Use of amomum villosum extract in preparation of drugs for preventing and treating chronic alcoholic liver and intestinal injury
By developing a method for preparing cardamom extract, we have solved the problem of poor efficacy of drugs in the treatment of chronic alcoholic liver and intestine injury. This method significantly reduces AST and ALT levels, improves liver and intestine tissue damage, restores mitochondrial function, regulates liver and intestine axis disorders, and provides an efficient, safe, and low-cost treatment option.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- KUNMING UNIV OF SCI & TECH
- Filing Date
- 2026-04-21
- Publication Date
- 2026-06-05
AI Technical Summary
Existing medications for chronic alcoholic liver and intestinal injury are ineffective, have limited action, and have side effects, making clinical management difficult, patient compliance poor, and screening tools with low sensitivity, leading to delayed diagnosis and poor prognosis.
The extract of Amomum tsao-ko was used to prepare Amomum tsao-ko powder by extraction with 70% ethanol. The powder was then extracted with ultrasound and freeze-dried to prepare a 70% ethanol solution extract of Amomum tsao-ko. This extract was used to prevent and treat chronic alcoholic liver and intestine injury, significantly reduce plasma AST and ALT levels, improve pathological damage to liver and intestine tissues, restore mitochondrial function, and regulate liver and intestine axis disorders.
The extract of Amomum tsao-ko significantly reduced the plasma AST and ALT levels in mice with alcoholic liver and intestine injury, improved the pathological damage of liver and intestine tissues, restored liver mitochondrial function, and regulated the expression of key factors in the liver-intestinal axis, providing a highly effective, safe, and low-cost liver and intestine protection effect.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of cardamom extract in the preparation of drugs for the prevention and treatment of chronic alcoholic liver and intestinal damage. Background Technology
[0002] Chronic alcoholic liver-intestinal injury is caused by long-term excessive alcohol consumption. After alcohol enters the digestive tract through the mouth, throat, and esophagus, it directly damages the intestinal mucosal barrier, leading to dysbiosis and increased intestinal permeability. This allows substances such as botulinum toxin and D-lactic acid to diffuse throughout the body via the bloodstream, further inducing or aggravating liver inflammation, creating a vicious cycle of liver-intestinal axis dysfunction. Currently available treatments for alcoholic liver injury, such as biphenyl diester, corticosteroids, and silymarin, mostly have certain side effects and limited action. Clinical management also faces challenges such as insufficient monitoring, poor patient compliance, and low sensitivity of screening tools, often leading to delayed diagnosis and poor prognosis.
[0003] Amomum tsao-ko, the dried, mature fruit of a perennial evergreen shrub belonging to the genus Amomum in the family Zingiberaceae, is mainly distributed in Yunnan, Guangxi, Guizhou, and Sichuan provinces of my country, with Yunnan being the primary source. Amomum tsao-ko contains abundant terpenes, phenolic acids, and organic acids. Regarding its biological activity, it has been reported to possess gastrointestinal regulating, antibacterial, and anti-inflammatory properties, and its extracts have no mutagenic effects. However, no research has been published domestically or internationally on the effects of Amomum tsao-ko extracts on chronic alcoholic liver and intestinal damage. Summary of the Invention
[0004] To address the problems of poor efficacy, limited action, and high production costs of existing drugs for the treatment of chronic alcoholic liver and intestinal injury, this invention provides a novel application of cardamom extract in the preparation of drugs for the prevention and treatment of chronic alcoholic liver and intestinal injury, achieving efficient, safe, and low-cost liver and intestinal protection.
[0005] To achieve the above objectives, the present invention provides the following technical solution: Application of cardamom extract in the preparation of a treatment to prevent liver and intestinal damage caused by long-term alcohol consumption.
[0006] The prevention and treatment of liver and intestinal damage caused by long-term alcohol consumption by the extract of the cardamom includes (1)-(4): (1) Significantly reduced plasma AST and ALT levels in mice with chronic alcohol-induced liver and intestinal injury, and significantly improved mitochondrial dysfunction and the expression of related factors (TLR4, ADRB2) in the model mice caused by long-term alcohol consumption; (2) Improves the pathological damage of liver and intestine tissues in mice with chronic alcoholic liver and intestine injury model; (3) Reduce the risk of developing chronic alcoholic liver and intestine injury; Specifically, the application of cardamom extract in the preparation of drugs for the prevention and treatment of chronic alcoholic liver and gut injury, wherein the chronic alcoholic liver and gut injury is caused by long-term alcohol consumption and is accompanied by liver and gut axis disorder.
[0007] Specifically, the herb extract can simultaneously protect liver and colon tissues, repair the intestinal mucosal barrier, and restore liver mitochondrial function.
[0008] Specifically, the herb extract can downregulate TLR4 expression and upregulate ADRB2 expression in liver tissue.
[0009] Specifically, the cardamom extract can reduce the levels of AST and ALT in the plasma of mice with an alcoholic liver and intestine injury model.
[0010] Specifically, the cardamom extract is an ethanol extract of cardamom; the cardamom extract is a 70% ethanol extract of cardamom.
[0011] This invention presents a series of studies on the effects of Amomum tsao-ko extract in preventing and treating alcoholic liver and intestinal damage caused by long-term alcohol consumption. The studies confirmed that Amomum tsao-ko extract can significantly reduce plasma AST and ALT levels; improve the pathological damage to the liver and intestines of mice with chronic alcoholic liver and intestinal damage; and regulate mitochondrial function and the expression of related factors in mice with chronic alcoholic liver and intestinal damage. Overall, it effectively improves the liver and intestines damage in mice with chronic alcoholic liver and intestinal damage. The Amomum tsao-ko or its extract provided by this invention has a significant effect on preventing and treating liver and intestinal damage caused by long-term alcohol consumption. Furthermore, Amomum tsao-ko is widely distributed, has high safety, is inexpensive, and the raw materials are readily available. Amomum tsao-ko or its extract has promising prospects for development and application in the prevention and treatment of chronic alcoholic liver and intestinal damage.
[0012] The cardamom extract was prepared by the following method: (1) Take mature cardamom, dry it in the sun, grind it, and sieve it to obtain cardamom powder; (2) Add 70% ethanol solution to the cardamom powder in step (1) for ultrasonic-assisted extraction, and then centrifuge to obtain cardamom residue and supernatant; (3) Add ethanol to the cardamom residue in step (2), extract with ultrasound 2-3 times, filter the combined supernatant to obtain filtrate; (4) The filtrate from step (3) is concentrated by rotary evaporation and the ethanol is recovered until an aqueous phase is obtained; the aqueous phase is further concentrated by rotary evaporation to obtain a concentrated solution; the concentrated solution is pre-frozen in a -80°C freezer and then freeze-dried at -70°C for 48 hours using a vacuum freeze dryer to obtain a 70% ethanol solution extract of cardamom (freeze-dried powder), which is then sealed and stored at 4°C.
[0013] Preferably, the sieving in step (1) is an 80-100 mesh sieve.
[0014] Preferably, the mass-to-volume ratio of the cardamom powder or residue to 70% ethanol in steps (2) and (3) is 1g:16ml.
[0015] Preferably, the centrifugation in steps (2) and (3) is performed by centrifuging at a speed of 4000 r / min for 10 to 20 minutes.
[0016] Preferably, the ultrasonic extraction in steps (2) and (3) is performed by ultrasonication at 70W power for 25-30 minutes.
[0017] Preferably, the rotary evaporation in step (4) is to concentrate the liquid into a paste at 45°C and 0.07~0.08 MPa.
[0018] Preferably, the dosage of the cardamom extract is 200 mg / kg to 600 mg / kg.
[0019] Preferably, the extract includes cardamom extract; oral formulations containing cardamom extract are ingested into the gastrointestinal tract of mammals.
[0020] This invention relates to the prevention and treatment of chronic alcoholic liver and intestine injury using an extract of Amomum villosum as the active ingredient. It can be used to prevent and treat chronic alcoholic liver and intestine injury by adding one or more formulation-acceptable excipients or by combining it with other active ingredients to achieve a synergistic effect in preventing and treating chronic alcoholic liver and intestine injury. The extract is an oral preparation, which can be a liquid preparation, granules, pills, tablets, capsules, powder, etc., and its application scope includes food and pharmaceuticals.
[0021] The beneficial effects of this invention are as follows: 1. The cardamom raw material used in this invention is a natural product, widely available, easy to collect, and inexpensive. The extraction method is simple, easy to implement, and highly reproducible, making it suitable for industrial promotion. Compared with existing drugs such as silymarin, which have single effects and side effects, the cardamom extract of this invention forms a multi-target hepato-gut axis protection network through the synergistic effect of compound active ingredients. It is more effective in improving hepato-gut axis disorders, repairing hepato-gut tissue damage, restoring liver mitochondrial function, and inhibiting inflammation. It provides a highly efficient, safe, and inexpensive new source of drugs for the prevention and treatment of alcoholic hepato-gut injury caused by chronic long-term drinking, which can significantly reduce the related public health burden and has important pioneering application value.
[0022] 2. The extract of Amomum villosum in this invention can significantly reduce the plasma AST and ALT levels in mice with long-term alcoholic liver and intestine injury model, improve pathological damage to liver and colon tissues, repair the intestinal mucosal barrier, correct liver-intestinal axis disorder, effectively restore liver mitochondrial function, increase ATP content, restore NAD+ / NADH ratio, reduce ROS level and restore mitochondrial membrane potential, and also regulate key factors of the liver-intestinal axis, significantly downregulate abnormally elevated TLR4 expression and upregulate abnormally decreased ADRB2 expression, comprehensively block alcohol-induced inflammatory response and signaling pathway abnormalities, and achieve synergistic protection of the liver-intestinal axis.
[0023] 3. The cardamom extract of the present invention provides a novel, efficient, safe, and low-cost source of drug for the prevention and treatment of alcoholic liver and intestinal damage caused by long-term drinking. It can effectively reduce the health hazards and public health burden caused by chronic alcoholic liver and intestinal damage, and has important clinical application value and market prospects. Attached Figure Description
[0024] Figure 1 This is the total ion chromatogram in negative ion mode for principal component analysis of the ethanol extract of Amomum villosum in this invention using UPLC-MS / MS. Figure 2 Venn diagram for blood component analysis of the ethanol extract of Amomum villosum in this invention; Figure 3 The figure shows the changes in AST and ALT concentrations in the plasma of model mice when the ethanol extract of Amomum villosum was applied; Figure A shows the plasma AST level, and Figure B shows the ALT level. Figure 4 The present invention relates to the detection results of ATP content and NAD+ / NADH ratio in liver tissue of model mice using the ethanol extract of Amomum villosum. Figure A shows the ATP content in liver tissue, and Figure B shows the NAD+ / NADH ratio. Figure 5 The figures show the changes in mitochondrial-related indicators in the liver of model mice when the ethanol extract of Amomum villosum was applied. Figure A shows the expression level of ROS in liver tissue, and Figure B shows the mitochondrial membrane potential (JC-1) in liver tissue. Figure 6 The images show the pathological results of liver and colon tissue sections of model mice using the ethanol extract of Amomum villosum from this invention; Figure A shows the H&E staining results of liver tissue from each group of mice, and Figure B shows the AB-PAS staining results of colon tissue from each group of mice. Figure 7 This study investigates the expression of relevant factors in the liver tissue of model mice when the ethanol extract of Amomum villosum var. mongolicum was applied. Detailed Implementation
[0025] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.
[0026] Example 1: Preparation and component analysis of cardamom extract (1) Preparation of ethanol extract of cardamom Mature cardamom fruits were harvested from Nujiang, Yunnan Province. After drying, grinding, and passing through an 80-mesh sieve, cardamom powder was obtained. 150g of the cardamom powder was weighed and added to 70% ethanol (the mass-to-volume ratio of cardamom powder to 70% ethanol was 1g:16ml). The mixture was ultrasonically extracted for 30 minutes at 70W, then soaked at 4℃ for 12 hours, followed by centrifugation for 10 minutes (4000r / min). The supernatant was retained, and the cardamom residue was extracted once more using the same method. The supernatants were combined. The combined supernatant was placed in a rotary evaporator and concentrated to a paste-like liquid at 45℃ and 0.07~0.08MPa. The concentrate was pre-frozen at -80℃ and then freeze-dried at -70℃ for 48 hours using a vacuum freeze dryer. This yielded the freeze-dried cardamom extract powder.
[0027] (2) Qualitative analysis and blood-entry component analysis of the main chemical components of the cardamom extract. The principal components of the cardamom extract were qualitatively analyzed using UPLC-MS / MS. An Agilent SB-C18 column (1.8 μm, 2.1 mm × 100 mm) was used; mobile phase A was ultrapure water (containing 0.1% formic acid), and mobile phase B was acetonitrile (containing 0.1% formic acid); the elution program was: 0 min, 5% B; 9 min, 95% B; 10 min, 95% B; 11 min, 5% B. The column temperature was 40℃; the injection volume was 2 μL, and the flow rate was 0.35 mL / min. The analytical results are as follows: Figure 1 As shown in the diagram; the analysis of the blood-borne components of the cardamom extract was presented in Venn diagram form, and the results are as follows. Figure 2 As shown. AKT refers to the ethanol extract of Amomum villosum; Control refers to the mouse gavage group administered pure water; AKTH refers to the mouse gavage group administered Amomum villosum extract.
[0028] Depend on Figure 1 It is known that the main compounds contained in the ethanol extract of Amomum villosum include protocatechuic acid, 2,3-dihydroxybenzoic acid, proanthocyanidin B3, (2S)-2-methoxy-3-phenylpropionic acid, (3R)-3-(4-hydroxyphenyl)butyric acid, proanthocyanidin B2, 2-phenylbut-3-en-2-ol, terpineic acid, etc., as well as other phenolic acids, tannins, organic acids and terpenes.
[0029] Depend on Figure 2 It is known that there are 169 blood-entering components in the ethanol extract of Amomum villosum. Some of these components are shown in Table 1.
[0030] Example 2: Evaluation of the activity of Amomum villosum extract in preventing and treating chronic alcoholic liver and intestinal injury. 2.1 Establishment and experimental procedure of a mouse model of chronic alcoholic liver and intestine injury C57BL / 6J male mice (8 weeks old, weighing approximately 20g) were used as the research subjects. Chronic alcoholic liver and intestine injury was induced by oral administration of 43% Hongxing Erguotou (10ml / kg) for 16 consecutive days to establish a mouse model of long-term alcohol-induced liver and intestine injury. Thirty minutes after the last administration, all mice except the blank group and negative control group received an equal volume of distilled water via gavage, while all other mice received 56% Hongxing Erguotou. The 70% ethanol extract of *Amomum villosum* obtained in Example 1 was used to evaluate its preventive and therapeutic effects on the alcohol-induced liver and intestine injury model in mice. Mice were acclimatized to a standard environment for 7 days after purchase and fed a standard diet. The specific experimental procedure and grouping are as follows: Control group: Administered an equal volume of distilled water by gavage for 23 consecutive days; Negative control group (NC): Administered 600 mg / kg of 70% ethanol extract of Amomum villosum via gavage for 23 consecutive days; Model group: Starting from day 8, mice were administered 10 ml / kg volume of 43% Hongxing Erguotou via gavage for 16 consecutive days. 30 min after the last administration, all mice were administered an equal volume of 56% Hongxing Erguotou via gavage. Low-dose group of Amomum tsao-ko (Model+AKT(200)): 200 mg / kg of 70% ethanol extract of Amomum tsao-ko was administered by gavage for 23 consecutive days. Starting from day 8, 10 ml / kg of 43% Hongxing Erguotou was administered by gavage for 16 consecutive days. 30 min after the last administration, all mice were administered an equal volume of 56% Hongxing Erguotou by gavage. High-dose group of Amomum tsao-ko (Model+AKT(600)): 600 mg / kg of 70% ethanol extract of Amomum tsao-ko was administered by gavage for 23 consecutive days. Starting from day 8, 10 ml / kg of 43% Hongxing Erguotou was administered by gavage for 16 consecutive days. 30 min after the last administration, all mice were administered an equal volume of 56% Hongxing Erguotou by gavage. Positive control group (Model+SILY): Mice were given 50 mg / kg silymarin by gavage for 23 consecutive days, and from day 8 onwards, they were given 10 ml / kg volume of 43% Hongxing Erguotou by gavage for 16 consecutive days. 30 minutes after the last administration, all mice were given an equal volume of 56% Hongxing Erguotou by gavage.
[0031] 2.2 Effects of active ingredients from cardamom on plasma AST and ALT levels in model mice 2.2.1 Blood was collected from the eyeballs of mice in each group using anticoagulant tubes. The supernatant plasma was collected after centrifugation at 2000 r / min for 10 min. The AST and ALT levels in the plasma of each group of mice were detected using AST and ALT kits.
[0032] 2.2.2 Experimental Results AST (aspartate aminotransferase) and ALT (alanine aminotransferase) are enzymes present in high concentrations within hepatocytes. When hepatocytes are damaged, the cell membrane ruptures, causing AST and ALT to be rapidly released into the bloodstream. Changes in their concentrations can reflect the degree of hepatocyte damage; therefore, measuring plasma AST and ALT concentrations can provide some insight into liver tissue damage. This invention evaluates the effects of *Amomum villosum* extract on a mouse model of chronic alcoholic liver and intestine injury by detecting the levels of AST and ALT in the plasma of various groups of mice using an AST and ALT kit.
[0033] The results are shown in the figure. Figure 3 It can be seen that, compared with the control group, the concentrations of AST and ALT in the model group were significantly increased. After administering high-dose and low-dose groups of Amomum tsao-ko extract, the concentrations of AST and ALT were significantly restored, with effects similar to those in the positive control group. This indicates that the active ingredients of Amomum tsao-ko can significantly improve liver tissue damage in mice with chronic alcoholic liver and intestine injury. It is statistically significant.
[0034] 2.3 Effects of active ingredients from cardamom on mitochondrial function and redox metabolic indices in liver tissue of model mice 2.3.1 Liver tissues from mice in each group were aseptically harvested, rinsed with sterile pre-cooled PBS, and 30 mg of tissue was collected. 100 μL of the corresponding extraction buffer from each kit was added, and the mixture was homogenized at 4°C. The supernatant was collected by centrifugation at 2000 r / min, and the levels of JC-1 and NAD+ in the liver tissues of each group of mice were detected using the corresponding kits. + The expression levels of NADH, ATP, and ROS were assessed. Specifically, the JC-1 assay for mitochondrial membrane potential in liver tissue involved aseptically collected fresh liver tissue from each group of mice, which was then used in this kit for detection.
[0035] 2.3.2 Experimental Results ATP (adenosine triphosphate) is the direct energy carrier for cellular life activities, and its level directly reflects the terminal production capacity of mitochondria in synthesizing bioenergy through oxidative phosphorylation. + NADH and its reduced form NADH are key coenzymes and electron carriers in energy metabolism, and the ratio of the two (NAD...) +NADH (nicotinamide adenine dinucleotide redox pair) is a core indicator reflecting cellular redox status and the driving force of the mitochondrial electron transport chain. In alcoholic liver injury, ethanol metabolism excessively depletes NAD. + This leads to NAD + A significantly decreased NADH ratio inhibits the tricarboxylic acid cycle and electron transport, weakens mitochondrial ATP synthesis, and leads to hepatocyte energy metabolism disorders. Therefore, by measuring the ATP and NAD ratio in liver tissue... + The NADH ratio can objectively and directly evaluate the functional status of mitochondria from two aspects: energy production and metabolic regulation.
[0036] Reactive oxygen species (ROS) are metabolic byproducts generated during electron transport in mitochondria, primarily synthesized in the mitochondria. In dysfunctional mitochondria, the electron transport chain is disrupted, leading to increased electron leakage and excessive ROS production. Therefore, elevated ROS levels in liver tissue are a direct consequence of mitochondrial dysfunction and a self-inflicted damage mediator, serving as a key indicator for assessing oxidative stress levels and the extent of mitochondrial damage.
[0037] JC-1 is a fluorescent probe sensitive to mitochondrial membrane potential. Normal mitochondria maintain a high inner membrane potential (ΔΨm), which is the electrochemical basis for driving ATP synthesis and a core marker of their functional integrity. A decrease or loss of membrane potential indicates severe impairment of mitochondrial energy conversion function and is usually an early key event leading to cellular failure.
[0038] The results are shown in Figure 4-5. (From...) Figure 4 and Figure 5 As can be seen from A, compared with the control group, the model group had higher levels of ATP and NAD. + NADH and JC-1 levels were significantly reduced. After administration of high-dose and low-dose groups of cardamom extract, ATP and NAD levels were found to be significantly reduced. + The significant recovery of NADH and JC-1 levels indicates that the active ingredients of *Amomum villosum* can significantly improve mitochondrial damage in a mouse model of chronic alcoholic liver and intestine injury; Figure 5 As shown in Figure B, the ROS level in the model group was significantly higher than that in the control group. After treatment with high-dose and low-dose groups of Amomum villosum extract, the ROS level was significantly reduced, indicating that the active ingredients of Amomum villosum can significantly regulate mitochondrial function and redox metabolism indicators in the liver tissue of mice with chronic alcoholic liver and intestine injury. It is statistically significant.
[0039] 2.4 Detection of the effects of active ingredients on pathological damage in liver and colon tissues of model mice 2.4.1 Take liver tissue from each group of mice, aseptically extract the liver tissue, rinse with sterile pre-cooled PBS, fix and prepare paraffin sections, stain with HE and observe the pathological changes of the tissue structure under a microscope.
[0040] Methods for preparing paraffin sections: Sampling: Fresh liver tissue was collected and fixed in 10% formalin solution for at least 24 hours. The liver tissue was removed from the fixative and trimmed in a fume hood using a scalpel. The trimmed tissue and corresponding labels were then placed in a dehydration box. Dehydration and wax impregnation: Place the dehydration box into the dehydrator and dehydrate it sequentially with alcohol in a gradient. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, molten paraffin I at 65℃ for 1 hour, molten paraffin II at 65℃ for 1 hour, molten paraffin III at 65℃ for 1 hour; Embedding: The tissue impregnated with wax is embedded in an embedding machine. First, the molten wax is placed into the embedding frame. Before the wax solidifies, the tissue is taken out from the dehydration box, placed into the embedding frame according to the requirements of the embedding surface, and labeled accordingly. It is then cooled on a -10℃ freezing stage. After the wax solidifies, the wax block is removed from the embedding frame and trimmed. Sectioning: Place the trimmed wax block on a paraffin microtome and section it to a thickness of 4μm; float the section on 40℃ warm water in a slide spreader to flatten the tissue, pick up the tissue on a glass slide, bake the slide in a 60℃ oven, dry the wax with water and bake it until it melts, then remove it and store it at room temperature for later use.
[0041] The HE staining method is as follows: Dewaxing paraffin sections to water: Immerse the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then rinse with tap water; Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, blue back solution, and rinse with running water. Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol solutions for 5 min each, and then stained in eosin staining solution for 5 min.
[0042] Dehydration and mounting: The sections were sequentially immersed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min. After clearing, the sections were mounted with neutral resin. Microscopic examination, image acquisition and analysis 2.4.2 For each group of mice, colon tissue was aseptically collected, rinsed with sterile pre-cooled PBS, fixed and prepared into paraffin sections, stained with AB-PAS and observed under a microscope for pathological changes in tissue structure.
[0043] The method for preparing paraffin sections of colon tissue is the same as that for preparing paraffin sections of liver tissue described above.
[0044] Dewaxing: Tissue sections are dewaxed from the paraffin block using a series of xylene and ethanol solutions, and then dehydrated to water using ethanol solutions of different concentrations. Alcian staining: Immerse the slides in Alcian blue staining solution for about 10-20 minutes to make the acidic polysaccharide turn blue; wash the slides with distilled water to remove excess Alcian blue dye; Oxidation treatment: The slices were immersed in periodic acid solution for about 5 minutes to oxidize some of the hydroxyl groups in glycogen and acidic mucopolysaccharide molecules; PAS staining: Transfer the sections to Schiff reagent for PAS staining for 10-20 min until glycogen and other neutral polysaccharides turn purple-red; rinse the sections with running water to remove excess Schiff reagent and neutralize excess Schiff reagent with acidic solution. Dehydration and mounting: After stepwise dehydration with conventional ethanol and clearing with xylene solution, the sections were mounted with neutral resin; Microscopic examination was performed to observe and record the staining results, and images were acquired and analyzed.
[0045] 2.4.3 Experimental Results The results are as follows Figure 6 As shown. By Figure 6 As shown in Figure A, compared with the control group, the model group showed significant fatty degeneration of hepatocytes, characterized by cytoplasm filled with vacuoles of varying sizes, nuclei compressed to the periphery, and some hepatocytes exhibiting ballooning swelling. Disordered hepatic cord structure and compression and narrowing of hepatic sinusoids were also observed. After administration of high-dose and low-dose groups of *Amomum tsao-ko* extract, significant improvement in liver tissue pathological damage was observed, along with a significant reduction in hepatocyte vacuolation and effective amelioration of hepatocyte fatty degeneration and swelling. This indicates that *Amomum tsao-ko* extract can significantly improve liver tissue pathological damage in mice with chronic alcoholic enterohepatitis model.
[0046] Depend on Figure 6As shown in Figure B, compared with the control group, the model group exhibited a significant decrease in the number and atrophy of goblet cells in the colonic mucosa, abnormal mucus composition, disordered colonic crypt structure, inflammatory cell infiltration within the lamina propria, and disruption of intestinal epithelial cell polarity and mucus barrier integrity. This reflects alcohol-induced intestinal mucus secretion dysfunction, inflammatory response, and barrier damage. After administration of high- and low-dose groups of *Amomum tsao-ko* extract, significant improvement in colonic histopathological damage was observed, with a significant increase in the number of colonic mucosal goblet cells and a decrease in inflammatory cell infiltration. This indicates that *Amomum tsao-ko* extract can significantly improve colonic histopathological damage in mice with chronic alcoholic liver and intestine injury.
[0047] 2.5 Detection of the expression of related factors in the liver tissue of model mice by the active ingredient. 2.5.1 The expression of related factors ADRB2 and TLR4 in the liver tissue of model mice was detected by Western blotting.
[0048] 2.5.2 Experimental Results TLR4 belongs to the Toll-like receptor family and is widely distributed in various tissue cells, including monocytes and macrophages. Studies have shown that TLR4 can initiate intracellular signal transduction through the NF-κB pathway, participating in inflammatory responses and immune regulation. ADRB2 receptor is a member of the G protein-coupled transmembrane receptor superfamily. It can specifically bind to catecholamines and other endogenous ligands and be activated by their agonists, producing a series of biological effects.
[0049] The results are as follows Figure 7 As shown. By Figure 7 It can be seen that, compared with the control group, the expression of TLR4 in the model group was significantly increased, and after administration of high-dose and low-dose groups of Amomum tsao-ko extract, their expression was significantly restored; compared with the control group, the expression of ADRB2 in the model group was significantly decreased, and after administration of high-dose and low-dose groups of Amomum tsao-ko extract, ADRB2 expression was significantly restored; indicating that the active ingredients of Amomum tsao-ko can significantly regulate the expression of liver tissue-related factors in mice with chronic alcoholic liver and intestine injury. It is statistically significant.
[0050] In summary, the cardamom extract significantly reduced the levels of AST and ALT in the plasma of mice with chronic alcoholic liver and intestinal injury; improved the pathological damage in the liver and colon tissues of the model mice; restored mitochondrial function in the liver tissue of the model mice; and regulated the expression of liver-related factors in the liver tissue of the model mice. The cardamom extract provided by this invention has a significant effect on preventing and treating alcohol-induced liver and intestinal injury. Furthermore, the raw material is derived from natural products, ensuring high safety and low cost, making it suitable for industrial production and market application.
[0051] In addition, the preparation method of the ethanol extract of Amomum tsao-ko also has the above-mentioned effects by changing the sieve mesh size of the mature Amomum tsao-ko powder to 90 mesh or 100 mesh and sonicating it at 70W power for 25 min or at 80W power for 28 min.
[0052] The specific embodiments of the invention have been described in detail above, but these are merely examples, and the invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications or substitutions to the invention are also within the scope of this invention. Therefore, all equivalent transformations, modifications, and improvements made without departing from the spirit and principles of this invention should be included within the scope of this invention.
Claims
1. The application of Amomum tsao-ko extract in the preparation of drugs for the prevention and treatment of chronic alcoholic liver and gut injury, wherein the chronic alcoholic liver and gut injury is caused by long-term alcohol consumption and is accompanied by liver and gut axis disorder.
2. The application as described in claim 1, characterized in that, The herb extract can simultaneously protect liver and colon tissues, repair the intestinal mucosal barrier, and restore liver mitochondrial function.
3. The application as described in claim 2, characterized in that, The herb extract can downregulate TLR4 expression and upregulate ADRB2 expression in liver tissue.
4. The application as described in claim 4, characterized in that, The cardamom extract can reduce the levels of AST and ALT in the plasma of mice with a chronic alcoholic liver and intestine injury model.
5. The application as described in claim 1, characterized in that, The cardamom extract is a 70% ethanol extract of cardamom.