Methods for the prevention and / or treatment of liver fibrosis using 6-methoxybenzoxazolinone and Coix lachryma-Jobi L. extract containing 6-methoxybenzoxazolinone

Abstract

The present invention provides a method for the prevention and / or treatment of liver fibrosis using 6-methoxybenzoxazolinone and a Coix lachryma-jobi L. extract containing 6-methoxybenzoxazolinone. The 6-methoxybenzoxazolinone and the Coix lachryma-jobi L. extract containing 6-methoxybenzoxazolinone of the present invention achieve the effect of preventing and / or treating liver fibrosis, as demonstrated in various efficacy experiments.

Description

INFORMATION ON THE SEQUENCE LISTING

[0001] The sequence listing associated with this application is provided in text form instead of a paper copy and is hereby incorporated into the description by reference. The name of the XML file containing the sequence listing is sl. The XML file is 22,101 bytes in size and was created on July 11, 2023. BACKGROUND OF THE INVENTION 1. Field of the Invention

[0002] The present invention relates to a method for the prevention and / or treatment of liver fibrosis using 6-methoxybenzoxazolinone (6-MBOA) and a Coix lachryma-jobi L. extract containing 6-methoxybenzoxazolinone. 2. State of the art

[0003] The liver is an important organ in the human body, composed of parenchymal cells and non-parenchymal cells. Parenchymal cells, also called hepatocytes, account for 60% of the cells in the total liver and comprise 80% of the liver's volume. Non-parenchymal cells consist of other biologically important cells, including sinus endothelial cells, Kupffer cells, hepatic stellate cells (HSCs), and hepatic natural killer cells, in which each type of cell disclosed above accounts for 3 to 20% of the number.

[0004] Liver fibrosis is characterized by excessive wound repair in injuries caused by viral infection, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), autoimmune liver disease (AILD), and cholestatic liver injury. Accompanied by redundant extracellular matrix (ECM) deposition, liver fibrosis not only alters liver structure but also undermines liver function. Hepatic stellate cell (HSC) activation plays an essential role because activated HSCs are the main producers of ECM in a damaged liver.

[0005] Currently, clinical drug treatment for liver fibrosis has limited efficacy and serious side effects, and many patients cannot be further treated. More importantly, these drugs only alleviate symptoms but fundamentally fail to solve the problem of neurodegeneration and death. Therefore, how to develop a new drug that can truly treat and / or prevent liver fibrosis is an important problem that the present invention aims to solve.

[0006] To solve the above-mentioned problems, the specialist urgently needs to develop a new composition for the prevention and / or treatment of liver fibrosis for the benefit of a large group of people who need it. SUMMARY OF THE INVENTION

[0007] A primary object of the present invention is to provide a method for preventing and / or treating liver fibrosis comprising administering to a subject in need thereof a composition containing an effective amount of 6-methoxybenzoxazolinone (6-MBOA).

[0008] According to one embodiment of the present invention, 6-MBOA reduces inflammatory infiltration, liver structural remodeling, collagen deposition, α-smooth muscle actin (α-SMA) expression level, liver hydroxyproline content, serum transforming growth factor-β1 (TGF-β1) concentration, liver weight index, reactive oxygen species (ROS) production, and TGF-β1-induced fibronectin expression level in the subject in need thereof.

[0009] According to one embodiment of the present invention, 6-MBOA negatively regulates the expression levels of phosphor-SMAD2 (p-SMAD2) and phosphor-SMAD3 (p-SMAD3) in a subject in need thereof.

[0010] According to one embodiment of the present invention, 6-MBOA increases superoxide dismutase (SOD) activity, catalase (CAT) activity and glutathione (GSH) content, decreases the expression of NADPH oxidase 4 (NOX4) and decreases malondialdehyde (MDA) content caused by oxidative stress.

[0011] According to one embodiment of the present invention, 6-MBOA inhibits hepatic stellate cell (HSC) activation and epithelial-mesenchymal transition (EMT), including inhibiting the expression of fibronectin and maintaining E-cadherin expression levels.

[0012] According to one embodiment of the present invention, 6-MBOA reduces the expression levels of zinc finger protein SNAIL1 / 2, TWIST1 / 2 and zinc finger E-box binding homeobox 1 (ZEB1).

[0013] Another object of the present invention is to provide a method for preventing and / or treating liver fibrosis comprising administering to a subject in need thereof a composition containing an effective amount of Coix lachryma-jobi L. extract containing 6-methoxybenzoxazolinone (6-MBOA).

[0014] According to one embodiment of the present invention, the Coix lachryma-jobi L. extract is obtained by extracting Coix lachryma-jobi L. with a solvent, and the solvent is water, alcohol, a mixture of alcohol and water, or a combination thereof.

[0015] According to one embodiment of the present invention, the Coix lachryma-jobi L. extract reduces the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), total glyceride (TG) and total cholesterol (TC) in the subject in need thereof.

[0016] According to one embodiment of the present invention, the Coix lachryma-jobi L. extract reduces the levels of gene expression and protein including smooth muscle actin (α-SMA), collagen type I alpha 1 (COL1A1), fibronectin, and maintains normal E-cadherin protein levels compared to the control, indicating that epithelial-mesenchymal transition (EMT) is prevented. Gene expression (mRNA) of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) is reduced. In contrast, the anti-inflammatory cytokine interleukin (IL)-10 is increased, indicating the anti-inflammatory properties of Coix lachryma-jobi L. extracts.

[0017] According to one embodiment of the present invention, the Coix lachryma-jobi L. is root, leaf or seed of the Coix lachryma-jobi L.

[0018] According to one embodiment of the present invention, the composition is a pharmaceutical composition, a food composition or a topical composition.

[0019] According to one embodiment of the present invention, the pharmaceutical composition is in a dosage form for oral administration.

[0020] According to one embodiment of the present invention, the pharmaceutical composition is in a dosage form for parenteral administration.

[0021] According to one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, adjuvant and / or food additive.

[0022] According to one embodiment of the present invention, the composition has a dosage form as a powder, granules, solution, jelly or paste.

[0023] In summary, 6-methoxybenzoxazolinone (6-MBOA) and the Coix lachryma-jobi L. extract containing 6-MBOA of the present invention achieve the effect of improving liver fibrosis in vivo and in vitro, accompanied by the downregulation of fibrotic and inflammatory factors. Furthermore, 6-MBOA improves oxidative stress in the liver and reduces the production of liver fibrotic biomarkers through the TGF-β / SMAD signaling pathway, inhibiting the further development of HSCs toward mesenchymal transition. Both 6-MBOA and the Coix lachryma-jobi L. extract containing 6-MBOA possess therapeutic potential for liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings are part of the present specification and are included here to further demonstrate some aspects of the present invention, which may be better understood by reference to one or more of these drawings, in combination with the detailed description of the embodiments presented herein. Fig.Figure 1 is a schematic diagram of an animal experiment design in Adlay groups. 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 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, during which the intraperitoneal injection of TAA was administered three times a week, the mice were finally sacrificed at 12 weeks of age. NT represents no treatment, ASP represents Adlay seed powder, ALP represents Adlay leaf powder, ASPS represents Adlay seed polysaccharide, and ASE represents Adlay seed ethanol extract. The Fig.Figures 2A–2F show the effects of Coix lachryma-jobi L. extract on liver histopathology and physiological parameters in TAA-induced fibrotic C57BL / 6J mice. (2A) Histopathological changes of the liver observed with hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining (original magnification × 200, and the scale bar is 100 µm). (2B) Quantifications of IHC staining of α-smooth muscle actin (α-SMA) performed using Image J software. (2C) Changes in liver hydroxyproline content. (2D, 2E) Changes in liver weights and body weights. (2F) Ratio of liver weight to body weight, ratio of change in liver weight divided by body weight. Data were presented as mean ± standard deviation (SD) (n = 6 per group).The bars are marked with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). The Fig.Figures 3A-3F show the effects of Coix lachryma-jobi L. extract on liver injury and nutrient metabolism in TAA-induced fibrotic C57BL / 6J mice. (3A-3C) Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) indicate liver injury. (3D-3F) Serum levels of total glyceride (TG), total cholesterol (TC), and glucose are used to observe nutrient metabolism. Data were presented as mean ± SD (n = 6 per group). Bars are labeled with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). The Fig.Figures 4A-4E show the effects of Coix lachryma-jobi L. extract on fibrotic and inflammatory gene levels in TAA-induced fibrotic C57BL / 6J mice. (4A, 4B) mRNA levels of fibrotic markers α-SMA and collagen type I alpha 1 (COL1A1). (4C-4E) mRNA levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10. Data were presented as mean ± SD (n = 6 per group). Bars are labeled with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). The Fig.Figures 5A-5H show the effects of different concentrations of Coix lachryma-jobi L. (adlay) extract on cell viability, fibrotic protein expression, and inflammatory gene levels in HSC-T6 cells. (5A) Schematic representation of drug treatment in HSC-T6 cells. (5B) Cell viability of HSC-T6 cells treated with different concentrations of adlay leaf, root, and seed extracts. (5C-5E) Western blot results of α-SMA and COL1A1 in HSC-T6 cells treated with TGF-β1 and adlay extracts, and quantification of the results using Image J software. (5F-5H) mRNA levels of inflammatory factors in HSC-T6 cells treated with TGF-β1 and adlay extracts. Data were presented as mean ± SD (n = 3 per group). The cell viability bars are * , 0 of the concentration vs. other concentrations marked, marked with ** (p < 0.01); ***(p < 0.001). The bars of the Western blot and real-time PCR are marked with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). In the Fig. 5C-5H the letters “L”, “R”, “S” represent extracts of leaf, root or seed respectively. Fig.Figure 6 is a schematic diagram of an animal experiment design in 6-methoxybenzoxazolinone (6-MBOA) (CX) groups. For animal experiments, 6-week-old C57BL / 6J mice were randomly divided into four groups, including NT, TAA, CX20 (TAA + 20 mg / kg CX), and CX50 (TAA + 50 mg / kg CX) (n = 8 per group). Mice received an intraperitoneal injection of TAA three times a week to induce liver fibrosis; they also received CX or the vehicle by oral gavage once a day. At the end of the animal experiments, mice were sacrificed at 12 weeks of age. The Fig.Figures 7A–7K show the effects of CX on liver histopathology and physiological parameters in TAA-induced fibrotic C57BL / 6J mice. (7A) Chemical structure of CX (C8H7NO3, MW = 165.15). (7B) Histological changes of the liver observed by H&E, Sirius Red, and IHC staining (original magnification × 200, and the scale bar is 100 µm). (7C) Quantifications of Sirius Red staining of collagen. (7D–7F) Quantifications of IHC staining of α-SMA, phospho-SMAD2 (phospho-SMAD2), and phospho-SMAD3 (phospho-SMAD3). (7G) Changes in liver hydroxyproline content. (7H) Expression of TGF-β1 in serum. (7I, 7J) Changes in liver and body weights. (7K) Liver weight to body weight ratio. Quantification of Sirius Red and IHC staining was achieved using Image J software. Data were presented as mean ± SD (n = 8 per group).The bars are marked with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). The Fig. Figures 8A–8D show the effects of CX on antioxidant enzymes and lipid peroxidation in TAA-induced fibrotic C57BL / 6J mice. (8A, 8B) Changes in the activity of superoxide dismutase (SOD) and catalase (CAT). (8C) Changes in the level of glutathione (GSH). (8D) Changes in the level of malondialdehyde (MDA). Data were presented as mean ± SD (n = 8 per group). Bars are labeled with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). The Fig.Figures 9A-9G show the effects of CX on liver injury and nutrient metabolism in TAA-induced fibrotic C57BL / 6J mice. (9A-9D) Serum levels of liver injury indicate ALT, AST, TBIL, and alkaline phosphatase (ALP). (9E-9G) Serum levels of TG, TC, and glucose are used to observe nutrient metabolism. Data were presented as mean ± SD (n = 8 per group). Bars are marked with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). ALP represents Adlay leaf powder. The Fig.Figures 10A–10E show the effects of CX on fibrotic and inflammatory gene levels in TAA-induced fibrotic C57BL / 6J mice. (10A, 10B) Liver mRNA levels of α-SMA and COL1A1 detected using real-time PCR. (10C–10E) Liver mRNA levels of inflammatory factors TNF-α, IL-6, and IL-10. Data were presented as mean ± SD (n = 8 per group). Bars are labeled with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). The Fig.Figures 11A-11F show the effects of CX on cell viability, inflammatory gene levels, and reactive oxygen species (ROS) in TGF-β1-activated HSC-T6 cells. (11A) Schematic representation for TGF-β1 and CX treatment in HSC-T6 cells. 6-MBOA represents 6-methoxybenzoxazolinone. (11B) Cell viability of HSC-T6 cells treated with different concentrations of CX. (11C-11E) mRNA levels of inflammatory factors in HSC-T6 cells treated with TGF-β1 and CX. (11F) ROS levels of HSC-T6 cells detected with DCFDA and normalized with protein content. Data were presented as mean ± SD (n = 3 per group). Cell viability bars are labeled * , 0 of the concentration vs. other concentrations marked, marked with ***(p < 0.001). The bars of the Western blot and real-time PCR are labeled with different letters; the same letter indicates no significant difference between the two samples (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). The Fig.Figures 12A-12G show the effects of CX on HSC activation and epithelial-mesenchymal transition (EMT) in TGF-β1-activated T6 HSC cells. (12A) Analysis elements include Western blot results of E-cadherin, fibronectin, α-SMA, and COL1A1. (12B-12E) Quantifications of Western blots of E-cadherin, fibronectin, α-SMA, and COL1A1 were performed using Image J software. (12F, 12G) Wound healing assay results were additionally measured at 0, 24, and 48 hours (magnification ×200, scale bar is 100 µm) and quantified using Image J software. Wound closure is defined as (wound area at 0 h - wound area at 24 or 48 h) / wound area at 0 h × 100%. Data were presented as mean ± SD (n = 3 per group).The data bars are marked with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). The Fig.Figures 13A–13J show effects of CX on TGF-β / SMAD signaling in TGF-β1-activated T6 HSC cells. (13A) Schematic representation of TGF-β1 pathway stimulation and CX treatment in T6 HSC cells. (13B) Western blot results of phospho-SMAD2 (p-SMAD2), phospho-SMAD3 (p-SMAD3), SMAD2 / 3, zinc finger protein SNAIL1 / 2, TWIST1 / 2, zinc finger E-box-binding homeobox 1 (ZEB1), and NADPH oxidase 4 (NOX4) normalized with α-tubulin. (13C-13I) Western blot quantifications of p-SMAD2, p-SMAD3, SMAD2 / 3, SNAIL1 / 2, TWIST1 / 2, ZEB1, and NOX4 using Image J software. Data were presented as mean ± SD (n = 3 per group). Data bars are labeled with different letters; the same letter indicates no significant difference between the two samples (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). Fig.Figure 13J illustrates the process of the present invention targeting signal transduction, gene transcription, translation, and protein expression of liver fibrosis, and the process that CX can completely inhibit liver fibrosis starting from the signal transduction of TGF-β1. The Fig.Figures 14A–14C show the effects of CX on phosphoinositide 3-kinase (PI3K) and the extracellular signal-regulated kinase (ERK) pathway in TGF-β1-activated HSC-T6 cells. (14A) Western blot results of p-PI3K and p-ERK1 / 2 normalized with α-tubulin. (14B, 14C) Western blot quantifications of p-PI3K and p-ERK1 / 2 using Image J. Data were presented as mean ± SD (n = 3 per sample). Data bars are labeled with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0025] In the following detailed description of the embodiments of the present invention, reference is made to the accompanying drawings, which are shown to illustrate the specific embodiments in which the present disclosure may be practiced. These embodiments are provided to enable those skilled in the art to practice the present disclosure. It is understood that other embodiments may be utilized and that changes may be made to the embodiments without departing from the scope of the present invention. The following description, therefore, is not to be considered limiting the scope of the present invention. definition

[0026] As used herein, the data provided represent experimental values ​​which may vary within a range of ±20%, preferably within ±10%, and most preferably within ±5%.

[0027] Unless the context indicates otherwise, “a,” “a,” “the,” and similar terms used in the description (particularly in the claims that follow) should be construed as including both singular and plural forms.

[0028] According to the present invention, Coix lachryma-jobi L. (e.g., Coix lachryma-jobi L. var. ma-yuen Stapf), also known as Adlay, Coix seed, Chinese pearl barley, Semen coicis, and Yokuinin, is an annual or perennial herb of the Gramineae family. Adlay is a popular herbal medicinal product and dietary supplement in Asia and is often taken to improve inflammatory diseases. It is about 1 to 1.8 meters tall, and the cultivation period is about 5 months. Furthermore, the Adlay seed consists of four parts from the outside to the inside, including the bran, the hull, the testa, and the endosperm (polished Adlay), each with its own biological activities.

[0029] According to the present invention, 6-methoxybenzoxazolinone (6-MBOA) (CX) (formula: C8H7NO3) is a compound from Gramineae plants and has anti-liver fibrosis and hormone modulating activities.

[0030] According to the present invention, the pharmaceutical composition may be prepared in a dosage form suitable for parenteral or oral administration using techniques well known to those skilled in the art, including, but not limited to, injection (e.g., sterile aqueous solution or dispersion), sterile powder, tablet, lozenge, pellet, capsule, dispersible powder or granules, solution, suspension, emulsion, syrup, elixir, slurry, and the like.

[0031] The pharmaceutical composition according to the present invention can be administered by 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.

[0032] The pharmaceutical composition according to the present invention may comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more reagents selected from the group consisting of solvent, emulsifier, suspending agent, disintegrator, binder, excipient, stabilizer, chelating agent, diluent, gelling agent, preservative, lubricant, absorption delaying agent, liposome, and the like. The selection and amount of these reagents are within the scope of the skill and routine techniques of the person skilled in the art.

[0033] 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.

[0034] 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'-dichlorofluorescin diacetate (DCFDA, purity ≥97.0%) were purchased from Sigma-Aldrich (Sigma-Aldrich, USA). Recombinant human transforming growth factor beta 1 (TGF-β1) was purchased from PeproTech (PeproTech, USA). TBARS and catalase (CAT) assay kits were purchased from Cayman Chemical (Cayman Chemical, USA). Reduced glutathione (GSH) and superoxide dismutase (SOD) assay kits were purchased from Abbkine (Abbkine Scientific, China). The hydroxyproline assay kit was purchased from Elabscience (Elabscience Biotechnology, China). The mouse TGF-β1 ELISA kit was purchased from Invitrogen (Thermo Fisher Scientific, USA).Antibodies against smooth muscle actin (α-SMA) (GTX100458), collagen, type I, alpha 1 (COL1A1) (GTX112731), fibronectin (GTX112794), E-cadherin (GTX629691), NADPH oxidase 4 (NOX4) (GTX121929), TWIST1 / 2 (GTX127310), zinc finger E-box binding homeobox 1 (ZEB1) (GTX105278), β-actin (GTX109639), α-tubulin (GTX628802), phospho-SMAD2 (phospho-2) (GTX133614), phospho-SMAD3 (phospho-3) (phosphoinositide 3-kinase (PI3K)) (GTX129841), phosphoinositide 3-kinase (ERK) (PI3K) (GTX132123), and horseradish peroxidase (HRP) (Gene Gene) (GTX111123) were purchased from GeneTex (GeneTex, USA). Zinc finger protein SNAIL1 / 2 (ab180714) was also purchased from Abcam (Abcam, UK).

[0035] According to the present invention, animals used in the following examples are described as follows. Male C57BL / 6J mice (5 weeks old) were obtained from the National Laboratory Animal Center (Taipei, Taiwan) and housed in a temperature-controlled room on a 12-h light / dark cycle with free access to food and drinking water at the Animal Center of National Yang Ming Chiao Tung University (Taipei, Taiwan). All experiments were approved by the Institutional Animal Care and Use Committee of National Yang Ming Chiao Tung University.

[0036] For testing Adlay extracts, mice were randomly divided into the following seven groups (n = 6 per group): (1) no treatment group (NT, normal diet), (2) thioacetamide group (TAA, 200 mg / kg, normal diet), (3) 10% Adlay seed powder (ASP) group, (4) 1% Adlay leaf powder (ALP) group, (5) 1% Adlay seed polysaccharide (ASPS) group, (6) 0.025% Adlay seed ethanol extract (ASE) group, and (7) 0.1% ASE group. The NT group received plain saline, and other groups were injected intraperitoneally with TAA dissolved in saline three times a week.

[0037] For CX testing, mice were randomly divided into the following four groups (n = 8 per 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 received pure saline, while other groups were intraperitoneally injected with TAA dissolved in saline three times a week. In addition, the NT group and TAA group also received 5% propylene glycol, while the CX group received CX dissolved in 5% propylene glycol (prepared in 0.9% saline) by oral gavage once a day.

[0038] At the end of the experiment, the mice were sacrificed. Serum was obtained from the mice's whole blood after centrifugation and stored at -80°C. Liver tissues were collected and used for tissue histological staining.

[0039] According to the present invention, the method 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 were presented as mean ± standard deviation (SD). Differences of P value < 0.05 were considered statistically significant. Example 1: Preparation of Coix lachryma-jobi L. extracts

[0040] Various parts of Adlay were used for cell experiments, including leaves (L), roots (R), and seeds (S). After drying and grinding, the Adlay was extracted with water, alcohol, a mixture of alcohol and water, or a combination thereof (preferably 70% ethanol), concentrated, and dried under reduced pressure. On the other hand, Adlay seed powder (ASP, normal diet with 10% ASP), Adlay leaf powder (ALP, normal diet with 1% ALP), Adlay seed polysaccharide (ASPS, normal diet with 1% ASPS), and Adlay seed ethanol extract (ASE, normal diet with 0.025% and 0.1% ASE) were used for animal experiments. All Adlay extracts were provided by Dr. Yu-Hsin Chen (Agricultural Improvement Center, Taiwan).

[0041] The procedure of the schematic diagram of an animal experimental design in Adlay groups is described as follows. To investigate the effects of Adlay on liver fibrosis, this example established a TAA-induced liver fibrotic C57BL / 6J mouse model. Fig.Figure 1 is a schematic diagram of an animal experiment design in Adlay groups. 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 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, during which the intraperitoneal injection of TAA was administered three times a week, the mice were finally sacrificed at 12 weeks of age. NT represents no treatment, ASP represents Adlay seed powder, ALP represents Adlay leaf powder, ASPS represents Adlay seed polysaccharide, and ASE represents Adlay seed ethanol extract. Six-week-old mice were randomly divided into seven groups, including the NT, TAA, 10% ASP, 1% ALP, 1% ASPS, 0.025% ASE, and 0.1% ASE groups (see Fig.1). The NT and TAA groups received a normal diet, while the Adlay groups had Adlay extracts added to the normal diet. In addition, TAA or saline was administered to the mice by intraperitoneal injection three times a week for six weeks. After six weeks, the mice were sacrificed, and their liver tissues and serum were collected for subsequent analysis. Example 2 Effects of Coix lachryma-jobi L. extract on liver histopathology and physiological values ​​in TAA-induced fibrotic liver mice

[0042] After six weeks of exposure, TAA-induced pathological changes were observed in hematoxylin and eosin (H&E) and immunohistochemistry (IHC)-stained liver tissue sections.

[0043] The procedure for histopathological observation of the liver is as follows. After fixation in 10% neutral buffered formalin solution, the liver tissues were embedded in paraffin and then cut into 4 µm-thick sections. Pathological sections were examined to assess the change in tissue architecture using the H&E staining kit (CIS-Biotechnology, Taiwan), the state of collagen deposition using the Sirius Red staining kit (CIS-Biotechnology, Taiwan), and the expression of specific proteins in the tissue using the immunohistochemistry kit (Agilent, USA) and phospho-SMAD2 (1:100; GeneTex, USA), phospho-SMAD3 (1:100; GeneTex, USA), and α-SMA (1:100; GeneTex, USA) antibodies. All stainings were observed using a light microscope (BEL Photonics, Italy).

[0044] The procedure for the hydroxyproline assay is as follows. Hydroxyproline, as an indirect index of collagen content in the liver, was determined using a hydroxyproline assay kit, and the testing procedures followed the manufacturer's protocol. Briefly, liver tissue samples were hydrolyzed with HCl at 95°C for 6 h, then the pH was adjusted to 7. Carbon powder was used to adsorb the impurities, centrifuged, and the supernatant was collected for analysis. Hydroxyproline content was measured at a wavelength of 558 nm using an ELISA reader (TECAN, Switzerland).

[0045] The Fig.Figures 2A–2F show the effects of Coix lachryma-jobi L. extract on liver histopathology and physiological parameters in TAA-induced fibrotic C57BL / 6J mice. (2A) Histopathological changes of the liver observed with hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining (original magnification × 200, and the scale bar is 100 µm). (2B) Quantifications of IHC staining of α-smooth muscle actin (α-SMA) performed using Image J software. (2C) Changes in liver hydroxyproline content. (2D, 2E) Changes in liver weights and body weights. (2F) Ratio of liver weight to body weight, ratio of change in liver weight divided by body weight. Data were presented as mean ± standard deviation (SD) (n = 6 per group).The bars are marked with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05).

[0046] Compared with the NT group, the TAA group induced inflammatory infiltration, deformation of liver architecture, and excessive expression of α-SMA ( Fig. 2A and Fig. 2B), while the Adlay groups showed significantly reduced inflammatory infiltration, improved liver deformation, and reduced α-SMA expression. To detect changes in collagen content in the liver, the hydroxyproline (collagen component) assay was performed. The results showed that the TAA group had higher hydroxyproline levels than the NT group, indicating an increase in collagen ( Fig.2C). On the other hand, all Adlay groups had significantly reduced hydroxyproline content, suggesting that Adlay reduced TAA-induced collagen deposition in the liver. Weight loss and liver tissue enlargement were observed in the TAA group compared with the NT group ( Fig. 2D and Fig. 2E). Among the Adlay groups, the 10% ASP, 1% ALP, 1% ASPS, and 0.1% ASE groups showed significantly reduced liver enlargement induced by TAA, but there was no obvious difference in body weight. To avoid individual differences, the changes in liver weight and body weight were corrected by the ratio of liver weight to body weight ( Fig. 2F). Compared with the TAA group, only the 0.025% ASE group showed no obvious change, while others showed a significantly decreased liver index. Example 3 Effects of Coix lachryma-jobi L. extract on biochemical characterization of the liver in TAA-induced fibrotic liver mice

[0047] The biochemical analysis procedure is as follows. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), total cholesterol (TC), total glyceride (TG), and glucose were measured using a chemistry analyzer. Briefly, after centrifugation of whole blood from mice, the serum was collected and dropped onto the tested chips for analysis using an automated clinical chemistry analyzer (Fuji, Japan).

[0048] The Fig.Figures 3A-3F show the effects of Coix lachryma-jobi L. extract on liver injury and nutrient metabolism in TAA-induced fibrotic C57BL / 6J mice. (3A-3C) Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) indicate liver injury. (3D-3F) Serum levels of total glyceride (TG), total cholesterol (TC), and glucose are used to observe nutrient metabolism. Data were presented as mean ± SD (n = 6 per group). Bars are labeled with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05).

[0049] Serum AST, ALT, and TBIL are indices of liver function, while serum TG, TC, and glucose are related to nutrient metabolism in the liver. Compared with the NT group, only the TAA group showed elevated AST, ALT, and TBIL levels ( Fig. 3A-3C). In contrast, the Adlay groups had downregulated TAA-induced upregulation of liver function indices, especially in the 10% ASP, 1% ALP, 1% ASPS, and 0.1% ASE groups. On the other hand, the TAA group showed significantly increased TG and TC levels and decreased glucose levels compared with the NT group ( Fig. 3D-3F). Treatment with Coix lachryma-jobi L. extract significantly improved TAA-induced TG and TC levels in all Adlay groups ( Fig. 3D-3E) and glucose levels in the 10% ASP and 1% ASPS groups remained comparable to those in the control group. Example 4 Effects of Coix lachryma-jobi L. extract on liver mRNA levels of fibrotic and inflammatory genes in TAA-induced fibrotic liver mice

[0050] The procedure for real-time PCR analysis is as follows. Total RNA was extracted using Trizol reagent (Ambion, USA) and then reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). PCR amplification was performed using SYBR Green PCR Master Mix (Applied Biosystems, USA), and gene mRNA expression was normalized to GAPDH. All primers were obtained from Genomics (Genomics, Taiwan). The primer sequences used in this example are listed in Table 1. Rat primer sequences are used for real-time PCR of HSC-T6 cells, and mouse primer sequences are used for real-time PCR of C57BL / 6J mice. Table 1 Primer name species Forward sequences Reverse sequences α-SMA Rats GCTCTGGTGTGTG ACAATGG (SEQ ID NO:1) CTTTTCCATGTC GTCCCAGT (SEQ ID NO:2) COL1A1 Rats TGTTCAGCTTTGTG GACCTC (SEQ ID NO:3) GCCATTGTGGC AGATACAGA (SEQ ID NO:4) GAPDH Rats GGCACAGTCAAGG CTGAGAATG (SEQ ID NO:5) ATGGTGGTGAA GACGCCAGTA (SEQ ID NO:6) IL-6 Rats ATTGTATGAACAG CGATGATGCAC (SEQ ID NO:7) CCAGGTAGAAA CGGAACTCCAG A (SEQ ID NO:8) IL-10 Rats GCGACGCTGTCAT CGATTTC (SEQ ID NO:9) GTAGATGCCGG GTGGTTCAA (SEQ ID NO:10) TNF-α Rats TCAGTTCCATGGC GTTGTCTTTGAG CCAGAC (SEQ ID NO:11) ATCCATGCCAT T (SEQ ID NO: 12) α-SMA Mice GTCCCAGACATCA GGGAGTAA (SEQ ID NO: 13) TCGGATACTTC AGCGTCAGGA (SEQ ID NO:14) COL1A1 Mice TGGATTCCCGTTCG AGTACG (SEQ ID NO:15) ATTAGGCGCAG GAAGGTCAG (SEQ ID NO:16) GAPDH Mice AACTTTGGCATTGT GGAAGG (SEQ ID NO:17) GGATGCAGGGA TGATGTTCT (SEQ ID NO: 18) IL-6 Mice CCAGTTGCCTTCTT GGGACT (SEQ ID NO:19) GGTCTGTTGGG AGTGGTATCC (SEQ ID NO:20) IL-10 Mice GCTCTTACTGACTG GCATGAG (SEQ ID NO:21) CGCAGCTCTAG GAGCATGTG (SEQ ID NO:22) TNF-α Mice TGGGAGTAGACAA GGTACAACCC CATCTTCTCAA AATTCGAGTGA (SEQ ID NO:23) CAA (SEQ ID NO:24)

[0051] To confirm changes in gene levels in fibrotic liver mice, mRNA levels of fibrotic genes were monitored using real-time PCR. Fig. Figures 4A-4E show the effects of Coix lachryma-jobi L. extract on fibrotic and inflammatory gene levels in TAA-induced fibrotic C57BL / 6J mice. (4A, 4B) mRNA levels of fibrotic markers α-SMA and collagen type I alpha 1 (COL1A1). (4C-4E) mRNA levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10. Data were presented as mean ± SD (n = 6 per group). Bars are labeled with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05).

[0052] Compared with the NT group, the TAA group showed significantly increased levels of fibrotic markers α-SMA and collagen type 1 (COL1A1) ( Fig. 4A and Fig. 4B). In contrast, all Adlay groups had significantly reduced α-SMA and COL1A1 levels, suggesting that Adlay reduced the expression of fibrotic markers. Inflammation is closely linked to fibrogenesis; therefore, 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 upregulation of TNF-α and IL-6 and downregulation of IL-10 in the TAA group compared to the NT group ( Fig.4C-4E). All Adlay groups had a reduced TAA-induced increase in TNF-α and IL-6 mRNA levels, as well as significantly upregulated IL-10 mRNA levels, although the 1% ALP, 0.025% ASE, and 0.1% ASE groups did not differ significantly from the TAA group. Example 5 Effects of Coix lachryma-jobi L. extract on cell viability, fibrotic protein expression, and inflammatory gene levels in HSC-T6 cells

[0053] The cell culture procedure is as follows. HSC-T6 cells are an immortalized cell line isolated from SV40 large T antigen-transformed primary stellate cells of male Sprague-Dawley rats. HSC-T6 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, USA) containing 8% fetal bovine serum (FBS, HyClone, USA), 100 U / ml penicillin, and 100 µg / ml streptomycin (Gibco, USA).

[0054] For hepatic stellate cell (HSC) activation, cells were cultured for 24 h and then replaced with serum-free medium for 24 h, followed by treatment with transforming growth factor beta 1 (TGF-β1) (10 ng / ml) in the presence or absence of CX (10-100 µM) or Adlay leaf, root, and seed extracts (25 and 50 µg / ml) for 24 h or 30 min for intracellular signaling pathways.

[0055] The procedure for the cell viability assay is as follows. HSC-T6 cells were cultured at 1.5 × 10 4Seeded into 96-well plates and incubated with CX (0-800 µM) or Adlays extracts (0-800 µg / ml) for 24 h. Cell viability was then determined using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (Sigma-Aldrich, USA). Briefly, MTT was added to each well at a final concentration of 0.5 µg / ml and incubated at 37 °C for 1 h. Next, MTT was removed and DMSO was added to dissolve MTT formazan crystals formed by metabolically viable cells. Finally, the absorbance of each well was detected by a spectrophotometer at a wavelength of 570 nm.

[0056] The procedure for Western blot analysis is as follows. Cells were lysed in RIPA buffer with protease inhibitor and then centrifuged at 4°C and 12,000 rpm for 10 min. The supernatant was collected, and the total protein concentration was quantified to 50 µg. After heating at 95°C for 10 min, the protein samples were separated by SDS-PAGE gel, transferred to PVDF membranes, followed by blocking with 6% milk, incubation with primary antibodies at 4°C overnight, and secondary antibodies at room temperature for 1 h. Blot images were visualized on an Amersham Imager 680 (Cytiva, USA) using ECL (GeneDireX, USA).

[0057] HSC activation is considered a primary event in liver fibrosis. This example used HSC-T6 cells as a cell model and added TGF-β1 to act as an inducer of HSC activation.

[0058] The Fig.Figures 5A-5H show the effects of Coix lachryma-jobi L. extract on cell viability, fibrotic protein expression, and inflammatory gene levels in HSC-T6 cells. (5A) Schematic representation of drug treatment in HSC-T6 cells. (5B) Cell viability of HSC-T6 cells treated with Adlay leaf, root, and seed extracts. (5C-5E) Western blot results of α-SMA and COL1A1 in HSC-T6 cells treated with TGF-β1 and Adlay extracts, and quantification of the results using Image J software. (5F-5H) mRNA levels of inflammatory factors in HSC-T6 cells treated with TGF-β1 and Adlay extracts. Data were presented as mean ± SD (n = 3 per group). Cell viability bars are labeled * , 0 of the concentration vs. other concentrations marked, marked with ** (p < 0.01); ***(p < 0.001). The bars of the Western blot and real-time PCR are marked with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). In Fig. 5C-5H the letters “L”, “R”, “S” represent extracts of leaf, root or seed respectively.

[0059] To determine the appropriate doses of Coix lachryma-jobi L. extract (leaf, root and seed) for cell assays and to investigate the effects of Coix lachryma-jobi L. extract on cell viability ( Fig. 5A). Cell viability showed that Adlay leaf and root extracts at a dose below 100 µg / ml and Adlay seed extract at a dose below 200 µg / ml had no significant effect ( Fig.5B). Accordingly, 20 and 50 µg / ml of Coix lachryma-jobi L. extracts were used for the subsequent tests. Fibrotic marker expression in HSC-T6 cells treated with TGF-β1 was detected using Western blot analysis. The results showed that all Coix lachryma-jobi L. extracts dose-dependently reduced the TGF-β1-induced expression of α-SMA and COL1A ( Fig. 5C-5E). Furthermore, for inflammation, the mRNA levels of TNF-α, IL-6, and IL-10 were assessed using real-time PCR. The results showed that all Coix lachryma-jobi L. extracts downregulated TGF-β1-induced TNF-α and IL-6 mRNA levels and upregulated IL-10 mRNA levels in a dose-dependent manner ( Fig. 5F-5H). Variations in fibrotic protein and inflammatory genes showed that Coix lachryma-jobi L. extract improved inflammation and HSC activation in HSC-T6 cells treated with TGF-β1. Example 6Schematic diagram of an animal experiment design in 6-methoxybenzoxazolinone (6-MBOA) (CX) groups

[0060] To investigate the effects of CX on liver fibrosis, this example established a TAA-induced fibrotic C57BL / 6J mouse model. Fig. Figure 6 is a schematic diagram of an animal experiment design in 6-methoxybenzoxazolinone (6-MBOA) (CX) groups. For animal experiments, 6-week-old C57BL / 6J mice were randomly divided into four groups, including NT, TAA, CX20 (TAA + 20 mg / kg CX), and CX50 (TAA + 50 mg / kg CX) (n = 8 per group). Mice received an intraperitoneal injection of TAA three times a week to induce liver fibrosis; they also received CX or the drug by oral gavage once a day. At the end of the animal experiments, mice were sacrificed at 12 weeks of age. Six-week-old mice were randomly divided into four groups, namely NT, TAA, CX20, and CX50 ( Fig.6). Among these groups, only the NT group was orally gavaged and administered with vehicle, the TAA group was orally gavaged with vehicle and administered with TAA, and the CX groups were orally gavaged with CX and administered with TAA. Oral gavage was performed once a day, while intraperitoneal injection was performed three times a week; both oral gavage and intraperitoneal injection were administered continuously for six weeks. Afterward, the mice were sacrificed, and their liver tissues and serum were collected for subsequent analysis. Example 7 Effects of 6-methoxybenzoxazolinone (6-MBOA) (CX) on liver histopathology and physiological values ​​in TAA-induced fibrotic liver mice

[0061] To observe the effects of CX on liver histopathology, fibrotic liver mice were treated with CX and liver tissue stained with H&E and Sirius red. The fibrotic marker α-SMA and TGF-β / SMAD pathway-related proteins p-SMAD2 and p-SMAD3 in the liver sections were also examined using IHC staining.

[0062] The procedure for the TGF-β1 enzyme-linked immunosorbent assay (ELISA) is as follows. TGF-β1 levels were detected in serum using commercial mouse ELISA kits (Invitrogen, USA), and the procedure followed a standard sandwich ELISA protocol. Briefly, serum was added to a microwell coated with TGF-β1 antibody, then sequentially incubated with biotin conjugate, streptavidin-HRP, substrate solution, and stop solution. After reaction with stop solution, the color intensity was immediately measured at 450 nm using an ELISA reader (TECAN, Switzerland).

[0063] The Fig.Figures 7A–7K show the effects of CX on liver histopathology and physiological parameters in TAA-induced fibrotic C57BL / 6J mice. (7A) Chemical structure of CX (C8H7NO3, MW = 165.15). (7B) Histological changes of the liver observed by H&E, Sirius Red, and IHC staining (original magnification × 200, and the scale bar is 100 µm). (7C) Quantifications of Sirius Red staining of collagen. (7D–7F) Quantifications of IHC staining of α-SMA, phospho-SMAD2 (phospho-SMAD2), and phospho-SMAD3 (phospho-SMAD3). (7G) Changes in liver hydroxyproline content. (7H) Expression of TGF-β1 in serum. (7I, 7J) Changes in liver and body weights. (7K) Liver weight to body weight ratio. Quantification of Sirius Red and IHC staining was achieved using Image J software. Data were presented as mean ± SD (n = 8 per group).The bars are marked with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (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 ( ). Fig.7B-7F). Both CX20 and CX50 groups showed significantly reduced inflammatory infiltration, liver structural deformation, collagen deposition, and α-SMA expression. Furthermore, the CX50 group negatively regulated p-SMAD2 and p-SMAD3 expression, implying that CX ameliorated TAA-induced injury through SMAD signaling pathways. The hydroxyproline content of the TAA group was significantly higher than that of the NT group, while the CX groups showed significantly lower hydroxyproline content than the TAA group, implying that CX treatment reduced collagen content ( Fig. 7G). TGF-β1 is an important cytokine associated with fibrosis; a change in TGF-β1 can be an indicator of the development of fibrosis. ELISA results showed that TGF-β1 expression significantly increased in the TAA group compared to the NT group, while CX significantly reduced the TAA-induced upregulation of TGF-β1 ( Fig.7H). Injection of TAA alone caused liver enlargement and body weight loss, while treatment with CX reduced liver weight but did not significantly improve the TAA-induced change in body weight ( Fig. 7I and Fig. 7J). To avoid individual differences in mice, liver and body weight were adjusted using the ratio of liver weight to body weight. The findings showed that the ratio of liver weight to body weight increased significantly in the TAA group compared with the NT group, while the ratio of liver weight to body weight decreased significantly in the CX group compared with the TAA group ( Fig. 7K). The current results showed that CX attenuated the classic characteristics of fibrosis, such as excessive α-SMA expression, collagen accumulation, and higher TGF-β1 levels, by affecting the expression of SMADs. Example 8 Effects of CX on oxidative stress and biochemical characterizations in TAA-induced fibrotic liver mice

[0064] Liver injury was caused by an active metabolite of TAA, which leads to oxidative stress. Organisms can scavenge oxidative free radicals through the 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. It is an indicator of oxidative stress in cells, tissues, organs, or organisms. It is used to detect oxidative stress in TAA-induced fibrotic mice. Elevated MDA levels indicate increased oxidative stress.

[0065] The procedure for oxidative stress indicator tests is as follows. The enzyme activities of catalase (CAT) and superoxide dismutase (SOD), the content of malondialdehyde (MDA), and the concentration of glutathione (GSH) in tissue were tested to investigate changes in oxidative stress levels in TAA-induced fibrotic liver mice. Hydroxyproline is the component of collagen precursor tested. All kits were commercially available, and the working protocol followed the manufacturer's instructions. Briefly, the liver tissue was homogenized in ice-cold buffer and centrifuged at 4°C. The supernatant was collected for analysis. Finally, the absorbance of the samples was measured using an ELISA reader (TECAN, Switzerland).

[0066] The Fig.Figures 8A–8D show the effects of CX on antioxidant enzymes and lipid peroxidation in TAA-induced fibrotic C57BL / 6J mice. (8A, 8B) Changes in the activity of superoxide dismutase (SOD) and catalase (CAT). (8C) Changes in the level of glutathione (GSH). (8D) Changes in the level of malondialdehyde (MDA). Data were presented as mean ± SD (n = 8 per group). Bars are marked with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). Compared with the TAA group, both the CX20 and CX50 groups showed increased CAT activities and GSH levels, but decreased MDA levels. Furthermore, SOD activities were significantly increased in the CX50 group, indicating that CX has the effect of improving oxidative stress.

[0067] The Fig.Figures 9A-9G show the effects of CX on liver injury and nutrient metabolism in TAA-induced fibrotic C57BL / 6J mice. (9A-9D) Serum levels of liver injury indicate ALT, AST, TBIL, and ALP. (9E-9G) Serum levels of TG, TC, and glucose were used to monitor nutrient metabolism. Data were presented as mean ± SD (n = 8 per group). Bars are labeled with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05). ALP represents Adlay leaf powder.

[0068] Regarding biochemical characterizations, the liver injury indices AST, ALT, TBIL and ALP increased significantly in the TAA group compared with the NT group, but decreased significantly in the CX groups compared with the TAA group ( Fig.9A-9D). In addition, TAA also caused a disturbance in nutrient metabolism. Compared with the NT group, the TAA group had significantly increased TG and TC levels but decreased glucose levels, while CX significantly reduced the changes in TG, TC, and glucose levels caused by TAA ( Fig. 9E-9G). These findings showed that CX can improve lipid metabolism ( Fig. 9E-9F), can improve abnormal liver damage caused by TAA and maintain normal glucose metabolism ( Fig. 9G). Example 9 Effects of CX on fibrotic and inflammatory gene levels in TAA-induced fibrotic liver mice

[0069] To confirm the changes in the fibrotic gene in mice, the mRNA levels of fibrotic genes were detected using real-time PCR. Fig.Figures 10A–10E show the effects of CX on fibrotic and inflammatory gene levels in TAA-induced fibrotic C57BL / 6J mice. (10A, 10B) Liver mRNA levels of α-SMA and COL1A1 detected using real-time PCR. (10C–10E) Liver mRNA levels of inflammatory factors TNF-α, IL-6, and IL-10. Data were presented as mean ± SD (n = 8 per group). Bars are labeled with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05).

[0070] Compared with the NT group, the TAA group had significantly upregulated α-SMA and COL1A1 mRNA levels, while the CX groups showed significantly reduced gene translation of TAA-induced fibrotic markers ( Fig. 10A and Fig.10B). Furthermore, inflammation is necessary for the development of fibrosis; therefore, mRNA levels of inflammatory genes TNF-α, IL-6, and the anti-inflammatory gene IL-10 were detected to understand the inflammatory status. Compared with the NT group, the TAA group showed induced upregulation of IL-6 and TNF-α and downregulation of IL-10. CX treatment significantly reduced TAA-induced upregulation of IL-6 and TNF-α levels and downregulation of IL-10 mRNA levels ( Fig. 10C-10E). These results demonstrated that CX reduced the expression of inflammatory gene mRNA in the gene transcription of TAA-induced fibrotic mice, thereby inhibiting the development of subsequent cellular fibrosis. Example 10 Effects of CX on cell viability, inflammatory gene levels, and ROS levels in HSC-T6 cells

[0071] To investigate the cell cytotoxicity of CX and determine the optimal doses for subsequent cell experiments, different doses of CX were tested in HSC-T6 cells using the MTT assay.

[0072] The procedure for the ROS detection assay is as follows. 2',7'-Dichlorofluorescein diacetate (DCFDA), a cell-permeable fluorogenic probe, is commonly used to detect ROS and nitric oxide in living cells. DCFDA rapidly dehydrates to form fluorescent 2',7'-dichlorofluorescein (DCF) when oxidized by ROS in cells. For ROS detection, cells were seeded in 96-well plates for 24 h, then changed to serum-free medium and drug treated. The medium was then changed to DCFDA solution for 30 min, and the excitation / emission spectra of 485 / 525 nm were detected using multimode microplate readers (TECAN, Switzerland).

[0073] The Fig.Figures 11A-11F show the effects of CX on cell viability, inflammatory gene levels, and reactive oxygen species (ROS) in TGF-β1-activated HSC-T6 cells. (11A) Schematic representation for TGF-β1 and CX treatment in HSC-T6 cells. 6-MBOA represents 6-methoxybenzoxazolinone. (11B) Cell viability of HSC-T6 cells treated with different concentrations of CX. (11C-11E) mRNA levels of inflammatory factors in HSC-T6 cells treated with TGF-β1 and CX. (11F) ROS levels of HSC-T6 cells detected with DCFDA and normalized with protein content. Data were presented as mean ± SD (n = 3 per group). Cell viability bars are labeled * , 0 of the concentration vs. other concentrations marked, marked with ***(p < 0.001). The bars of the Western blot and real-time PCR are labeled with different letters; the same letter indicates no significant difference between the two samples (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05).

[0074] The results showed no significant effects on cell viability at a dose below 400 µM (i.e., no toxicity); therefore, 10, 20, 50, and 100 µM were used for subsequent cell assays ( Fig. 11B). Similarly, the mRNA levels of TNF-α, IL-6, and IL-10 were detected in HSC-T6 cells treated with TGF-β1 using real-time PCR. 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. Furthermore, ROS levels were detected in HSC-T6 cells using the DCFDA assay. The findings showed that TGF-β1 induced ROS production, while CX reduced TGF-β1-induced ROS levels with increasing doses ( Fig. 11F). These results demonstrated that TGF-β1 promoted inflammation and the production of ROS, while CX showed the potential to reduce inflammation and ROS in HSC-T6 cells activated with TGF-β1. Example 11 Effects of CX on HSC activation and epithelial-mesenchymal transition (EMT) in HSC-T6 cells

[0075] To understand the effects of CX on HSC activation and EMT in TGF-β1-activated HSC-T6 cells, the expressions of fibrotic and EMT-related proteins were examined using Western blot analysis.

[0076] The wound healing assay procedure is as follows. HSC-T6 cells were seeded onto 6-well plates for 24 h. Monolayer cells were scraped with a 200 µL pipette tip, and non-adherent cells were washed away with PBS. Serum-free medium was changed after 24 h. Then, the cells were treated with TGF-β1 and CX for 0, 24, and 48 h. Cell migration images were observed using inverted microscopy (Olympus, Japan), and wound areas were quantified using Image J software.

[0077] The Fig.Figures 12A-12G show the effects of CX on HSC activation and epithelial-mesenchymal transition (EMT) in TGF-β1-activated T6 HSC cells. (12A) Analysis elements include Western blot results of E-cadherin, fibronectin, α-SMA, and COL1A1. (12B-12E) Quantifications of Western blots of E-cadherin, fibronectin, α-SMA, and COL1A1 were performed using Image J software. (12F, 12G) Results of the wound healing assay at 0, 24, and 48 hours (magnification ×200, scale bar is 100 µm) were quantified using Image J software. Wound closure is defined as (wound area at 0 h - wound area at 24 or 48 h) / wound area at 0 h × 100%. Data were presented as mean ± SD (n = 3 per group).The data bars are marked with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05).

[0078] Markers for epithelial-mesenchymal transition or fibrosis include α-SMA, COL1A1, and fibronectin, cell injury, and mesenchymal replacement of normal tissue architecture, which can ultimately lead to organ failure. TGF-β1 treatment can upregulate the expression of α-SMA, COL1A1, and the mesenchymal marker fibronectin, as well as the downregulated epithelial marker E-cadherin ( Fig.12A-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. When EMT occurs, cell migration ability may be enhanced. Therefore, migration changes were examined using a wound healing assay. The percentage of wound closure increased significantly in the TGF-β1 group compared to the NT group ( Fig. 12F and Fig. 12G). TGF-β1-induced wound closure at 24 h was significantly reduced by CX treatment at a dose of 100 µM, whereas TGF-β1-induced wound closure at 48 h was reduced by CX treatment at a dose of 20–100 µM. These results suggested that CX can inhibit HSC activation and EMT in HSC-T6 cells treated with TGF-β1. Example 12 Effects of CX on TGF-β / SMAD and mesenchymal transcriptional signaling pathway in HSC-T6 cells

[0079] The mechanisms of CX on HSC activation and EMT were further investigated using Western blot analysis.

[0080] The Fig.Figures 13A–13J show effects of CX on TGF-β / SMAD signaling in TGF-β1-activated T6 HSC cells. (13A) Schematic representation of TGF-β1 pathway stimulation and CX treatment in T6 HSC cells. (13B) Western blot results of phospho-SMAD2 (p-SMAD2), phospho-SMAD3 (p-SMAD3), SMAD2 / 3, zinc finger protein SNAIL1 / 2, TWIST1 / 2, zinc finger E-box-binding homeobox 1 (ZEB1), and NADPH oxidase 4 (NOX4) normalized with α-tubulin. (13C-13I) Western blot quantifications of p-SMAD2, p-SMAD3, SMAD2 / 3, SNAIL1 / 2, TWIST1 / 2, ZEB1, and NOX4 using Image J software. Data were presented as mean ± SD (n = 3 per group). Data bars are labeled with different letters; the same letter indicates no significant difference between the two samples (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05).

[0081] Overall, when the liver is damaged, stellate cells secrete TGF-β1 hormone, which is phosphorylated in p-SMAD2 and p-SMAD3 by SMAD pathway-related proteins ( Fig. 12C-12D) and transmits signals to the nucleus to promote gene transcription of nuclear EMT-related proteins SNAIL1 / 2, TWIST1 / 2 and ZEB1 ( Fig. 13F-13H). At this time, the expression of cadherin ( Fig. 12B) and the expression of fibronectin increased ( Fig. 12C). Liver collagen COL1A1 and tubulin α-SMA increase ( Fig. 12D-12E) and eventually lead to liver fibrosis ( Fig. 7B). TAA induces liver damage and simultaneously causes ROS ( Fig. 11F) and inflammatory response. The main markers include the increase in the two inflammatory hormones TNF-α and IL-6 ( Fig. 11C-11D) and the decrease of the anti-inflammatory hormone IL-10 ( Fig.11E). When an inflammatory response occurs, the expression of the related protein NOX4 ( Fig. 13I), the expression of the antioxidant stress proteins SOD, CAT and GSH decreases ( Fig. 8A-8C) and the oxidative stress metabolite MDA also increases ( Fig. 8D). The combination of the above reactions ultimately leads to liver fibrosis ( Fig. 7B). In the TGF-β1 group, the above side effects were significantly higher than those in the NT group, indicating that TGF-β1 could phosphorylate SMAD2 and SMAD3, then activate the downstream proteins SNAIL1 / 2, TWIST1 / 2, ZEB1, and NOX4, further causing ROS production and triggering EMT.

[0082] In contrast, upon treatment with CX, TGF-β1-stimulated SMAD phosphorylation and downstream fibrotic protein expression decreased with increasing dose, indicating that CX inhibited SMAD2 and SMAD3 phosphorylation ( Fig.13B-13D), which reduced gene transcription of SNAIL1 / 2, TWIST1 / 2 and ZEB1 ( Fig. 13F-13H), the cadherin content was maintained ( Fig. 12B), fibronectin decreased and EMT prevented liver fibrosis ( Fig. 12C). At the same time, CX also reduced the inflammatory response and the effects included reducing the expression of the inflammatory hormones TNF-α and IL-6 ( Fig. 11C-11D), increasing the expression of the anti-inflammatory hormone IL-10 ( Fig. 11E), reducing the expression of NOX4 ( Fig. 13I) and further preventing EMT and ROS production.

[0083] The Fig.Figures 14A–14C show the effects of CX on phosphoinositide 3-kinase (PI3K) and the extracellular signal-regulated kinase (ERK) pathway in TGF-β1-activated HSC-T6 cells. (14A) Western blot results of p-PI3K and p-ERK1 / 2 normalized with α-tubulin. (14B, 14C) Western blot quantifications of p-PI3K and p-ERK1 / 2 using Image J. Data were presented as mean ± SD (n = 3 per sample). Data bars are labeled with different letters; the same letter indicates no significant difference between the two groups (p > 0.05), while different letters indicate a significant difference between the two groups (p < 0.05).

[0084] Regarding the non-SMAD pathway, the results showed that CX did not cause significant changes in the phosphorylation of PI3K and ERK, suggesting that CX has little effect on the ERK and PI3K signaling pathways ( Fig. 14A-14C).

[0085] In summary, 6-MBOA (CX) and the Coix lachryma-jobi L. extract containing 6-MBOA of the present invention achieve the effect of preventing liver fibrosis in vivo and in vitro, accompanied by the downregulation of fibrotic and inflammatory factors. Furthermore, 6-MBOA ameliorates oxidative stress in the liver, reduces the production of liver fibrotic biomarkers, and promotes the loss of mesenchymal identity of HSCs through the TGF-β / SNLAD signaling pathway. Both 6-MBOA and the Coix lachryma-jobi L. extract containing 6-MBOA possess therapeutic potential for liver fibrosis.

[0086] Although the present invention has been described with reference to the preferred embodiments, it will be apparent to those skilled in the art that various modifications and changes in form and detail may be made therein without departing from the scope of the present invention as defined by the appended claims. This is followed by a sequence listing as an electronic document. This can be accessed both in DEPATISnet and in the DPMA register.

Claims

[1] A method for preventing and / or treating liver fibrosis comprising administering to a subject in need thereof a composition containing an effective amount of 6-methoxybenzoxazolinone (6-MBOA). [2] The method of claim 1, wherein the 6-MBOA reduces inflammatory infiltration, liver structural deformation, collagen deposition, α-smooth muscle actin (α-SMA) expression level, liver hydroxyproline content, serum transforming growth factor-β1 (TGF-β1) concentration, liver weight, liver index, reactive oxygen species (ROS) production, and TGF-β1-induced fibronectin expression level in the subject in need thereof. [3] The method of claim 1, wherein the 6-MBOA negatively regulates the expression levels of phospho-SMAD2 (p-SMAD2) and phospho-SMAD3 (p-SMAD3) in the subject in need thereof. [4] The method of claim 1, wherein the 6-MBOA increases superoxide dismutase (SOD) activity, catalase (CAT) activity and glutathione (GSH) content, decreases the expression of NADPH oxidase 4 (NOX4) and decreases malondialdehyde (MDA) content caused by oxidative stress. [5] The method of claim 1, wherein the 6-MBOA inhibits hepatic stellate cell (HSC) activation and epithelial-mesenchymal transition (EMT), including inhibiting the expression of fibronectin and maintaining E-cadherin expression levels. [6] The method of claim 1, wherein the 6-MBOA reduces the expression levels of zinc finger protein SNAIL1 / 2, TWIST1 / 2 and zinc finger E-box binding homeobox 1 (ZEB1). [7] The method of claim 1, wherein the composition is a pharmaceutical composition, a food composition or a topical composition. [8] The method of claim 7, wherein the pharmaceutical composition is in a dosage form for oral administration. [9] The method of claim 7, wherein the pharmaceutical composition is in a dosage form for parenteral administration. [10] The method of claim 7, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, adjuvant and / or food additive. [11] A method according to claim 1, wherein the composition has a dosage form as a powder, granules, solution, jelly or paste. [12] A method for preventing and / or treating liver fibrosis comprising administering to a subject in need thereof a composition containing an effective amount of Coix lachryma-jobi L. extract containing 6-methoxybenzoxazolinone (6-MBOA). [13] The process according to claim 12, wherein the Coix lachryma-jobi L. extract is obtained by extracting Coix lachryma-jobi L. with a solvent, and the solvent is water, alcohol, a mixture of alcohol and water, or a combination thereof. [14] The method of claim 12, wherein the Coix lachryma-jobi L. extract reduces serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), total glyceride (TG) and total cholesterol (TC) in the subject in need thereof. [15] The method of claim 12, wherein the Coix lachryma-jobi L. extract reduces the levels of gene expression as well as protein including α-smooth muscle actin (α-SMA), collagen type I alpha 1 (COLIAI), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). [16] A method according to claim 13, wherein the Coix lachryma-jobi L. is root, leaf or seed of Coix lachryma-jobi L. [17] The method of claim 12, wherein the composition is a pharmaceutical composition, a food composition or a topical composition. [18] The method of claim 17, wherein the pharmaceutical composition is in a dosage form for oral administration. [19] The method of claim 17, wherein the pharmaceutical composition is in a dosage form for parenteral administration. [20] The method of claim 17, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, adjuvant and / or food additive.

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