Application of 6-methoxybenzoxazolinone and coix seed extract containing 6-methoxybenzoxazolinone in prevention and / or treatment of hepatic fibrosis

Abstract

The invention provides 6-methoxy benzoxazolinone and application of a coix seed extract containing the 6-methoxy benzoxazolinone to prevention and / or treatment of hepatic fibrosis. According to the 6-methoxy benzoxazolinone and the coix seed extract containing the 6-methoxy benzoxazolinone, disclosed by the invention, the 6-methoxy benzoxazolinone and the coix seed extract containing the 6-methoxy benzoxazolinone achieve the effect of preventing and / or treating the hepatic fibrosis through a plurality of efficacy experiments.

Description

Technical Field

[0001] The present invention relates to use of 6-methoxybenzoxazolinone (6-MBOA) and a coix seed (Coix lachryma-jobi L.var.ma-yuen Stapf) extract containing 6-methoxybenzoxazolinone for preventing and / or treating liver fibrosis. Background Art

[0002] The liver is an important organ in the human body, which is composed of parenchymal cells and non-parenchymal cells. Parenchymal cells, also known as hepatocytes, account for 60% of the cells in the liver and occupy 80% of the liver volume. Non-parenchymal cells are composed of other biologically important cells, including sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells (HSCs) and hepatic natural killer cells, each of which accounts for 3 to 20% of the number.

[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 injury. Accompanied by excessive accumulation of extracellular matrix (ECM), liver fibrosis not only changes the liver structure but also impairs liver function. The activation of hepatic stellate cells is considered to be a central event. When the liver is damaged, activated hepatic stellate cells are the main producers of extracellular matrix.

[0004] At present, clinical drugs for treating liver fibrosis have limited effects and serious side effects, and many patients cannot continue treatment. More importantly, drugs only relieve symptoms but fail to fundamentally solve the problem, so how to develop a new drug that can effectively treat and / or prevent liver fibrosis is an important issue that the present invention hopes to solve.

[0005] In order to solve the above problems, technicians in this field are in urgent need of developing novel compositions for preventing and / or treating liver fibrosis to benefit the vast population in need. Summary of the invention

[0006] In view of this, an object of the present invention is to provide a use of 6-methoxybenzoxazolinone (6-MBOA) for preparing 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 level, liver hydroxyproline content, serum transforming growth factor beta 1 (TGF-β1) upregulation, liver weight, liver index, TGF-β1-induced reactive oxygen species (ROS) production and TGF-β1-induced fibronectin expression level in individuals who need to prevent and / or treat liver fibrosis.

[0008] In one embodiment of the present invention, the 6-MBOA negatively regulates the expression levels of phosphorylated maternal DPP homolog 2 (phosphor-Mothers against decapentaplegic homolog 2, p-SMAD2) and phosphorylated maternal DPP homolog 3 (phosphor-Mothers against decapentaplegichomolog 3, p-SMAD3) in individuals who need to be prevented and / or treated for liver fibrosis.

[0009] In one embodiment of the present invention, the 6-MBOA increases superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione (GSH) content and E-cadherin (E-cadherin) expression level, and reduces malondialdehyde (MDA) content in an individual who needs to prevent and / or treat liver fibrosis.

[0010] In one embodiment of the present invention, the 6-MBOA inhibits hepatic stellate cell (HSC) activation and epithelial-mesenchymal transition (EMT).

[0011] In one embodiment of the present invention, the negative regulation is achieved by 6-MBOA reducing the expression levels of zinc finger proteins SNAIL1 / 2, TWIST1 / 2, zinc finger E-box-binding homeobox 1 (ZEB1) and NADPH oxidase 4 (NOX4).

[0012] Another object of the present invention is to provide a use of an extract of Coix lachryma-jobi L.var.ma-yuen Stapf containing 6-methoxybenzoxazolinone (6-MBOA) for preparing a composition for preventing and / or treating liver fibrosis.

[0013] In one embodiment of the present invention, the coix seed extract is prepared by extracting coix seed with a solvent, and the solvent is water, alcohol, a mixture of alcohol and water or a combination thereof.

[0014] In one embodiment of the present invention, the coix seed 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 who needs to be prevented and / or treated for liver fibrosis, and up-regulates serum glucose levels in an individual who needs to be prevented and / or treated for liver fibrosis.

[0015] In one embodiment of the present invention, the coix seed extract reduces the levels of α-smooth muscle actin (α-SMA) gene, type I collagen alpha 1 (COL1A1) gene, tumor necrosis factor-α (TNF-α) gene and interleukin-6 (IL-6) gene in the liver of an individual who needs to be prevented and / or treated for liver fibrosis.

[0016] In one embodiment of the present invention, the coix seed is the root, leaf or seed of coix 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 present invention, the pharmaceutical composition is in a dosage form for oral administration.

[0019] In one embodiment of the present 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 pharmaceutically acceptable excipient, carrier, adjuvant and / or food additive.

[0021] In one embodiment of the present invention, the composition has a powder, a granule, a solution, a gel, or a paste form.

[0022] In summary, the efficacy of 6-methoxybenzoxazolinone and the coix seed extract containing 6-methoxybenzoxazolinone of the present invention is that: it can improve liver fibrosis accompanied by the decrease of fibrosis and inflammatory factors, 6-MBOA improves the oxidative stress of the liver, and reduces the production of liver fibrosis biomarkers and promotes the loss of mesenchymal characteristics of liver stellate cells by regulating the TGF-β / SMAD signaling pathway, highlighting the therapeutic potential of 6-methoxybenzoxazolinone and the coix seed extract containing 6-methoxybenzoxazolinone on liver fibrosis.

[0023] The following will further illustrate the implementation mode of the present invention. The following examples are used to illustrate the present invention, not to limit the scope of the present invention. Any person skilled in the art may make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the content defined in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the design of the animal experiment for the coix seed group. In this experiment, C57BL / 6J mice were injected intraperitoneally with thioacetamide (TAA) to induce liver fibrosis. At six weeks of age, the 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 6 weeks of co-feeding, TAA was injected intraperitoneally three times a week, and TAA (200 mg / kg) was injected intraperitoneally three times a week for 6 weeks, and the mice were finally sacrificed at 12 weeks of age. NT means no treatment, ASP means coix seed powder, ALP means coix seed leaf powder, ASPS means coix seed polysaccharide, and ASE means coix seed ethanol extract.

[0025] Figures 2A to 2F The effects of coix seed extract on liver histopathology and physiological values ​​in TAA-induced liver fibrosis C57BL / 6J mice were shown. Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining were used to observe the changes in liver histopathology (original magnification ×200, scale bar 100μm) (2A); α-smooth muscle actin (α-SMA) was quantified by IHC staining using Image J software (2B); changes in liver hydroxyproline content (2C); changes in liver weight and body weight (2D, 2E); liver index, the ratio of liver weight change divided by body weight (2F). Data are expressed as mean ± standard deviation (SD) (n = 6 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0026] Figures 3A to 3F The effects of coix seed extract on liver function and nutrient metabolism in TAA-induced liver fibrosis C57BL / 6J mice were shown. Serum liver function indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) levels (3A-3C). Serum total glyceride (TG), total cholesterol (TC), and glucose levels were used to observe nutrient metabolism (3D-3F). Data are expressed as mean ± standard deviation (SD) (n = 6 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0027] Figures 4A to 4EThe effects of coix seed extract on fibrotic and inflammatory gene levels in TAA-induced liver fibrosis C57BL / 6J mice. The mRNA levels of fibrotic markers α-smooth muscle actin (α-SMA) and collagen, type I, alpha 1 (COL1A1) (4A and 4B). The mRNA levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 (4C to 4E). Data are expressed as mean ± standard deviation (SD) (n = 6 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0028] Figures 5A to 5H The effects of coix seed extract on HSC-T6 cell viability, fibrotic protein expression, and inflammatory gene levels are shown. Schematic diagram of drug treatment of HSC-T6 cells (5A). Cell viability of HSC-T6 cells treated with coix seed leaf, root, and seed extracts (5B). Western blot results of α-SMA and COL1A1 in HSC-T6 cells treated with TGF-β1 and coix seed extract, and quantification of the results using Image J software (5C to 5E). mRNA levels of inflammatory factors in HSC-T6 treated with TGF-β1 and coix seed extract (5F to 5H). Data are expressed as mean ± standard deviation (SD) (n = 3 per group). Cell viability bars are marked with *, and concentration 0 is compared with other concentrations, marked with ** (p < 0.01); *** (p < 0.001). The bar graphs of Western blot and real-time PCR are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05). Figure 5C and 5F The L in ~5H represents leaf, Figure 5D and 5F The R in ~5H stands for root, and Figures 5E to 5H The S stands for seed.

[0029] Figure 6Schematic diagram of the design of animal experiments for the 6-methoxybenzoxazolinone (6-MBOA) (CX) group. For animal experiments, 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 per group). Mice were injected intraperitoneally with TAA three times a week to induce liver fibrosis. They also received CX or vehicle once a day through a feeding tube. TAA (200mg / kg) was injected intraperitoneally three times a week, and CX (20 or 50mg / kg) was fed once a day through a feeding tube for 6 weeks. At the end of the animal experiment, the mice were sacrificed at 12 weeks old.

[0030] Figures 7A to 7K The effects of CX on the histopathological and physiological values ​​of liver in C57BL / 6J mice with TAA-induced liver fibrosis are shown. Chemical structure of CX (C8H7NO3, MW = 165.15) (7A). H&E, Sirius red and IHC staining were used to observe the histopathological changes in the liver (original magnification × 200, scale bar 100 μm) (7B). Quantification of Sirius red staining of collagen (7C). Quantification of IHC staining of α-SMA, phosphor-Mothers against decapentaplegichomolog 2 (p-SMAD2) and phosphor-Mothers against decapentaplegic homolog 3 (p-SMAD3) (7D to 7F). Changes in liver hydroxyproline content (7G). Expression of TGF-β1 in serum (7H). Changes in liver and body weight (7I and 7J). Liver index, the ratio of liver weight change divided by body weight (7K). Quantification of Sirius Red and IHC staining was achieved using Image J. Data are expressed as mean ± standard deviation (SD) (n = 8 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0031] Figures 8A to 8DThe effects of CX on antioxidant enzymes and lipid peroxidation in TAA-induced liver fibrosis C57BL / 6J mice are shown. Changes in superoxide dismutase (SOD) and catalase (CAT) activities (8A and 8B). Changes in glutathione (GSH) content (8C). Changes in malondialdehyde (MDA) content (8D). Data are expressed as mean ± standard deviation (SD) (n = 8 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0032] Figures 9A to 9G The effects of CX on liver function and nutrient metabolism in TAA-induced liver fibrosis C57BL / 6J mice were shown. Serum liver function indicators ALT, AST, TBIL, and ALP levels (9A-9D). Serum TG, TC, and glucose levels (9E-9G) were used to observe nutrient metabolism. Data are expressed as mean ± standard deviation (SD) (n = 8 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05). ALP represents coix seed leaf powder.

[0033] Figures 10A to 10E The effects of CX on fibrotic and inflammatory gene levels in TAA-induced liver fibrosis C57BL / 6J mice are shown. Liver mRNA levels of α-SMA and COL1A1 were detected using real-time PCR (10A and 10B). Liver mRNA levels of inflammatory factors TNF-α, IL-6, and IL-10 (10C to 10E). Data are expressed as mean ± standard deviation (SD) (n = 8 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0034] Figures 11A to 11FThe effects of CX on the viability, inflammatory gene levels, and reactive oxygen species (ROS) of HSC-T6 cells activated by TGF-β1 are shown. Schematic diagram of TGF-β1 and CX treatment in HSC-T6 cells (11A). Cell viability of HSC-T6 cells treated with different concentrations of CX, 6-MBOA represents 6-methoxybenzoxazolinone (11B). mRNA levels of inflammatory factors in HSC-T6 cells treated with TGF-β1 and CX (11C-11E). ROS levels of HSC-T6 were detected by DCFDA and normalized by protein content. Data are expressed as mean ± standard deviation (SD) (n = 3 per group). Cell viability bars are marked with *, and concentration 0 is marked with *** compared with other concentrations (p < 0.001). The bar graphs of Western blot and real-time PCR are labeled with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0035] Figures 12A to 12G The effect of CX on HSC activation and epithelial-mesenchymal transition (EMT) in TGF-β1-activated HSC-T6 cells is shown. Western blot results of E-cadherin, fibronectin, α-SMA and COL1A1 (12A). Western blots of E-cadherin, fibronectin, α-SMA and COL1A1 were quantified using Image J software (12B to 12E). Wound healing assay results at 0, 24 and 48 hours (magnification × 200, scale bar 100 μm), and quantified using image J software (12F and 12G). Wound closure was defined as (wound area at 0 hours - wound area at 24 or 48 hours) / wound area at 0 hours × 100%. Data are expressed as mean ± standard deviation (SD) (n = 3 per group). The bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0036] Figures 13A to 13IThe effect of CX on the TGF-β / SMAD signaling pathway in TGF-β1-activated HSC-T6 cells is shown. Schematic diagram of the TGF-β1 pathway and CX treatment in HSC-T6 cells (13A). Western blot results of phosphorylated mother DPP homolog 2 (phosphor-Mothers against decapentaplegic homolog 2, p-SMAD2), phosphorylated mother DPP homolog 3 (phosphor-Mothers against decapentaplegic homolog 3, p-SMAD3), SMAD2 / 3, zinc finger protein SNAIL1 / 2, TWIST1 / 2, zinc finger E-box-binding homeobox 1 (ZEB1), and NADPH oxidase 4 (NADPH oxidase 4, NOX4) were normalized with α-tubulin (13B). Western blots of p-SMAD2, p-SMAD3, SMAD2 / 3, SNAIL1 / 2, TWIST1 / 2, ZEB1, and NOX4 were quantified (13CI) using image J software. Data are expressed as mean ± standard deviation (SD) (n = 3 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0037] Figures 14A to 14C The effects of CX on phosphorylated phosphoinositide 3-kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (ERK) signaling pathways in TGF-β1-activated HSC-T6 cells are shown. Western blot results of p-PI3K and p-ERK1 / 2 normalized with α-tubulin (14A). Western blot quantification of p-PI3K and p-ERK1 / 2 was performed using Image J (14B and 14C). Data are expressed as mean ± standard deviation (SD) (n = 3 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05). DETAILED DESCRIPTION

[0038] definition

[0039] The numerical values ​​used in this article are approximate values, and all experimental data are expressed in the range of ±20%, preferably in the range of ±10%, and most preferably in the range of ±5%.

[0040] Unless otherwise specified herein, the terms "a", "an", "the" and similar terms used in this specification (especially in the scope of the following patent application) should be understood to include both singular and plural forms.

[0041] According to the present invention, coix seed (Coix lachryma-jobi L.var.ma-yuen Stapf, Adlay), also known as coix seed, coix seed, and coix seed, is an annual or perennial herb of the Poaceae family. It is a common herb and food supplement in Asia and is often used to improve inflammatory diseases. It is about 1 to 1.8 meters tall, generally blooms from July to September, and bears fruit from September to October. In addition, coix seed is composed of four parts from the outside to the inside, namely, bran, shell, seed coat, and endosperm (polished coix seed), each of which has its own biological activity.

[0042] According to the present invention, 6-methoxybenzoxazolinone (6-MBOA) (CX) (chemical formula C8H7NO3) is a compound in the grass family, which has anti-diabetic and hormone regulating activities.

[0043] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for parenteral or oral administration using techniques well known to those skilled in the art, including, but not limited to: injections [e.g., sterile aqueous solutions or dispersions], sterile powders, tablets, troches, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries and the like.

[0044] The pharmaceutical composition according to the present invention can be administered via a 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.

[0045] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier widely used in drug manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of: solvent, emulsifier, suspending agent, decomposer, binder, excipient, stabilizer, chelating agent, diluent, gelling agent, preservative, lubricant, absorption delaying agent, liposome and the like. The selection and amount of these agents fall within the professional qualities and routine technical scope of those skilled in the art.

[0046] According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), sugar solution, aqueous solution containing alcohol, and combinations thereof.

[0047] According to the present invention, the chemicals and reagents used in the following examples are described as follows. 6-Methoxybenzoxazolinone (6-MBOA) (CX) (purity ≧96.5%), thioacetamide (TAA) (purity ≧99.0%) and 2′,7′-dichlorofluorescindiacetate (DCFDA, purity ≧97.0%) were purchased from Sigma-Aldrich (USA). Recombinant human transforming growth factor beta 1 (TGF-β1) was purchased from PeproTech (PeproTech, USA). TBARS and catalase (CAT) detection kits were purchased from Cayman Chemical (USA). Reduced glutathione (GSH) and superoxide dismutase (SOD) detection kits were purchased from Abbkine (Abbkine Scientific, China). The hydroxyproline assay kit was purchased from Elabscience (Elabscience Biotechnology, China) and the mouse TGF-β1 ELISA kit was purchased from Invitrogen (Thermo Fisher Scientific, USA).Targeting α-smooth muscleactin (α-SMA) (GTX100458), collagen, type I, alpha 1 (COL1A1) (GTX112731), fibronectin (GTX112794), E-cadherin (GTX629691), NADPH oxidase 4 (NADPH oxidase 4, NOX4) (GTX121929), TWIST1 / 2 (GTX127310), zinc finger E-box-binding homeobox 1 (GTX1127311), 1, ZEB1)(GTX105278), β-actin(GTX109639), α-tubulin(GTX628802), phosphor-Mothers against decapentaplegichomolog 2(p-SMAD2)(GTX133614), phosphor-Mothers against decapentaplegic homolog 3(p-SMAD3)(GTX129841), phosphorylated extracellular signal-regulated kinase 1 / 2(ERK1 / 2)(GTX635617), phosphorylated phosphoinositide 3-kinase(PI3K)(GTX132597), SMAD2 / 3(GTX111123) and horseradish peroxidase(HRP) The antibody against peroxidase (HRP) was purchased from GeneTex (USA). In addition, the zinc finger protein SNAIL1 / 2 (ab180714) was purchased from Abcam (UK).

[0048] According to the present invention, the animals used in the following examples are described as follows: Male C57BL / 6J mice (5 weeks old) were obtained from the Experimental Animal Center of Taiwan, China, and were housed in a temperature-controlled room with a 12-hour light-dark cycle and free access to food and drinking water (Animal Center of Yang-Ming Chiao Tung University, Taiwan, China).

[0049] For the test of coix seed extract, mice were randomly divided into the following seven groups (n=6 in each group): (1) no treatment group (NT, normal diet), (2) thioacetamide group (TAA, 200 mg / kg, normal diet), (3) 10% coix seed powder (ASP) group, (4) 1% coix leaf powder (ALP) group, (5) 1% coix seed polysaccharide (ASPS) group, (6) 0.025% coix seed ethanol extract (ASE) group and (7) 0.1% ASE group. The NT group was given pure saline, and the other groups were intraperitoneally injected with TAA dissolved in saline three times a week.

[0050] For CX testing, mice were randomly divided into the following four groups (n = 8 in each group): (1) NT group, (2) TAA group (200 mg / kg), (3) CX group (CX20, 20 mg / kg), and (4) CX group (CX50, 50 mg / kg). The NT group was given pure saline, and the other groups were intraperitoneally injected with TAA dissolved in saline three times a week. In addition, the NT and TAA groups were also given 5% propylene glycol, while the CX group was given CX dissolved in 5% propylene glycol (prepared with 0.9% saline) and then fed by feeding tube once a day.

[0051] At the end of the experiment, the mice were sacrificed, the whole blood of the mice was centrifuged and serum was obtained and stored at -80°C, and the liver tissue was collected for histological staining.

[0052] According to the present invention, the process of statistical analysis is as follows. Statistical analysis was performed using one-way ANOVA, followed by Tukey's multiple comparison test in GraphPad Prism 7. Cell viability data were analyzed using two-way ANOVA, followed by Dunnett's multiple comparison test. All data are expressed as mean ± standard deviation (SD). Differences with a P value < 0.05 were considered statistically significant.

[0053] The present invention is further illustrated by the following examples, which are provided for illustration only and are not intended to limit the scope of the present invention, which is defined in the appended claims.

[0054] Example 1. Preparation of Coix lachryma-jobi L. var. ma-yuen Stapf extract

[0055] For cell experiments, different parts of coix seed, including leaves (L), roots (R), and seeds (S), were used. The dried and ground coix seeds were extracted with 70% ethanol, concentrated, and dried under reduced pressure. On the other hand, coix seed powder (ASP, normal diet containing 10% ASP), coix leaf powder (ALP, normal diet containing 1% ALP), coix seed polysaccharide (ASPS, normal diet containing 1% ASPS), and coix seed ethanol extract (ASE, normal diet containing 0.025% and 0.1% ASE) were used for animal experiments. All coix seed extracts were kindly provided by Dr. Yu-Hsin Chen.

[0056] The process of the design diagram of the coix seed group animal experiment is described as follows: In order to detect the effect of coix seed on liver fibrosis, this example established a thioacetamide (TAA)-induced liver fibrosis C57BL / 6J mouse model. Figure 1 Schematic diagram of the animal experiment design for the coix seed group. In this experiment, C57BL / 6J mice were intraperitoneally injected with TAA to induce liver fibrosis. At six weeks of age, the 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 6 weeks of co-feeding, TAA was injected intraperitoneally three times a week, and the mice were finally sacrificed at 12 weeks of age. NT means no treatment, ASP means coix seed powder, ALP means coix seed leaf powder, ASPS means coix seed polysaccharide, and ASE means coix seed ethanol extract. Six-week-old mice were randomly divided into seven groups, including NT, TAA, 10% ASP, 1% ALP, 1% ASPS, 0.025% ASE and 0.1% ASE groups (see Figure 1 ). The NT and TAA groups received a normal diet, while the coix seed group received coix seed extract added to the normal diet. In addition, TAA or saline was intraperitoneally injected into the mice three times a week for six weeks. The mice were sacrificed after six weeks, and their liver tissues and serum were collected for subsequent analysis.

[0057] Example 2. Effects of coix seed extract on liver histopathology and physiological value in TAA-induced liver fibrosis mice

[0058] After six weeks of exposure, TAA-induced pathological changes in liver tissue sections stained with hematoxylin and eosin (H&E) and immunohistochemistry (IHC) were observed.

[0059] The procedure for liver histopathological observation is as follows. Liver tissues were fixed with 10% neutral formalin buffer, embedded in paraffin, and cut into 4 μm thick sections. Pathological sections were examined using an H&E staining kit (CIS-Biotechnology) to evaluate changes in tissue structure, a Sirius red staining kit (CIS-Biotechnology) to evaluate collagen deposition, and immunohistochemistry kits (Agilent, USA), phosphorylated SMAD2 (1:100; GeneTex, USA), phosphorylated SMAD3 (1:100; GeneTex, USA), and α-smooth muscle actin (α-SMA) (1:100; GeneTex, USA) antibodies to evaluate the expression of specific proteins in tissues. All staining was observed using an optical microscope (BEL Photonics, Italy).

[0060] The procedure for hydroxyproline determination is as follows. Hydroxyproline, as an indirect indicator of collagen content in the liver, was determined by a hydroxyproline determination kit, and the test procedure followed the manufacturer's guidelines. Briefly, liver tissue samples were hydrolyzed with HCl at 95°C for 6 h, then the pH was adjusted to 7, impurities were adsorbed with carbon powder, centrifuged, and the supernatant was collected for analysis. Hydroxyproline content was measured at a wavelength of 558 nm on an ELISA reader (TECAN, Switzerland).

[0061] Figures 2A to 2F The effects of coix seed extract on the pathological and physiological values ​​of liver tissue in C57BL / 6J mice with TAA-induced liver fibrosis were shown. H&E and IHC staining were used to observe the pathological changes of liver tissue (original magnification ×200, scale bar 100μm) (2A); α-smooth muscle actin (α-SMA) was quantified by IHC staining using Image J software (2B); changes in liver hydroxyproline content (2C); changes in liver weight and body weight (2D, 2E); liver index, the ratio of liver weight change divided by body weight (2F). Data are expressed as mean ± standard deviation (SD) (n = 6 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0062] Compared with the NT group, the TAA group induced inflammatory infiltration, liver structural deformation, and excessive expression of α-SMA ( Figure 2A and 2B), while the coix seed group had significantly reduced inflammatory infiltration, improved liver deformation, and decreased α-SMA expression. In order to detect changes in collagen content in the liver, hydroxyproline (a component of collagen) was determined. The results showed that the hydroxyproline content in the TAA group was higher than that in the NT group, indicating that collagen increased ( Figure 2C On the other hand, the hydroxyproline content was significantly decreased in all coix seed groups, indicating that coix seed reduced TAA-induced hepatic collagen deposition. Compared with the NT group, the TAA group had a decrease in body weight and an increase in liver tissue size ( Figure 2D and 2E ). In the coix seed group, the liver enlargement caused by TAA was significantly reduced in the 10% ASP, 1% ALP, 1% ASPS, and 0.1% ASE groups, but there was no significant difference in body weight. To avoid individual differences, changes in liver weight and body weight were corrected by liver index ( Figure 2F ). Compared with the TAA group, only the 0.025% ASE group showed no significant change, while other liver indexes decreased significantly.

[0063] Example 3. Effects of coix seed extract on biochemical characteristics of liver in mice with TAA-induced liver fibrosis

[0064] The process of biochemical analysis is as follows. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), total cholesterol (TC), total glyceride (TG) and glucose levels were measured using a biochemical analyzer. In short, the whole blood of the mice was centrifuged, the serum was collected, the serum was dropped on the test chip, and the analysis was performed using an automatic clinical chemistry analyzer (Fuji, Japan).

[0065] Figures 3A to 3F The effects of coix seed extract on liver function and nutrient metabolism in TAA-induced liver fibrosis C57BL / 6J mice were shown. Serum liver function indicators ALT, AST, TBIL levels (3A-3C). Serum TG, TC, glucose levels were used to observe nutrient metabolism (3D-3F). Data are expressed as mean ± standard deviation (SD) (n = 6 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0066] Serum AST, ALT, and TBIL are indicators of liver function, and serum TG, TC, and glucose are related to liver nutrient metabolism. Compared with the NT group, only the TAA group had increased AST, ALT, and TBIL levels ( Figures 3A to 3C ). In contrast, the coix seed group downregulated the upregulation of liver function indices caused by TAA, especially in the 10% ASP, 1% ALP, 1% ASPS, and 0.1% ASE groups. On the other hand, compared with the NT group, the TAA group showed significantly increased TG and TC levels and decreased glucose values ​​( Figure 3D~3F Treatment with coix seed extract significantly improved TAA-induced TG and TC levels in all coix seed groups and significantly increased glucose values ​​in the 10% ASP and 1% ASPS groups.

[0067] Example 4. Effects of coix seed extract on liver fibrosis and inflammatory gene mRNA levels in TAA-induced liver fibrosis mice

[0068] The process of real-time polymerase chain reaction (real-time PCR) analysis is as follows. Total RNA was extracted with Trizol reagent (Ambion, USA) and then reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (ThermoScientific, USA). PCR amplification was completed using SYBR Green PCR Master Mix (Applied Biosystems, USA), and the mRNA expression of the gene was standardized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All primers were obtained from Genomics. The primer sequences used in this example are shown in Table 1. Rat primer sequences were used for real-time PCR of HSC-T6 cells, and mouse primer sequences were used for real-time PCR of C57BL / 6J mice.

[0069] Table 1

[0070]

[0071]

[0072] To confirm the gene level changes in liver fibrosis mice, real-time PCR was used to observe the mRNA levels of fibrosis genes. Figures 4A to 4EThe effects of coix seed extract on fibrotic and inflammatory gene levels in TAA-induced liver fibrosis C57BL / 6J mice. The mRNA levels of fibrotic markers α-smooth muscle actin (α-SMA) and collagen, type I, alpha 1 (COL1A1) (4A and 4B). The mRNA levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 (4C to 4E). Data are expressed as mean ± standard deviation (SD) (n = 6 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0073] Compared with the NT group, the levels of fibrosis markers α-SMA and COL1A1 were significantly increased in the TAA group ( Figure 4A and 4B ). In contrast, the levels of α-SMA and COL1A1 were significantly decreased in all coix seed groups, indicating that coix seed extract reduced the expression of fibrosis markers. Inflammation is closely related to fibrosis, therefore, the mRNA levels of inflammatory genes were also detected. Tumor necrosis factor-α (TNF-α) and IL-6 are pro-inflammatory cytokines, while IL-10 is an anti-inflammatory cytokine. Real-time PCR results showed that TNF-α and IL-6 were upregulated and IL-10 was downregulated in the TAA group compared with the NT group ( Figures 4C to 4E All coix seed extract groups reduced TAA-induced increases in TNF-α and IL-6 mRNA levels and significantly upregulated IL-10 mRNA levels, although 1% ALP, 0.025% ASE, and 0.1% ASE groups showed no significant differences from the TAA group.

[0074] Example 5. Effects of coix seed extract on HSC-T6 cell viability, fibrosis protein expression and inflammatory gene levels

[0075] The cell culture process is as follows. HSC-T6 cells are immortalized cell lines isolated from primary stellate cells transformed with SV40 large T antigen from male Sprague-Dawley rats. HSC-T6 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich, USA) containing 8% fetal bovine serum (FBS, HyClone, USA), 100 U / mL penicillin and 100 μg / mL streptomycin (Gibco, USA).

[0076] For hepatic stellate cell (HSC) activation, cells were cultured for 24 h and then replaced with serum-free medium for 24 h, after which they were treated with transforming growth factor beta 1 (TGF-β1) (10 ng / mL) for 24 h or intracellular signaling pathways for 30 min in the presence or absence of CX (10-100 μM) or coix seed extracts (25 and 50 μg / mL).

[0077] The procedure for cell viability analysis is as follows. HSC-T6 cells were cultured at 1.5×10 4 Cells were seeded in 96-well plates and treated with CX (0-800 μM) or coix seed extract (0-800 μg / mL) for 24 hours. Cell viability was then determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA). Briefly, MTT was added to each well to a final concentration of 0.5 μg / mL and incubated at 37°C for 1 hour. Next, MTT was removed and dimethyl sulfoxide (DMSO) was added to dissolve MTT-formazan crystals formed by metabolically active cells. Finally, the absorbance of each well was measured at a wavelength of 570 nm using a spectrophotometer.

[0078] The procedure for western blot analysis is as follows. Cells were lysed in RIPA buffer containing protease inhibitors and then centrifuged at 4°C, 12,000 rpm for 10 minutes. The supernatant was collected and the total protein concentration was quantified to 50 μg. After heating at 95°C for 10 minutes, the protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred to PVDF membrane, blocked with 6% milk, and the primary antibody was allowed to act overnight at 4°C, and the secondary antibody was allowed to act for 1 hour at room temperature. The ink dot images were displayed on Amersham Imager 680 (Cytiva, USA) using ECL reagent (GeneDireX, USA).

[0079] HSC activation is considered to be the main event in liver fibrosis. In this example, HSC-T6 cells were used as a cell model, and TGF-β1 was added as an inducer of HSC activation.

[0080] Figures 5A to 5H The effects of coix seed extract on HSC-T6 cell viability, fibrotic protein expression, and inflammatory gene levels are shown. Schematic diagram of drug treatment of HSC-T6 cells (5A). Cell viability of HSC-T6 cells treated with coix seed leaf, root, and seed extracts (5B). Western blot results of α-SMA and COL1A1 in HSC-T6 cells treated with TGF-β1 and coix seed extract, and quantification of the results using Image J software (5C to 5E). mRNA levels of inflammatory factors in HSC-T6 treated with TGF-β1 and coix seed extract (5F to 5H). Data are expressed as mean ± standard deviation (SD) (n = 3 per group). Cell viability bars are marked with *, and concentration 0 is compared with other concentrations, marked with ** (p < 0.01); *** (p < 0.001). The bar graphs of Western blot and real-time PCR are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05). Figure 5C and 5F The L in ~5H represents leaf, Figure 5D and 5F The R in ~5H stands for root, and Figures 5E to 5H The S stands for seed.

[0081] To determine the appropriate dosage of coix seed extract (leaf, root and seed) for cell testing and to examine the effect of coix seed extract on cell viability ( Figure 5A Cell viability showed that the extracts of coix seed leaf and root at doses below 100 μg / mL and the extract of coix seed at doses below 200 μg / mL had no significant effect ( Figure 5B). Therefore, subsequent tests used 20 and 50 μg / mL of coix seed extracts. Western blot analysis was used to detect the expression of fibrotic markers in HSC-T6 cells treated with TGF-β1. The results showed that all coix seed extracts dose-dependently reduced the expression of TGF-β1-induced α-SMA and COL1A ( Figures 5C to 5E ). In addition, for inflammation, mRNA levels of TNF-α, IL-6, and IL-10 were evaluated using real-time PCR. The results showed that all coix seed extracts down-regulated TGF-β1-induced TNF-α and IL-6 mRNA levels and up-regulated IL-10 mRNA levels in a dose-dependent manner ( Figures 5F to 5H ). Changes in fibrotic proteins and inflammatory genes indicated that coix seed extract improved inflammation and HSC activation in HSC-T6 cells treated with TGF-β1.

[0082] Example 6. Schematic diagram of the animal experiment design for the 6-methoxybenzoxazolinone (6-MBOA) (CX) group

[0083] In order to detect the effect of CX on liver fibrosis, this example established a TAA-induced liver fibrosis C57BL / 6J mouse model. Figure 6 Schematic diagram of the animal experiment design for the CX group. For 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). The mice were injected intraperitoneally with TAA three times a week to induce liver fibrosis. They also received CX or vehicle through a feeding tube once a day. At the end of the animal experiment, the mice were sacrificed at 12 weeks of age. Six-week-old mice were randomly divided into four groups, namely NT, TAA, CX20 and CX50 ( Figure 6 ). Among these groups, only the NT group was tube-fed and given the vehicle, the TAA group was tube-fed and given the vehicle and TAA, and the CX group was tube-fed and given CX and TAA; tube-fed once a day and intraperitoneally injected three times a week; both tube-fed and intraperitoneally injected were administered for six weeks. Afterwards, the mice were sacrificed, and their liver tissues and serum were collected for subsequent analysis.

[0084] Example 7. Effects of 6-methoxybenzoxazolinone (6-MBOA) (CX) on liver histopathology and physiological values ​​in TAA-induced liver fibrosis mice

[0085] To observe the effect of CX on liver histopathology, mice with hepatic fibrosis were treated with CX, and liver tissues were stained with H&E and Sirius red. The fibrosis marker α-SMA and TGF-β / SMAD pathway-related proteins p-SMAD2 and p-SMAD3 were also examined in liver sections under IHC staining.

[0086] The process of TGF-β1 enzyme-linked immunosorbent assay (ELISA) is as follows. The level of TGF-β1 in serum was detected using a commercial mouse ELISA kit (Invitrogen, USA), and the working procedure followed the standard sandwich ELISA protocol. In short, serum was added to the microwells coated with TGF-β1 antibody, and then reacted with biotin conjugate, streptavidin-HRP, substrate solution and stop solution in sequence. After reaction with the stop solution, the color intensity was immediately measured at 450nm using an ELISA reader (TECAN, Switzerland).

[0087] Figures 7A to 7K The effects of CX on the histopathological and physiological values ​​of liver in C57BL / 6J mice with TAA-induced liver fibrosis are shown. Chemical structure of CX (C8H7NO3, MW = 165.15) (7A). H&E, Sirius red and IHC staining were used to observe the histopathological changes in the liver (original magnification × 200, scale bar 100 μm) (7B). Quantification of Sirius red staining of collagen (7C). Quantification of IHC staining of α-SMA, p-SMAD2 and p-SMAD3 (7D to 7F). Changes in liver hydroxyproline content (7G). Expression of TGF-β1 in serum (7H). Liver and body weight changes (7I and 7J). Liver index, the ratio of liver weight change divided by body weight (7K). Quantification of Sirius red and IHC staining was achieved using Image J. Data are expressed as mean ± standard deviation (SD) (n = 8 per group). The bars are marked with different letters; the same letters indicate that the difference between the two groups was not significant (p>0.05), and different letters indicate that the difference between the two groups was significant (p<0.05). Compared with the NT group, the TAA group showed inflammatory infiltration, liver structural deformation, collagen deposition, and excessive expression of α-SMA and p-SMAD2 / 3 (7B-7F). Both the CX20 and CX50 groups showed significantly reduced inflammatory infiltration, liver structural deformation, collagen deposition, and α-SMA expression. In addition, the CX50 group negatively regulated the expression of p-SMAD2 and p-SMAD3, indicating that CX improved TAA-induced damage through the SMAD signaling pathway. The hydroxyproline content in the TAA group was significantly higher than that in the NT group, while the hydroxyproline content in the CX group was significantly lower than that in the TAA group, indicating that CX treatment reduced the collagen content ( Figure 7G). TGF-β1 is an important cytokine related to fibrosis, and changes in TGF-β1 may be an indicator of fibrosis development. ELISA results showed that compared with the NT group, TAA group showed a significant increase in TGF-β1 expression, while CX significantly reduced the upregulation of TGF-β1 caused by TAA ( Figure 7H TAA alone resulted in liver enlargement and body weight loss, whereas CX treatment reduced liver weight but did not significantly improve TAA-induced changes in body weight ( Fig.7I and 7J ). In order to avoid individual differences in mice, liver index was used to adjust liver and body weight. The results showed that the liver index of the TAA group was significantly increased compared with the NT group, while the liver index of the CX group was significantly decreased compared with the TAA group ( Figure 7K The current results suggest that CX, by affecting the expression of SMADs, alleviates typical features of fibrosis, such as excessive α-SMA expression, collagen accumulation, and higher levels of TGF-β1.

[0088] Example 8. Effects of CX on oxidative stress and biochemical characteristics of TAA-induced liver fibrosis mice

[0089] Liver damage is caused by TAA reactive metabolites that lead to oxidative stress. Antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), as well as lipid peroxidation product malondialdehyde (MDA), were used to detect oxidative stress in TAA-induced fibrotic mice.

[0090] The process of oxidative stress index testing is as follows. The enzymatic activities of catalase (CAT) and superoxide dismutase (SOD), malondialdehyde (MDA) content and glutathione (GSH) concentration in tissues were detected to detect changes in oxidative stress levels in TAA-induced liver fibrosis mice. Hydroxyproline is a component of collagen precursors. All test kits are commercial products, and the work protocol follows the manufacturer's guidelines. In brief, liver tissues were homogenized in ice-cold buffer and centrifuged at 4°C, and then the supernatant was collected for analysis, and finally the absorbance of the samples was read using an ELISA reader (TECAN, Switzerland).

[0091] Figures 8A to 8DFigure 8 shows the effect of CX on antioxidant enzymes and lipid peroxidation in TAA-induced liver fibrosis C57BL / 6J mice. Changes in the activities of SOD and CAT (8A and 8B). Changes in the content of GSH (8C). Changes in the content of MDA (8D). Data are expressed as mean ± standard deviation (SD) (n = 8 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate a significant difference between the two groups (p<0.05). Compared with the NT group, the activities of SOD, CAT, and GSH content in the TAA group were decreased, but the MDA content was increased ( Fig. 8A ~D). Compared with the TAA group, the CAT activity and GSH content in the CX20 and CX50 groups increased, but the MDA content decreased. In addition, the SOD activity was significantly increased in the CX50 group.

[0092] Figures 9A to 9G The effects of CX on liver function and nutrient metabolism in TAA-induced liver fibrosis C57BL / 6J mice were shown. Serum liver function indicators ALT, AST, TBIL, and ALP levels (9A-9D). Serum TG, TC, and glucose levels (9E-9G) were used to observe nutrient metabolism. Data are expressed as mean ± standard deviation (SD) (n = 8 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0093] In terms of biochemical characteristics, the liver function indicators AST, ALT, TBIL, and ALP in the TAA group were significantly higher than those in the NT group, and significantly lower in the CX group than in the TAA group ( Figures 9A to 9D ). In addition, TAA also caused nutritional metabolic disorders. Compared with the NT group, the TG and TC levels in the TAA group were significantly increased, and the glucose level was decreased, while CX significantly reduced the changes in TG, TC, and glucose levels caused by TAA ( Figures 9E to 9G ). These findings suggest that CX alleviates oxidative stress in the liver and improves liver function abnormalities caused by TAA.

[0094] Example 9. Effects of CX on fibrosis and inflammatory gene levels in TAA-induced liver fibrosis mice

[0095] To confirm the changes in mouse fibrosis genes, real-time PCR was used to detect the mRNA levels of fibrosis genes. Figures 10A to 10EThe effects of CX on fibrotic and inflammatory gene levels in TAA-induced liver fibrosis C57BL / 6J mice are shown. Liver mRNA levels of α-SMA and COL1A1 were detected using real-time PCR (10A and 10B). Liver mRNA levels of inflammatory factors TNF-α, IL-6, and IL-10 (10C to 10E). Data are expressed as mean ± standard deviation (SD) (n = 8 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0096] Compared with the NT group, the TAA group significantly upregulated α-SMA and COL1A1 mRNA levels, while the CX group significantly reduced TAA-induced upregulation of fibrotic markers ( Fig. 10A and 10B ). Inflammation is necessary for the development of fibrosis; therefore, inflammatory gene mRNA levels were detected. Compared with the NT group, the TAA group induced upregulation of IL-6 and TNF-α, and downregulation of IL-10. CX treatment significantly reduced the TAA-induced upregulation of IL-6 and TNF-α levels and downregulation of IL-10 mRNA levels ( Figures 10C to 10E ). These results suggest that CX reduces inflammation and fibrosis at the genetic level in mice with TAA-induced fibrosis.

[0097] Example 10. Effects of CX on HSC-T6 cell viability, inflammatory gene levels and reactive oxygen species (ROS) levels

[0098] To examine the cytotoxicity of CX and determine the optimal dose for subsequent cell experiments, various doses of CX were tested in HSC-T6 cells using the MTT assay.

[0099] The process of ROS detection analysis is as follows. 2',7'-Dichlorofluorescein diacetate (DCFDA) is a cell-permeable fluorescent probe commonly used to detect ROS and nitric oxide in living 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 hours, followed by starvation and drug treatment, and then the medium was replaced with DCFDA solution for 30 minutes, and the excitation / emission spectra were detected at 485 / 525 nm by multimode microplate readers (TECAN, Switzerland).

[0100] Figures 11A to 11F The effects of CX on TGF-β1-activated HSC-T6 cell viability, inflammatory gene levels, and ROS are shown. Schematic diagram of TGF-β1 and CX treatment in HSC-T6 cells (11A). Cell viability of HSC-T6 cells treated with different concentrations of CX (11B). mRNA levels of inflammatory factors in HSC-T6 cells treated with TGF-β1 and CX (11C-11E). ROS levels in HSC-T6 were detected with DCFDA and normalized with protein content. Data are expressed as mean ± standard deviation (SD) (n = 3 per group). Cell viability bars are marked with *, and concentration 0 is marked with *** compared with other concentrations (p < 0.001). Bar graphs of Western blot and real-time PCR are labeled with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p < 0.05).

[0101] The results showed no significant effects at doses below 400 μM; therefore, 10, 20, 50, and 100 μM were used in subsequent cell assays ( Fig. 11B ). Similarly, real-time PCR was used to detect the mRNA levels of TNF-α, IL-6, and IL-10 in HSC-T6 cells treated with TGF-β1. The results showed that TGF-β1 treatment significantly increased TNF-α and IL-6, and decreased IL-10 mRNA levels ( Fig. 11C ~E), while CX dose-dependently reduced TGF-β1-induced TNF-α and IL-6, and significantly increased IL-10 mRNA levels at the highest dose. In addition, the DCFDA assay was used to detect ROS levels in HSC-T6 cells. The results showed that TGF-β1 induced ROS production, while CX reduced TGF-β1-induced ROS levels with increasing doses ( Fig.11F ). These results suggest that TGF-β1 promotes inflammation and ROS production, whereas CX shows the potential to reduce inflammation and ROS in HSC-T6 cells activated with TGF-β1.

[0102] Example 11. Effect of CX on activation of hepatic stellate cells (HSC) and epithelial-mesenchymal transition (EMT) in HSC-T6 cells

[0103] To understand the effects of CX on HSC activation and EMT in TGF-β1-activated HSC-T6 cells, the expression of fibrosis- and EMT-related proteins was examined using western blot analysis.

[0104] The procedure for wound healing assay is as follows. HSC-T6 cells were seeded in a 6-well plate for 24 hours, the monolayer was scraped with a 200 μL micropipette tip, non-attached cells were washed with PBS, serum-free medium was replaced for 24 hours, and then TGF-β1 and CX were treated for 0, 24 and 48 hours. Cell migration images were observed using an inverted microscope (Olympus, Japan), and the wound area was quantified using Image J software.

[0105] Figures 12A to 12G The effect of CX on HSC activation and EMT in TGF-β1-activated HSC-T6 cells is shown. Western blot results of E-cadherin, fibronectin, α-SMA and COL1A1 (12A). Western blots of E-cadherin, fibronectin, α-SMA and COL1A1 were quantified using Image J software (12B to 12E). Wound healing assay results at 0, 24 and 48 hours (magnification × 200, scale bar 100 μm), and quantified using image J software (12F and 12G). Wound closure was defined as (wound area at 0 hours - wound area at 24 or 48 hours) / wound area at 0 hours × 100%. Data are expressed as mean ± standard deviation (SD) (n = 3 per group). Bars are marked with different letters; the same letters indicate that there is no significant difference between the two groups (p>0.05), and different letters indicate that there is a significant difference between the two groups (p<0.05).

[0106] TGF-β1 treatment upregulated the expression of α-SMA, COL1A1, and the mesenchymal marker fibronectin, and downregulated the epithelial marker E-cadherin ( Figures 12A to 12E ). Treatment with CX reduced TGF-β1-induced α-SMA, COL1A1, and fibronectin expression, but significantly enhanced E-cadherin expression even at the lowest dose. As EMT occurs, cell migration is enhanced. Therefore, a wound healing assay was used to examine migratory changes. The percentage of wound closure was significantly increased in the TGF-β1 group compared with the NT group ( Fig.12F and 12G CX treatment at a dose of 100 μM significantly reduced TGF-β1-induced wound closure for 24 hours, while CX treatment at doses of 20 to 100 μM reduced TGF-β1-induced wound closure for 48 hours. These results suggest that CX inhibits HSC activation and EMT in HSC-T6 cells treated with TGF-β1.

[0107] Example 12. Effect of CX on TGF-β / Mothers against decapentaplegic homolog (SMAD) pathway in HSC-T6 cells

[0108] Western blot analysis was used to further explore the mechanism of CX on HSC activation and EMT.

[0109] Figures 13A to 13I The effect of CX on the TGF-β / SMAD signaling pathway in TGF-β1-activated HSC-T6 cells is shown. Schematic diagram of the stimulation of the TGF-β1 pathway and CX treatment in HSC-T6 cells (13A). Western blot results of p-SMAD2, p-SMAD3, SMAD2 / 3, SNAIL1 / 2, TWIST1 / 2, ZEB1, and NOX4 normalized with α-tubulin (13B). Western blots of p-SMAD2, p-SMAD3, SMAD2 / 3, SNAIL1 / 2, TWIST1 / 2, ZEB1, and NOX4 were quantified using imageJ software (13CI). Data are expressed as mean ± standard deviation (SD) (n = 3 per group). Bars are marked with different letters; the same letters indicate that the difference between the two groups was not significant (p>0.05), and different letters indicate that the difference between the two groups was significant (p<0.05).

[0110] The expression of SMAD pathway-related proteins p-SMAD2, p-SMAD3, EMT-related proteins SNAIL1 / 2, TWIST1 / 2, ZEB1 and ROS-related protein NOX4 in the TGF-β1 group was significantly higher than that in the NT group ( Figures 13B to 13I ), indicating that TGF-β1 can phosphorylate SMAD2 and SMAD3, and then activate downstream proteins SNAIL1 / 2, TWIST1 / 2, ZEB1 and NOX4, further leading to ROS production and triggering EMT. When treated with CX, TGF-β1-stimulated SMAD phosphorylation and downstream protein expression decreased with increasing doses, indicating that CX reduced the expression of SNAIL1 / 2, TWIST1 / 2, ZEB1 and NOX4, and further prevented EMT and ROS production by inhibiting SMAD2 and SMAD3 phosphorylation.

[0111] Figures 14A to 14CThe effects of CX on phosphorylated phosphoinositide 3-kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (ERK) signaling pathways in TGF-β1-activated HSC-T6 cells are shown. Western blot results of p-PI3K and p-ERK1 / 2 normalized with α-tubulin (14A). Western blot quantification of p-PI3K and p-ERK1 / 2 was performed using Image J (14B and 14C). Data are expressed as mean ± standard deviation (SD) (n = 3 per group). Bars are marked with different letters; the same letters indicate no significant difference between the two groups (p>0.05), and different letters indicate significant difference between the two groups (p<0.05).

[0112] For non-SMAD pathways, the results showed that CX did not cause significant changes in PI3K and ERK phosphorylation, suggesting that CX had little effect on the ERK and PI3K signaling pathways ( Figures 14A to 14C ).

[0113] In summary, the 6-methoxybenzoxazolinone and the coix seed extract containing 6-methoxybenzoxazolinone of the present invention can improve liver fibrosis accompanied by the decrease of fibrosis and inflammatory factors, 6-MBOA improves the oxidative stress of the liver, and reduces the production of liver fibrosis biomarkers and promotes the loss of mesenchymal characteristics of liver stellate cells by regulating the TGF-β / SMAD signaling pathway, highlighting the therapeutic potential of 6-methoxybenzoxazolinone and the coix seed extract containing 6-methoxybenzoxazolinone on liver fibrosis.

[0114] The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or changes made thereto without departing from the spirit and scope of the present invention shall be included in the appended claims.

Claims

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

2. The use according to claim 1, wherein the 6-MBOA reduces inflammatory infiltration, liver structural deformation, collagen deposition, expression level of α-SMA, liver hydroxyproline content, upregulation of serum transforming growth factor-β1, liver weight, liver index, TGF-β1-induced reactive oxygen species production and TGF-β1-induced fibronectin expression level in individuals who need to prevent and / or treat liver fibrosis.

3. The use according to claim 1, wherein the 6-MBOA negatively regulates the expression levels of phosphorylated maternal DPP homolog 2 and phosphorylated maternal DPP homolog 3 in an individual who needs to be prevented and / or treated for liver fibrosis.

4. The use according to claim 1, wherein the 6-MBOA increases superoxide dismutase activity, catalase activity, glutathione content and E-cadherin expression level, and reduces malondialdehyde content in an individual who needs to be prevented and / or treated for liver fibrosis.

5. The use according to claim 1, wherein the 6-MBOA inhibits hepatic stellate cell activation and epithelial-mesenchymal transition.

6. The use according to claim 3, wherein the negative regulation is achieved by reducing the expression levels of zinc finger proteins SNAIL1 / 2, TWIST1 / 2, zinc finger structure E-box-binding homeobox 1 and NADPH oxidase 4 through the 6-MBOA.

7. Use of an extract of Coix lachryma-jobi L. var. ma-yuenStapf containing 6-methoxybenzoxazolinone for preparing a composition for preventing and / or treating liver fibrosis.

8. The use according to claim 7, wherein the coix seed extract is obtained by extracting coix seed with a solvent, and the solvent is water, alcohol, a mixture of alcohol and water, or a combination thereof.

9. The use according to claim 7, wherein the coix seed extract down-regulates serum alanine aminotransferase, serum aspartate aminotransferase, serum total bilirubin, serum total glyceride and serum total cholesterol levels in individuals who need to be prevented and / or treated for liver fibrosis, and up-regulates serum glucose levels in individuals who need to be prevented and / or treated for liver fibrosis.

10. The use according to claim 7, wherein the coix seed extract reduces the levels of α-smooth muscle actin gene, type I collagen α1 gene, tumor necrosis factor-α gene and interleukin-6 gene in the liver of an individual who needs to be prevented and / or treated for liver fibrosis. The use according to claim 8, wherein the coix seed is the root, leaf or seed of coix seed.

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 pharmaceutically acceptable excipient, carrier, adjuvant and / or food additive.

16. The use according to claim 1 or 7, wherein the composition has a powdery, granular, solution, gel, or paste dosage form.

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