D-7F show the results of quantifying IHC staining of α-SMA, p-SMAD2 and p-SMAD3. Figure 7G shows the changes in the content of hepatic hydroxyproline.

[0080] Figure 7H shows the expression of TGF-β1 in serum. Figures 7I and 7J show liver and body weight changes. Figure 7K shows the ratio of liver weight change divided by body weight, i.e., liver-body weight ratio. Image J is used to quantify picrosirius red and IHC staining. Data are shown as mean ± standard deviation (SD) (n=8 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p>0.05), and different English letters indicate that there was a significant difference between the two groups (p<0.05).

[0081] As shown in Figures 7B-7F, compared with the NT group, the TAA group exhibited inflammatory infiltration, liver structure deformation, collagen deposition, and overexpression of α-SMA and p-SMAD2 / 3. The CX20 and CX50 groups showed significant reductions in inflammatory infiltration, liver structure deformation, collagen deposition, and expression of α-SMA. The CX50 group down-regulated the expression of p-SMAD2 and p-SMAD3. This indicates that CX ameliorates TAA-induced injury via the SMAD signaling pathway.

[0082] As shown in Figure 7G, the hydroxyproline content in the TAA group was higher than that in the NT group, and the hydroxyproline content in the CX group was lower than that in the TAA group, indicating that CX treatment could reduce the collagen content. TGF-β1 is an important cytokine related to fibrosis. The change in TGF-β1 is considered to be an indicator of fibrosis progression. As shown in Figure 7H, the ELISA results showed that compared with the NT group, the expression of TGF-β1 in the TAA group was significantly improved, and CX could significantly reduce TAA-induced TGF-β1 upregulation.

[0083] As can be seen from Figure 7I and Figure 7J, TAA injection alone causes liver hypertrophy and body weight loss, and CX treatment reduces liver weight, but there is no obvious improvement in body weight change caused by TAA. To avoid individual differences in mice, liver weight and body weight are adjusted using liver-to-body weight ratio. As shown in Figure 7K, compared with the NT group, the liver-to-body weight ratio of the TAA group is significantly improved, and compared with the TAA group, the liver-to-body weight ratio of the CX group is significantly decreased. This result suggests that CX reduces typical characteristics of fibrosis, such as overexpression of α-SMA, accumulation of collagen, and higher levels of TGF-β1, by affecting the expression of SMADs.

[0084] Example 8. Effects of CX on oxidative stress and biochemical characteristics of TAA-induced liver fibrosis mice Liver injury is caused by oxidative stress due to TAA active metabolites. Organisms can scavenge oxidative free radicals by antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). The lipid peroxidation product malondialdehyde (MDA), which is generated after oxidation of polyunsaturated fatty acids, is an indicator of oxidative stress in cells, tissues, organs, or organisms, and can be used to detect oxidative stress in TAA-induced fibrotic mice, with increased MDA content indicating increased oxidative pressure.

[0085] The flow of detection of indicators of oxidative stress is described below. Changes in the level of oxidative stress in TAA-induced liver fibrosis mice are detected by detecting the enzyme activity of catalase (CAT) and superoxide dismutase (SOD), the content of malondialdehyde (MDA), and the concentration of glutathione (GSH) in the tissue. Hydroxyproline is a component of collagen precursors. All the measurement kits are commercially available and are operated according to the manufacturer's guidelines. That is, the liver tissue is homogenized in ice-cold buffer, centrifuged at 4°C, and the supernatant is collected. Finally, the absorbance of the sample is measured using an ELISA reader (TECAN, Switzerland).

[0086] Figures 8A to 8D show the effects of CX on antioxidant enzymes and lipid peroxidation in TAA-induced hepatic fibrosis C57BL / 6J mice. Figures 8A and 8B show the changes in SOD and CAT activity. Figure 8C shows the changes in GSH content. Figure 8D shows the changes in MDA content. Data are shown as mean ± standard deviation (SD) (n = 8 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p > 0.05), and different English letters indicate that there was a significant difference between the two groups (p < 0.05).

[0087] As shown in Figures 8A to 8D, compared with the NT group, the SOD, CAT activity, and GSH content in the TAA group are decreased, but the MDA content is increased. Compared with the TAA group, the CAT activity and GSH content in the CX20 and CX50 groups are increased, but the MDA content is decreased. In addition, the SOD activity in the CX50 group is significantly increased, indicating that CX has the effect of improving oxidative stress.

[0088] Figures 9A to 9G show the effect of CX on liver function damage and nutritional metabolism in TAA-induced liver fibrosis C57BL / 6J mice. Figures 9A to 9D show serum levels of ALT, AST, TBIL, and ALP, which are indicators of liver function damage. Figures 9E to 9G show serum levels of TG, TC, and glucose to observe nutritional metabolism. Data are shown as mean ± standard deviation (SD) (n = 8 in each group). For the English letters in the bar graphs, the same English letter indicates that there was no significant difference between the two groups (p > 0.05), and different English letters indicate that there was a significant difference between the two groups (p < 0.05). ALP indicates powder of Job's tears leaves.

[0089] Regarding biochemical characteristics, as shown in Figures 9A-9D, the AST, ALT, TBIL, and ALP, which are indicators of liver function damage, in the TAA group were higher than those in the NT group, and significantly lower in the CX group than those in the TAA group. In addition, TAA also causes nutritional metabolic disorders. As can be seen from Figures 9E-9G, compared with the NT group, the TG and TC levels in the TAA group were significantly improved and the glucose level was reduced, and CX significantly reduced the changes in TG, TC, and glucose levels caused by TAA. As can be seen from these findings, CX can improve lipid metabolism ability, as shown in Figures 9E-9F, and improve liver function damage and abnormality caused by TAA, as shown in Figure 9G, and maintain normal glucose metabolism.

[0090] Example 9. Effects of CX on fibrosis and inflammatory gene levels in TAA-induced liver fibrosis mice To confirm the changes in fibrotic genes in mice, real-time PCR is used to detect the mRNA levels of fibrotic genes. Figures 10A-10E show the effects of CX on fibrosis and inflammatory gene levels in TAA-induced liver fibrosis C57BL / 6J mice. Figures 10A and 10B show the results of detecting hepatic mRNA levels of α-SMA and COL1A1 using real-time PCR. Figures 10C-10E show hepatic mRNA levels of inflammatory factors TNF-α, IL-6, and IL-10.

[0091] Data are shown as mean ± standard deviation (SD) (n = 8 in each group). The same alphabetical letter in the bar graph indicates that there was no significant difference between the two groups (p > 0.05), and different alphabetical letters indicate that there was a significant difference between the two groups (p < 0.05).

[0092] As shown in Figures 10A and 10B, compared with the NT group, the TAA group significantly upregulates the mRNA levels of α-SMA and COL1A1, and the CX group significantly reduces the gene translation of TAA-induced fibrotic markers. In addition, since inflammation is necessary for the progression of fibrosis, the inflammation situation can be grasped by detecting the mRNA levels of inflammatory genes TNF-α and IL-6 and anti-inflammatory gene IL-10. Compared with the NT group, the TAA group induces an increase in IL-6 and TNF-α and a decrease in IL-10. As can be seen from Figures 10C to 10E, CX treatment significantly reduces the upregulation of TAA-induced IL-6 and TNF-α levels and the downregulation of IL-10 mRNA levels. As can be seen from these results, CX reduces the expression of inflammatory gene mRNA in gene translation in TAA-induced fibrotic mice, suppressing the progression of subsequent cell fibrosis.

[0093] Example 10. Effects of CX on HSC-T6 cell viability, inflammatory gene levels, and reactive oxygen species (ROS) levels To verify the cytotoxicity of CX and to identify the optimal dose for subsequent cell experiments, each dose of CX is tested by MTT assay on HSC-T6 cells.

[0094] The procedure for ROS detection is described below. 2',7'-Dichlorofluorescein diacetate (DCFDA) is a cell-permeable fluorescent probe used to detect ROS and nitric oxide in live cells. When DCFDA is oxidized by ROS in cells, it is rapidly deesterified to form fluorescent 2',7'-dichlorofluorescein (DCF). For ROS detection, cells were seeded in 96-well plates for 24 h, changed to serum-free medium, and treated with drugs. After that, the medium was replaced with DCFDA solution for 30 min, and the excitation / emission spectrum at 485 / 525 nm was detected by a multimode microplate reader (TECAN, Switzerland).

[0095] Figures 11A-11F show the effects of CX on cell viability, inflammatory gene levels and ROS of TGF-β1-activated HSC-T6. Figure 11A shows a schematic diagram of TGF-β1 and CX treatment on HSC-T6 cells. Figure 11B shows cell viability of HSC-T6 cells treated with different concentrations of CX. Figures 11C-11E show mRNA levels of inflammatory factors in HSC-T6 cells treated with TGF-β1 and CX. Figure 11F shows the results of detecting ROS levels of HSC-T6 by DCFDA and normalizing with protein content.

[0096] Data are shown as mean ± standard deviation (SD) (n=3 per group). * in the cell viability bar graph indicates that the concentration is 0 compared to other concentrations, and *** indicates p<0.001. For the English letters in the bar graphs of Western blotting and real-time PCR, the same English letter indicates that there was no significant difference between the two groups (p>0.05), and different English letters indicate that there was a significant difference between the two groups (p<0.05).

[0097] As shown in Figure 11B, doses below 400 μM show no significant effect on cell viability, i.e., no toxicity, so 10, 20, 50 and 100 μM are used for subsequent cell measurements. Similarly, real-time PCR is used to detect the mRNA levels of TNF-α, IL-6 and IL-10 in HSC-T6 cells treated with TGF-β1. As shown in Figures 11C-11E, TGF-β1 treatment significantly increases TNF-α and IL-6, and decreases IL-10 mRNA levels.

[0098] CX dose-dependently reduces TGF-β1-induced TNF-α and IL-6, and at the highest dose significantly enhances IL-10 mRNA levels. DCFDA assay is used to detect ROS levels in HSC-T6 cells. As shown in Figure 11F, TGF-β1 induces ROS production, and CX reduces TGF-β1-induced ROS levels with increasing doses. These results suggest that TGF-β1 induces inflammation and ROS production, while CX may reduce inflammation and ROS in TGF-β1-activated HSC-T6 cells.

[0099] Example 11. Effect of CX on hepatic stellate cell (HSC) activation and epithelial-mesenchymal transition (EMT) in HSC-T6 cells To confirm the effect of CX on HSC activation and EMT in TGF-β1-activated HSC-T6 cells, Western blotting is used to detect the expression of fibrosis- and EMT-related proteins.

[0100] The procedure for measuring wound healing is as follows: HSC-T6 cells were seeded on a 6-well plate for 24 hours, the monolayer cells were detached with a 200 μL micropipette tip, and unattached cells were washed away with PBS. After 24 hours, the medium was replaced with serum-free medium, and the cells were treated with TGF-β1 and CX for 0, 24, and 48 hours. Images of cell migration were observed using an inverted microscope (Olympus, Japan), and the wound area was quantified using Image J software.

[0101] Figures 12A-G show the effect of CX on HSC activation and EMT in TGF-β1-activated HSC-T6 cells. Figure 12A shows the results of Western blotting for E-cadherin, fibronectin, α-SMA, and COL1A1. Figures 12B-E show the results of quantifying Western blotting for E-cadherin, fibronectin, α-SMA, and COL1A1 by Image J software. Figures 12F and 12G show the results of quantifying wound healing test results at 0, 24, and 48 hours (magnification: ×200, scale bar: 100 μm) by Image J software.

[0102] Wound healing was defined as (wound area at 0 hours - wound area at 24 hours or 48 hours) / wound area at 0 hours x 100%. Data are shown as mean ± standard deviation (SD) (n = 3 in each group). For the English letters in the bar graphs, the same English letters indicate that there was no significant difference between the two groups (p > 0.05), and different English letters indicate that there was a significant difference between the two groups (p < 0.05).

[0103] Markers of epithelial-mesenchymal transition or fibrosis include α-SMA, COL1A1, and fibronectin. Normal tissue architecture is replaced by cell damage and mesenchymal transition, which may ultimately lead to organ failure. As shown in Figures 12A-E, TGF-β1 treatment up-regulates the expression of α-SMA, COL1A1, and the mesenchymal marker fibronectin, and down-regulates the epithelial marker E-cadherin.

[0104] CX treatment reduces TGF-β1-induced α-SMA, COL1A1 and fibronectin expression, but significantly enhances E-cadherin expression even at the lowest dose. As EMT occurs, cell migration ability is enhanced. Therefore, we use wound healing assay to observe the changes in migration.

[0105] As shown in Figures 12F and 12G, the wound healing percentage of the TGF-β1 group is significantly improved compared with the NT group. CX treatment at a dose of 100 μM significantly reduces TGF-β1-induced wound healing at 24 hours, and CX treatment at doses of 20-100 μM reduces TGF-β1-induced wound healing at 48 hours. As can be seen from these results, CX can inhibit HSC activation and epithelial-mesenchymal transition (EMT) in HSC-T6 cells treated with TGF-β1.

[0106] Example 12. Effect of CX on TGF-β / SMAD homolog (Mothers against decapentaplegic homolog, SMAD) and stromal transition transcriptional signaling pathways in HSC-T6 cells Western blotting will be used to study the mechanism of CX on HSC activation and EMT.

[0107] Figures 13A-13I show the effect of CX on the TGF-β / SMAD signaling pathway in TGF-β1-activated HSC-T6 cells. Figure 13A is a schematic diagram showing the results of stimulation of the TGF-β1 pathway in HSC-T6 cells and CX treatment. Figure 13B shows the results of Western blotting of p-SMAD2, p-SMAD3, SMAD2 / 3, SNAIL1 / 2, TWIST1 / 2, ZEB1 and NOX4 normalized with α-tubulin. Figures 13C-13I show the results of Western blotting of p-SMAD2, p-SMAD3, SMAD2 / 3, SNAIL1 / 2, TWIST1 / 2, ZEB1 and NOX4 quantified by Image J software.

[0108] Data are shown as mean ± standard deviation (SD) (n = 3 in each group). The same alphabetical letter in the bar graph indicates that there was no significant difference between the two groups (p > 0.05), and different alphabetical letters indicate that there was a significant difference between the two groups (p < 0.05).

[0109] Generally speaking, as shown in Figures 12C-12D, when the liver is damaged, astrocytes secrete TGF-β1 hormone, which is phosphorylated by SMAD pathway-related proteins to become p-SMAD2 and p-SMAD3. As shown in Figures 13F-13H, it transmits signals to the cell nucleus and promotes the transcription of EMT-related proteins SNAIL1 / 2, TWIST1 / 2, and ZEB1 genes in the cell nucleus. At this time, as shown in Figure 12B, the expression of cadherin decreases, as shown in Figure 12C, the expression of fibronectin increases, as shown in Figures 12D-12E, collagen COL1A1 and tubulin α-SMA in the liver increase, and finally causes liver fibrosis as shown in Figure 7B. As shown in Figure 11F, TAA induces liver injury and simultaneously causes ROS and inflammatory responses. The main markers include TNF-α and IL-6.

[0110] As shown in Figure 11C-11D, these two inflammatory hormones increase, and as shown in Figure 11E, the anti-inflammatory hormone IL-10 decreases. At the same time as the inflammatory reaction occurs, the expression of the related protein NOX4 increases, as shown in Figure 13I, the expression levels of the antioxidant stress proteins SOD, CAT, and GSH decrease, as shown in Figure 8A-8C, and the oxidative stress metabolite MDA also increases, as shown in Figure 8D. The combination of the above reactions ultimately causes liver fibrosis, as shown in Figure 7B. As can be seen from Figure 13B-13I, compared with the NT group, the poor reactions of the above reactions in the TGF-β1 group are significantly improved, which indicates that TGF-β1 can phosphorylate SMAD2 and SMAD3, activate the downstream proteins SNAIL1 / 2, TWIST1 / 2, ZEB1, and NOX4, and further generate ROS to induce EMT.

[0111] In contrast to the above, when treated with CX, increasing the dose of CX reduces the phosphorylation of SMAD and the expression of downstream proteins by TGF-β1 stimulation. From this, CX suppresses the phosphorylation of SMAD2 and SMAD3 as shown in Figures 13B to 13D, reduces the gene transcription of SNAIL1 / 2, TWIST1 / 2, and ZEB1 as shown in Figures 13F to 13H, maintains the content of cadherin as shown in Figure 12B, reduces the content of fibronectin as shown in Figure 12C, inhibits epithelial-mesenchymal transition, and finally avoids liver fibrosis. At the same time, CX also reduces the inflammatory response, and as a result, the expression of inflammatory hormones TNF-α and IL-6 is reduced as shown in Figures 11C to 11D, the expression of anti-inflammatory hormone IL-10 is increased as shown in Figure 11E, the expression of NOX4 is reduced as shown in Figure 13I, and further prevents EMT and ROS generation.

[0112] Figure 14A-C show the effect of CX on the signaling pathways of phosphorylated phosphoinositide 3-kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (ERK) in TGF-β1-activated HSC-T6 cells. Figure 14A shows the results of Western blotting in which p-PI3K and p-ERK1 / 2 were normalized with α-tubulin. Figure 14B and Figure 14C show the results of quantifying p-PI3K and p-ERK1 / 2 by Image J Western blotting.

[0113] Data are shown as mean ± standard deviation (SD) (n = 3 in each group). The same alphabetical letter in the bar graph indicates that there was no significant difference between the two groups (p > 0.05), and different alphabetical letters indicate that there was a significant difference between the two groups (p < 0.05).

[0114] As shown in Figures 14A to 14C, for the non-SMAD pathway, CX did not induce significant changes in the phosphorylation of PI3K and ERK, and had little effect on the signaling pathways of ERK and PI3K.

[0115] In summary, the Job's tears extract containing 6-methoxybenzoxazolinone and 6-methoxybenzoxazolinone according to the present invention has the effect of improving liver fibrosis by reducing fibrotic and inflammatory factors. 6-MBOA improves oxidative stress in the liver, reduces the production of biomarkers of liver fibrosis by regulating the TGF-β / SMAD signaling pathway, and inhibits the mesenchymal transition of hepatic stellate cells. The Job's tears extract containing 6-methoxybenzoxazolinone and 6-methoxybenzoxazolinone shows the possibility of treating liver fibrosis.

[0116] The above description is merely illustrative and not restrictive. Any equivalent modifications or variations made without departing from the spirit and scope of the present invention shall be included in the scope of the attached patent application.

Claims

1. Use of 6-methoxybenzoxazolinone (6-MBOA) for the manufacture of a composition for preventing and / or treating liver fibrosis.

2. 2. The use according to claim 1, wherein the 6-MBOA reduces inflammatory infiltration, liver structure deformation, collagen deposition, expression levels of α-SMA, liver hydroxyproline content, upregulation of serum transforming growth factor beta 1 (TGF-β1), liver weight, liver function parameters, TGF-β1-induced reactive oxygen species (ROS) production, and TGF-β1-induced fibronectin expression levels in an individual for preventing and / or treating liver fibrosis.

3. The use according to claim 1, wherein the 6-MBOA negatively regulates the expression levels of phosphorylated SMAD2 (p-SMAD2) and phosphorylated SMAD3 (p-SMAD3) in an individual for preventing and / or treating liver fibrosis.

4. The use according to claim 1, wherein the 6-MBOA increases the activity of superoxide dismutase (SOD), the activity of catalase (CAT) and the content of glutathione (GSH), and decreases the expression level of NADPH oxidase 4 (NOX4) and the content of malondialdehyde (MDA) caused by oxidative stress in an individual for preventing and / or treating liver fibrosis.

5. The use according to claim 1, wherein the 6-MBOA inhibits hepatic stellate cell (HSC) activation and epithelial-mesenchymal transition (EMT), including suppressing fibronectin expression and maintaining E-cadherin expression levels.

6. The use according to claim 3, wherein the negative regulation is achieved by 6-MBOA decreasing the expression levels of zinc finger proteins SNAIL1 / 2, TWIST1 / 2, and zinc finger E-box-binding homeobox 1 (ZEB1).

7. Use of an extract of Job's tears (Coix lachryma-jobi L.) containing 6-methoxybenzoxazolinone (6-MBOA) for the manufacture of a composition for preventing and / or treating liver fibrosis.

8. The Job's tears extract is obtained by extracting Job's tears with a solvent, The use according to claim 7, wherein the solvent is water, alcohol, an alcohol-water mixture, or a combination thereof.

9. The use according to claim 7, wherein the Job's Tears extract down-regulates serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum total bilirubin (TBIL), serum total glyceride (TG), and serum total cholesterol (TC) levels in an individual for preventing and / or treating liver fibrosis, and up-regulates serum glucose levels in an individual for preventing and / or treating liver fibrosis.

10. The use according to claim 7, wherein the Job's Tears extract reduces the levels of liver α-smooth muscle actin (α-SMA) gene, Collagen type I alpha 1 (COL1A1) gene, tumor necrosis factor-α (TNF-α) gene, and interleukin-6 (IL-6) gene in an individual for preventing and / or treating liver fibrosis.

11. The use according to claim 8, wherein the Job's tears is a root, leaf or seed of Job's tears.

12. The use according to claim 1 or 7, wherein the composition is a pharmaceutical composition, a food composition, or a topical composition.

13. The use according to claim 12, wherein the pharmaceutical composition is in a dosage form for oral administration.

14. The use according to claim 12, wherein the pharmaceutical composition is in a dosage form for parenteral administration.

15. The use according to claim 12, wherein the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient, vector, adjuvant, and / or food additive.

16. The use according to claim 1 or 7, wherein the composition is in the form of a powder, granule, solution, gel, or paste.

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