WO2017215349A1 - 罗汉果醇在制备肝纤维化药物中的应用 - Google Patents

罗汉果醇在制备肝纤维化药物中的应用 Download PDF

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WO2017215349A1
WO2017215349A1 PCT/CN2017/081423 CN2017081423W WO2017215349A1 WO 2017215349 A1 WO2017215349 A1 WO 2017215349A1 CN 2017081423 W CN2017081423 W CN 2017081423W WO 2017215349 A1 WO2017215349 A1 WO 2017215349A1
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mogroside
preparation
liver fibrosis
medicament
preventing
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French (fr)
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谢海峰
张朝凤
胡云岭
谢期林
罗明锋
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成都普睿法药物研发有限公司
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)

Definitions

  • the invention belongs to the technical field of prevention and treatment of liver fibrosis, and particularly relates to the application of mogroside in the preparation of a medicament for preventing and/or treating liver fibrosis.
  • Hepatic fibrosis refers to the excessive deposition of diffuse extracellular matrix (especially collagen) in the liver. It is a kind of damage repair reaction after chronic liver injury caused by various diseases. It is cirrhosis. Early reversible phase. If left untreated, it may progress to decompensated cirrhosis and various complications of end-stage liver disease. Continued development of liver fibrosis can lead to cirrhosis and even liver cancer. Hepatocyte inflammation and necrosis are the initiating factors of liver fibrosis, and they are also the driving factors for their forward development. Because the mechanism is not clear, it is subject to certain restrictions on the treatment of drugs, and the traditional Chinese medicine has the advantages of overall regulation and clear anti-oxidation, enzyme-lowering liver protection, low toxicity and low cost.
  • HSC hepatic stellate cells
  • ECM extracellular matrix
  • Deposition mainly including type I collagen, type III collagen type IV collagen.
  • Activated HSC can also synthesize and secrete a large amount of TGF- ⁇ 1 , which continuously increases the level of TGF- ⁇ 1.
  • activated HSC proliferates and secretes large amounts of ECM, which causes liver fibrosis to gradually worsen.
  • the activation of HSC is a central link in the process of liver fibrosis.
  • TGF- ⁇ is a key factor in the activation of HSC and plays an important role in liver fibrosis.
  • Activated HSC is the main cell producing ECM, which is central to ECM metabolism and the production of various cellular mediators.
  • ⁇ -SMA is one of the hallmarks of HSC activation.
  • CCl 4 induces liver fibrosis in mice is a classic in vivo pharmacodynamic model for screening anti-hepatic fibrosis drugs. CCl 4 can cause oxidation and anti-oxidation imbalance in the body, and increase the production of oxygen free radicals, thereby initiating lipid peroxidation and reducing its TGF- ⁇ 1 content can slow the progression of liver fibrosis in mice.
  • Mangosteen is a mature fruit of the genus Cucurbitaceae, Siratia grosvenorii (Swingle) C. Jeffre y . It is mainly produced in Guangxi Yongfu, Lingui and Longsheng counties, and is a famous specialty in Guangxi. Mangosteen is cool, sweet, and belongs to the lungs and large intestine. It has the functions of moistening the lungs and relieving cough, clearing away heat and relieving heat, clearing the throat and opening the sound, cooling blood and smoothing the intestines. It is a unique economic crop of homologous medicines and foods in China.
  • the total glycosides of Siraitia grosvenii are the main active ingredients in Siraitia grosvenii, and have a wide range of biological and pharmacological values. Modern medical research has proved that the total glycosides of Siraitia grosvenii not only have the effects of antitussive, antiasthmatic, antispasmodic, anti-inflammatory, and regulate digestive tract function, but also enhance immunity, protect liver and reduce enzymes, treat acute lung injury, anti-oxidation and anti-aging. .
  • the main component of Luo Han Guo extract is mogroside.
  • the total glycosides in Luo Han Guo are cucurbitane triterpenoids, including Mogroside V; Mogroside IVe; Mogroside IIIe; Mogroside II A2 (Mogroside II A2); Mogroside III A1; Mogroside IVa; Mogroside VI; Siamenoside I; 11-O- Luo Han Guo Glycoside V (11-Oxomogroside V) and the like.
  • Mogroside is a aglycon of a mogroside compound and can be obtained by acid hydrolysis.
  • the technical problem to be solved by the present invention is to provide a new medical use of rohanol, that is, the use of rohanol in the treatment and/or prevention of liver fibrosis drugs and/or health care products.
  • rohanol in the preparation of a medicament and/or a health care product for preventing and/or treating liver fibrosis.
  • the mogroside is obtained by acid hydrolysis of the Luo Han Guo extract, and the structural formula is as follows:
  • the mogroside is replaced by a chemically modified mogroin derivative, which includes a halide, an esterified product, an amine compound, an ether compound, and a salt of mogroside of mogroside.
  • a chemically modified mogroin derivative which includes a halide, an esterified product, an amine compound, an ether compound, and a salt of mogroside of mogroside.
  • the application comprises use in the treatment of acute liver injury, liver cirrhosis, viral hepatitis, liver cancer.
  • the application comprises reducing collagen deposition, reducing inflammatory factor release, reducing the expression of ⁇ -SMA in liver tissue, and inhibiting proliferation of liver fibroblasts.
  • the medicament and/or health care product is a preparation prepared by using mogroside as a main active ingredient.
  • the formulation contains a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier may comprise from 0.1 to 99.9% by weight of the formulation.
  • the pharmaceutically acceptable carrier comprises the following materials: mannitol, sorbitol, sodium metabisulfite, sodium hydrogen sulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA , EDTA calcium sodium, monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, Glucose, fructose, dextran, glycine, starch, cane Sugar, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerin, earth temperature 80, agar, calcium carbonate, calcium hydrogencarbonate, surfactant, polyethylene
  • the dosage form of the preparation includes a tablet, a capsule, an injection, an oral solution, a pill, and a powder.
  • the mogroside is obtained by extracting mogroside, hydrolyzed with 5% sulfuric acid, separated by silica gel column, purified and purified, and the purity is over 95%.
  • Liver fibrosis is a common pathological change in the development of various liver diseases or liver damage to a certain period.
  • the continuous development of liver fibrosis can lead to subsequent complications such as liver cirrhosis and liver cancer.
  • Due to the multiple links and complexity of the pathological mechanism of liver fibrosis the effect of single link or targeted intervention is not satisfactory, and there is still a lack of specific effective drugs in clinical practice.
  • the inventors have demonstrated in vitro and in vivo experiments that mogroside can significantly improve the degree of liver fibrosis in mice, reduce collagen deposition, and attenuate TGF- ⁇ 1-induced liver fibrosis in vitro by HSC-T6, showing a model of liver fibrosis in vivo and in vitro. Intervention.
  • the mogroside alcohol of the invention has the characteristics of clear structure, good stability and easy control of quality, and can be used for preparing drugs for preventing or/and treating liver fibrosis.
  • the experimental materials involved in the invention are derived from the original plants, and the original plants have a wide range, low cost, clear extract activity, and wide practical value.
  • Figure 2 The effect of MG and Mogrol on the degree of inflammation in each group (HE staining ⁇ 200), and the effect of MG IVE and Mogrol on the degree of liver fibrosis in each group (Masson staining ⁇ 200).
  • Figure 4 shows the effect of MG and Mogrol on the content of ⁇ -SMA protein in HSC-T6 induced by TGF- ⁇ 1. ## p ⁇ 0.01,versus control; ** p ⁇ 0.01, *** p ⁇ 0.001versus model.
  • Figure 5 Effect of MG and Mogrol on ⁇ -SMA protein in mice with liver fibrosis induced by CCl 4 .
  • FIG. 6 Effect of MG and Mogrol on the stimulation of ⁇ -SMA protein in HSC-T6 cells by TGF- ⁇ 1. ** p ⁇ 0.01, *** p ⁇ 0.001 compared to the TGF- ⁇ 1 model group; # p ⁇ 0.05 compared to the TGF- ⁇ 1 + MG group.
  • rohanol in the preparation of a medicament and/or a health care product for preventing and/or treating liver fibrosis.
  • the mogroside is obtained by acid hydrolysis of the Luo Han Guo extract, and the structural formula is as follows:
  • the mogroside is obtained by using a commercially available Luo Han Guo extract, hydrolyzed with 5% sulfuric acid, separated by silica gel column, purified and purified, and the purity is 95% or more.
  • the mogroside is replaced by a chemically modified mogroin derivative, which includes a halide, an esterified product, an amine compound, an ether compound, and a salt of mogroside of mogroside.
  • a chemically modified mogroin derivative which includes a halide, an esterified product, an amine compound, an ether compound, and a salt of mogroside of mogroside.
  • the use comprises the preparation of a medicament for the treatment of liver fibrosis-related diseases and/or health supplements.
  • the application comprises use in the treatment of acute liver injury, liver cirrhosis, viral hepatitis, liver cancer.
  • the application comprises reducing collagen deposition, reducing inflammatory factor release, reducing the expression of ⁇ -SMA in liver tissue, and inhibiting proliferation of liver fibroblasts.
  • the medicament and/or health care product is a preparation prepared by using mogroside as a main active ingredient.
  • the preparation method of the preparation of the mogroside of the present invention is a conventional one, which can be obtained from the prior patents, publicly known documents, or prepared according to the disclosed pharmaceutical standards.
  • the dosage form of the drug and/or health care product includes a tablet, a capsule, an injection, an oral solution, a pill, and a powder.
  • the mogroside preparation of the present invention may be in any pharmaceutically acceptable dosage form, and includes: tablets, sugar-coated tablets, film-coated tablets, enteric coated tablets, capsules, hard capsules, soft capsules, oral liquids. Oral preparation, granules, granules, pills, powders, ointments, granules, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, pills, Patch.
  • the rohanol preparation is a pharmaceutical composition prepared by using mogroside as a main active ingredient, the group
  • the pharmaceutically acceptable carrier may contain a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier may be present in the formulation in an amount of from 0.1 to 99.9% by weight.
  • the preparation of the mogroside preparation of the present invention may optionally be carried out by adding a suitable pharmaceutically acceptable carrier, including but not limited to the following, selected from the group consisting of: mannitol, sorbitol, sodium metabisulfite. , sodium hydrogen sulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium EDTA, monovalent alkali metal carbonate, acetate, phosphate or Aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derived , cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycer
  • the medicine or health care product according to the present invention refers to a medicine or a health care product capable of reducing the amount of collagen accumulation in the interstitial tissue of liver fibrosis.
  • the medicament or health care product of the present invention refers to a drug or a health care product which can inhibit the release of inflammatory factors, reduce the degree of inflammation, inhibit collagen formation, and protect liver tissue from liver fibrosis.
  • the medicament or health care product according to the present invention refers to a drug or a health care product which exerts anti-liver fibrosis by anti-inflammatory and inhibits proliferation of hepatocytes.
  • Example 1 Inosanol Improves CCl 4 Induced Liver Fibrosis in Mice
  • MG total glycoside
  • Mogrol rohanol
  • ICR mice female, weighing 18-22 g, were provided by the Qinglongshan animal breeding ground in Nanjing. Wahaha pure water; ⁇ -SMA monoclonal antibody (abcam); goat anti-rabbit IgG (bioworld).
  • mice ICR female mice were divided into blank group, model group, MG group (250 mg/kg) and Mogrol group (10 mg/kg), and the drugs were administered by intragastric administration, with 20 rats in each group.
  • the mice were intraperitoneally injected with 40% CCl4 (olive oil suspension) 2 ml/kg twice a week to establish a mouse model of liver fibrosis; the blank group was intraperitoneally injected with olive oil 2 ml/kg twice a week. Molding and simultaneous administration of drugs for treatment.
  • the mice were sacrificed at the 6th week after modeling, and the blood was taken. After centrifugation, the upper serum was aspirated and stored at -80 °C.
  • liver tissue damage and fibrosis were observed.
  • the other liver tissues were frozen at -80 °C to detect ⁇ -SMA protein content.
  • mice in the model group had the most weight loss compared with the mice in the blank group, and the weight of the mice in the MG and Mogrol groups increased, which was higher than that in the model group (Fig. 1). ), indicating that total glucosides of Siraitia grosvenorii and mogroside can improve the weight loss of mice caused by liver fibrosis to some extent.
  • liver tissue structure of the blank group was intact, no inflammatory cell infiltration, no fibroblast proliferation, and a small amount of blue staining in the liver tissue of the control mice.
  • Collagen fibers are a major component of the extracellular matrix.
  • the hepatocyte structure was destroyed, necrosis, calcification, fibroblast proliferation, massive collagen deposition, and liver fibrosis.
  • Masson staining a large number of densely stained collagen fibers were obtained, which were bundled or flaky, which basically conformed to the characteristics of liver fibrosis, indicating that the experimental liver fibrosis model was successful.
  • liver tissue structure of the mice was intact, the inflammatory cell infiltration and fibroblast proliferation were lighter than the model group, and the collagen deposition was reduced. Compared with the model group, the degree of liver fibrosis was reduced in all groups administered, and Mogrol treatment was better (Fig. 2).
  • ⁇ -SMA is a marker of liver fibrosis, so the degree of liver fibrosis can be judged by detecting the content of ⁇ -SMA protein.
  • ⁇ -SMA protein was detected by Western Blotting method.
  • 50 mg of liver tissue was taken and cut into fine fragments, and 1 mL of RIPA lysate and 10 ⁇ L of PMSF (100 mM) were added. Homogenize with a glass homogenizer until fully cleaved (cleaved for 1 h). 12000 g was centrifuged at 4 ° C for 15 minutes, the supernatant was taken, and the protein content was measured by BCA. Mix 4:1 with the loading buffer, denature at 100 ° C for 10 min, centrifuge at 3000 g for 10 minutes, and store in a -80 ° C refrigerator.
  • the denatured protein was dispensed and separated by polyacrylamide gel electrophoresis (5% of stacked gel and 12% of separated gel), the loading amount was 70 ug; the accumulated glue voltage was 80 V for 35 min, and the separation voltage was 120 V.
  • the film transfer time can be adjusted according to the current; the PVDF membrane is blocked with 10% skim milk powder for 120min; TBST is washed once; ⁇ -SMA monoclonal antibody (with closed Diluted in liquid, ratio 1:1000) overnight at 4 °C; TBST wash 3 times; goat anti-rabbit IgG secondary antibody (diluted with TBST, ratio 1:3000) was incubated for 120 min at room temperature; TBST was washed 3 times; ECL chemiluminescence was used for color development, and the dark chamber was exposed to a tablet. The gray scale of the Western blot strip was detected using ⁇ -actin as an internal reference.
  • results were expressed as mean ⁇ SD, ## p ⁇ 0.01, compared with the blank control group; *** p ⁇ 0.001 compared with the CCl 4 model group.
  • HSC-T6 is an effector cell of liver fibrosis.
  • Transforming growth factor (TGF- ⁇ ) is a key factor in promoting liver fibrosis, which activates HSC-T6.
  • Activated HSC-T6 releases cytokines through autocrine or paracrine forms, and continues to deposit large amounts of ECM, eventually causing liver. Fibrosis.
  • the degree of liver fibrosis was determined by detecting the content of ⁇ -SMA protein.
  • HSC-T6 was purchased from Nanjing Kaiji Biotechnology Development Co., Ltd. HSC-T6 was cultured in a DMEM medium containing 10% domestic calf serum at 37 ° C in a 5% CO 2 incubator. The medium was changed once every 2 days, and the cells were digested with 0.25% trypsin when grown at a density of 90%, and the cells were collected and passaged.
  • results were expressed as mean ⁇ SD, ## p ⁇ 0.01, compared with the blank control group; ** p ⁇ 0.01, compared with the TGF- ⁇ 1 model group.
  • results were expressed as mean ⁇ SD, ### p ⁇ 0.001, compared with the blank control group; ** p ⁇ 0.01, compared with the TGF- ⁇ 1 model group.
  • Example 4 Effects of total glycosides and mogrosides on the expression of ⁇ -SMA protein in HSC-T6 induced by TGF- ⁇ 1
  • HSC-T6 was purchased from Nanjing Kaiji Biotechnology Development Co., Ltd. HSC-T6 was cultured in a DMEM medium containing 10% domestic calf serum at 37 ° C in a 5% CO 2 incubator. The medium was changed once every 2 days, and the cells were digested with 0.25% trypsin when grown at a density of 90%, and the cells were collected and passaged.

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Abstract

罗汉果醇在制备治疗和/或预防肝纤维化的药物和/或保健品中的用途。体内外实验证明罗汉果醇可显著改善体内小鼠肝组织纤维化程度，降低胶原沉积，减弱TGF-β1诱导HSC-T6的体外肝纤维化，显示对体内外肝纤维化模型的干预作用。

Description

罗汉果醇在制备肝纤维化药物中的应用 技术领域
本发明属于肝纤维化的防治技术领域，具体涉及罗汉果醇在制备预防和/或治疗肝纤维化的药物中的应用。
背景技术
肝纤维化(hepatic fibrosis，HF)是指肝脏内弥漫性细胞外基质(特别是胶原物质)过度沉积，是机体对各种病因引起的慢性肝损伤后的一种损伤修复反应，是肝硬化的早期可逆阶段。如不及时治疗则可能进展成为失代偿期肝硬化并出现各种终末期肝病并发症。肝纤维化持续发展可导致肝硬化、甚至肝癌。肝细胞炎症、坏死是肝纤维化的始动因素，又是其向前发展的推动因素。由于对其机制尚未明确，因此，在治疗药物方面受到一定的限制，而中医药具有整体调节及明确的抗氧化、降酶护肝、毒性低、价廉的优点而逐渐被重视。
在肝纤维化过程中，各种致病因子通过TGF-β/Smad途径促进肝星状细胞(hepatic stellate cell，HSC)激活，在HSC活化早期刺激细胞外基质(extracellular matrix，ECM)的合成与沉积，主要包括I型胶原、III型胶原IV型胶原。活化的HSC也能合成分泌大量的TGF-β1，使TGF-β1水平持续增高。在持续阶段，活化的HSC不断增殖，分泌大量的ECM，促使肝纤维化逐渐加重。在肝纤维化过程中HSC的活化是中心环节，TGF-β是活化HSC的关键因素，在肝纤维化过程中扮演重要角色。活化的HSC是产成ECM的主要细胞，它在ECM代谢和各种细胞介质的产生过程中处于中心地位，α-SMA是HSC的活化的标志之一。CCl4诱导小鼠肝纤维化是经典的筛选抗肝纤维化药物的体内药效模型，CCl4可引起体内氧化与抗氧化失衡，氧自由基产生增多，从而启动脂质过氧化反应，降低其TGF-β1含量可减缓小鼠肝纤维化进程。
罗汉果为葫芦科(Cucurbitaceae)植物罗汉果(Siratia grosvenorii(Swingle)C.Jeffrey)的成熟果实，主产于广西永福、临桂和龙胜等县，为广西著名特产。罗汉果性凉、味甘，归肺、大肠经，具有润肺止咳、清热解暑、利咽开音、凉血滑肠的功效，是我国特有的药食同源性经济作物，同时被收载于中华人民共和国药典，作为常用中药使用，在治疗咽喉炎、百日咳、急慢性气管炎、胃肠疾病方面疗效显著。罗汉果总苷是罗汉果中主要的有效成分，具有广泛的生物特性和药理价值。现代医学研究证明，罗汉果总苷不仅具有镇咳、平喘、祛痰、抗炎、调节消化道功能之功效，还能增强免疫力、保肝降酶、治疗急性肺损伤、抗氧化以及防衰老。罗汉果提取物的主要成分为罗汉果苷，罗汉果中的总苷为葫芦烷型三萜苷类化合物，包括罗汉果苷V(Mogroside V)；罗汉果苷IVe(Mogroside IVe)；罗汉果苷IIIe(Mogroside IIIe)；罗汉 果苷II A2(Mogroside II A2)；罗汉果苷III A1(Mogroside III A1)；罗汉果苷IVa(Mogroside IVa)；罗汉果苷VI(Mogroside VI)；赛门苷I(Siamenoside I)；11-O-罗汉果苷V(11-Oxomogroside V)等。罗汉果醇是罗汉果苷类化合物的苷元，可以通过酸水解得到。
迄今为止，尚无罗汉果醇在抗肝纤维化药物中的应用报道。
发明内容
本发明要解决的技术问题是提供罗汉果醇的医药新用途，即罗汉果醇在治疗和/或预防肝纤维化的药物和/或保健品中的用途。
为解决上述技术问题本发明的技术方案如下：
罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用。
进一步地，所述罗汉果醇，是由罗汉果提取物经酸水解得到，且结构式如下所示：
Figure PCTCN2017081423-appb-000001
优选的，所述罗汉果醇使用经化学修饰得到的罗汉果醇衍生物代替，所述罗汉果醇衍生物包括罗汉果醇的卤化物、酯化物、胺基化合物、醚化物以及罗汉果醇的盐。
优选的，所述应用包括在抗急性肝损伤、肝硬化、病毒性肝炎、肝癌方面的应用。
优选的，所述应用包括降低胶原蛋白沉积，减少炎性因子释放，降低肝组织中α-SMA的表达，抑制肝脏成纤维细胞增殖。
进一步地，所述药物和/或保健品是以罗汉果醇为主要活性成分制备成的制剂。
优选的，所述制剂含有药物可接受的载体，药物可接受的载体在制剂中所占重量百分比可以是0.1-99.9％。
优选的，所述药物可接受的载体包括以下物质：甘露醇、山梨醇、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素C、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗 糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、土温80、琼脂、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β-环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁等。
进一步地，所述制剂的剂型包括片剂、胶囊剂、注射剂、口服液、丸剂、散剂。
优选的，所述罗汉果醇是采用罗汉果提取物，用5％的硫酸水解，经硅胶柱分离、精制、纯化得到，纯度95％以上。
有益效果：
肝纤维化是多种肝部疾病或肝损伤发展到一定时期的一种常见的病理变化，肝纤维化持续发展可导致肝硬化、肝癌等后续并发疾病。由于肝纤维化病理机制的多环节、复杂性，单一环节或靶向干预的效果尚不理想，临床上仍然缺乏特异有效药物。本发明人通过体内外实验证明罗汉果醇可显著改善改善体内小鼠肝组织纤维化程度，降低胶原沉积，减弱TGF-β1诱导HSC-T6的体外肝纤维化，显示对体内外肝纤维化模型的干预作用。
本发明的罗汉果醇具有结构明确、稳定性好、质量易控制等特点，可以用于制备预防或/和治疗肝纤维化相关药物。本发明所涉及实验材料来自原植物，原植物分别范围广，成本低，提取物活性明确，具有广泛的实用价值。
附图说明
图1MG、Mogrol对CCl4诱导肝纤维化小鼠体重变化的影响。
图2MG、Mogrol对各组肝组织炎症程度的影响(HE染色×200)，以及MG IVE、Mogrol对各组肝组织纤维化程度的影响(Masson染色×200)。
图3MG、Mogrol对CCl4诱导肝纤维化小鼠中α-SMA蛋白含量影响。##p<0.01，versus control；**p<0.01，***p<0.001versus model。
图4MG、Mogrol对TGF-β1诱导HSC-T6中α-SMA蛋白含量影响。##p<0.01，versus control；**p<0.01，***p<0.001versus model。
图5MG、Mogrol对CCl4诱导肝纤维化小鼠中α-SMA蛋白的影响。*p<0.05，**p<0.01，***p<0.001，与CCl4模型组相比；#p<0.05，与CCl4+MG组相比。
图6MG、Mogrol对TGF-β1刺激HSC-T6细胞中α-SMA蛋白的影响。**p<0.01，***p<0.001，与TGF-β1模型组相比；#p<0.05，与TGF-β1+MG组相比。
具体实施方式
罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用。
进一步地，所述罗汉果醇，是由罗汉果提取物经酸水解得到，且结构式如下所示：
Figure PCTCN2017081423-appb-000002
优选的，罗汉果醇是采用市售罗汉果提取物，用5％的硫酸水解，经硅胶柱分离、精制、纯化得到，纯度95％以上。
优选的，所述罗汉果醇使用经化学修饰得到的罗汉果醇衍生物代替，所述罗汉果醇衍生物包括罗汉果醇的卤化物、酯化物、胺基化合物、醚化物以及罗汉果醇的盐。
优选的，所述应用包括制备用于肝纤维化相关的疾病药物和/或保健品中的应用。
优选的，所述应用包括在抗急性肝损伤、肝硬化、病毒性肝炎、肝癌方面的应用。
优选的，所述应用包括降低胶原蛋白沉积，减少炎性因子释放，降低肝组织中α-SMA的表达，抑制肝脏成纤维细胞增殖。
优选的，所述药物和/或保健品，是以罗汉果醇为主要活性成分制备成的制剂。
本发明罗汉果醇的制剂制备方法为现有常规技术，可以从现有专利、公知文献中获得，或者根据公开的药品标准制备。所述药物和/或保健品的剂型包括片剂、胶囊剂、注射剂、口服液、丸剂、散剂。
本发明的罗汉果醇制剂可以是任何可药用的剂型，这些剂型包括：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、口服液、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、滴丸剂、贴剂。
进一步地，所述罗汉果醇制剂，是以罗汉果醇为主要有效成分制成的药物组合物，该组 合物可以根据需要含有药物可接受的载体，药物可接受的载体在制剂中所占重量百分比可以是0.1-99.9％。
本发明的罗汉果醇制剂，在制备成药剂时可选择性的加入适合的药物可接受的载体，所述药物可接受的载体包括但不限于以下物质，选自：甘露醇、山梨醇、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素C、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、土温80、琼脂、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β-环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁等。
本发明所述药物或保健品，是指能够降低肝纤维化组织间质中胶原堆积量的药物或保健品。
本发明所述药物或保健品，是指能抑制炎性因子释放，减轻炎症程度，抑制胶原形成、保护肝组织的抗肝纤维化的药物或保健品。
本发明所述药物或保健品，是指通过抗炎及抑制肝细胞增殖而发挥抗肝纤维化的药物或保健品。
下面结合实施例对本发明的具体实施方式做进一步的描述，并不因此将本发明限制在所述的实施例范围之中。
实施例1罗汉果醇改善CCl4诱导小鼠的肝纤维化
取罗汉果总苷(以下简写MG)、罗汉果醇(以下简写Mogrol)进行下述体内药效学研究。CCl4诱导小鼠肝纤维化模型是一种经典方法，而且与人类肝纤维化相近。
1.材料与仪器
ICR小鼠，雌性，体重18-22g，由南京市青龙山动物繁殖场提供。娃哈哈纯净水；α-SMA单克隆抗体(abcam)；羊抗兔IgG(bioworld)。
2.实验方法
ICR雌性小鼠，分空白组、模型组、MG组(250mg/kg)、Mogrol组(10mg/kg)，药物分别灌胃给药，每组各20只。小鼠腹腔注射40％的CCl4(橄榄油悬浊液)2ml/kg，每周2次，建立肝纤维化小鼠模型；空白组腹腔注射橄榄油2ml/kg，每周2次。造模同时灌胃药物进行治疗。于造模后第6周处死小鼠，取血，离心后吸取上层血清分装冻存于-80℃；取肝脏，将部分肝放入4％中性甲醛固定，逐级酒精脱水，二甲苯透明，浸蜡，石蜡包埋后，常规切片， HE染色，Masson染色，观察肝组织损伤及纤维化变化，其余肝组织分装冻存于-80℃，用以检测α-SMA蛋白含量。CCl4诱导肝纤维化小鼠体重变化，结果如图1，造模6周后肝组织形态学观察，结果如图2。
所有数据均用均数±SD(x±s)表示。应用SPSS11.5统计软件处理，统计采用单因素方差分析(one-way ANOVA)，P<0.05表示差异有统计学意义。
3.实验结果
3.1罗汉果醇对肝纤维化小鼠体重的影响
由CCl4诱导肝纤维化小鼠体重变化可知，较空白组小鼠，模型组小鼠体重下降最多，在给予MG、Mogrol药物组小鼠体重均有所增加，并高于模型组(图1)，说明罗汉果总苷及罗汉果醇均能一定程度上改善因肝纤维化引起的小鼠体重下降。
3.2罗汉果醇对肝纤维化小鼠肝组织形态的影响
病理组织切片经HE、Masson染色，结果表明空白组小鼠肝组织结构完整较清晰，未见炎性细胞浸润，无成纤维细胞增生，对照组小鼠的肝组织内可见少量染成蓝色的胶原纤维，是细胞外基质的主要组成部分。模型组小鼠肝细胞结构破坏，坏死、钙化，成纤维细胞增生，大量胶原沉积，形成肝纤维化。Masson染色后可见多量致密被染成蓝色的胶原纤维，呈束状或片状沉积，基本符合肝纤维化的特点，则说明实验小鼠肝纤维化模型造模成功。经MG、Mogrol治疗后，可见小鼠肝组织结构完整较清晰，炎性细胞浸润及成纤维细胞增生程度均比模型组轻，并且胶原沉积均减少。给药各组与模型组相比，肝纤维化程度均有所减轻，且Mogrol治疗效果更好(图2)。
3.3罗汉果醇对模型小鼠肝组织中α-SMA蛋白含量的影响。
α-SMA是肝纤维化的标志，所以可通过检测α-SMA蛋白含量来判断肝纤维化的程度。
本实验采用Western Blotting方法检测α-SMA蛋白含量，首先取肝组织50mg，剪切成细小的碎片，加入1mL的RIPA裂解液和10μL的PMSF(100mM)。用玻璃匀浆器匀浆，直至充分裂解(裂解1h)。12000g 4℃离心15分钟，取上清，分装用BCA测蛋白含量。按4:1与上样缓冲液混匀，100℃水浴变性10min，3000g离心10分钟后，分装保存于-80℃冰箱。然后取分装的变性蛋白，用聚丙烯酰胺凝胶电泳法(堆积胶为5％，分离胶为12％)分离，上样量为70ug；堆积胶电压80V，持续35min，分离胶电压120V，持续90min；用恒压110V湿法转模45min，PVDF膜，转膜时间依据电流可作调整；将PVDF膜用10％脱脂奶粉封闭120min；TBST洗涤1次；α-SMA单克隆抗体(用封闭液稀释，比例1:1000)4℃孵育过夜；TBST洗涤 3次；羊抗兔IgG二抗(用TBST稀释，比例1:3000)室温孵育120min；TBST洗涤3次；ECL化学发光法显色，暗室中压片曝光。以β-actin作为内参，检测蛋白质印迹条带灰度。
实验结果表明，与空白组小鼠肝组织相比，模型组小鼠肝组织中α-SMA蛋白含量显著增加(P<0.01)，MG、Mogrol药物组肝组织中α-SMA蛋白含量均显著降低(P<0.01或P<0.001)，且Mogrol治疗肝纤维化作用更好(图3，表2和表3)，提示MG、Mogrol可以减轻CCl4诱导小鼠体内肝纤维化。
表2罗汉果总苷对肝纤维化小鼠α-SMA蛋白的影响
Figure PCTCN2017081423-appb-000003
结果用mean±SD表示，##p<0.01，与空白对照组相比；**p<0.01，与CCl4模型组相比。
表3罗汉果醇对肝纤维化小鼠α-SMA蛋白的影响
Figure PCTCN2017081423-appb-000004
结果用mean±SD表示，##p<0.01，与空白对照组相比；***p<0.001，与CCl4模型组相比。
实施例2罗汉果醇对TGF-β1诱导HSC-T6中α-SMA蛋白表达影响
HSC-T6是肝纤维化的效应细胞。转化生长因子(TGF-β)是促进肝纤维化的关键因子，其可活化HSC-T6，活化的HSC-T6可通过自分泌或旁分泌的形式释放细胞因子，持续ECM大量沉积，最终引起肝纤维化。本实验通过检测α-SMA蛋白含量来判断肝纤维化的程度。
HSC-T6购于南京凯基生物科技发展有限公司。HSC-T6采用含有10％国产小牛血清的DMEM培养基，置37℃，5％CO2培养箱中培养。每2天换1次培养基，细胞生长90％密度时用0.25％胰蛋白酶消化，收集细胞并传代。
取对数生长细胞，胰蛋白酶消化，将细胞浓度调整为2×105/ml，2ml/孔接种于6孔板中，培养24h后去除培养液，分别设置空白组，TGF-β1模型组(10ng/ml)，药物组同时分别给予MG(40μM)、Mogrol(10μM)。培养24h后，拿出置于冰盒上弃培养基，预冷PBS洗两遍并弃掉，每孔加入100μl上样缓冲液，静置2-3min后，用细胞刮轻轻刮取细胞至500μl ep管中，100℃水浴变性10min，得变性后的蛋白分装保存于-80℃冰箱。同样按照上述WB法测各组细胞α-SMA蛋白。
实验结果表明，与空白组相比，TGF-β1模型组中细胞α-SMA蛋白含量显著增加(P<0.01)，MG、Mogrol药物组中α-SMA蛋白含量均显著降低(P<0.01或P<0.001)，且Mogrol治疗肝纤维化作用更好(图4，表4和表5)，提示MG、Mogrol也可以减轻TGF-β1诱导HSC-T6体外肝纤维化。
表4罗汉果总苷对TGF-β1刺激HSC-T6细胞中α-SMA蛋白的影响
Figure PCTCN2017081423-appb-000005
结果用mean±SD表示，##p<0.01，与空白对照组相比；**p<0.01，与TGF-β1模型组相比。
表5罗汉果醇对TGF-β1刺激HSC-T6细胞中α-SMA蛋白的影响
Figure PCTCN2017081423-appb-000006
结果用mean±SD表示，###p<0.001，与空白对照组相比；**p<0.01，与TGF-β1模型组相比。
以上实验结果表明，在MG、Mogrol给药组中，HE、Masson染色病理切片显示给药组肝纤维化程度均明显改善，提示MG、Mogrol具有抑制胶原形成、保护肝组织的作用。在CCl4诱导小鼠体内肝纤维化和TGF-β1诱导HSC-T6体外肝纤维化实验中，MG、Mogrol药物组均可以降低α-SMA蛋白表达，证实了MG、Mogrol的抗肝纤维药物用途，且Mogrol治疗肝纤维化作用更好。
实施例3罗汉果总苷和罗汉果醇对CCl4诱导小鼠肝纤维化组织中α-SMA蛋白表达影响
材料、仪器以及实验方法均同于实施例1.
实验结果表明，与空白组小鼠肝组织相比，模型组小鼠肝组织中α-SMA蛋白含量显著增加(P<0.01)，MG、Mogrol药物肝组织中α-SMA蛋白含量均显著降低(P<0.05或0.001)，Mogrol治疗肝纤维化作用较MG更好(P<0.05)(图5，表6)。
表6罗汉果总苷和罗汉果醇对肝纤维化小鼠α-SMA蛋白的影响
Figure PCTCN2017081423-appb-000007
结果用mean±SD表示，*p<0.05，**p<0.01，***p<0.001，与CCl4模型组相比；#p<0.05，与CCl4+MG组相比。
实施例4罗汉果总苷和罗汉果醇对TGF-β1诱导HSC-T6中α-SMA蛋白表达的影响
HSC-T6购于南京凯基生物科技发展有限公司。HSC-T6采用含有10％国产小牛血清的DMEM培养基，置37℃，5％CO2培养箱中培养。每2天换1次培养基，细胞生长90％密度时用0.25％胰蛋白酶消化，收集细胞并传代。
取对数生长细胞，胰蛋白酶消化，将细胞浓度调整为2×105/ml，2ml/孔接种于6孔板中，培养24h后去除培养液，分别设置空白组，TGF-β1模型组(10ng/ml)，药物组同时分别给予MG(5μg/ml)、Mogrol(5μg/ml)。培养24h后，拿出置于冰盒上弃培养基，预冷PBS洗两遍并弃掉，每孔加入100μl上样缓冲液，静置2-3min后，用细胞刮轻轻刮取细胞至500μl离心管中，100℃ 水浴变性10min，得变性后的蛋白分装保存于-80℃冰箱。同样按照上述WB法测各组细胞α-SMA蛋白。实验结果表明，与空白组相比，TGF-β1模型组中细胞α-SMA蛋白含量显著增加(P<0.001)，MG、Mogrol药物组中α-SMA蛋白含量均显著降低(P<0.01或0.001)，且苷元Mogrol治疗肝纤维化作用较MG更好(P<0.05)(图6，表7)。
表7罗汉果总苷和罗汉果醇对TGF-β1刺激HSC-T6细胞中α-SMA蛋白的影响
Figure PCTCN2017081423-appb-000008
结果用mean±SD表示，**p<0.01，***p<0.001，与TGF-β1模型组相比；#p<0.05，与TGF-β1+MG组相比。

Claims (10)

  1. 罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用。
  2. 根据权利要求1所述的罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用，其特征在于，所述罗汉果醇，是由罗汉果提取物经酸水解得到，且结构式如下所示：
    Figure PCTCN2017081423-appb-100001
  3. 根据权利要求2所述的罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用，其特征在于，所述罗汉果醇使用经化学修饰得到的罗汉果醇衍生物代替，所述罗汉果醇衍生物包括罗汉果醇的卤化物、酯化物、胺基化合物、醚化物以及罗汉果醇的盐。
  4. 根据权利要求3所述的罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用，其特征在于，所述应用包括在抗急性肝损伤、肝硬化、病毒性肝炎或肝癌方面的应用。
  5. 根据权利要求1所述的罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用，其特征在于，所述应用包括降低胶原蛋白沉积，减少炎性因子释放，降低肝组织中α-SMA的表达，抑制肝脏成纤维细胞增殖。
  6. 根据权利要求1所述的罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用，其特征在于，所述药物和/或保健品是以罗汉果醇为主要活性成分制备成的制剂。
  7. 根据权利要求6所述的罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用，其特征在于，所述制剂含有药物可接受的载体，药物可接受的载体在制剂中所占重 量百分比可以是0.1-99.9％。
  8. 根据权利要求7所述的罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用，其特征在于，所述药物可接受的载体包括以下物质：甘露醇、山梨醇、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素C、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、土温80、琼脂、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β-环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙或硬脂酸镁等。
  9. 根据权利要求6所述的罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用，其特征在于，所述制剂的剂型包括片剂、胶囊剂、注射剂、口服液、丸剂或散剂。
  10. 根据权利要求2所述的罗汉果醇在制备预防和/或治疗肝纤维化的药物和/或保健品中的应用，其特征在于，所述罗汉果醇是采用罗汉果提取物，用5％的硫酸水解，经硅胶柱分离、精制、纯化得到，纯度95％以上。
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