WO2017113649A1 - 罗汉果提取物抗肺纤维化的应用 - Google Patents

罗汉果提取物抗肺纤维化的应用 Download PDF

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WO2017113649A1
WO2017113649A1 PCT/CN2016/086831 CN2016086831W WO2017113649A1 WO 2017113649 A1 WO2017113649 A1 WO 2017113649A1 CN 2016086831 W CN2016086831 W CN 2016086831W WO 2017113649 A1 WO2017113649 A1 WO 2017113649A1
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pulmonary fibrosis
extract
mogroside
hydrolysate
mice
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French (fr)
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谢海峰
张朝凤
谢期林
胡云岭
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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
    • 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 pulmonary fibrosis, and relates to the application of a momordica grosvenori extract in preparing medicines and/or health care products for preventing and/or treating pulmonary fibrosis diseases.
  • Pulmonary fibrosis is caused by a variety of pathogenic factors inside and outside the lung. It is the result of a series of chronic lung injury or disease progression to the advanced stage, which is a serious hazard to human health.
  • the causes of pulmonary fibrosis include genetic immune dysfunction, viral infections, drugs and chemicals, radiation, air pollution (haze, smoking, dust) and other factors.
  • the pathophysiological process of pulmonary fibrosis is complicated. In the early stage, pulmonary inflammation is the main cause, the alveolar wall is diffusely thickened, a large number of inflammatory cells infiltrate, and a large amount of collagen fibers are formed and deposited in the middle and late stages. Alveolar deformation and occlusion are observed.
  • the normal lung tissue structure is Destruction, loss of function.
  • the clinical manifestations of pulmonary fibrosis include irritating dry cough, restrictive ventilatory dysfunction, progressive dyspnea, decreased diffuse function, and hypoxemia.
  • pulmonary fibrosis is still a disease with high mortality rate, and there is no effective treatment in clinic.
  • Traditional medicines still rely on anti-inflammatory, immunosuppressive and anti-coagulation, and glucocorticoids are commonly used in clinic. Cyclophosphamide, cyclosporine, colchicine and penicillamine, but with poor efficacy and side effects. Therefore, the search for effective anti-pulmonary fibrosis drugs has become an urgent need of current medicine.
  • TGF- ⁇ 1 transforming growth factor- ⁇ 1
  • CTGF connective tissue growth factor
  • PDGF vascular endothelial growth factor
  • TGF- ⁇ 1 plays a very important role in promoting the phenotypic transformation of myofibroblasts, stimulating the synthesis and secretion of various cytokines, regulating cell proliferation and differentiation, etc.
  • ECM extracellular matrix
  • the animal model of pulmonary fibrosis induced by tracheal instillation of bleomycin is a classic animal model for studying pulmonary fibrosis drugs at home and abroad.
  • the animal model of pulmonary fibrosis replicated by this method is similar to human pulmonary fibrosis and can be used in a short time.
  • pulmonary fibrosis is mainly glucocorticoids, cytotoxic drugs, immunosuppressants, and immunomodulators, but there is no specific drug. In view of this, it is necessary to develop a new type of highly effective and safe drug for the treatment of pulmonary fibrosis.
  • Mangosteen is a mature fruit of the genus Cucurbitaceae (Siratia grosvenorii (Swingle) C. Jeffrey). 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.
  • the content of mogroside V is the highest in the total extract of Siraitia grosvenii.
  • the extract containing the ratio of different mogrosides can be obtained according to the length of enzymatic hydrolysis.
  • Mogroside is a aglycon of a mogroside compound and can be obtained by acid hydrolysis.
  • the present invention discloses a new medical use of Mangosteen extract, that is, its use for the preparation of a medicament and/or a health care product for preventing and/or treating pulmonary fibrosis diseases.
  • Mangosteen extract for the preparation of a medicament and/or health care product for preventing and/or treating pulmonary fibrosis diseases.
  • the mass percentage of the total glycoside of the Luo Han Guo extract in the Luo Han Guo extract is ⁇ 50%.
  • the total mass percentage of the monosaccharide of Luo Han Guo is ⁇ 80%.
  • hydrolysate or hydrolysate of Momordica grosvenori extract for the preparation of a medicament and/or health care product for preventing and/or treating pulmonary fibrosis diseases.
  • the hydrolysate or hydrolyzate of the Luo Han Guo extract contains at least one or more of mogroside IIIe, mogroside IVe or mogroside.
  • the enzymatic hydrolysate of the Luo Han Guo extract contains at least mogroside IIIe:
  • the hydrolysate of the Luo Han Guo extract contains at least romanol:
  • the enzymatic hydrolysate of the Luo Han Guo extract contains at least mogroside IVe:
  • Lo Han Guo extract or its hydrolysate or hydrolyzate may be formulated into tablets, granules, decoctions or capsules with other human acceptable pharmaceutical excipients.
  • the medicine or health care product is a medicine or a health care product for reducing the amount of collagen accumulation in the interstitial tissue of pulmonary fibrosis.
  • the drug or health care product is a drug or a health care product which can reduce the degree of inflammation, inhibit collagen formation, and protect lung tissue against pulmonary fibrosis.
  • the drug or health care product is a drug or a health care product that exerts anti-pulmonary fibrosis by anti-inflammatory and inhibits interstitialization of alveolar epithelial cells.
  • Pulmonary fibrosis is a common pathological change in the development of various lung diseases or lung injury.
  • the clinical results show that there is no significant change in the survival rate of patients with pulmonary fibrosis after glucocorticoid treatment. There is no clear treatment.
  • the invention is prepared from the traditional Chinese medicine for treating lung and relieving cough.
  • mogrosides can be used for the treatment of pulmonary fibrosis.
  • the inventors have demonstrated in vivo that the aglycones of mogroside IIIe, mogroside IVe and mogrosides can be used as the aglycones of mogroside and mogroside.
  • the invention relates to the experimental material from the original plant, the original plant has a wide range, the cost is low, the extract activity is clear, and has wide practical value.
  • Figure 1 Trend of body weight change in mice with bleomycin-induced pulmonary fibrosis induced by total glucosides of Siraitia grosvenii. Compared with the control group, #p ⁇ 0.05; *p ⁇ 0.05, **p ⁇ 0.01 compared with the model group; positive control drug: prednisone acetate.
  • Fig. 2 Effect of total glycosides of Siraitia grosvenii on the changes of lung coefficient in mice with bleomycin-induced pulmonary fibrosis model. Compared with the control group, ## p ⁇ 0.001; compared with the model group, *p ⁇ 0.05, **p ⁇ 0.01; positive control drug: prednisone acetate.
  • Fig. 3 Effect of total glycosides of Siraitia grosvenii on the degree of fibrosis in lung tissue of mice with bleomycin-induced pulmonary fibrosis model (Masson staining). Compared with the control group, ## p ⁇ 0.01; compared with the model group, *p ⁇ 0.05, **p ⁇ 0.01; positive control drug: prednisone acetate.
  • Figure 4 Effect of total glycosides of Siraitia grosvenii on HYP content in lung tissue of mice with pulmonary fibrosis on day 14 after bleomycin induction. Compared with the control group, ## p ⁇ 0.01; compared with the model group, **p ⁇ 0.01.
  • Fig. 5 Effect of total glycosides of Siraitia grosvenii on the expression of ⁇ -SMA protein in lung tissue of bleomycin-induced pulmonary fibrosis mice. Compared with the control group, # p ⁇ 0.05; compared with model group, * p ⁇ 0.05.
  • Figure 6 Trend of body weight change in mice with bleomycin-induced pulmonary fibrosis induced by mogroside IIIe. Compared with the control group, #p ⁇ 0.05; *p ⁇ 0.05, **p ⁇ 0.01 compared with the model group; positive control drug: prednisone acetate.
  • Fig. 7 Effect of mogroside IIIe on the changes of lung coefficient in mice with bleomycin-induced pulmonary fibrosis model. Compared with the control group, ## p ⁇ 0.001; compared with model group, * p ⁇ 0.05, ** p ⁇ 0.01; positive control: prednisone.
  • Figure 8 Effect of mogroside IIIe on the degree of pulmonary fibrosis in mice with bleomycin-induced pulmonary fibrosis model (Masson staining). Compared with the control group, ## p ⁇ 0.01; compared with the model group, *p ⁇ 0.05, **p ⁇ 0.01; positive control drug: prednisone acetate.
  • Figure 9 Effect of mogroside IIIe on HYP content in lung tissue of bleomycin-induced pulmonary fibrosis in mice. Compared with the control group, # p ⁇ 0.05; compared with model group, * p ⁇ 0.05.
  • Figure 10 Effect of mogroside IIIe on the expression of ⁇ -SMA in human alveolar type II epithelial cells induced by TGF- ⁇ 1. Compared with the control group, ## p ⁇ 0.01; compared with the model group, *p ⁇ 0.05.
  • Figure 11 is a graph showing the trend of body weight change in mice with bleomycin-induced pulmonary fibrosis induced by mogroside IVe. Compared with the control group, #p ⁇ 0.05; *p ⁇ 0.05, **p ⁇ 0.01 compared with the model group; positive control drug: prednisone acetate.
  • Figure 12 Effect of mogroside IVe on the changes of lung coefficient in mice with bleomycin-induced pulmonary fibrosis model. Compared with the control group, ## p ⁇ 0.001; compared with the model group, *p ⁇ 0.05, **p ⁇ 0.01; positive control drug: prednisone acetate.
  • Figure 13 Effect of mogroside IVe on pulmonary fibrosis (Masson staining) in mice with bleomycin-induced pulmonary fibrosis model. Compared with the control group, ## p ⁇ 0.01; compared with the model group, *p ⁇ 0.05, **p ⁇ 0.01; positive control drug: prednisone acetate.
  • Figure 14 Trend of body weight change in mice treated with bleomycin-induced pulmonary fibrosis. Compared with the control group, #p ⁇ 0.05; *p ⁇ 0.05, **p ⁇ 0.01 compared with the model group; positive control drug: prednisone acetate.
  • Figure 15 Effect of mogroside on lung fibrosis (Masson staining) in lung tissue of mice exposed to bleomycin-induced pulmonary fibrosis. Compared with the control group, ## p ⁇ 0.01; compared with the model group, *p ⁇ 0.05, **p ⁇ 0.01; positive control drug: prednisone acetate.
  • the Luo Han Guo extract that is, Luo Han Guo, is extracted by the prior art.
  • the mass percentage of the total glycoside of the Luo Han Guo extract in the Luo Han Guo extract is ⁇ 50%.
  • the total mass percentage of the monosaccharide of Luo Han Guo is ⁇ 80%.
  • hydrolysate or hydrolysate of Momordica grosvenori extract for the preparation of a medicament and/or health care product for preventing and/or treating pulmonary fibrosis diseases.
  • the hydrolysate or hydrolyzate of the Luo Han Guo extract contains at least one or more of mogroside IIIe, mogroside IVe or mogroside.
  • the enzymatic hydrolysate of the Luo Han Guo extract contains at least mogroside IIIe.
  • the hydrolysate of the Luo Han Guo extract contains at least romanol.
  • the enzymatic hydrolysate of the Luo Han Guo extract contains at least mogroside IVe.
  • Lo Han Guo extract or its hydrolysate or hydrolyzate may be formulated into tablets, granules, decoctions or capsules with other human acceptable pharmaceutical excipients.
  • the medicine or health care product is a medicine or a health care product for reducing the amount of collagen accumulation in the interstitial tissue of pulmonary fibrosis.
  • the drug or health care product is a drug or a health care product which can reduce the degree of inflammation, inhibit collagen formation, and protect lung tissue against pulmonary fibrosis.
  • the drug or health care product is a drug or a health care product that exerts anti-pulmonary fibrosis by anti-inflammatory and inhibits interstitialization of alveolar epithelial cells.
  • the total glycosides of Siraitia grosvenii can be extracted by the existing method.
  • the present invention has been researched to prove that broccoli total glycoside extract can improve bleomycin-induced pulmonary fibrosis in mice.
  • the pathological sections of lung tissue after Masson staining were observed in vivo.
  • the lung coefficient and body weight of each group were measured and the expression of HYP and ⁇ -SMA in lung tissue of each group were detected at different stages.
  • In vitro experiments showed that mogrosides significantly inhibited the release of NO from mouse macrophage RAW264.7 induced by LPS.
  • the experimental results show that the total extract of Siraitia grosvenii in the present invention can be It can improve the degree of fibrosis in the lung tissue of model mice, reduce the collagen deposition in the lung tissue of the model, improve the interstitialization of epithelial cells, and show the therapeutic effect on the pulmonary fibrosis model of mice. It has a new medical use for treating pulmonary fibrosis diseases.
  • the present invention uses Luo Han Guo total glycoside (Example 1), Mogroside IIIe (Example 2), Mogroside IVe (Example 3) and Mogroside (Example 4) as examples to verify the anti-pulmonary fibrosis disease of Luo Han Guo extract. Part of the pharmacodynamic test and results.
  • the total glycosides of Siraitia grosvenii are commercially available as Luoguoguo extract, which is purchased from Guilin Rhein Biotechnology Co., Ltd. and produces 80% of the monosaccharide extract of Mogroside (Mogrosides > 80%, product code MOV 09).
  • Mogroside IIIe is obtained by commercially available Luo Han Guo extract, separated by macroporous resin, and separated by high pressure reverse phase preparative chromatography. The purity is 95% or more. Or referring to the method of preparing mogroside IV according to Chinese Patent No. 2010105610299, the commercially available Luo Han Guo extract is hydrolyzed by ⁇ -glucosidase, separated by macroporous resin, and separated by high pressure reverse phase preparative chromatography. The purity is 95% or more.
  • the mogroside IVe is obtained by using a commercially available Luo Han Guo extract, which is separated by macroporous resin and separated by high pressure reverse phase preparative chromatography.
  • the purity is 95% or more.
  • the commercially available Luo Han Guo extract is hydrolyzed by ⁇ -glucosidase, separated by macroporous resin, and separated by high pressure reverse phase preparative chromatography. The purity is 95% or more.
  • the mogroside is commercially available as a extract of Siraitia grosvenii, hydrolyzed with 5% sulfuric acid, and purified by silica gel. The purity is 95% or more.
  • Example 1 80% Siraitia chinensis total glycoside extract improves bleomycin-induced mouse lung fiber
  • the experimental animal is administered in an amount of 50 mg to 100 mg/kg, and is set as the total glycoside-H and L groups of Luo Han Guo.
  • Mogrosides also known as mogrosides, is an effective part of the homologous plant Luo Han Guo.
  • the total glucosides of Momordica grosvenii include Mogroside V; Mogroside IV; Mogroside IIeI; Mogroside II A2; Mogroside III A1; Mogroside IVa; Mogroside VI; Siamenoside I; 11-O-Romane glycoside V (11-Oxomogroside V) and other cucurbitane-type triterpenoid glycosides.
  • ICR mice male, weighing 25-30 g, 100, were provided by the Yangzhou Comparative Medical Center.
  • mice were used as a blank control group (control group in Fig. 1), and the other 80 were used for modeling. All the above mice were intraperitoneally injected with 4% chloral hydrate for anesthesia, and the injection volume was 10 ml/kg. After anesthesia, the mice were fixed and the neck of the mice was sterilized; the neck skin of the mice was cut longitudinally with scissors, and the trachea was exposed; the syringe was inserted into the trachea, and the mice in the blank control group were injected into the physiology.
  • mice were injected with bleomycin (5 mg/kg); then the rats were quickly erected, the rats were rotated, the mice were observed for breathing, the wound was sutured, and 1-2 drops of penicillin injection was dispensed at the suture.
  • the post-operative mice were returned to the dry and clean squirrel cage for rest, waiting for recovery, and awakened after about 1-2 hours, and then reared normally.
  • mice On the 7th day after model establishment, the other 80 mice were randomly divided into a model group, a positive drug (prednisone acetate) group, and a high dose group of Luo Han Guo total glycoside extract (100 mg/kg, Luo Han Guo total glycoside-H). Low dose group of Luo Han Guo total glycoside extract (50 mg/kg, Luo Han Guo total glycoside-L), 20 in each group.
  • the blank control group and the model group were intragastrically administered with normal saline every day.
  • the positive drug group was intragastrically administered with 6.67 mg/kg/d prednisone acetate, and the total extract of Luo Han Guo total glycoside was divided into high and low dose groups, and 100 mg/kg/d and 50 mg were administered respectively.
  • lung coefficient lung weight (mg) / body weight (g).
  • the left small lung of the mice was fixed in 4% neutral formaldehyde for 28 days, dehydrated with graded alcohol, transparent with xylene, dipped in wax, embedded in paraffin, routinely sectioned, Masson stained, and observed lung tissue morphology and lung injury. The degree of pulmonary fibrosis. Other lobes were preserved and used for the determination of HYP content.
  • the body weight of the high and low dose groups of the total extract of Siraitia grosvenorii and the positive drug (prednisone acetate) increased significantly after the 14th and 28th days of the experiment, with significant difference (P ⁇ 0.01). It is suggested that the total glycosides of Siraitia grosvenii can improve the physique of bleomycin-induced pulmonary fibrosis mice at different doses of 100 mg/kg and 50 mg/kg, and slow down the weight loss of mice with pulmonary fibrosis (Fig. 1).
  • the lung tissue damage caused by bleomycin increases the number of white blood cells, especially neutrophil infiltration, causing alveolar inflammation, inflammatory cells release inflammatory mediators NO, TNF- ⁇ and various cytokines.
  • the experiment was carried out according to the requirements of the kit, and the number of white blood cells in the bronchoalveolar lavage fluid of each group of mice at 14 days was detected by colorimetry (as shown in Table 1).
  • BLM bleomycin
  • neutrophils and lymphocytes in the alveolar lavage fluid of mice showed significant aggregation.
  • the total number of cells and the number of neutrophils in the BLM group were significantly increased ( ## P ⁇ 0.01).
  • the lung coefficient of the model group was significantly increased and the difference was statistically significant (P ⁇ 0.01 or 0.05).
  • the total glycosides of Luo Han Guo were high and low after 14 and 21 days of administration.
  • the lung coefficient of the dose group and the positive drug (prednisone acetate) group decreased significantly, with significant difference (P ⁇ 0.01). It is suggested that the total glycosides of Siraitia grosvenii can alleviate the degree of pulmonary fibrosis development in model mice at different doses of 100 mg/kg and 50 mg/kg (Fig. 2).
  • the lung pathological sections were stained by Masson.
  • the results showed that the lung tissue of the blank control group was intact and clear, the alveolar septum was not thickened, the alveolar cavity was translucent, no obvious exudate was found in the cavity, and no fibroblast proliferation was observed.
  • the blank control group a small amount of collagen fibers stained blue was observed in the lung tissue, which was the main component of the extracellular matrix.
  • the alveolar structure of the mice was destroyed, the alveolar space was widened, a large number of inflammatory cells infiltrated into acute fibroblasts, a large amount of collagen was deposited, and pulmonary fibrosis was formed.
  • HYP Hydroxyproline
  • connective tissue protein which accounts for about 14% of the weight of collagen. It plays a key role in the stability of collagen. Since collagen is the only protein containing more HYP, Determination of HYP content can reflect changes in the total amount of tissue collagen. The content of HYP in lung tissue was detected by digestion. Compared with the blank control group, the HYP content in the lung tissue of the model group was significantly increased at 28 days (P ⁇ 0.01). Compared with the model group, the drug significantly reduced the HYP content in the lung tissue (P ⁇ 0.05). It is suggested that the total glycoside of Siraitia grosvenum can improve bleomycin-induced pulmonary fibrosis in mice at a dose of 100 mg/kg (Fig. 4).
  • Alveolar epithelial cells themselves can be used as an important source of lung fibroblasts and myofibroblasts through a process called epithelial-mesenchymal transition (EMT). Alveolar epithelialization is considered the key to fibrosis One of the links. Fibroblasts and myofibroblasts differentiated from epithelial cells are often obtained through morphological changes, fibroblast or myofibroblast-specific markers (eg, high expression of actin a-SMA), loss of epithelial signatures (eg, Epithelial cadherin (E-cadherin and tight junction protein) is integrated with these epithelial tissues. The expression level of ⁇ -SMA in lung tissue of each group was analyzed by Western blot.
  • EMT epithelial-mesenchymal transition
  • the expression level of HYP ⁇ -SMA in the lung tissue of the model group was significantly increased at 28 days (P ⁇ 0.01).
  • the total glycoside extract of Siraitia grosvenii could significantly reduce the expression level of ⁇ -SMA in lung tissue. (P ⁇ 0.05), the results suggest that total glycosides of Siraitia grosvenii can reduce the expression of ⁇ -SMA in lung tissue and improve the lung fiber level induced by BLM (Fig. 5).
  • the high and low doses of Siraitia sinensis could significantly reduce lung index, HYP content and ⁇ -SMA expression in lung tissue (p ⁇ 0.05or 0.01), and pathological results showed lung tissue structure of total glycosides of Siraitia grosvenorii.
  • the number of neutrophils in the alveolar lavage fluid of the model group was significantly increased, indicating that bleomycin caused an inflammatory reaction in the model group, thereby initiating an inflammatory cascade in vivo.
  • the degree of inflammation and fibrosis of the mice was alleviated to some extent, suggesting that the drug can protect the lung cells from damage and prevent pulmonary fibrosis.
  • inosin IIIe was selected for the following in vivo pharmacodynamic studies. Intratracheal injection of bleomycin-induced pulmonary fibrosis in mice is a commonly used method in the world and is similar to human pulmonary interstitial fibrosis.
  • ICR mice male, weighing 25-30 g, 100, were provided by the Yangzhou Comparative Medical Center.
  • mice were used as a blank control group (control group in Fig. 6), and the other 80 were used for modeling. All the above mice were intraperitoneally injected with 4% chloral hydrate for anesthesia, and the injection volume was 10 ml/kg. After anesthesia, the mice were fixed and the neck of the mice was sterilized; the neck skin of the mice was cut longitudinally with scissors, and the trachea was exposed; the syringe was inserted into the trachea, the mice in the blank control group were injected with physiological saline, and the other mice were injected with bleomycin.
  • mice were returned to the dry and clean squirrel cage for rest, waiting for recovery, and awakened after about 1-2 hours, and then reared normally.
  • mice On the 7th day after modeling, the other 80 mice were randomly divided into a model group, a positive drug (prednisone acetate) group, a high dose group of mogroside IIIe (50 mg/kg, mogroside IIIe-H), and mogroside. Low dose group IIIe (10 mg/kg, mogroside IIIe-L), 20 in each group.
  • the blank control group and the model group were intragastrically administered with normal saline every day.
  • the positive drug group was intragastrically administered with 6.67 mg/kg/d prednisone acetate, and Luo Han Guo.
  • the glycosides IIIe were divided into high and low dose groups, and were intragastrically administered with 50 mg/kg/d and 10 mg/kg/d respectively.
  • the left small lung was fixed in 4% neutral formaldehyde, dehydrated step by step, transparent in xylene, dipped in wax, embedded in paraffin, routinely sectioned, Masson stained, and observed the morphology of lung tissue, lung injury and pulmonary fibrosis. Other lobes were preserved and used for the determination of HYP content.
  • Bleomycin-induced lung injury in mice the number of white blood cells increased, especially neutrophil infiltration, causing alveolar inflammation, inflammatory cells release inflammatory transmitters NO, TNF- ⁇ and various cytokines, on the one hand aggravated Lung tissue damage, on the other hand, promotes excessive collagen production through various growth factors.
  • the experiment was performed according to the requirements of the kit, and the number of white blood cells in the bronchoalveolar lavage fluid of each group of mice at 14 days was detected by colorimetry (as shown in Table 2). On day 14 after treatment with bleomycin (BLM), neutrophils and lymphocytes in the alveolar lavage fluid of mice showed significant aggregation.
  • the total number of cells and the number of neutrophils in the alveolar lavage fluid of the BLM group were significantly increased (compared with the blank control group, ## P ⁇ 0.01).
  • the number of cells in the alveolar lavage fluid and the number of neutrophils were significantly lower than those in the BLM group alone (*P ⁇ 0.05 compared with the BLM group). It is suggested that mogroside IIIe can reduce BLM-induced inflammatory cell exudation.
  • the lung coefficient of the model group was significantly increased and the difference was statistically significant (P ⁇ 0.01 or 0.05).
  • the mogroside IIIe drug was administered.
  • the lung coefficients of the group and the prednisone acetate group were significantly decreased, with significant difference (P ⁇ 0.01).
  • the lung coefficient of the model group was significantly increased and the difference was statistically significant (P ⁇ 0.01 or 0.05).
  • the mogroside IIIe was high and low after 14 and 21 days of administration.
  • the lung coefficient of the dose group and the positive drug (prednisone acetate) group decreased significantly, with significant difference (P ⁇ 0.01). It is suggested that mogroside IIIe can improve bleomycin-induced pulmonary fibrosis in mice at different doses of 20 mg/kg and 10 mg/kg, and slow down the development of pulmonary fibrosis in model mice (see Figure 7).
  • the lung pathological sections were stained by Masson.
  • the results showed that the lung tissue of the blank control group was intact and clear, the alveolar septum was not thickened, the alveolar cavity was translucent, no obvious exudate was found in the cavity, and no fibroblast proliferation was observed.
  • the blank control group a small amount of collagen fibers stained blue was observed in the lung tissue, which was the main component of the extracellular matrix.
  • the alveolar structure of the mice was destroyed, the alveolar space was widened, a large number of inflammatory cells infiltrated into acute fibroblasts, a large amount of collagen was deposited, and pulmonary fibrosis was formed.
  • Hydroxyproline is an amino acid derived from the hydrolysis of connective tissue protein, which accounts for about 14% of the weight of collagen. It plays a key role in the stability of collagen. Since collagen is the only protein containing more HYP, Determination of HYP content can reflect changes in the total amount of tissue collagen. The content of HYP in lung tissue was detected by digestion. Compared with the blank control group, the HYP content in the lung tissue of the model group was significantly increased at 28 days (P ⁇ 0.01). Compared with the model group, the drug could significantly reduce lung tissue. The content of HYP in the middle (P ⁇ 0.05). It is suggested that mogroside IIIe can improve the lung fibrosis induced by bleomycin in mice at different doses of 50mg/kg. Mogroside IIIe can reduce the collagen fiber content of lung tissue and slow down the development of pulmonary fibrosis in model mice. Figure 9).
  • Alveolar epithelial cells can be obtained as an important source of lung fibroblasts and myofibroblasts through an epithelial-metaplasmic transformation (EMT) process to obtain a mesenchymal phenotype.
  • EMT epithelial-metaplasmic transformation
  • alveolar epithelialization should be considered as one of the key aspects of fibrosis.
  • damage can induce the transformation of epithelial cells into a stromal cell phenotype, thus contributing to the fibrosis of many organs.
  • Fibroblasts and myofibroblasts differentiated from epithelial cells are often obtained by morphological changes (eg, changes from cubic cell morphology to elongated or fusiform), fibroblasts or myofibroblast-specific markers (eg, alpha - SMA), loss of epithelial markers (eg, epithelial cadherin (E-cadherin and tight junction protein), and these epithelial tissues are integrated.
  • epithelial-mesenchymal transition EMT was induced by TGF- ⁇ 1 in A549 cells.
  • actin a-SMA was analyzed by Western blot.
  • the signal transduction pathway of EMT was investigated. The significance of the pathogenesis of pulmonary fibrosis.
  • the well-grown A549 cells subcultured for 2-4 passages were passaged at 1 ⁇ 10 5 and divided into four groups, and the serum-free DMEM culture solution was starved for 12 hours to make the cells at the same growth level, and the model group was TGF- ⁇ 1 was added to the serum medium at a concentration of 5 ng/mL, and the positive drug group and the mogroside IIIe group (100 ⁇ M) were added with a concentration of 5 ng/mL of TGF- ⁇ 1, and the corresponding drug was cultured for 48 hours, under an inverted microscope. The changes of lung epithelial cells were observed.
  • the high and low doses of mogroside IIIe significantly reduced the lung index, and the HYP content in the lung tissue of the high dose group decreased significantly (p ⁇ 0.05), and the pathological results showed the lung tissue structure of the mogroside IIIe drug group.
  • the number of neutrophils in the alveolar lavage fluid of the model group was significantly increased, indicating that bleomycin caused an inflammatory reaction in the model group, thereby initiating an inflammatory cascade in vivo.
  • ICR mice male, weighing 25-30 g, 100, were provided by the Yangzhou Comparative Medical Center.
  • mice were used as a blank control group, and the other 80 were used for modeling. All the above mice were intraperitoneally injected with 4% chloral hydrate for anesthesia, and the injection volume was 10 ml/kg. After anesthesia, the mice were fixed and disinfected. The neck of the mouse; the neck skin of the mouse was cut longitudinally with scissors, the fascia and muscle were bluntly removed by forceps, and the trachea was exposed; the syringe was inserted into the trachea, and the mice in the blank control group were injected with physiological saline, and the other mice were injected.
  • mice On the 7th day after modeling, the other 80 mice were randomly divided into a model group, a positive drug (prednisone acetate) group, a high dose group of mogroside IVe (50 mg/kg, mogroside IVe-H), and mogroside. IVe low dose group (50 mg/kg, mogroside IVe-L), 20 in each group.
  • the blank control group and the model group were intragastrically administered with normal saline daily.
  • the positive drug group was intragastrically administered with 6.67 mg/kg/d prednisone acetate, and the mogroside IVe was divided into high and low dose groups.
  • the two groups were intragastrically administered with 50 mg/kg/d (high).
  • the dose group) and 20 mg/kg/d (low dose group) were continuously gavage until the 28th day, and the weight was recorded.
  • the mice were sacrificed on 28 days, the lung tissues were dissected, and the lung coefficient was calculated.
  • the lung coefficient lung weight (mg) ) / weight (g).
  • the left small lung was fixed in 4% neutral formaldehyde, dehydrated step by step, transparent in xylene, dipped in wax, embedded in paraffin, routinely sectioned, Masson stained, and observed the morphology of lung tissue, lung injury and pulmonary fibrosis.
  • the weight of the mice in the model group decreased significantly and the difference was statistically significant (P ⁇ 0.01 or 0.05).
  • the fermented IVe IVe was high and low after 14 and 28 days of administration.
  • the body weight of the dose group and the positive drug (prednisone acetate) group increased significantly, with significant difference (P ⁇ 0.01).
  • Prompt mogroside IVe at 50mg/kg At the dose of 20 mg/kg, the physique of bleomycin-induced pulmonary fibrosis in mice was improved to a certain extent, and the degree of weight loss in lung model mice was slowed down (Fig. 11).
  • Bleomycin-induced lung injury in mice the number of white blood cells increased, especially neutrophil infiltration, causing alveolar inflammation, inflammatory cells release inflammatory transmitters NO, TNF- ⁇ and various cytokines, on the one hand aggravated Lung tissue damage, on the other hand, promotes excessive collagen production through various growth factors.
  • the experiment was performed according to the requirements of the kit, and the number of white blood cells in the bronchoalveolar lavage fluid of each group of mice at n14 days was measured by colorimetry (results are shown in Table 3). On day 14 after treatment with bleomycin (BLM), neutrophils and lymphocytes in the alveolar lavage fluid of mice showed significant aggregation.
  • the lung coefficient was measured on the 28th day after modeling. Compared with the blank control group, the lung coefficient of the model group was significantly increased and the difference was statistically significant (P ⁇ 0.01 or 0.05). Compared with the lung coefficient of the model group, the mogroside IVe drug group and the prednisone acetate group The lung coefficient was significantly decreased, with a significant difference (P ⁇ 0.01). Compared with the blank control group, the lung coefficient of the model group was significantly increased and the difference was statistically significant (P ⁇ 0.01 or 0.05). Compared with the body weight of the model group, the mogroside IVe high and low dose groups and positive drugs ( The lung coefficient of prednisone acetate group decreased significantly, with significant difference (P ⁇ 0.01). It is suggested that mogroside IVe can improve bleomycin-induced pulmonary fibrosis in mice at different doses of 50 mg/kg and 20 mg/kg, and slow down the development of pulmonary fibrosis in model mice (Fig. 12).
  • the pathological tissue sections were stained by Masson. The results showed that the lung tissue of the blank control group was intact and clear, the alveolar septum was not thickened, the alveolar cavity was translucent, and no fibroblasts were proliferated. In the blank control group, a small amount of dyeing was observed in the lung tissue. Blue collagen fibers are a major component of the extracellular matrix. In the model group, the alveolar structure of the mice was destroyed, and the alveolar space was widened and large. Collagen deposition, pulmonary fibrosis, Masson staining can be seen in a number of densely stained blue collagen fibers, which are bundled or flaky, basically consistent with the characteristics of pulmonary fibrosis, indicating the preparation of experimental lung fibrosis model success.
  • the high and low doses of mogroside IVe significantly reduced the lung index, and the pathological results showed that the lung tissue structure of the mogroside IVe and mogroside groups was significantly improved.
  • the number of neutrophils in the alveolar lavage fluid of the model group was significantly increased, indicating that bleomycin caused an inflammatory reaction in the model group, thereby initiating an inflammatory cascade in vivo.
  • the degree of inflammation and fibrosis in the mice was alleviated to varying degrees, suggesting that the drug can protect the lung cells from damage and prevent pulmonary fibrosis.
  • ICR mice male, weighing 25-30 g, 100, were provided by the Yangzhou Comparative Medical Center.
  • mice were used as a blank control group, and the other 80 were used for modeling. All the above mice were intraperitoneally injected with 4% chloral hydrate for anesthesia, and the injection volume was 10 ml/kg. After anesthesia, the mice were fixed and disinfected. The neck of the mouse; the neck skin of the mouse was cut longitudinally with scissors, the fascia and muscle were bluntly removed by forceps, and the trachea was exposed; the syringe was inserted into the trachea, and the mice in the blank control group were injected with physiological saline, and the other mice were injected.
  • mice were returned to the dry and clean squirrel cage for rest, waiting for recovery, and awakened after about 1-2 hours, and then reared normally.
  • the other mice were randomly divided into a model group, a positive drug (prednisone acetate) group, a high dose group of mogroside (50 mg/kg, mogroside-H), and a low dose group of mogroside. 20 mg/kg, mogroside-L), 20 in each group.
  • the blank control group and the model group were intragastrically administered with normal saline every day.
  • the positive drug group was intragastrically administered with 7.0 mg/kg/d prednisone acetate, and the high dose and low dose group of Luo Hanokol was continuously administered until the 28th day.
  • the mice were sacrificed on 28 days; the lung tissues were dissected, fixed in 4% neutral formaldehyde, dehydrated in grades, transparent in xylene, dipped in wax, embedded in paraffin, routinely sectioned, Masson stained, observed lung tissue morphology, lung injury And the degree of pulmonary fibrosis.
  • the high and low dose groups of mogroside significantly reduced the lung index, and the pathological results showed that the lung tissue structure of the mogroside group was significantly improved.
  • the number of neutrophils in the alveolar lavage fluid of the model group was significantly increased, indicating that bleomycin caused an inflammatory reaction in the model group, and the degree of inflammation and fibrosis in the mice after administration of the drug.
  • the drug can protect lung cells from damage and prevent lung fibrosis.
  • Inosine as a aglycone of cucurbitane-type tetracyclic triterpenoids, which has a common parent nucleus in the structural skeleton of such compounds.
  • the experimental results show that Luo Han Guo extract, Luo Han Guo IVe or Luo Han Guo alcohol obtained from the extraction of Luo Han Guo extract and Luo Han Guo extract can improve lung tissue inflammation and pulmonary fibrosis in bleomycin-induced pulmonary fibrosis model.
  • the degree, as well as the production of collagen in lung tissue, has a new use for the treatment of pulmonary fibrosis.

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Abstract

提供了罗汉果提取物或其酶解物或水解物在制备治疗和/或预防肺纤维化疾病的药物或保健品中的用途，其中，罗汉果提取物中总苷质量百分比≥50%，优选≥80%,罗汉果酶解物或水解物中至少含有罗汉果苷Ⅲe、罗汉果苷Ⅳ或罗汉果醇中的一种或多种。

Description

罗汉果提取物抗肺纤维化的应用 技术领域
本发明属于肺纤维化的防治技术领域，涉及一种罗汉果提取物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用。
背景技术
肺纤维化(pulmonary fibrosis,PF)是由各种肺内外致病因素所致的，是一系列慢性肺部损伤或疾病发展到晚期的结果，严重危害人类健康。肺纤维化的病因包括遗传性免疫功能异常、病毒感染、药物及化学品、放射线、空气污染(雾霾、吸烟、粉尘)等因素。肺纤维化的病理生理过程复杂，早期以肺部炎症为主，肺泡壁弥漫性增厚，大量炎性细胞浸润，中晚期胶原纤维大量生成、沉积，肺泡变形、闭索，正常肺组织结构被破坏，功能丧失。肺纤维化的临床表现主要有刺激性干咳、限制性通气功能障碍、进行性呼吸困难、弥散功能降低和低血氧症等。近年来，肺纤维化仍是一种高死亡率的疾病，临床上缺乏有效的治疗手段；传统治疗药物仍以抗炎、免疫抑制、抗凝血为主，临床常用的有糖皮质类激素、环磷酰胺、环孢霉素、秋水仙碱和青霉胺等，但疗效不佳且副作用大。因此，寻找有效抗肺纤维化药物成为当前医学的迫切需要。
相关研究表明，体内许多细胞因子均能够影响肺纤维化的形成发展，如可以促进纤维化形成的转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)、血管内皮生长因子(PDGF)等。其中TGF-β1扮演了很重要的角色，促进肌成纤维细胞的表型转化，刺激多种细胞因子的合成、分泌，调控细胞的增殖与分化等，是一种强力的致纤维化因子，可以刺激细胞合成并分泌细胞外基质(ECM)，还可以改变基质降解酶成分的活性,直接加剧ECM的沉积。肺纤维化的发病机制中，起到关键性作用的是肺成纤维细胞的活化、增殖，同时释放大量促纤维因子，进而增加平滑肌肌动蛋白α-SMA的表达和胶原蛋白的蓄积，导致ECM的沉积，最终引发肺纤维化。以抑制促纤维化因子为切入点开发治疗肺纤维化的药物是目前的研究热点。
气管滴注博莱霉素诱导鼠肺纤维化的动物模型是国内外研究肺纤维化药物的经典动物模型，该法复制的肺纤维化动物模型与人肺纤维化近似，并可在短时间内诱导产生组织炎症及纤维化，引起体内氧化与抗氧化失衡，促纤维化标志物TGF-β1、α-SMA等的表达，导致细胞外基质沉积及纤维组织增生形成肺纤维化。目前肺纤维化的治疗药物以糖皮质激素、细胞毒性药物、免疫抑制剂、免疫调节剂为主，但尚无特效药。鉴于此，有必要研制新型的高效、安全的治疗肺纤维化的药物。
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。天然提取的罗汉果总苷里面罗汉果苷V的含量最高，罗汉果总苷采用β-葡萄糖苷酶水解之后，根据酶解时间长短可以得到含有不同罗汉果苷成分比例的提取物。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，尚无罗汉果提取物在抗肺纤维化中的应用报道。
发明内容
本发明公开了一种罗汉果提取物的医药新用途，即其可用于制备预防和/或治疗肺纤维化疾病的药物和/或保健品的用途。
本发明的技术方案：
罗汉果提取物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用。
所述罗汉果提取物中罗汉果总苷的质量百分比≥50％。优选罗汉果总苷质量百分比≥80％。
罗汉果提取物的酶解物或水解物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用。
所述罗汉果提取物的酶解物或水解物中至少含有罗汉果苷IIIe、罗汉果苷IVe或罗汉果醇中的一种或多种。
优选的，所述的罗汉果提取物的酶解物中至少含有罗汉果苷IIIe：
Figure PCTCN2016086831-appb-000001
优选的，所述的罗汉果提取物的水解物中至少含有罗汉果醇：
Figure PCTCN2016086831-appb-000002
优选的，所述的罗汉果提取物的酶解物中至少含有罗汉果苷IVe：
Figure PCTCN2016086831-appb-000003
进一步，可将罗汉果提取物或其酶解物或水解物与其他人体可接受的药用辅料制成片剂、颗粒剂、汤剂或胶囊。
进一步，所述药物或保健品为降低肺纤维化组织间质中胶原堆积量的药物或保健品。
所述药物或保健品为能减轻炎症程度，抑制胶原形成、保护肺组织的抗肺纤维化的药物或保健品。
所述药物或保健品为通过抗炎及抑制肺泡上皮细胞间质化而发挥抗肺纤维化的药物或保健品。
有益效果：
肺纤维化是多种肺部疾病或肺损伤发展到晚期的一种常见的病理变化，临床研究结果显示糖皮质激素治疗肺纤维化后患者生存率无明显改变，目前尚无明确治疗药物。本发明从传统的润肺止咳中药罗汉果中制备得到。目前，没有任何研究报道罗汉果苷类成分可用于肺纤维化的治疗，本发明人通过体内实验证明罗汉果苷IIIe，罗汉果苷IVe和罗汉果苷类成分的苷元——罗汉果醇、罗汉果总苷均能显著改善博莱霉素诱导的小鼠肺纤维化；且罗汉果苷IIIe，罗汉果苷IVe、罗汉果醇和罗汉果总苷均具有较好的稳定性，可以用于制备治疗相应疾病的药物；由此得出：罗汉果提取物具有抗肺纤维化的作用。
本发明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1罗汉果总苷对博来霉素诱导的肺纤维化模型小鼠体重变化趋势图。与对照组相比，#p<0.05；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋酸泼尼松。
图2罗汉果总苷对博来霉素诱导的肺纤维化模型小鼠小鼠肺系数变化的影响。与对照组相比，##p<0.001；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋酸泼尼松。
图3罗汉果总苷对博来霉素诱导肺纤维化模型各组小鼠肺组织纤维化程度的影响(Masson染色)。与对照组相比，##p<0.01；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋酸泼尼松。
图4罗汉果总苷对博来霉素诱导后第14天的肺纤维化小鼠肺组织中HYP含量的影响。与对照组相比，##p<0.01；与模型组相比，**p<0.01。
图5罗汉果总苷对博来霉素诱导的肺纤维化小鼠肺组织中α-SMA蛋白表达水平的影响。与对照组相比，#p<0.05；与模型组相比，*p<0.05。
图6罗汉果苷IIIe对博来霉素诱导的肺纤维化模型小鼠体重变化趋势图。与对照组相比，#p<0.05；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋酸泼尼松。
图7罗汉果苷IIIe对博来霉素诱导的肺纤维化模型小鼠小鼠肺系数变化的影响。与对照组相比，##p<0.001；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋酸泼尼松。
图8罗汉果苷IIIe对博来霉素诱导肺纤维化模型各组小鼠肺组织纤维化症程度的影响(Masson染色)。与对照组相比，##p<0.01；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋酸泼尼松。
图9罗汉果苷IIIe对博来霉素诱导的肺纤维化小鼠肺组织中HYP含量的影响。与对照组相比，#p<0.05；与模型组相比，*p<0.05。
图10罗汉果苷IIIe对TGF-β1诱导后人肺泡II型上皮细胞中α-SMA表达量的影响。与对照组相比，##p<0.01；与模型组相比，*p<0.05。
图11罗汉果苷IVe对博来霉素诱导的肺纤维化模型小鼠体重变化趋势图。与对照组相比，#p<0.05；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋酸泼尼松。
图12罗汉果苷IVe对博来霉素诱导的肺纤维化模型小鼠小鼠肺系数变化的影响。与对照组相比，##p<0.001；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋酸泼尼松。
图13罗汉果苷IVe对博来霉素诱导肺纤维化模型各组小鼠肺纤维化程度(Masson染色)的影响。与对照组相比，##p<0.01；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋 酸泼尼松。
图14罗汉果醇对博来霉素诱导的肺纤维化模型小鼠体重变化趋势图。与对照组相比，#p<0.05；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋酸泼尼松。
图15罗汉果醇对博来霉素诱导肺纤维化模型各组小鼠肺组织肺纤维化程度(Masson染色)的影响。与对照组相比，##p<0.01；与模型组相比，*p<0.05,**p<0.01；阳性对照药物:醋酸泼尼松。
具体实施方式
罗汉果提取物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用。本发明中，罗汉果提取物即罗汉果采用现有技术提取得到的物质。
所述罗汉果提取物中罗汉果总苷的质量百分比≥50％。优选罗汉果总苷质量百分比≥80％。
罗汉果提取物的酶解物或水解物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用。
所述罗汉果提取物的酶解物或水解物中至少含有罗汉果苷IIIe、罗汉果苷IVe或罗汉果醇中的一种或多种。
优选的，所述的罗汉果提取物的酶解物中至少含有罗汉果苷IIIe。
优选的，所述的罗汉果提取物的水解物中至少含有罗汉果醇。
优选的，所述的罗汉果提取物的酶解物中至少含有罗汉果苷IVe。
进一步，可将罗汉果提取物或其酶解物或水解物与其他人体可接受的药用辅料制成片剂、颗粒剂、汤剂或胶囊。
进一步，所述药物或保健品为降低肺纤维化组织间质中胶原堆积量的药物或保健品。
所述药物或保健品为能减轻炎症程度，抑制胶原形成、保护肺组织的抗肺纤维化的药物或保健品。
所述药物或保健品为通过抗炎及抑制肺泡上皮细胞间质化而发挥抗肺纤维化的药物或保健品。
罗汉果总苷采用现有方法提取即可。
本发明是经过研究证明罗汉果总苷提取物可改善博来霉素诱导小鼠肺纤维化。体内实验观察Masson染色后肺组织病理切片、测定各组肺脏系数、体重变化及在不同时期检测各组小鼠肺组织中HYP含量及α-SMA表达情况。体外实验考察罗汉果苷类化合物可显著抑制LPS诱导小鼠巨噬细胞RAW264.7的NO释放。实验结果表明：本发明中的罗汉果总苷提取物可 改善模型小鼠肺组织纤维化程度，降低模型肺组织胶原沉积，改善上皮间质化，显示对小鼠肺纤维化模型的治疗作用，具有可用于治疗肺纤维化疾病的新医药用途。
本发明以罗汉果总苷(实施例1)、罗汉果苷IIIe(实施例2)，罗汉果苷IVe(实施例3)和罗汉果醇(实施例4)为例来验证罗汉果提取物抗肺纤维化疾病的部分药效学试验及结果。
罗汉果总苷采用市售罗汉果提取物，购自桂林莱茵生物科技股份有限公司生产的80％罗汉果总苷提取物(Mogrosides>80％，产品代码MOV 09)。
本发明罗汉果苷IIIe，罗汉果苷IVe和罗汉果醇的制备：
罗汉果苷IIIe采用市售罗汉果提取物，经大孔树脂分离，高压反相制备色谱分离得到。纯度95％以上。或参考中国专利2010105610299制备罗汉果苷Ⅳ的方法，将市售罗汉果提取物用β-葡萄糖苷酶水解后，经大孔树脂分离，高压反相制备色谱分离得到。纯度95％以上。
罗汉果苷IVe采用市售罗汉果提取物，经大孔树脂分离，高压反相制备色谱分离得到。纯度95％以上。或参考中国专利2010105610299制备罗汉果苷Ⅳ的方法，将市售罗汉果提取物用β-葡萄糖苷酶水解后，经大孔树脂分离，高压反相制备色谱分离得到。纯度95％以上。
罗汉果醇采用市售罗汉果提取物，用5％的硫酸水解，经硅胶纯化得到。纯度95％以上。
实施例1 80％罗汉果总苷提取物改善博莱霉素诱导的小鼠肺纤维
选取桂林莱茵生物科技股份有限公司生产的80％罗汉果总苷提取物(即罗汉果总苷质量占提取物总质量80％的罗汉果提取物)(Mogrosides>80％，产品代码MOV 09)进行下述体内药效学研究。气管内注入博来霉素诱导小鼠肺纤维化模型,是国际上普遍采用的一种方法，而且与人类肺间质纤维化疾病相近。
罗汉果总苷用于抗肺纤维化使用时，实验动物给药量为50mg～100mg/kg，分别设定为罗汉果总苷-H、L组。罗汉果总苷(Mogrosides)又称为罗汉果甜苷，是药食同源植物罗汉果的有效部位。罗汉果总苷包含了包括罗汉果苷V(Mogroside V)；罗汉果苷IVe(Mogroside IV)；罗汉果苷IIIe(Mogroside IIeI)；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)等葫芦烷型三萜苷类化合物。
1.1实验方法
ICR小鼠，雄性，体重25-30g，100只，由扬州比较医学中心提供。
将20只小鼠作为空白对照组(图1中的对照组)，其他80只用来造模，将上述所有小鼠腹腔注射4％的水合氯醛进行麻醉，注射体积10ml/kg，小鼠麻醉后，固定小鼠，消毒小鼠颈部；用剪刀纵向剪开小鼠颈部皮肤，暴露气管；注射器刺入气管，空白对照组小鼠注入生理 盐水，其余小鼠均注入博来霉素(5mg/kg)；然后迅速将鼠板直立，旋转鼠板，观察小鼠呼吸情况，缝合伤口，并在缝合处滴1-2滴青霉素注射液。将术后小鼠放回干燥洁净的鼠笼休息，等待苏醒，大约l-2h后苏醒，之后正常饲养。
造模后第7天开始，将其他80只小鼠随机分为模型组、阳性药(醋酸泼尼松)组、罗汉果总苷提取物高剂量组(100mg/kg,罗汉果总苷-H)、罗汉果总苷提取物低剂量组(50mg/kg,罗汉果总苷-L)，每组各20只。空白对照组、模型组每天灌胃生理盐水，阳性药组灌胃6.67mg/kg/d醋酸泼尼松，罗汉果总苷提取物分高、低剂量组，分别灌胃100mg/kg/d和50mg/kg/d，连续灌胃至第28天，于14、28天处死部分小鼠。称体重记录，解剖取出肺组织，计算肺系数，肺系数＝肺重(mg)/体重(g)。将实验28天小鼠左小肺放入4％中性甲醛中固定，逐级酒精脱水，二甲苯透明，浸蜡，石蜡包埋后，常规切片，Masson染色，观察肺组织形态、肺损伤及肺纤维化程度。其他肺叶分叶保存，用于HYP含量的测定。
所有数据均用均数±S.D(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。
1.2实验结果
1.2.1罗汉果总苷对模型小鼠体重的影响
与模型组小鼠体重相比较，实验第14、28天后罗汉果总苷提取物的高、低剂量组和阳性药物(醋酸泼尼松)组的体重均有明显上升，具有显著性差异(P<0.01)。提示罗汉果总苷在100mg/kg和50mg/kg剂量下可不同程度的改善博莱霉素诱导肺纤维化小鼠的体质，减缓肺纤维化模型小鼠体重下降程度(图1)。
1.2.2罗汉果总苷对模型小鼠支气管肺泡灌洗液中白细胞数量的影响
博来霉素引起的小鼠肺组织损伤，则白细胞数量增加，尤其是中性粒细胞浸润，引起肺泡炎症，炎症细胞释放炎症递质NO、TNF-α等及各种细胞因子。本实验按照试剂盒的要求规范操作，比色法检测14天时各组小鼠支气管肺泡灌洗液中白细胞数量(如表1所示)。博来霉素(BLM)处理后第14天小鼠肺泡灌洗液中中性粒细胞和淋巴细胞出现明显的聚集。与空白对照组相比较，BLM组肺泡灌洗液中的细胞总数和中性粒细胞的数量均显著升高(##P<0.01)。经罗汉果总苷高剂量(100mg/kg)处理后，肺泡灌洗液中细胞总数和中性粒细胞的数量的数量较单独给予BLM组明显下降(*P<0.05)。提示罗汉果总苷能够减少BLM诱导的炎症细胞的渗出。
表1 对博来霉素诱导后小鼠支气管肺泡灌洗液中的细胞总数及中性粒细胞数量的影响(×104，n＝5)
组别 细胞总数 中性粒细胞数
空白对照组 12.17±3.98 1.32±0.21
BLM组 55.38±21.46## 19.32±6.22##
罗汉果总苷-H(100mg/kg) 30.81±17.53** 10.18±4.32*
罗汉果总苷-L(50mg/kg) 41.65±13.12* 14.18±6.43
1.2.3罗汉果总苷对模型小鼠肺系数的影响
与空白对照组比较，模型组小鼠肺系数明显增高且差异均有统计学意义(P<0.01或0.05)；与模型组小鼠体重相比较，给药14、21天后罗汉果总苷高、低剂量组和阳性药物(醋酸泼尼松)组的肺系数均有明显下降，具有显著性差异(P<0.01)。提示罗汉果总苷在100mg/kg和50mg/kg剂量下可不同程度的减缓模型小鼠肺纤维化发展程度(图2)。
1.2.4罗汉果总苷对模型小鼠肺组织的影响
取第28天肺病理组织切片经Masson染色，结果表明空白对照组的小鼠肺组织结构完整清晰，肺泡间隔未增厚，肺泡腔透亮，腔内未见明显渗出物，无成纤维细胞增生；空白对照组小鼠的肺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。模型组小鼠肺泡结构破坏，肺泡间隔增宽，大量炎性细胞浸润急成纤维细胞增生，大量胶原沉积，肺纤维化形成，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，基本符合肺纤维化的特点，则说明实验小鼠肺纤维化模型制备成功。经药物治疗后，可见小鼠肺组织结构完整清晰，肺泡间隔略增厚，炎性细胞浸润及成纤维细胞增生程度均比模型组轻。给药各组及阳性药组与模型组相比，纤维化程度均减轻(图3)。
1.2.5罗汉果总苷对模型小鼠肺组织HYP含量所产生的影响
羟脯氨酸(HYP)是由结缔组织蛋白质水解所得的一种氨基酸，约占胶原重量的14％，对胶原蛋白的稳定性起关键作用，由于胶原是唯一含HYP较多的蛋白质，因此，测定HYP含量能反映组织胶原的总量变化。采用消化法检测肺组织中HYP的含量。与空白对照组相比，28天时模型组肺组织HYP含量显著增加(P<0.01)，与模型组相比，药物可明显降低肺组织中HYP含量(P<0.05)。提示罗汉果总苷在100mg/kg剂量下可不同程度的改善博莱霉素诱导的小鼠肺纤维化(图4)。
1.2.6罗汉果总苷对模型小鼠肺组织α-SMA水平的影响
肺泡上皮细胞本身就可以通过一个叫做上皮细胞一间质转化(EMT)的过程获得间质细胞表型而作为肺成纤维细胞和肌纤维母细胞的重要来源。肺泡上皮间质化被看作纤维化的关键 环节之一。从上皮细胞分化过来的成纤维细胞和肌纤维母细胞常常通过形态变化，成纤维细胞或肌纤维母细胞特异标记的获得(例如，肌动蛋白a-SMA高表达)，上皮特征标记的丢失(例如，上皮细胞钙粘蛋白〔E-cadherin和紧密连接蛋白)和这些上皮组织融为一体。采用Western blot发分析各组肺组织中α-SMA表达水平。与空白对照组相比，28天时模型组肺组织HYP含量α-SMA表达水平显著增加(P<0.01)，与模型组相比，罗汉果总苷提取物可明显降低肺组织中α-SMA表达水平(P<0.05)，结果提示罗汉果总苷可降低模型肺组织α-SMA表达水平，改善BLM诱导的小鼠肺纤维程度(如图5)。
1.3讨论
与模型组相比，罗汉果总苷高、低剂量组能明显降低肺脏指数，肺组织中HYP含量、α-SMA表达水平(p<0.05or 0.01)，且病理结果显示罗汉果总苷的肺脏组织结构明显改善，肺泡结构受损及肺泡间隔增厚程度都明显减轻，炎性细胞浸润减少，胶原纤维含量减少。与空白对照组相比,模型组小鼠肺泡灌洗液中中性粒细胞数量显著增加，表明在模型组中博莱霉素引起炎性反应，从而启动体内炎症级联反应。而在给予罗汉果总苷提取物后，小鼠炎症及纤维化程度有不同程度的减轻，提示药物可以保护肺细胞免受损伤从而防治肺纤维化。
实施例2 罗汉果苷IIIe改善博莱霉素诱导的小鼠肺纤维
选取上述罗汉果苷IIIe进行下述体内药效学研究。气管内注入博来霉素诱导小鼠肺纤维化模型,是国际上普遍采用的一种方法，而且与人类肺间质纤维化相近。
2.1实验方法
ICR小鼠，雄性，体重25-30g，100只，由扬州比较医学中心提供。
将20只小鼠作为空白对照组(图6中的对照组)，其他80只用来造模，将上述所有小鼠腹腔注射4％的水合氯醛进行麻醉，注射体积10ml/kg，小鼠麻醉后，固定小鼠，消毒小鼠颈部；用剪刀纵向剪开小鼠颈部皮肤，暴露气管；注射器刺入气管，空白对照组小鼠注入生理盐水，其余小鼠均注入博来霉素(5mg/kg)；然后迅速将鼠板直立，旋转鼠板，观察小鼠呼吸情况，缝合伤口，并在缝合处滴1-2滴青霉素注射液。将术后小鼠放回干燥洁净的鼠笼休息，等待苏醒，大约l-2h后苏醒，之后正常饲养。
造模后第7天开始，将其他80只小鼠随机分为模型组、阳性药(醋酸泼尼松)组、罗汉果苷IIIe高剂量组(50mg/kg，罗汉果苷IIIe-H)、罗汉果苷IIIe低剂量组(10mg/kg，罗汉果苷IIIe-L)，每组各20只。
空白对照组、模型组每天灌胃生理盐水，阳性药组灌胃6.67mg/kg/d醋酸泼尼松，罗汉果 苷IIIe分高、低剂量组，分别灌胃50mg/kg/d和10mg/kg/d，连续灌胃至第28天，于14、28天处死小鼠。称体重记录，解剖取出肺组织，冰生理盐水洗净，吸水纸吸干后称重，计算肺系数，肺系数＝肺重(mg)/体重(g)。将左小肺放入4％中性甲醛中固定，逐级酒精脱水，二甲苯透明，浸蜡，石蜡包埋后，常规切片，Masson染色，观察肺组织形态、肺损伤及肺纤维化程度。其他肺叶分叶保存，用于HYP含量的测定。
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。
2.2实验结果
2.2.1罗汉果苷IIIe对模型小鼠体重的影响
与空白对照组比较，模型组小鼠体重明显下降且差异均有统计学意义(P<0.01或0.05)；与模型组小鼠体重相比较，给药14、28天后罗汉果苷IIIe高、低剂量组和阳性药物(醋酸泼尼松)组的体重均有明显上升，具有显著性差异(P<0.01)。提示罗汉果苷IIIe在20mg/kg和10mg/kg剂量下可不同程度的改善博莱霉素诱导肺纤维化小鼠的体质，减缓肺纤维化模型小鼠体重下降程度，结果如图6所示。
2.2.2罗汉果苷IIIe对模型小鼠支气管肺泡灌洗液中白细胞数量的影响
博来霉素引起的小鼠肺组织损伤，则白细胞数量增加，尤其是中性粒细胞浸润，引起肺泡炎症，炎症细胞释放炎症递质NO、TNF-α等及各种细胞因子，一方面加重肺组织损伤，另一方面又通过各种生长因子促进胶原产生过度增多。本实验按照试剂盒的要求规范操作，比色法检测14天时各组小鼠支气管肺泡灌洗液中白细胞数量(如表2所示)。博来霉素(BLM)处理后第14天小鼠肺泡灌洗液中中性粒细胞和淋巴细胞出现明显的聚集。与空白对照组相比较，BLM组肺泡灌洗液中的细胞总数和中性粒细胞的数量均显著升高(与空白对照组相比，##P<0.01)。经罗汉果苷IIIe处理后，肺泡灌洗液中细胞总数和中性粒细胞的数量的数量较单独给予BLM组明显下降(与BLM组相比，*P<0.05)。提示罗汉果苷IIIe能够减少BLM诱导的炎症细胞的渗出。
表2 对博来霉素诱导后小鼠支气管肺泡灌洗液中的细胞总数及中性粒细胞数量的影响(×104，n＝5)
Figure PCTCN2016086831-appb-000004
2.2.3罗汉果苷IIIe对模型小鼠肺系数的影响
与空白对照组比较，模型组小鼠肺系数明显增高且差异均有统计学意义(P<0.01或0.05)；与模型组小鼠肺系数相比较，给药14、28天后，罗汉果苷IIIe药物组和醋酸泼尼松组的肺系数均有明显下降，具有显著性差异(P<0.01)。与空白对照组比较，模型组小鼠肺系数明显增高且差异均有统计学意义(P<0.01或0.05)；与模型组小鼠体重相比较，给药14、21天后罗汉果苷IIIe高、低剂量组和阳性药物(醋酸泼尼松)组的肺系数均有明显下降，具有显著性差异(P<0.01)。提示罗汉果苷IIIe在20mg/kg和10mg/kg剂量下可不同程度的改善博莱霉素诱导的小鼠肺纤维化，减缓模型小鼠肺纤维化发展程度(参见图7)。
2.2.4罗汉果苷IIIe对模型小鼠肺组织的影响
取第28天肺病理组织切片经Masson染色，结果表明空白对照组的小鼠肺组织结构完整清晰，肺泡间隔未增厚，肺泡腔透亮，腔内未见明显渗出物，无成纤维细胞增生；空白对照组小鼠的肺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。模型组小鼠肺泡结构破坏，肺泡间隔增宽，大量炎性细胞浸润急成纤维细胞增生，大量胶原沉积，肺纤维化形成，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，基本符合肺纤维化的特点，则说明实验小鼠肺纤维化模型制备成功。经罗汉果苷IIIe治疗后，可见小鼠肺组织结构完整清晰，肺泡间隔略增厚，成纤维细胞增生程度均比模型组轻。经阳性药物醋酸泼尼松治疗后，阳性组小鼠肺泡间隔较宽，肺泡腔变窄，较多成纤维细胞增生，病变程度较模型组减轻。给药各组及阳性药组与模型组相比，纤维化程度均减轻(参见图8)。
2.2.5罗汉果苷IIIe对模型小鼠肺组织HYP含量所产生的影响。
羟脯氨酸(HYP)是由结缔组织蛋白质水解所得的一种氨基酸，约占胶原重量的14％，对胶原蛋白的稳定性起关键作用，由于胶原是唯一含HYP较多的蛋白质，因此，测定HYP含量能反映组织胶原的总量变化。采用消化法检测肺组织中HYP的含量。与空白对照组相比，28天时模型组肺组织HYP含量显著增加(P<0.01)，与模型组相比，药物可明显降低肺组织 中HYP含量(P<0.05)。提示罗汉果苷IIIe在50mg/kg剂量下可不同程度的改善博莱霉素诱导的小鼠肺纤维化，罗汉果苷IIIe可降低模型肺组织胶原纤维含量，减缓模型小鼠肺纤维化发展程度(如图9)。
2.2.6罗汉果苷IIIe对TGF-β1诱导的人肺泡II型上皮细胞α-SMA水平的影响
肺泡上皮细胞可以通过上皮细胞一间质转化(EMT)的过程获得间质细胞表型而作为肺成纤维细胞和肌纤维母细胞的重要来源。在这个新的模式中，肺泡上皮间质化应该被看作纤维化的关键环节之一。在成熟细胞中，损伤可以诱导上皮细胞向间质细胞表型的转化，因此促成了很多器官的纤维化。从上皮细胞分化过来的成纤维细胞和肌纤维母细胞常常通过形态变化(例如，从立方细胞形态向长条形或者梭形的改变)，成纤维细胞或肌纤维母细胞特异标记的获得(例如，α-SMA)，上皮特征标记的丢失(例如，上皮细胞钙粘蛋白〔E-cadherin和紧密连接蛋白)，和这些上皮组织融为一体。通过体外试验，以TGF-β1诱导A549细胞出现上皮细胞-间质细胞转化(EMT)，采用Western blot方法分析肌动蛋白a-SMA表达水平，探讨EMT过程的信号转导途径中罗汉果苷IIIe对肺纤维化发病机制的研究意义。
本发明中将传代培养2-4代的生长良好的A549细胞以1×lO5传代，分成四组，加无血清的DMEM培养液饥饿处理12小时，使细胞处于同一生长水平，模型组在无血清培养基中加入浓度为5ng/mL的TGF-β1，阳性药物组和罗汉果苷IIIe组(100μM)加入浓度为5ng/mL的TGF-β1,以及相应药物，培养48小时之后，在倒置显微镜下观察肺上皮细胞变化，Western blot检测TGF一β1对A549细胞上皮转化成间质细胞的标志物蛋白α-SMA表达影响。实验结果表明：与空白对照组相比，A549细胞在加入TGF-β1后，间质细胞标记物α-SMA表达上调(P<0.01)，而罗汉果苷IIIe在50μM浓度下可显著抑制TGF-β1引起的α-SMA(如图10)。
2.2.7讨论：
与模型组相比，罗汉果苷IIIe高、低剂量组能明显降低肺脏指数，高剂量组的肺组织中HYP含量明显下降(p<0.05)，且病理结果显示罗汉果苷IIIe药物组的肺脏组织结构明显改善，肺泡结构受损及肺泡间隔增厚程度都明显减轻，炎性细胞浸润减少，胶原纤维含量减少。与空白对照组相比,模型组小鼠肺泡灌洗液中中性粒细胞数量显著增加，表明在模型组中博莱霉素引起炎性反应，从而启动体内炎症级联反应。而在给予药物后，小鼠炎症及纤维化程度有不同程度的减轻，肺组织中HYP含量显著下降，提示药物可以保护肺细胞免受损伤从而防治肺纤维化。体外实验结果表明，罗汉果苷IIIe在50μM浓度可抑制TGF-β1所诱导的上皮间质化，降低肺成纤维母细胞标志物α-SMA。
综上所述，体内外实验结果表明罗汉果苷IIIe可改善博莱霉素小鼠肺纤维化模型中肺组织炎症及肺纤维化程度，以及肺组织中胶原蛋白的生成，并有效抑制细胞生长因子TGF-β1所引起的人肺泡II上皮细胞间质化，罗汉果苷IIIe具有治疗肺纤维化疾病的新用途。
实施例3 罗汉果苷IVe改善博莱霉素诱导引起的小鼠肺纤维
选取上述罗汉果苷IVe(式IV)进行下述体内药效学研究。
3.1实验方法
ICR小鼠，雄性，体重25-30g，100只，由扬州比较医学中心提供。
将20只小鼠作为空白对照组，其他80只用来造模，将上述所有小鼠腹腔注射4％的水合氯醛进行麻醉，注射体积10ml/kg，小鼠麻醉后，固定小鼠，消毒小鼠颈部；用剪刀纵向剪开小鼠颈部皮肤，用镊子纵向钝性撕开筋膜与肌肉，暴露气管；注射器刺入气管，空白对照组小鼠注入生理盐水，其余小鼠均注入博来霉素(5mg/kg)；然后迅速将鼠板直立，旋转鼠板，观察小鼠呼吸情况，旋转后用75％酒精棉消毒颈部伤口，缝合伤口，并在缝合处滴1-2滴青霉素注射液。将术后小鼠放回干燥洁净的鼠笼休息，等待苏醒，大约l-2h后苏醒，之后正常饲养。
造模后第7天开始，将其他80只小鼠随机分为模型组、阳性药(醋酸泼尼松)组、罗汉果苷IVe高剂量组(50mg/kg,罗汉果苷IVe-H)、罗汉果苷IVe低剂量组(50mg/kg,罗汉果苷IVe-L)，每组各20只。
空白对照组、模型组每天灌胃生理盐水，阳性药组灌胃6.67mg/kg/d醋酸泼尼松，罗汉果苷IVe分高、低剂量组，两者分别灌胃50mg/kg/d(高剂量组)和20mg/kg/d(低剂量组)，连续灌胃至第28天，称体重记录；于28天处死小鼠，解剖取出肺组织，计算肺系数，肺系数＝肺重(mg)/体重(g)。将左小肺放入4％中性甲醛中固定，逐级酒精脱水，二甲苯透明，浸蜡，石蜡包埋后，常规切片，Masson染色，观察肺组织形态、肺损伤及肺纤维化程度。
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。
3.2实验结果
3.2.1、罗汉果苷IVe对肺纤维化模型小鼠体重的影响
与空白对照组比较，模型组小鼠体重明显下降且差异均有统计学意义(P<0.01或0.05)；与模型组小鼠体重相比较，给药14、28天后，罗汉果苷IVe高、低剂量组和阳性药物(醋酸泼尼松)组的体重均有明显上升，具有显著性差异(P<0.01)。提示罗汉果苷IVe在50mg/kg 和20mg/kg剂量下可不同程度的改善博莱霉素诱导肺纤维化小鼠的体质，减缓肺模型小鼠体重下降程度(图11)。
3.2.2、罗汉果苷IVe对模型小鼠支气管肺泡灌洗液中白细胞数量的影响
博来霉素引起的小鼠肺组织损伤，则白细胞数量增加，尤其是中性粒细胞浸润，引起肺泡炎症，炎症细胞释放炎症递质NO、TNF-α等及各种细胞因子，一方面加重肺组织损伤，另一方面又通过各种生长因子促进胶原产生过度增多。本实验按照试剂盒的要求规范操作，比色法检测n14天时各组小鼠支气管肺泡灌洗液中白细胞数量(结果如表3)。博来霉素(BLM)处理后第14天小鼠肺泡灌洗液中中性粒细胞和淋巴细胞出现明显的聚集。与空白对照组相比较，BLM组肺泡灌洗液中的细胞总数和中性粒细胞的数量均显著升高(与空白对照组相比，##P<0.01)。经处理后，肺泡灌洗液中细胞总数和中性粒细胞的数量的数量较单独给予BLM组明显下降(与BLM组相比，*P<0.05)。提示罗汉果苷IVe能够减少BLM诱导的炎症细胞的渗出。
表3 对博来霉素诱导后小鼠支气管肺泡灌洗液中的细胞总数及中性粒细胞数量的影响(×104，n＝5)
组别 细胞总数 中性粒细胞数
空白对照组 12.26±3.74 1.36±0.23
BLM组 58.93±21.47## 19.26±9.65##
罗汉果苷IVe高剂量组(50mg/kg) 30.84±16.49* 3.97±3.89*
3.2.3罗汉果苷IVe对模型小鼠肺系数的影响
造模之后的第28天测定肺系数。与空白对照组比较，模型组小鼠肺系数明显增高且差异均有统计学意义(P<0.01或0.05)；与模型组小鼠肺系数相比较，罗汉果苷IVe药物组和醋酸泼尼松组的肺系数均有明显下降，具有显著性差异(P<0.01)。与空白对照组比较，模型组小鼠肺系数明显增高且差异均有统计学意义(P<0.01或0.05)；与模型组小鼠体重相比较，罗汉果苷IVe高、低剂量组和阳性药物(醋酸泼尼松)组的肺系数均有明显下降，具有显著性差异(P<0.01)。提示罗汉果苷IVe在50mg/kg和20mg/kg剂量下可不同程度的改善博莱霉素诱导的小鼠肺纤维化，减缓模型小鼠肺纤维化发展程度(图12)。
3.2.4罗汉果苷IVe对模型小鼠肺组织的影响
病理组织切片经Masson染色，结果表明空白对照组的小鼠肺组织结构完整清晰，肺泡间隔未增厚，肺泡腔透亮，无成纤维细胞增生；空白对照组小鼠的肺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。模型组小鼠肺泡结构破坏，肺泡间隔增宽，大 量胶原沉积，肺纤维化形成，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，基本符合肺纤维化的特点，则说明实验小鼠肺纤维化模型制备成功。经罗汉果苷IVe治疗后，可见小鼠肺组织结构完整清晰，肺泡间隔略增厚，成纤维细胞增生程度均比模型组轻。给药各组及阳性药组与模型组相比，纤维化程度均减轻(图13)。
3.2.5、讨论：
与模型组相比，罗汉果苷IVe高、低剂量组能明显降低肺脏指数，病理结果显示罗汉果苷IVe和罗汉果醇药物组的肺脏组织结构明显改善。与空白对照组相比，模型组小鼠肺泡灌洗液中中性粒细胞数量显著增加，表明在模型组中博莱霉素引起炎性反应，从而启动体内炎症级联反应。而在给予药物后，小鼠炎症及纤维化程度有不同程度的减轻，提示药物可以保护肺细胞免受损伤从而防治肺纤维化。体内实验结果表明罗汉果苷IVe可改善博莱霉素小鼠肺纤维化模型中肺组织炎症及肺纤维化程度，以及肺组织中胶原蛋白的生成，罗汉果苷IVe具有治疗肺纤维化疾病的新用途。
实施例4 罗汉果醇改善博莱霉素诱导的小鼠肺纤维
选取上述罗汉果醇(式IX)进行下述体内药效学研究。
4.1实验方法
ICR小鼠，雄性，体重25-30g，100只，由扬州比较医学中心提供。
将20只小鼠作为空白对照组，其他80只用来造模，将上述所有小鼠腹腔注射4％的水合氯醛进行麻醉，注射体积10ml/kg，小鼠麻醉后，固定小鼠，消毒小鼠颈部；用剪刀纵向剪开小鼠颈部皮肤，用镊子纵向钝性撕开筋膜与肌肉，暴露气管；注射器刺入气管，空白对照组小鼠注入生理盐水，其余小鼠均注入博来霉素(5mg/kg)；然后迅速将鼠板直立，旋转鼠板，观察小鼠呼吸情况，旋转后用75％酒精棉消毒颈部伤口，缝合伤口，并在缝合处滴1-2滴青霉素注射液。将术后小鼠放回干燥洁净的鼠笼休息，等待苏醒，大约l-2h后苏醒，之后正常饲养。造模后第7天开始，将其他小鼠随机分为模型组、阳性药(醋酸泼尼松)组、罗汉果醇高剂量组(50mg/kg,罗汉果醇-H)、罗汉果醇低剂量组(20mg/kg,罗汉果醇-L)，每组各20只。
空白对照组、模型组每天灌胃生理盐水，阳性药组灌胃7.0mg/kg/d醋酸泼尼松，罗汉果醇高、低剂量组，连续灌胃至第28天，称体重记录。于28天处死小鼠；解剖取出肺组织，4％中性甲醛中固定，逐级酒精脱水，二甲苯透明，浸蜡，石蜡包埋后，常规切片，Masson染色，观察肺组织形态、肺损伤及肺纤维化程度。
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。
4.2实验结果
4.2.1、罗汉果醇对肺纤维化模型小鼠体重的影响
与模型组小鼠体重相比较，罗汉果醇高、低剂量组和阳性药物(醋酸泼尼松)组的体重均有明显上升。提示罗汉果醇在50mg/kg和20mg/kg剂量下可不同程度的改善博莱霉素诱导肺纤维化小鼠的体质，减缓模型小鼠体重下降程度(图14)。
4.2.2、罗汉果醇对模型小鼠支气管肺泡灌洗液中白细胞数量的影响
博来霉素引起的小鼠肺组织损伤，则白细胞数量增加，尤其是中性粒细胞浸润，引起肺泡炎症，炎症细胞释放炎症递质NO、TNF-α等及各种细胞因子，一方面加重肺组织损伤，另一方面又通过各种生长因子促进胶原产生过度增多。本实验按照试剂盒的要求规范操作，比色法检测14天时各组小鼠支气管肺泡灌洗液中白细胞数量(结果如表4)。与空白对照组相比较，博来霉素诱导后模型组小鼠肺泡灌洗液中的细胞总数和中性粒细胞的数量均显著升高(与空白对照组相比，##P<0.01)。经罗汉果醇(50mg/kg)处理后，肺泡灌洗液中细胞总数和中性粒细胞的数量的数量较单独给予BLM组明显下降(与BLM组相比，*P<0.05)。提示罗汉果醇能够减少BLM诱导的炎症细胞的渗出。
表4 对博来霉素诱导后小鼠支气管肺泡灌洗液中的细胞总数及中性粒细胞数量的影响(×104，n＝5)
组别 细胞总数 中性粒细胞数
空白对照组 13.16±3.74 1.96±0.65
BLM组 65.13±21.57## 18.26±9.45##
罗汉果醇高剂量组(50mg/kg) 34.72±17.83** 4.16±4.26**
4.2.3罗汉果醇对模型小鼠肺组织的影响
空白对照组小鼠的肺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。模型组小鼠肺泡结构破坏，肺泡间隔增宽，大量胶原沉积，肺纤维化形成，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，基本符合肺纤维化的特点，则说明实验小鼠肺纤维化模型制备成功。经罗汉果醇高、低剂量(50、20mg/kg)治疗后，可见小鼠肺组织结构完整清晰，肺泡间隔略增厚，成纤维细胞增生程度均比模型组轻。给药各组与模型组相比，纤维化程度均减轻(图15)。
4.2.5、讨论：
与模型组相比，罗汉果醇高、低剂量组能明显降低肺脏指数，病理结果显示罗汉果醇药物组的肺脏组织结构明显改善。与空白对照组相比，模型组小鼠肺泡灌洗液中中性粒细胞数量显著增加，表明在模型组中博莱霉素引起炎性反应，而在给予药物后，小鼠炎症及纤维化程度有不同程度的减轻，提示药物可以保护肺细胞免受损伤从而防治肺纤维化。罗汉果醇作为葫芦烷型四环三萜罗汉果苷类化合物的苷元，具有此类化合物结构骨架中共同母核体内实验结果揭示了体内实验结果表明罗汉果醇可改善博莱霉素小鼠肺纤维化模型中肺组织肺纤维化程度，预示这此类成分在制备预防或治疗肺纤维化疾病药物中的新用途。
综上所述，实验结果表明罗汉果提取物以及罗汉果提取物进水解处理得到的罗汉果IIIe、罗汉果IVe或罗汉果醇均可改善博莱霉素小鼠肺纤维化模型中肺组织炎症及肺纤维化程度，以及肺组织中胶原蛋白的生成，罗汉果总苷具有治疗肺纤维化疾病的新用途。

Claims (10)

  1. 罗汉果提取物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用。
  2. 根据权利要求1所述的罗汉果提取物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用，其特征在于，所述罗汉果提取物中罗汉果总苷的质量百分比≥50％。
  3. 根据权利要求2所述的罗汉果提取物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用，其特征在于，所述罗汉果提取物中罗汉果总苷的质量百分比≥80％。
  4. 罗汉果提取物的酶解物或水解物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用。
  5. 根据权利要求4所述的罗汉果提取物的酶解物或水解物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用，其特征在于：所述罗汉果提取物的酶解物或水解物中至少含有罗汉果苷IIIe、罗汉果苷IV或罗汉果醇中的一种或多种。
  6. 根据权利要求5所述的罗汉果提取物的酶解物或水解物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用，其特征在于，所述的罗汉果提取物的酶解物中至少含有罗汉果苷IIIe：
    Figure PCTCN2016086831-appb-100001
  7. 根据权利要求5所述的罗汉果提取物的酶解物或水解物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用，其特征在于，所述的罗汉果提取物的水解物中至少含有罗汉果醇：
    Figure PCTCN2016086831-appb-100002
  8. 根据权利要求5所述的罗汉果提取物的酶解物或水解物在制备预防和/或治疗肺纤维化疾病的药物和/或保健品中的应用，其特征在于，所述的罗汉果提取物的酶解物中至少含有罗汉果苷IVe：
    Figure PCTCN2016086831-appb-100003
  9. 根据权利要求1～3任一项所述的罗汉果提取物的应用，或权利要求4～8任一项所述的罗汉果提取物的酶解物或水解物的应用，其特征在于，将罗汉果提取物或者其酶解物或水解物与其他人体可接受的药用辅料制成片剂、颗粒剂、汤剂或胶囊。
  10. 根据权利要求权利要求1～3任一项所述的罗汉果提取物的应用，或权利要求4～8任一项所述的罗汉果提取物的酶解物或水解物的应用，其特征在于，所述药物或保健品为降低肺纤维化组织间质中胶原堆积量的药物或保健品；或
    所述药物或保健品为能减轻炎症程度，抑制胶原形成、保护肺组织的抗肺纤维化的药物或保健品或
    所述药物或保健品为通过抗炎及抑制肺泡上皮细胞间质化而发挥抗肺纤维化的药物或保健品。
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