Use of 6-methoxybenzoxazolinone and adlay extract containing 6-methoxybenzoxazolinone for preventing and / or treating liver fibrosis

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 Coix lachryma-jobi L. containing 6-methoxybenzoxazolinone for preventing and / or treating liver fibrosis.

Background Art

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

[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 the accumulation of excessive extracellular matrix (ECM) occurs, the structure of the liver changes, further causing damage to liver function. The activation of hepatic stellate cells is regarded as a serious situation. When the liver is damaged, the activated hepatic stellate cells become the main source of extracellular matrix production.

[0004] In current clinical practice, the effect of drug treatment for liver fibrosis is limited and serious side effects occur, so many patients cannot receive continuous treatment. More importantly, drugs can only relieve 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 to be solved by the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0005] To solve the above problems, those skilled in the art focus on the development of new compositions for preventing and / or treating liver fibrosis in order to help the large number of people in trouble.

Means for Solving the Problems

[0006] In view of this, an object of the present invention is to provide the use of 6-methoxybenzoxazolinone (6-MBOA) for producing 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, upregulation of serum transforming growth factor beta 1 (TGF-β1), liver weight, liver function values, TGF-β1-induced reactive oxygen species (ROS) production, and TGF-β1-induced fibronectin expression level in an individual preventing and / or treating liver fibrosis.

[0008] In one embodiment of the present invention, 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.

[0009] In one embodiment of the present invention, the 6-MBOA improves the activities of superoxide dismutase (SOD), catalase (CAT), and the content of glutathione (GSH) in an individual for preventing and / or treating liver fibrosis, 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, the 6-MBOA suppresses the activation and epithelial-mesenchymal transition (EMT) of hepatic stellate cells (HSCs), including suppressing the expression of fibronectin and maintaining the expression level of E-cadherin.

[0011] In one embodiment of the present invention, the negative regulation is achieved by the 6-MBOA reducing 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 is to provide the use of a Coix lachryma-jobi L. extract containing 6-methoxybenzoxazolinone (6-MBOA) for producing a composition for preventing and / or treating liver fibrosis.

[0013] In one embodiment of the present invention, the adlay extract is obtained by extracting adlay 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 adlay extract down-regulates the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), total glyceride (TG), and total cholesterol (TC) in the serum of an individual who prevents and / or treats liver fibrosis, and up-regulates the glucose level in the serum of an individual who prevents and / or treats liver fibrosis.

[0015] In one embodiment of the present invention, the adlay 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 who prevents and / or treats liver fibrosis.

[0016] In one embodiment of the present invention, the adlay is the root, leaf, or seed of adlay.

[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, vector, adjuvant, and / or food additive.

[0021] In one embodiment of the present invention, the composition is in a powdery, granular, solution, gel, or paste dosage form.

Advantages of the Invention

[0022] In summary, the effects of the 6-methoxybenzoxazolinone and the adlay extract containing 6-methoxybenzoxazolinone according to the present invention are to improve liver fibrosis, reduce fibrosis and inflammatory factors, improve the oxidative stress of the liver by 6-MBOA, and reduce the generation of biomarkers of liver fibrosis by regulating the TGF-β / SMAD signaling pathway, suppress the progression of hepatic stellate cell mesenchymal transition, indicating the possibility of treating liver fibrosis with 6-methoxybenzoxazolinone and the adlay extract containing 6-methoxybenzoxazolinone.

[0023] Hereinafter, the embodiments for carrying out the invention will be further described. However, the examples listed below are for explaining the present invention and are not intended to limit the scope of the present invention. Those skilled in the art can make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is regarded as being determined by the scope of the patent application attached later.

Brief Description of the Drawings

[0024]

Figure 1

Figures 2A - 2F

Figures 3A - 3F

Figures 4A - 4E

Figures 5A - 5H

Figure 6

Figures 7A - 7K

Figures 8A - 8D

Figures 9A - 9G

Figures 10A - 10E

Figures 11A - 11F

Figures 12A - 12G

Figures 13A - 13J

Figures 14A - 14C

Mode for Carrying Out the Invention

[0025] Definitions The numerical values described in this specification are approximate values. All experimental data indicate that they are in the range of ±20%, preferably in the range of ±10%, more preferably in the range of ±5%.

[0026] In this specification (especially the claims), unless otherwise specified, the terms "one," "the," and similar terms include both the singular and plural forms.

[0027] Job's tears (Coix lachryma-jobi L., Adlay) (for example, Coix lachryma-jobi L. var. ma-yuen Stapf) according to the present invention, also known as yokuinin, shikoku wheat, and Indian millet, is an annual or perennial herbaceous plant of the Poaceae family, a commonly seen traditional Chinese medicine and dietary supplement in Asia, used for improving inflammatory diseases, and has a height of about 1 to 1.8 m and a cultivation period of about 5 months. It should be noted that Job's tears is composed of four parts in order from the outside to the inside: bran, shell, seed coat, and endosperm (polished Job's tears), and the four parts have their respective biological activities.

[0028] 6-Methoxybenzoxazolinone (6-MBOA) (CX) (chemical formula C8H7NO3) according to the present invention is a compound in gramineous plants and has activities of anti-hepatic fibrosis and hormone regulation.

[0029] The pharmaceutical composition according to the present invention may be manufactured into dosage forms for parenteral or oral administration by techniques well known to those skilled in the art. For example, 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, but are not limited thereto.

[0030] The pharmaceutical composition according to 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 according to the present invention is widely used in pharmaceutical manufacturing technology and may contain one pharmaceutically acceptable vector. The pharmaceutically acceptable vector may include, for example, one or more reagents selected from the group consisting of a solvent, an emulsifier, a suspending agent, a decomposer, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a lubricant, an absorption delaying agent, a liposome, and the like. The selection and quantity of these reagents are within the scope of the specialized knowledge and routine techniques of those skilled in the art.

[0032] The pharmaceutically acceptable vector according to the present invention may contain one solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), a sugar solution, an aqueous solution containing alcohol, and combinations thereof.

[0033] Hereinafter, the chemicals and reagents used in the following examples according to the present invention will be described. 6-Methoxybenzoxazolinone (6-MBOA) (CX) (purity ≥ 96.5%), thioacetamide (TAA) (purity ≥ 99.0%), 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). The TBARS and catalase (CAT) detection kits are purchased from Cayman Chemical (USA). The reduced glutathione (GSH) and superoxide dismutase (SOD) detection kits are purchased from Abbkine (Abbkine Scientific, China).

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

[0036] Antibodies against α-smooth muscle actin (α-SMA) (GTX100458), type I collagen α1 (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 (ZEB1) (GTX105278), β-actin (GTX109639), α-Tubulin (GTX628802), phosphorylated SMAD homolog 2 (phosphor-Mothers against decapentaplegic homolog 2, p-SMAD2) (GTX133614), phosphorylated SMAD homolog 3 (phosphor-Mothers against decapentaplegic homolog 3, p-SMAD3) (GTX129841), phosphorylated extracellular signal-regulated kinase 1 / 2 (extracellular signal-regulated kinase 1 / 2, ERK1 / 2) (GTX635617), phosphorylated phosphoinositide 3-kinase (phosphoinositide 3-kinase, PI3K) (GTX132597), SMAD2 / 3 (GTX111123), and horseradish peroxidase (HRP) are purchased from GeneTex (USA).

[0037] Note that the Zinc finger protein SNAIL1 / 2 (ab180714) is purchased from Abcam (UK).

[0038] The animals used in the following examples according to the present invention will be described below. Male C57BL / 6J mice (5 weeks old) are obtained from the National Laboratory Animal Center (Taipei, Taiwan). The mice are bred in a temperature-controlled room with a 12-hour light-dark cycle, and food and water are freely available (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 test of the adlay extract, the mice are randomly divided into the following seven groups (n = 6 for each group). (1) No treatment group (NT, normal diet), (2) Thioacetamide group (TAA, 200 mg / kg, normal diet), (3) 10% adlay powder (ASP) group, (4) 1% adlay leaf powder (ALP) group, (5) 1% adlay polysaccharide (ASPS) group, (6) 0.025% ethanol extract of adlay seeds (ASE) group, and (7) 0.1% ASE group. Pure physiological saline is given to the NT group, and physiological saline in which TAA is dissolved is intraperitoneally injected three times a week into the other groups.

[0040] For the test of CX, the mice are randomly divided into the following four groups (n = 8 for 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). Pure physiological saline is given to the NT group, and physiological saline in which TAA is dissolved is intraperitoneally injected three times a week into the other groups. In addition, 5% propylene glycol is further administered once a day via a feeding tube to the NT group and the TAA group, and 5% propylene glycol (prepared with 0.9% physiological saline) in which CX is dissolved is administered to the CX group.

[0041] When the experiment is completed, the mice are euthanized. The whole blood of the mice is centrifuged to obtain serum, which is stored at -80°C, and liver tissues are collected for histological staining.

[0042] The following describes the flow of statistical analysis according to the present invention. Statistical analysis is performed by one-way ANOVA, and Tukey's multiple comparison test is performed using GraphPad Prism 7. For cell viability data, two-way ANOVA is performed, and Dunnett's multiple comparison test is performed. All data are presented as mean ± standard deviation (SD). A difference with P < 0.05 is considered statistically significant.

[0043] The present invention will be further described 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 indicated in the patent application scope.

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

[0045] The following describes the flow of the schematic diagram of the animal experiment design of the Coix group. To verify the effect of Coix 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 Coix group. In this experiment, TAA is injected intraperitoneally into C57BL / 6J mice to induce liver fibrosis. Mice at 6 weeks of age are randomly divided into 7 groups and given different diets (NT, TAA, 10% ASP, 1% ALP, 1% ASPS, 0.025% ASE, and 0.1% ASE, n = 6).

[0046] After co-breeding for 6 weeks, TAA is injected intraperitoneally 3 times a week, and finally, the mice are euthanized at 12 weeks of age. NT indicates no treatment, ASP indicates the powder of Coix, ALP indicates the powder of the leaves of Coix, ASPS indicates the polysaccharide of Coix, and ASE indicates the ethanol extract of the seeds of Coix. Mice at 6 weeks of age are randomly divided into 7 groups: NT, TAA, 10% ASP, 1% ALP, 1% ASPS, 0.025% ASE, and 0.1% ASE groups (see Figure 1). The NT and TAA groups are given normal diet, and the Coix groups are given normal diet added with Coix extract. In addition, TAA or physiological saline is injected into the mice intraperitoneally 3 times a week for 6 weeks. After 6 weeks, the mice are euthanized, and liver tissues and sera are collected for later analysis.

[0047] Example 2. Effect of Coix extract on the pathological and physiological values of liver tissues of TAA-induced liver fibrosis mice After 6 weeks of exposure, the pathological changes of liver tissue sections stained with TAA-induced hematoxylin and eosin (H&E) and immunohistochemistry (IHC) are observed.

[0048] The following describes the procedure for pathological observation of liver tissue. After fixing the liver tissue in 10% neutral buffered formalin solution, it is embedded in paraffin and cut into sections with a thickness of 4 μm. A pathological section is examined using an H&E staining kit (CIS-Biotechnology, Taiwan) to evaluate changes in tissue structure. The situation of collagen deposition is evaluated using a Picrosirius red staining kit (CIS-Biotechnology, Taiwan). An immunohistochemistry kit (Agilent, USA), antibodies against phosphorylated SMAD2 (1:100, GeneTex, USA), phosphorylated SMAD3 (1:100, GeneTex, USA), and α-smooth muscle actin (α-SMA) (1:100, GeneTex, USA) are used to evaluate the expression of specific proteins in the tissue. All stainings are observed using an optical microscope (BEL Photonics, Italy).

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

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

[0051] Data are shown as mean ± standard deviation (SD) (n = 6 for each group). For the English letters described in the bar graph, the same English letter indicates that no significant difference was observed between the two groups (p > 0.05), and different English letters indicate that a significant difference was observed 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 shows inflammatory infiltration, liver structure deformation, and overexpression of α-SMA, while the Job's tears group shows a significant decrease in inflammatory infiltration, improvement in liver deformation, and a decrease in the expression of α-SMA. To detect the changes in the collagen content in the liver, hydroxyproline (a collagen component) is measured. As can be seen from Figure 2C, the hydroxyproline content in the TAA group is higher than that in the NT group, indicating an increase in collagen. On the other hand, the hydroxyproline content in all Job's tears groups significantly decreases, indicating that Job's tears can reduce TAA-induced liver collagen deposition.

[0053] As can be seen from FIGS. 2D and 2E, compared with the NT group, the TAA group shows a decrease in body weight and an increase in liver tissue. In the Coix group, the TAA-induced hepatomegaly in the 10% ASP, 1% ALP, 1% ASPS, and 0.1% ASE groups was significantly reduced, but no significant difference was observed in body weight. In FIG. 2F, to avoid individual differences, the changes in liver weight and body weight are corrected by the liver-to-body weight ratio. Compared with the TAA group, only the 0.025% ASE group shows no obvious changes, and the numerical values of liver function in other groups are significantly decreased.

[0054] Example 3. Effect of Coix extract on the biochemical properties of the liver in TAA-induced liver fibrosis mice The following describes the process of biochemical analysis. Using a biochemical analyzer, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), total cholesterol (TC), total glyceride (TG), and glucose level in serum are measured. That is, after centrifuging the whole blood of mice, the serum is collected and dropped onto a test chip, and analyzed by an automatic clinical chemistry analyzer (Fuji, Japan).

[0055] FIGS. 3A-3F show the effects of Coix extract on liver function damage and nutritional metabolism in TAA-induced liver fibrosis C57BL / 6J mice. FIGS. 3A-3C show the levels of ALT, AST, and TBIL, which are indicators of liver function damage in serum. FIGS. 3D-3F show the TG, TC, and glucose levels in serum for observing nutritional metabolism.

[0056] Data are shown as mean ± standard deviation (SD) (n = 6 for each group). For the English letters described in the bar graph, the same English letter indicates that no significant difference was observed between the two groups (p > 0.05), and different English letters indicate that a significant difference was observed 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 - 3C, compared with the NT group, only the levels of AST, ALT, and TBIL in the TAA group are elevated. In contrast, especially in the 10% ASP, 1% ALP, 1% ASPS, and 0.1% ASE groups, an up - regulation of the indicators of liver function damage due to the down - regulation of TAA in the Coix group is observed. On the other hand, as shown in Figures 3D - 3F, compared with the NT group, the levels of TG and TC in the TAA group are significantly elevated, and the glucose value is decreased. According to the treatment with Coix extract, the levels of TAA - induced TG and TC in all Coix groups are significantly improved, and the glucose values in the 10% ASP and 1% ASPS groups are maintained at the same level as the control group.

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

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

[0060]

Table 1

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

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

[0063] As shown in Figures 4A and 4B, compared with the NT group, the levels of the fibrosis markers α-SMA and COL1A1 in the TAA group were significantly increased. Conversely, the levels of α-SMA and COL1A1 in all Job's tears groups were significantly decreased. From this result, it can be seen 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 were 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 results of real-time PCR shown in FIGS. 4C to 4E, compared with the NT group, TNF-α and IL-6 in the TAA group were upregulated, and IL-10 was downregulated. There was no significant difference between the 1% ALP, 0.025% ASE, and 0.1% ASE groups and the TAA group, but all Job's tears extract groups decreased the mRNA levels of TAA-induced TNF-α and IL-6 and significantly upregulated the mRNA level of IL-10.

[0065] Example 5. Effects of Job's tears extract on HSC-T6 cell viability, fibrotic protein expression, and inflammatory gene levels The following describes the process of cell culture. HSC-T6 cells are an immortalized cell line isolated from primary stellate cells transformed with male Sprague-Dawley rat-transformed SV40 large T antigen. 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] Hepatic stellate cells (HSCs) are activated, and after culturing the cells for 24 hours, they are replaced with serum-free medium for 24 hours. Then, 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), they are treated with transforming growth factor beta 1 (TGF-β1) (10 ng / mL) for 24 hours, or the intracellular signaling pathway is carried out for 30 minutes.

[0067] The following describes the process of cell viability analysis. 1.5×10 4Inoculate HSC-T6 cells into a 96-well plate and incubate them with CX (0 - 800 μM) or Coix seed extract (0 - 800 μg / mL) for 24 hours. Then, use 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, USA) to measure the cell viability. That is, add MTT to each well to a final concentration of 0.5 μg / mL and react at 37°C for 1 hour. Then, remove the MTT and add dimethyl sulfoxide (DMSO) to dissolve the MTT-formazan crystals formed from metabolically active cells. Finally, use a spectrophotometer to detect the absorbance of each well at a wavelength of 570 nm.

[0068] The following describes the process of Western blotting analysis. Dissolve the cells in RIPA buffer containing protease inhibitors and centrifuge at 4°C, 12,000 rpm for 10 minutes. Collect the supernatant, quantify the total protein concentration to 50 μg, heat at 95°C for 10 minutes, then separate the protein samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transfer them to a PVDF membrane, block with 6% milk, react with the primary antibody overnight at 4°C, and react with the secondary antibody for 1 hour at room temperature. Use an ECL reagent (GeneDireX, USA) and display the image with an Amersham Imager 680 (Cytiva, USA).

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

[0070] Figures 5A - 5H show the effects of Job's tears extract on HSC-T6 cell viability, fibrotic protein expression, and inflammatory gene levels. Figure 5A is a schematic diagram showing the drug treatment of HSC-T6 cells. Figure 5B shows the cell viability of HSC-T6 cells treated with extracts from the leaves, roots, and seeds of Job's tears. 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 quantified by Image J software.

[0071] Figures 5F - 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 a concentration of 0 compared to other concentrations, ** indicates p < 0.01, and *** indicates p < 0.001.

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

[0073] Figure 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 Figure 5B, when the amount of the extract from the leaves and roots of Job's tears is less than 100 μg / mL and the amount of the extract from the seeds of Job's tears is less than 200 μg / mL, it has no significant effect on cell viability. Therefore, in the following experiments, Job's tears extracts at 20 and 50 μg / mL 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 FIGS. 5C to 5E, all adlay extracts dose-dependently decreased the expression of TGF-β1-induced α-SMA and COL1A. Regarding inflammation, real-time PCR was used to evaluate the mRNA levels of TNF-α, IL-6, and IL-10. As a result, as shown in FIGS. 5F to 5H, all adlay extracts downregulated the mRNA levels of TGF-β1-induced TNF-α and IL-6 and upregulated the mRNA level of IL-10 in a dose-dependent manner. As can be seen from the changes in fibrotic proteins and inflammatory genes, adlay extracts improve the inflammation and HSC activation of HSC-T6 cells treated with TGF-β1.

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

[0076] In FIG. 6, 6-week-old mice are randomly divided into four groups: NT, TAA, CX20, and CX50. The NT group is given vehicle via a feeding tube. The TAA group is administered vehicle and TAA via a feeding tube. The CX group is administered CX and TAA via a feeding tube. It is administered once a day via a feeding tube for 6 weeks, and mice are injected intraperitoneally three times a week. Then, the mice are euthanized, and liver tissues and sera are collected for later analysis.

[0077] Example 7. Effects of 6-methoxybenzoxazolinone (6-MBOA) (CX) on the pathological and physiological values of liver tissues of TAA-induced liver fibrosis mice To observe the effect of CX on liver tissue pathology, liver fibrosis mice are treated with CX, and liver tissues are stained with H&E and picrosirius red. Furthermore, α-SMA, a fibrosis marker of liver sections, and p-SMAD2 and p-SMAD3, proteins related to the TGF-β / SMAD pathway, are detected by IHC staining.

[0078] The following describes the procedure for the enzyme-linked immunosorbent assay (ELISA) of TGF-β1. A commercially available mouse ELISA kit (Invitrogen, USA) is used to detect TGF-β1 levels in serum. The operation 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 sequence. Immediately after reacting with the stop solution, the color intensity at 450 nm is measured by an ELISA reader (TECAN, Switzerland).

[0079] Figures 7A to 7K show the effects of CX on the pathological and physiological values of liver tissues of 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 pathological histological changes of the liver by H&E, picrosirius red, and IHC staining (original magnification is ×200, scale bar is 100 μm). Figure 7C shows the results of quantifying collagen picrosirius red staining. Figures 7D to 7F show the results of quantifying IHC staining of α-SMA, p-SMAD2, and p-SMAD3. Figure 7G shows the change in the content of liver 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 the change in liver weight divided by body weight, that is, the liver-to-body weight ratio. Picrosirius red and IHC staining are quantified using Image J. Data are shown as mean ± standard deviation (SD) (n = 8 for each group). For the English letters described in the bar graph, the same English letter indicates that no significant difference was observed between the two groups (p > 0.05), and different English letters indicate that a significant difference was observed between the two groups (p < 0.05).

[0081] As shown in Figures 7B - 7F, compared with the NT group, the TAA group shows inflammatory infiltration, liver structural deformation, collagen deposition, and overexpression of α-SMA and p-SMAD2 / 3. The CX20 and CX50 groups show a significant decrease in inflammatory infiltration, liver structural deformation, collagen deposition, and the expression of α-SMA. In addition, the CX50 group negatively regulates the expression of p-SMAD2 and p-SMAD3. From this, it is shown that CX improves TAA-induced injury through the SMAD signaling pathway.

[0082] As shown in Figure 7G, since the hydroxyproline content in the TAA group is higher than that in the NT group, and the hydroxyproline content in the CX group is lower than that in the TAA group, it can be seen that CX treatment reduces the collagen content. TGF-β1 is an important cytokine related to fibrosis. Changes in TGF-β1 are considered indicators of fibrosis progression. As shown in Figure 7H, as can be seen from the ELISA results, compared with the NT group, the TGF-β1 expression in the TAA group is significantly improved, and CX can significantly reduce TAA-induced TGF-β1 upregulation.

[0083] As can be seen from FIGS. 7I and 7J, single injection of TAA causes liver hypertrophy and weight loss, and CX treatment reduces liver weight, but no obvious improvement is seen in the weight change caused by TAA. To avoid individual differences in mice, the liver weight ratio is used to adjust liver weight and body weight. As shown in FIG. 7K, compared with the NT group, the liver weight ratio of the TAA group is significantly improved, and compared with the TAA group, the liver weight ratio of the CX group is significantly decreased. From this result, CX reduces typical features 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. Effect of CX on Oxidative Stress and Biochemical Characteristics in TAA-Induced Liver Fibrosis Mice Liver injury is caused by oxidative stress due to the active metabolite of TAA. Organisms can remove oxidative free radicals by antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). The lipid peroxidation product malondialdehyde (MDA) is produced after the oxidation of polyunsaturated fatty acids and is an indicator of oxidative stress in cells, tissues, organs, or organisms. It is used to detect the oxidative stress of TAA-induced fibrotic mice, and an increase in the content of MDA indicates an increase in oxidative pressure.

[0085] The following describes the procedure for detecting the indicators of oxidative stress. By detecting the enzyme activities of catalase (CAT) and superoxide dismutase (SOD) in tissues, the content of malondialdehyde (MDA), and the concentration of glutathione (GSH), the change in the level of oxidative stress in TAA-induced liver fibrosis mice is detected. Hydroxyproline is a component of collagen precursors. All test kits are commercially available products and work according to the manufacturer's guidelines. That is, liver tissue is homogenized with ice-cold buffer, centrifuged at 4°C, and then the supernatant is collected. Finally, the absorbance of the sample is measured using an ELISA reader (TECAN, Switzerland).

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

[0087] As shown in Figures 8A - 8D, compared with the NT group, the activities of SOD and CAT and the content of GSH in the TAA group decrease, while the content of MDA increases. Compared with the TAA group, the activities of CAT and the content of GSH in the CX20 and CX50 groups increase, while the content of MDA decreases. In addition, since the activity of SOD is significantly improved in the CX50 group, it indicates that CX has the effect of improving oxidative stress.

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

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

[0090] Example 9. Effect 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 was used to detect the mRNA levels of fibrotic genes. Figures 10A to 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 the mRNA levels of α-SMA and COL1A1 in the liver using real-time PCR. Figures 10C to 10E show the mRNA levels of the inflammatory factors TNF-α, IL-6, and IL-10 in the liver.

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

[0092] As shown in FIGS. 10A and 10B, compared with the NT group, the TAA group significantly upregulated the mRNA levels of α-SMA and COL1A1, and the CX group significantly decreased the gene translation of TAA-induced fibrosis markers. Since inflammation is required for the progression of fibrosis, the inflammatory status can be grasped by detecting the mRNA levels of the inflammatory genes TNF-α and IL-6 and the anti-inflammatory gene IL-10. Compared with the NT group, the TAA group induced an increase in IL-6 and TNF-α and a decrease in IL-10. As can be seen from FIGS. 10C to 10E, CX treatment significantly reduced the upregulation of TAA-induced IL-6 and TNF-α levels and the downregulation of the mRNA level of IL-10. As can be seen from these results, CX reduces the expression of the mRNA of inflammatory genes in the gene translation of TAA-induced fibrotic mice and suppresses the subsequent progression of cell fibrosis.

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

[0094] The following describes the process of ROS detection. 2’,7’-Dichlorofluorescein diacetate (DCFDA) is a cell-permeable fluorescent probe and is used for the detection of ROS and nitric oxide in living cells. When DCFDA is oxidized by ROS in cells, it is rapidly de-esterified to form fluorescent 2’,7’-dichlorofluorescein (DCF). For ROS detection, cells were seeded in a 96-well plate for 24 hours, changed to serum-free medium for drug treatment, and then the medium was replaced with DCFDA solution for 30 minutes. The excitation / emission spectra at 485 / 525 nm were detected using multimode microplate readers (TECAN, Switzerland).

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

[0096] Data are shown as the mean ± standard deviation (SD) (n = 3 for each group). * in the bar graph of cell viability indicates a concentration of 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 letters indicate that no significant difference was observed between the two groups (p > 0.05), and different English letters indicate that a significant difference was observed between the two groups (p < 0.05).

[0097] As shown in Fig. 11B, at doses less than 400 μM, there is no significant effect on cell viability, that is, it shows no toxicity. Therefore, for subsequent cell measurements, 10, 20, 50, and 100 μM are used. 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 Figs. 11C to 11E, TNF-α and IL-6 are significantly increased by TGF-β1 treatment, and the mRNA level of IL-10 is decreased.

[0098] CX dose-dependently decreases TGF-β1-induced TNF-α and IL-6, and at the highest dose, significantly improves the mRNA level of IL-10. In addition, the DCFDA assay is used to detect the ROS level in HSC-T6 cells. As shown in Fig. 11F, TGF-β1 induces the production of ROS, and CX decreases the TGF-β1-induced ROS level with the increase in dose. As can be seen from these results, TGF-β1 induces inflammation and the production of ROS, while CX shows the potential to reduce inflammation and ROS in HSC-T6 cells activated by TGF-β1.

[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 following describes the process of measuring wound healing. HSC-T6 cells are inoculated in a 6-well plate for 24 hours, the monolayer cells are detached with a 200 μL micropipette tip, the unattached cells are washed away with PBS, and after 24 hours, the medium is replaced with serum-free medium and treated with TGF-β1 and CX for 0, 24, and 48 hours. An inverted microscope (Olympus, Japan) is used to observe the images of cell migration, and the wound area is quantified by Image J software.

[0101] Figures 12A - 12G 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 of E-cadherin, fibronectin, α-SMA, and COL1A1. Figures 12B - 12E show the results of quantifying the Western blotting of E-cadherin, fibronectin, α-SMA, and COL1A1 by Image J software. Figures 12F and 12G show the results of quantifying the results of the wound healing assay at 0 h, 24 h, and 48 h (magnification is ×200, scale bar is 100 μm) by Image J software.

[0102] The definition of wound healing is (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 for each group). For the English letters described in the bar graph, 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).

[0103] Markers for epithelial-mesenchymal transition or fibrosis include α-SMA, COL1A1, and fibronectin. Normal tissue structure may be replaced by cell injury and mesenchymal transition, ultimately leading to organ failure. As shown in Figures 12A - 12E, TGF-β1 treatment upregulates the expression of α-SMA, COL1A1, and fibronectin, which are mesenchymal markers, and downregulates E-cadherin, which is an epithelial marker.

[0104] CX treatment reduces the expression of TGF-β1-induced α-SMA, COL1A1, and fibronectin, but even at the lowest dose, it significantly improves the expression of E-cadherin. As EMT progresses, cell migration ability increases. Therefore, the wound healing assay is used to observe changes in migration.

[0105] As shown in FIGS. 12F and 12G, compared with the NT group, the wound healing percentage of the TGF-β1 group is significantly improved. 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. Effects of CX on the TGF-β / SMAD homolog (Mothers against decapentaplegic homolog, SMAD) and stromal migration transcriptional signaling pathway of HSC-T6 cells Use Western blotting to study the mechanism of CX on HSC activation and EMT.

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

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

[0109] Generally speaking, as shown in FIGS. 12C to 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 FIGS. 13F to 13H, the signal is transmitted to the cell nucleus, promoting the transcription of EMT-related proteins in the cell nucleus, namely SNAIL1 / 2, TWIST1 / 2, and ZEB1 genes. At this time, as shown in FIG. 12B, the expression of cadherin decreases, as shown in FIG. 12C, the expression of fibronectin increases, as shown in FIGS. 12D to 12E, the collagen COL1A1 and tubulin α-SMA in the liver increase, and as shown in FIG. 7B, ultimately liver fibrosis is caused. As shown in FIG. 11F, TAA induces liver damage and simultaneously causes ROS and an inflammatory response. The main markers are TNF-α and IL-6.

[0110] As shown in FIGS. 11C to 11D, these two inflammatory hormones increase, and as shown in FIG. 11E, the anti-inflammatory hormone IL-10 decreases. Simultaneously with the occurrence of the inflammatory response, as shown in FIG. 13I, the expression of the related protein NOX4 increases, as shown in FIGS. 8A to 8C, the expression levels of the antioxidant stress proteins SOD, CAT, and GSH decrease, and as shown in FIG. 8D, the metabolite MDA of oxidative stress also increases. By combining the above reactions, as shown in FIG. 7B, ultimately liver fibrosis is caused. As can be seen from FIGS. 13B to 13I, compared with the NT group, the adverse reactions of the above reactions in the TGF-β1 group are significantly improved. From this, it is shown that TGF-β1 can phosphorylate SMAD2 and SMAD3, activate downstream proteins SNAIL1 / 2, TWIST1 / 2, ZEB1, and NOX4, and further generate ROS to induce EMT.

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

[0112] FIGS. 14A to 14C show the effect of CX on the signal transduction pathways of phosphorylated phosphoinositide 3-kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (ERK) in TGF-β1-activated HSC-T6 cells. FIG. 14A shows the results of Western blotting with α-tubulin used to normalize p-PI3K and p-ERK1 / 2. FIGS. 14B and 14C show the results of quantifying the Western blotting of p-PI3K and p-ERK1 / 2 using Image J.

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

[0114] As shown in FIGS. 14A to 14C, for the non-SMAD pathway, CX does not cause significant changes in the phosphorylation of PI3K and ERK and has little effect on the signal transduction pathways of ERK and PI3K.

[0115] Summarizing the above, the 6-methoxybenzoxazolinone and the adlay extract containing 6-methoxybenzoxazolinone according to the present invention have the effect of improving liver fibrosis by reducing fibrosis 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 6-methoxybenzoxazolinone and the adlay extract containing 6-methoxybenzoxazolinone show the potential to treat liver fibrosis.

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

Claims

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

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

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

5. The 6-MBOA according to claim 1, wherein the 6-MBOA suppresses the activation and epithelial-mesenchymal transition (EMT) of hepatic stellate cells (HSCs), including suppression of fibronectin expression and maintenance of E-cadherin expression level, in an individual for preventing and / or treating liver fibrosis.

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

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