WO2026007239A1 - 灵芝酸a在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用 - Google Patents

灵芝酸a在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用

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Publication number
WO2026007239A1
WO2026007239A1 PCT/CN2024/119772 CN2024119772W WO2026007239A1 WO 2026007239 A1 WO2026007239 A1 WO 2026007239A1 CN 2024119772 W CN2024119772 W CN 2024119772W WO 2026007239 A1 WO2026007239 A1 WO 2026007239A1
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Prior art keywords
alcoholic fatty
liver disease
ganoderic acid
fatty liver
acid
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PCT/CN2024/119772
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English (en)
French (fr)
Inventor
杨宝学
卢烽
李敏
张航
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Medical Innovation Center Taizhou Of Peking University
Peking University
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Medical Innovation Center Taizhou Of Peking University
Peking University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • This invention belongs to the field of pharmaceutical technology, specifically relating to the application of ganoderic acid A in the preparation of products for the prevention and/or treatment of non-alcoholic fatty liver disease.
  • Nonalcoholic fatty liver disease is the most common liver disease worldwide and the leading cause of disability and life expectancy impairment. It can lead to a series of complications, including liver inflammation and fibrosis, eventually developing into nonalcoholic steatohepatitis. Poor dietary habits, a sedentary lifestyle, and aging all increase the risk of NAFLD. According to literature, the global prevalence of NAFLD increased from 25.3% in 1990-2006 to 38.0% in 2016-2019. my country is also a high-incidence area for NAFLD, with an incidence rate between 23.8% and 29% as of 2018. NAFLD seriously threatens human health and quality of life, imposing a huge economic and medical burden on my country.
  • Ganoderma lucidum is widely used in Asia as a traditional Chinese medicine.
  • Ganoderic acids are an important class of active substances in Ganoderma lucidum extracts. They are tetracyclic triterpenoids composed of 30 carbon atoms with a lanane skeleton. Based on the number and position of double bonds in the parent ring, ganoderic acids can be divided into different monomers.
  • ganoderic acid extracts ganoderic acid A is present in higher amounts than other ganoderic acid monomers and possesses various pharmacological effects, such as anti-inflammatory and antioxidant properties, and can also alleviate diseases caused by lipid metabolism disorders.
  • the effect and mechanism of ganoderic acid A in improving NAFLD have not yet been reported.
  • Nonalcoholic fatty liver disease is the most common liver disease worldwide and the leading cause of disability and life expectancy impairment.
  • This invention provides the application of ganoderic acid A in the preparation of products for the prevention and/or treatment of NAFLD.
  • Ganoderic acid A has a significant effect in alleviating NAFLD.
  • Ganoderic acid A can prevent and treat NAFLD by inhibiting bile acid reabsorption and the expression of genes related to cholesterol accumulation in the liver, and can be used in multiple ways for the prevention and treatment of NAFLD.
  • Ganoderic acid A has the advantages of being safe, effective, and suitable for long-term use, and has good application prospects.
  • this invention provides the application of ganoderic acid A in the preparation of products for the prevention and/or treatment of non-alcoholic fatty liver disease.
  • ganoderic acid A can be the sole active ingredient or one of the active ingredients.
  • the present invention also provides the use of compositions containing ganoderic acid A in the preparation of products for the prevention and/or treatment of non-alcoholic fatty liver disease.
  • composition further comprises pharmaceutically acceptable excipients and/or drugs compatible with ganoderic acid A.
  • the prevention and/or treatment of non-alcoholic fatty liver disease is manifested in at least one of the following aspects:
  • liver damage caused by non-alcoholic fatty liver disease is specifically reflected in the fact that ganoderic acid A can significantly reduce the elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) caused by non-alcoholic fatty liver disease.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • the non-alcoholic fatty liver disease is caused by a high-fat diet.
  • the product may be a drug, health product, and/or functional food.
  • the drug contains an effective dose of ganoderic acid A.
  • the effective dose is a unit dose (e.g., the content in one tablet) or a unit dose for the treated patient (e.g., a dose per unit body weight).
  • the drug is used to treat humans or mammals (e.g., mice).
  • the dosage form of the drug described in this invention is not limited, as long as it enables the active ingredient to be effectively delivered into the body, including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, lozenges, granules, powders, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches, etc.
  • the present invention also provides a method for preventing and/or treating non-alcoholic fatty liver disease.
  • the method for preventing and/or treating non-alcoholic fatty liver disease includes the following steps: administering ganoderic acid A or the composition containing ganoderic acid A to a recipient animal or human to prevent and/or treat non-alcoholic fatty liver disease.
  • This invention utilizes a high-fat diet-induced C57BL/6J mouse model to discover the alleviating effect of ganoderic acid A supplementation on non-alcoholic fatty liver disease.
  • the high-fat group mice showed significantly increased body weight, liver index, and white adipose tissue index.
  • the mice after daily oral administration of 20 mg/kg body weight of ganoderic acid to mice for 16 weeks, the mice showed significantly decreased body weight, liver index, and white adipose tissue index.
  • This invention through HE staining of liver tissue sections, observed significantly enlarged hepatocytes in mice in the high-fat group, with intracellular lipid accumulation and the formation of fat vesicles. After intervention with ganoderic acid A, the volume of fat vesicles within hepatocytes significantly decreased. Oil Red O staining of the liver showed that ganoderic acid A significantly reduced lipid deposition in the liver. Using a kit, this invention revealed that ganoderic acid A significantly reduced hepatic triglyceride and cholesterol levels. These findings suggest that ganoderic acid A can effectively alleviate the development and progression of non-alcoholic fatty liver disease.
  • ganoderic acid A can reduce lipid accumulation in the liver of mice caused by a high-fat diet.
  • ganoderic acid A can reduce the increase in triglyceride and cholesterol content in mouse liver tissue induced by a high-fat diet.
  • This invention by detecting metabolites in the terminal ileum of mice in a blank group, a high-fat group, and a high-fat group + ganoderic acid A group, found that ganoderic acid A significantly affected cholesterol/bile acid metabolism in mice and reduced the levels of some long-chain fatty acids. Simultaneously, using a kit, it was found that ganoderic acid A significantly increased the content of bile acids in feces. Furthermore, the kit also showed that ganoderic acid A significantly increased the lipid content in feces. These findings suggest that ganoderic acid A can inhibit lipid absorption by regulating bile acid/cholesterol metabolism and bile acid reabsorption in mice.
  • ganoderic acid A can lower levels of harmful bile acids such as taurocholic acid and glycochenodeoxycholic acid, inhibiting bile acid reabsorption. Ganoderic acid A also inhibits the absorption of triglycerides and cholesterol.
  • This invention uses real-time quantitative PCR technology to detect genes related to cholesterol breakdown in the livers of mice in the blank group, high-fat group, and high-fat + ganoderic acid A group.
  • ganoderic acid A promotes cholesterol breakdown in the liver and reduces cholesterol accumulation in the liver by increasing the expression of the cholesterol 7 ⁇ -hydroxylase (CYP7A1) gene.
  • This invention has discovered new natural active substances for the prevention and treatment of non-alcoholic fatty liver disease. It has the advantages of being safe, effective and suitable for long-term use, and has good application prospects.
  • Ganoderma lucidum acid A can prevent and treat non-alcoholic fatty liver disease by inhibiting the reabsorption of bile acids and the expression of genes related to cholesterol accumulation in the liver. It can be used in various ways to prevent and treat non-alcoholic fatty liver disease.
  • Figure 1 shows the effect of ganoderic acid A on the body weight of mice.
  • Figure 2 shows the effects of ganoderic acid A on liver index and fat index.
  • Figure 3 shows the effect of ganoderic acid A on the concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse blood.
  • Figure 4 shows mouse liver sections stained with HE and Oil Red O.
  • Figure 5 shows the effect of ganoderic acid A on the triglyceride and cholesterol content in mouse liver tissue.
  • Figure 6 shows the differential bile acid metabolites in mouse ileum tissue.
  • Figure 7 shows the differential long-chain fatty acids in mouse ileum tissue.
  • Figure 8 shows the effects of ganoderic acid A on triglycerides, cholesterol, and total bile acids in mouse feces.
  • Figure 9 shows the effect of ganoderic acid A on the expression level of Cyp7a1 gene in mouse liver tissue.
  • the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.
  • mice Eighteen 8-week-old C57BL/6J mice were acclimatized for one week and then randomly divided into three groups of six mice each: a control group, a high-fat group, and a high-fat + ganoderic acid A group.
  • the control group was fed a normal diet, while the high-fat and high-fat + ganoderic acid A groups were fed a high-fat diet.
  • the control and high-fat groups were administered 0.5% sodium carboxymethyl cellulose saline solution by gavage, while the high-fat + ganoderic acid A group was administered ganoderic acid A (20 mg/kg, dissolved in 0.5% sodium carboxymethyl cellulose saline solution) by gavage once daily for 16 weeks.
  • the normal and high-fat diets were purchased from Beijing Keao Xieli Feed Co., Ltd.
  • Example 1 The effect of ganoderic acid A in alleviating non-alcoholic fatty liver disease
  • ALT and AST serum alanine aminotransferase
  • AST aspartate aminotransferase
  • mice were dissected, liver tissue was collected, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 ⁇ m. Pathological evaluation was performed using hematoxylin and eosin (H&E) and Oil Red O staining.
  • H&E hematoxylin and eosin
  • Triglyceride and cholesterol content detection After the last administration experiment, liver tissue of mice was taken, and the triglyceride and cholesterol content of the liver was detected using the Triglyceride and Cholesterol Detection Kit of Prilex Company, in accordance with the instructions.
  • ganoderic acid A can significantly reduce the increase in triglyceride and cholesterol content in mouse liver tissue induced by a high-fat diet.
  • Example 2 Effect of ganoderic acid A on lipid absorption in mice on a high-fat diet
  • Non-targeted metabolomics assay After the last administration experiment, the mice were dissected and the contents of the ileum were collected. Metabolomics analysis was performed using liquid chromatography-tandem mass spectrometry to detect the components of the mouse ileum contents.
  • the levels of triglycerides, cholesterol, and bile acids in feces were measured. Feces were collected from mice after the last administration of the drug. Triglyceride and cholesterol levels were measured using a kit from Prilex Pharmaceuticals, and bile acid levels were measured using a kit from Nanjing Jiancheng Pharmaceutical Co., Ltd., following the manufacturer's instructions.
  • Example 3 Mechanism of action of ganoderic acid A in alleviating hepatic lipid accumulation
  • the expression level of Cyp7a1 gene in liver tissue was detected by real-time quantitative PCR.
  • ganoderic acid A significantly increased the expression level of Cyp7a1 , which is responsible for converting excess liver cholesterol into bile acids and alleviating liver lipid accumulation.
  • This invention provides the application of ganoderic acid A in the preparation of products for the prevention and/or treatment of non-alcoholic fatty liver disease (NAFLD).
  • Ganoderic acid A can prevent and treat NAFLD by inhibiting the reabsorption of bile acids and the expression of genes related to cholesterol accumulation in the liver, and can be used in various ways to prevent and treat NAFLD.
  • Ganoderic acid A has the advantages of being safe, effective, and suitable for long-term use, and has good application prospects.

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Abstract

灵芝酸A在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用。灵芝酸A可以调节高脂饮食诱导的C57BL/6J小鼠的胆汁酸重吸收，减少甘油三酯和胆固醇的吸收。灵芝酸A还可以增强肝脏胆固醇分解的关键酶胆固醇7α-羟化酶基因的表达，从而促进肝脏胆固醇转化为胆汁酸，缓解肝脏胆固醇积累。灵芝酸A通过调节胆汁酸代谢、抑制脂质吸收以及减轻肝脏脂质堆积，对高脂饮食诱发的非酒精性脂肪肝病具有显著的预防和改善作用。因此灵芝酸A可以作为保健品、功能性产品或药物用于非酒精性脂肪肝病的防治。

Description

灵芝酸A在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用 技术领域
本发明属于医药技术领域，具体涉及灵芝酸A在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用。
 背景技术
非酒精性脂肪性肝病（NAFLD）是目前世界范围内最常见的肝病，也是导致伤残改变寿命最多的肝病。其会继发引起肝脏炎症、纤维化等一系列病变，发展成为非酒精性脂肪肝炎。不良饮食习惯、久坐缺乏运动的生活方式以及衰老等都会增加患NAFLD的风险。据文献报道，NAFLD的全球患病率从1990-2006年的25.3%增加到2016-2019年的38.0%。我国亦是NAFLD的高发区，截止2018年我国NAFLD的发病率在23.8%至29%之间。NAFLD严重威胁人类健康和生活质量，给我国造成了巨大的经济和医疗负担。尽管NAFLD发病率居高不下且逐年递增，但迄今为止仍缺乏治疗NAFLD的安全有效药物。靶向脂质代谢被认为是NAFLD的主要治疗策略。干预脂代谢紊乱的药物常被用于改善NAFLD，例如减肥药和降脂药等，但长期使用这些药物会产生严重不良反应。因此，开发出安全有效、可长期使用的防治NAFLD的药物或保健品具有重要意义。
灵芝（ Ganoderma lucidum）作为传统中药在亚洲地区被广泛使用。而灵芝酸化合物是灵芝提取物中一类重要的活性物质，是由30个碳原子组成的四环三萜类化合物，具有羊毛烷类骨架。根据母环的双键数目和位置的区别，可以将灵芝酸分为不同的单体。在灵芝酸提取物中，同其他灵芝酸单体相比，灵芝酸A含量较高，并且具有多种药理作用，如抗炎、抗氧化等，还可以缓解由脂代谢紊乱引起的疾病。但灵芝酸A改善NAFLD的效果与机理仍未见报道。
发明概述
非酒精性脂肪性肝病（NAFLD）是目前世界范围内最常见的肝病，也是导致伤残改变寿命最多的肝病。本发明提供了灵芝酸A在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用。灵芝酸A缓解非酒精性脂肪肝病的作用显著。灵芝酸A可以通过抑制胆汁酸的重吸收和肝脏胆固醇堆积相关基因的表达来防治非酒精性脂肪肝的形成，可通过多种方式用于预防和治疗非酒精性脂肪肝病。
灵芝酸A具有安全有效适于长期服用的优点，具有良好的应用前景。
技术解决方案
为了解决上述技术问题，本发明提供了一种灵芝酸A在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用。
所述灵芝酸A的化学结构式如式I所示：
所述产品中，灵芝酸A可以作为唯一活性成分，也可以作为活性成分之一。
本发明还提供了包含灵芝酸A的组合物在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用。
上述的应用中，所述组合物还包含药学上可接受的辅料和/或与灵芝酸A可配伍的药物。
本发明中，所述预防和/或治疗非酒精性脂肪肝病体现在下述至少一方面：
1）抑制非酒精性脂肪肝病导致的体重、肝脏比重和/或白色脂肪比重的增加；
2）改善非酒精性脂肪肝病导致的肝脏损伤；
3）减轻非酒精性脂肪肝病导致的肝脏脂质堆积；
4）调节胆汁酸重吸收、抑制脂质（甘油三酯和胆固醇）的吸收；
5）增强肝脏胆固醇分解的关键酶胆固醇7α-羟化酶（CYP7A1）基因的表达。
进一步地，所述改善非酒精性脂肪肝病导致的肝脏损伤具体体现在：灵芝酸A能明显减轻非酒精性脂肪肝病导致的谷丙转氨酶和谷草转氨酶水平升高。
本发明中，所述非酒精性脂肪肝病是由高脂饮食引起的。
本发明中，所述产品可为药物、保健品和/或功能性食品。
所述药物含有有效剂量的灵芝酸A。有效剂量为单位给药剂量形式（如一片药物中的含量）或治疗的患者的单位剂量（如单位体重剂量）。在本发明中，药物治疗的对象为人或哺乳动物类（如小鼠等）。
本发明中所述的药物的剂型不限，只要能够使活性成分有效达到体内的剂型都可以，包括：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、滴剂、贴剂等。
本发明还提供了一种预防和/或治疗非酒精性脂肪肝病的方法。
本发明所提供的预防和/或治疗非酒精性脂肪肝病的方法，包括如下步骤：给受体动物或人施用灵芝酸A或所述包含灵芝酸A的组合物以预防和/或治疗非酒精性脂肪肝病。
本发明利用高脂饮食诱导的C57BL/6J小鼠模型，发现补充灵芝酸A对非酒精性脂肪肝病的缓解作用。与空白组相比，高脂组小鼠的体重、肝脏指数和白色脂肪指数均显著增加。与高脂组相比，每日经口灌胃给予小鼠20 mg/kg体重的灵芝酸16周后，小鼠的体重、肝脏指数和白色脂肪指数均显著下降。
本发明通过测定空白组、高脂组、高脂+灵芝酸A组小鼠血生化指标，显示灵芝酸A可以显著降低高脂饮食小鼠血清中的谷丙转氨酶和谷草转氨酶浓度，提示灵芝酸A可以改善高脂饮食诱发的肝脏损伤。
本发明通过肝脏组织切片HE染色，观察到高脂组小鼠肝细胞明显增大，细胞内充积脂质，形成脂肪泡。灵芝酸A干预后，肝细胞内脂肪泡的体积显著减小。肝脏油红O染色结果表明灵芝酸A可显著减少肝脏中脂质沉积。本发明使用试剂盒检测发现灵芝酸A可显著降低肝脏甘油三酯与胆固醇水平。提示灵芝酸A可以有效缓解非酒精性脂肪肝的发生发展。
具体的，灵芝酸A具有减轻高脂饮食引起的小鼠肝脏脂质堆积。
具体的，灵芝酸A可以降低高脂饮食引起的小鼠肝脏组织中甘油三酯与胆固醇含量增加。
本发明通过检测空白组、高脂组、高脂+灵芝酸A组小鼠回肠末端代谢产物，发现灵芝酸A显著影响了小鼠胆固醇/胆汁酸代谢，降低了一些长链脂肪酸水平。同时使用试剂盒检测发现灵芝酸A可显著增加粪便中胆汁酸的含量。并且使用试剂盒检测发现灵芝酸A可显著增加粪便中脂质含量。提示灵芝酸A可以通过调控小鼠的胆汁酸/胆固醇代谢与胆汁酸重吸收，进而抑制脂质吸收。
具体的，灵芝酸A可以降低有害胆汁酸如牛磺胆酸与甘氨鹅脱氧胆酸水平，抑制了胆汁酸重吸收。灵芝酸A抑制了甘油三酯与胆固醇的吸收。
本发明应用荧光定量PCR技术，检测空白组、高脂组、高脂+灵芝酸A组小鼠肝脏胆固醇分解相关基因。
具体的，灵芝酸A通过提高肝脏中胆固醇7α-羟化酶（CYP7A1）基因表达，促进肝脏胆固醇分解，减少肝脏胆固醇堆积。
有益效果
（1）本发明发掘了新的防治非酒精性脂肪肝病的天然活性物质，具有安全有效适于长期服用的优点，具有良好的应用前景。
（2）灵芝酸A缓解非酒精性脂肪肝病的作用显著。
（3）灵芝酸A可以通过抑制胆汁酸的重吸收和肝脏胆固醇堆积相关基因的表达来防治非酒精性脂肪肝的形成，可通过多种方式用于预防和治疗非酒精性脂肪肝病。
 附图说明
图1为灵芝酸A对小鼠体重的影响。
图2为灵芝酸A对肝脏指数和脂肪指数的影响。
图3为灵芝酸A对小鼠血液中谷丙转氨酶和谷草转氨酶浓度的影响。
图4为小鼠肝脏切片HE染色、油红O染色。
图5为灵芝酸A对小鼠肝脏组织甘油三酯、胆固醇含量的影响。
图6为小鼠回肠组织中差异胆汁酸代谢产物。
图7为小鼠回肠组织中差异长链脂肪酸。
图8为灵芝酸A对小鼠粪便中甘油三酯、胆固醇与总胆汁酸的影响。
图9为灵芝酸A对小鼠肝脏组织 Cyp7a1基因表达水平的影响。
本发明的实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。如无特殊说明，以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例的实验分组与给药：
将18只8周龄C57BL/6J小鼠适应性喂养一周后，随机分成3组，每组6只，即空白组、高脂组和高脂+灵芝酸A组，空白组喂养正常饲料，高脂组和高脂+灵芝酸A组喂养高脂饲料，空白组和高脂组每次灌胃给药0.5%羧甲基纤维素钠生理盐水溶液、高脂+灵芝酸A组每次灌胃给药灵芝酸A（20mg/kg，溶于0.5%羧甲基纤维素钠生理盐水溶液），灌胃频率为每天给药1次，共16周。所述正常、高脂饲料购于北京科澳协力饲料有限公司。
实施例1：灵芝酸A缓解非酒精性脂肪肝病的作用
每周对小鼠称重并记录各组小鼠的体重。待末次给药实验结束后，解剖小鼠取出肝脏、白色脂肪。称取肝脏、白色脂肪重量，并计算肝脏比重与白色脂肪比重（公式为：肝脏比重=肝脏重量/体重*100%；白色脂肪比重=白色脂肪重量/体重*100%）。
实验结果：见图1和图2。灵芝酸A可显著抑制高脂饮食引起的体重、肝脏比重和白色脂肪比重的增加。
血清中谷丙转氨酶与谷草转氨酶的检测：待末次给药实验结束后，小鼠禁食12 h后，取血，3000 r/min离心10min，分离血清待检，测定血清谷丙转氨酶和谷草转氨酶水平。
实验结果：见图3。灵芝酸A明显减轻高脂饮食引起的谷丙转氨酶和谷草转氨酶水平升高，表明灵芝酸A显著改善了高脂饮食引起的肝脏损伤。
HE染色、油红O染色：待末次给药实验结束后，解剖小鼠，取小鼠肝脏组织，用4%多聚甲醛固定组织，然后包埋石蜡，切片厚度为4 μm。采用苏木精素与伊红（H&E）和油红O染色进行病理评价。
实验结果：见图4。通过肝脏组织切片HE染色可观察到，高脂组小鼠肝细胞明显增大，细胞内充积脂质，形成脂肪泡。灵芝酸A干预后表现出明显的缓解作用，肝细胞内脂肪泡的体积显著减小。肝脏油红O染色结果表明灵芝酸A可显著减少肝脏中脂质沉积。
甘油三酯和胆固醇含量检测：待末次给药实验结束后，取小鼠肝脏组织，使用普利莱公司甘油三酯与胆固醇检测试剂盒，按照说明书操作检测肝脏甘油三酯与胆固醇含量。
实验结果：见图5，灵芝酸A可显著降低高脂饮食引起的小鼠肝脏组织甘油三酯与胆固醇含量增加。
实施例2：灵芝酸A对高脂饮食小鼠脂质吸收的影响
非靶向代谢组检测：待末次给药实验结束后，解剖小鼠并收集小鼠回肠内容物，使用液相色谱-串联质谱技术进行代谢组学分析，检测小鼠回肠内容物的成分。
实验结果：见图6与图7。高脂组与高脂+灵芝酸A组相比，结果表明灵芝酸A显著影响了小鼠胆固醇/胆汁酸代谢通路，降低了有害胆汁酸如牛磺胆酸（导致过度脂质吸收）与甘氨鹅脱氧胆酸（可导致糖脂代谢紊乱）水平。同时灵芝酸A降低了回肠中多种长链脂肪酸水平。以上结果提示灌胃灵芝酸A可影响小鼠的胆汁酸/胆固醇代谢，并通过改变胆汁酸代谢而影响脂质吸收。
粪便中甘油三酯、胆固醇和胆汁酸的含量。待末次给药实验结束后，收集小鼠粪便。甘油三酯、胆固醇含量使用普利莱公司试剂盒检测，胆汁酸含量使用南京建成公司试剂盒检测，按照说明书进行操作。
实验结果：见图8。灵芝酸A显著增加了粪便中甘油三酯、胆固醇和胆汁酸的水平，表明灵芝酸A促进了脂质和胆汁酸的排泄，减少了脂质的吸收。
实施例3：灵芝酸A缓解肝脏脂质堆积作用机理
荧光定量PCR法检测肝脏组织 Cyp7a1基因水平表达量。
实验结果：见图9，灵芝酸A显著提高了 Cyp7a1的表达水平，其负责将过量的肝脏胆固醇转化为胆汁酸，并缓解肝脏脂质堆积。
相关申请的交叉引用：
本申请要求申请日为2024年7月3日的中国专利申请(申请号为: 202410887701.5)的优先权，上述专利申请的全部内容在此被援引加入本文。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
 工业实用性
本发明提供了灵芝酸A在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用。灵芝酸A可以通过抑制胆汁酸的重吸收和肝脏胆固醇堆积相关基因的表达来防治非酒精性脂肪肝的形成，可通过多种方式用于预防和治疗非酒精性脂肪肝病。灵芝酸A具有安全有效适于长期服用的优点，具有良好的应用前景。

Claims (9)

  1. 灵芝酸A在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用。
  2.  包含灵芝酸A的组合物在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用。
  3.  根据权利要求2所述的应用，其特征在于：所述组合物还包含药学上可接受的辅料和/或与灵芝酸A可配伍的药物。
  4.  根据权利要求1-3中任一项所述的应用，其特征在于：所述预防和/或治疗非酒精性脂肪肝病体现在下述至少一方面：
    1）抑制非酒精性脂肪肝病导致的体重、肝脏比重和/或白色脂肪比重的增加；
    2）改善非酒精性脂肪肝病导致的肝脏损伤；
    3）减轻非酒精性脂肪肝病导致的肝脏脂质堆积；
    4）调节胆汁酸重吸收、抑制脂质的吸收；
    5）增强肝脏胆固醇分解的关键酶胆固醇7α-羟化酶（CYP7A1）基因的表达。
  5.  根据权利要求4所述的应用，其特征在于：所述改善非酒精性脂肪肝病导致的肝脏损伤具体体现在：灵芝酸A能明显减轻非酒精性脂肪肝病导致的谷丙转氨酶和谷草转氨酶水平升高。
  6. 根据权利要求1-5中任一项所述的应用，其特征在于：所述产品可为药物和/或保健品和/或功能性食品。
  7.  灵芝酸A在预防和/或治疗非酒精性脂肪肝病中的应用。
  8.  包含灵芝酸A的组合物在预防和/或治疗非酒精性脂肪肝病中的应用。
  9.  一种预防和/或治疗非酒精性脂肪肝病的方法，包括下述步骤：给受体动物或人施用灵芝酸A或包含灵芝酸A的组合物以预防和/或治疗非酒精性脂肪肝病。
PCT/CN2024/119772 2024-07-03 2024-09-19 灵芝酸a在制备预防和/或治疗非酒精性脂肪肝病的产品中的应用 Pending WO2026007239A1 (zh)

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