WO2017152796A1 - 黄柏酮在制备防治肺损伤和肺纤维化的药物中的应用 - Google Patents

黄柏酮在制备防治肺损伤和肺纤维化的药物中的应用 Download PDF

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WO2017152796A1
WO2017152796A1 PCT/CN2017/075179 CN2017075179W WO2017152796A1 WO 2017152796 A1 WO2017152796 A1 WO 2017152796A1 CN 2017075179 W CN2017075179 W CN 2017075179W WO 2017152796 A1 WO2017152796 A1 WO 2017152796A1
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pulmonary fibrosis
drug
use according
injection
obacunone
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PCT/CN2017/075179
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French (fr)
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徐洋
徐胜梅
陈为民
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广东省中医院
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Priority to US16/068,014 priority Critical patent/US10632100B2/en
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • 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
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
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    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4866—Organic macromolecular compounds
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the present invention relates to pharmaceutical preparations containing organic active ingredients, and in particular to anti-pulmonary damage and anti-pulmonary fibrosis activity of a compound containing a lactone ring.
  • Lung injury is a common disease, and acute lung injury seriously affects human health.
  • Acute lung injury is an injury of alveolar epithelial cells and capillary endothelial cells caused by various direct and indirect injury factors, resulting in diffuse pulmonary interstitial and alveolar edema, leading to acute hypoxic respiratory insufficiency. It is an early stage of acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • Pulmonary fibrosis (PF), also known as interstitial lung disease is a type of interstitial disease that has a very poor prognosis and seriously affects the quality of life of patients. It is also one of the most serious diseases of the respiratory system.
  • pulmonary fibrosis is the result of lung injury, characterized by fibroblast migration, lactation, and proliferation in the alveolar space.
  • the early mortality rate of ALI has been significantly improved, but the late pulmonary fibrosis process directly and indirectly leads to the death of patients.
  • the lung biopsy ALI death found that 55% had severe pulmonary fibrosis.
  • White fresh skin is a traditional Chinese medicine. It is a dry root bark of the white genus of the genus Rutaceae. It has a strong special odor. White fresh skin tastes bitter, cold, spleen, stomach, bladder, with hurricane dehumidification, heat detoxification, itching, insecticidal and other effects.
  • Obacunone is a limonoid compound isolated and extracted from white fresh skin, and has a molecular formula of C 26 H 30 O 7 , and its chemical structure is represented by the following formula (I).
  • the technical problem to be solved by the present invention is to provide a new new use of corkone, that is, a new application in pharmaceuticals.
  • cedarone in the preparation of a medicament for the prevention and treatment of lung injury and pulmonary fibrosis.
  • the drug consists of corkone and a medically acceptable adjuvant, wherein the percentage of the content of the ketone in the drug is 3-25%.
  • the drug may be an injection or a common oral preparation such as a tablet or a capsule.
  • the corkone of the present invention can be extracted from the traditional Chinese medicine white fresh skin or other plants by a conventional method, or can be obtained by synthetic or other methods.
  • the compound phellodone which embodies the use can inhibit Bleomycin-induced lung injury and pulmonary fibrosis in mice, and regulate the expression of inflammatory-related lymphokines in Bleomycin-induced lung injury tissues of mice, not only prevention and treatment
  • the effects of lung injury and pulmonary fibrosis are remarkable, and it also has the advantages of low toxicity and no damage to liver and kidney.
  • Figure 2 is a bar graph showing the results of inflammatory-associated lymphokine expression in rabbit lung injury tissues induced by Bleomycin in Bleomycin.
  • Figure 3 is a photomicrograph of the results of inhibition of Bleomycin-induced pulmonary fibrosis ( ⁇ -SMA expression) by cedarone.
  • Figure 4 is a photomicrograph of the results of no damage of liver and kidney in mice.
  • Huangbai Ketone was purchased from Tianjin Shilan Technology Co., Ltd. Corn oil (Guangdong Changxing Food Trading Co., Ltd.); DMSO (Sigma); phosphate buffer (PBS, Hyclone Development Company); HE dye solution (Google Bio); Masson trichrome staining kit (Fuzhou Maixin); Water ethanol (Damao); xylene (Damao).
  • Inverted phase contrast microscope (CKX41 Olympus); automatic cell counter (Countstar IC-100); biological safety cabinet (BSC-1000IIAC); fully automatic dyeing and sealing integrated workstation (LEICA ST5020), tissue embedding machine (HisTOSTAR ), fully automatic closed tissue dewatering machine (SHANDON PATACENTRE), paraffin slicer (LEICA RM2245).
  • Bleomycin was intraperitoneally injected with B6 mice at 8 weeks of age at a dose of 2 mg/dose, three times a week, once a week. B6 was pre-injected intraperitoneally with corn oil or 10 mg/kg of cedarone 48 hours prior to each injection of Bleomycin. Seven days after the last injection of Bleomycin, lung tissue was isolated, fixed, sectioned, HE stained (process detailed in Experiment 8) and Masson staining.
  • HE staining showed that compared with the lung tissue sections of normal B6 mice, a large number of alveolar atrophy was observed in the lung tissue of Bleomycin group, inflammatory cells infiltrated in the alveolar space, and some alveolar walls were thickened and broken, forming large alveolar cells and a large number of pulmonary interstitial cells. Hyperplasia; and the above-mentioned lesions in the lung tissue of the pre-injected cedarone group showed a significant improvement (Fig. 1A).
  • Huangbai Ketone was purchased from Tianjin Shilan Technology Co., Ltd. Corn oil (Guangdong Changxing Food Trading Co., Ltd.); DMSO (Sigma); TRIzol (Life); Primer (Hua Da Gene Company).
  • Desktop low speed automatic balance centrifuge DT5-3
  • CFX96 Touch TM quantitative PCR detection system Bio-Rad Corporation.
  • Bleomycin significantly induced changes in the expression of related cytokines such as IL-6, TGF- ⁇ , IL-17, IFN- ⁇ and MCP-1 in lung tissue.
  • IL-6, TGF- ⁇ , IL-17, and MCP-1 were significantly increased, while IFN- ⁇ was significantly decreased.
  • IFN- ⁇ was significantly decreased.
  • TGF- ⁇ is also associated with the induction of tissue fibrosis. While cedarone has largely corrected these changes induced by Bleomycin (Figure 2).
  • TGF- ⁇ induces the transformation of interstitial fibroblasts into myofibroblasts, and the marker of myofibroblasts expresses ⁇ -smooth muscle actin ( ⁇ -SMA). Therefore, this study provides a basis for the application of corkone in the prevention and treatment of pulmonary fibrosis by studying the effect of ketone on the expression of ⁇ -SMA in pulmonary fibroblasts.
  • Phellodone was purchased from Tianjin Silan Technology Co., Ltd. Corn oil (Guangdong Changxing Food Trading Co., Ltd.); DMSO (Sigma); phosphate buffer (PBS, Hyclone Development Company); HE dye solution (Valley Song Bio); absolute ethanol (Dalma); xylene (Dalma).
  • Inverted phase contrast microscope (CKX41 Olympus); automatic cell counter (Countstar IC-100); biological safety cabinet (BSC-1000IIAC); fully automatic dyeing and sealing integrated workstation (LEICA ST5020), tissue embedding machine (HisTOSTAR ), fully automatic closed tissue dewatering machine (SHANDON PATACENTRE), paraffin slicer (LEICA RM2245).
  • liver slices showed complete morphological structure of hepatic sinus and hepatic cord, no nuclear edema in the liver, no cytoplasmic lesions; glomerular structure was intact in the kidney sections, and the number was consistent with the control group, and no lesions were observed in the cytoplasm and nucleus.
  • results show that: phellodone has a significant role in inhibiting lung inflammation and fibrosis, and has no obvious side effects, thus showing good resistance to lung injury and pulmonary fibrosis.
  • the oil phase is slowly added to the aqueous phase, and stirred at high speed for 8 minutes with a high-speed disperser (rotation speed 2000 rpm /
  • the colostrum was prepared by homogenizing the colostrum with a high-pressure homogenizer (pressure 65 MPa, temperature 75 ° C) 7 times, diluted with water for injection to 1000 ml, filtered with a 0.45 ⁇ m microporous membrane, and then filled with nitrogen. Sealed, autoclaved (121 ° C, 15 min), that is.
  • Each 1ml, intramuscular injection, once a day, 2 times each time, 10 to 20 days for a course of treatment can be used continuously for 2 to 3 courses.

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Abstract

黄柏酮在制备防治肺损伤和肺纤维化的药物中的应用，该药物由黄柏酮和药学上可接受的辅料和添加剂组成，其中黄柏酮在药物中的质量百分含量为3-25％。该药物中的黄柏酮可抑制Bleomycin诱导的小鼠肺损伤及肺纤维化，调节Bleomycin诱导的小鼠肺损伤组织的炎性相关的淋巴因子表达。

Description

黄柏酮在制备防治肺损伤和肺纤维化的药物中的应用 技术领域
本发明涉及含有机有效成分的医药配制品，具体涉及含有内酯环的化合物的抗肺损伤和抗肺纤维化的活性。
背景技术
肺损伤为常见的疾患，急性肺损伤严重影响着人类的健康。急性肺损伤(acute lung injury,ALI)是各种直接和间接致伤因素导致的肺泡上皮细胞及毛细血管内皮细胞损伤，造成弥漫性肺间质及肺泡水肿，导致急性低氧性呼吸功能不全，是急性呼吸窘迫综合症(acute respiratory distress syndrome,ARDS)的早期阶段。肺纤维化(pulmonary fibrosis,PF)又称间质性肺疾病，是一类预后极差、严重影响患者生存质量的肺间质疾病，也是呼吸系统最严重的疾病之一。目前研究表明肺纤维化是肺损伤的结果，其特点是成纤维细胞在肺泡腔内迁移、乳附、增殖。近几年ALI的早期病死率得到明显的改善，但后期肺纤维化过程直接和间接导致患者死亡，肺活检ALI死亡者发现55％有严重肺纤维化。
白鲜皮为传统中药，是芸香科植物白藓属白鲜的干燥根皮，具有浓烈的特殊气味。白鲜皮味苦、寒，归脾、胃、膀胱经，具有祛风除湿、清热解毒、止痒、杀虫等功效。黄柏酮(Obacunone)是从白鲜皮中提取分离得到的一种柠檬苦素类化合物，其分子式为C26H30O7，化学结构如下式(Ⅰ)所示。
Figure PCTCN2017075179-appb-000001
目前的研究表明，黄柏酮具有抗肿瘤的作用，在对人结肠癌细胞(SW480)研究中表明，黄柏酮通过激活细胞色素C以诱导细胞凋亡，并通过激活p21使细胞周期阻滞在G2/M和G1阶段以抑制肿瘤细胞生长；在对人胰腺癌细胞(Panc-28)研究中表明，黄柏酮通过激活p53以诱导肿瘤细胞的凋亡。另外，黄柏酮还具有通过增强内皮型一氧化氮合酶二聚体的稳定性以抑制精氨酸酶活性并增加NO的产生的作用，因此，对于由于内皮功能紊乱而导致的心血 管疾病黄柏酮可以产生一定的治疗效果。但黄柏酮在防治肺损伤和肺纤维化方面的功能尚未见报道。
发明内容
本发明所要解决的技术问题是提供黄柏酮新的新用途，即在制药中的新应用。
上述在制药中的新应用为，黄柏酮在制备防治肺损伤和肺纤维化的药物中的应用。
上述应用中，所述的药物由黄柏酮和医学上可接受的辅料组成，其中，黄柏酮在药物中的质量百分含量为3-25％。所述药物可以是注射剂，也可以是常见的口服制剂，如片剂或胶囊剂。
本发明所述的黄柏酮可以采用常规的方法从中药白鲜皮或其它植物中提取得到，也可以由合成或其他方法制得。
本发明所述的应用，其中体现所述用途的化合物黄柏酮可抑制Bleomycin诱导的小鼠肺损伤及肺纤维化，调节Bleomycin诱导的小鼠肺损伤组织的炎性相关的淋巴因子表达，不仅防治肺损伤和肺纤维化的效果显著，而且还具有毒性低，对肝、肾无损伤优点。
附图说明
图1是黄柏酮抑制Bleomycin诱导的小鼠肺损伤病理结果的显微照片。
图2是黄柏酮调节Bleomycin诱导的小鼠肺损伤组织的炎性相关的淋巴因子表达结果的条形图。
图3是黄柏酮抑制Bleomycin诱导的肺纤维化(α-SMA表达)结果的显微照片。
图4是黄柏酮对小鼠肝、肾无损伤结果的显微照片。
具体实施方式
一、药效实验例
实验一：黄柏酮抑制Bleomycin诱导的肺损伤和纤维化的病理学改变
1.实验材料
1.1实验动物 小鼠动物8周龄的C57BL/6小鼠18只，体重18～22g，由广州中医药大学实验动物中心提供，合格证号44007200023897。
1.2药材、试剂和仪器 黄柏酮，购于天津士兰科技有限公司。玉米油(广东长兴食品贸易有限公司)；DMSO(Sigma公司)；磷酸盐缓冲液(PBS，Hyclone开发公司)；HE染液(谷歌生物)；Masson三色染色试剂盒(福州迈新)；无水乙醇(大茂)；二甲苯(大茂)。倒置相差显微镜(CKX41奥林巴斯)；自动细胞计数仪(Countstar IC-100)；生物安全柜(BSC-1000IIAC)；全自动染色封片一体化工作站(LEICA ST5020),组织包埋机(HisTOSTAR)，全自动封闭式组织脱水机(SHANDON PATACENTRE)，石蜡切片机 (LEICA RM2245)。
2.实验方法与结果
Bleomycin腹腔注射8周龄B6小鼠，剂量2mg/只，共注射三次，每周一次。每次注射Bleomycin前48小时，B6预先腹腔注射玉米油或10mg/kg黄柏酮。最后一次注射Bleomycin后7天，分离肺部组织，固定，制成切片，HE染色(过程在实验八中详述)和Masson染色。
HE染色显示，与正常B6小鼠肺组织切片比较，Bleomycin组肺组织切片可见大量肺泡萎缩，肺泡腔可见炎症细胞浸润，部分肺泡壁出现增厚，断裂，形成肺大泡，肺间质细胞大量增生；而预注射黄柏酮组鼠肺组织上述病变呈显著改善(图1A)。
Masson染色显示Bleomycin组视野内大部分区域被苯胺蓝染色，说明胶原、纤维连接蛋白等基质大量沉积。而经腹腔预注射黄柏酮组，上述病变呈现显著改善(图1B)。
实验二：黄柏酮抑制了Bleomycin诱导的炎性因子的表达
1.实验材料
1.1实验动物 小鼠动物8周龄的C57BL/6小鼠18只，体重18～22g，由广州中医药大学实验动物中心提供，合格证号44007200023897。
1.2药材、试剂和仪器 黄柏酮，购于天津士兰科技有限公司。玉米油(广东长兴食品贸易有限公司)；DMSO(Sigma公司)；TRIzol(Life公司)；引物(华大基因公司)。低速台式自动平衡离心机(DT5-3)；CFX96 TouchTM荧光定量PCR检测系统(Bio-Rad公司)。
2.实验方法与结果
定量PCR分析淋巴因子IL-6，TGF-β，IL-17，IFN-γ和MCP-1的mRNA水平。*P<0.05，**P<0.01表示BLM注射组与正常组有显著差异；#P<0.05，##P<0.01表示BLM注射组与BLM+黄柏酮注射组存在显著差异。
Bleomycin显著诱导了肺组织中相关细胞因子如IL-6，TGF-β，IL-17，IFN-γ和MCP-1的表达变化。其中IL-6，TGF-β，IL-17，MCP-1显著增加，而IFN-γ显著减少。这些因子是与炎性相关的，其中TGF-β还与诱导组织纤维化相关。而黄柏酮很大程度中修复了Bleomycin诱导的这些变化(图2)。
实验三：黄柏酮抑制了Bleomycin诱导的肺纤维化α-SMA的表达
1.实验材料
1.1实验动物 小鼠动物8周龄的C57BL/6小鼠18只，体重18～22g，由广州中医药大学实验动物中心提供，合格证号44007200023897。
1.2药材、试剂和仪器 黄柏酮，购于天津士兰科技有限公司。玉米油(广东长兴食品贸易有 限公司)；DMSO(Sigma公司)；无水乙醇(大茂)；二甲苯(大茂)；DAB显色试剂盒(福州迈新)；免疫组化染色试剂盒(福州迈新)；倒置相差显微镜(CKX41奥林巴斯)；生物安全柜(BSC-1000IIAC)；全自动染色封片一体化工作站(LEICA ST5020),组织包埋机(HisTOSTAR)，全自动封闭式组织脱水机(SHANDON PATACENTRE)，石蜡切片机(LEICA RM2245)。
2.实验方法与结果
肺纤维化时TGF-β诱导肺间质成纤维细胞转化为肌成纤维细胞，肌成纤维细胞的标志是表达α-平滑肌肌动蛋白(α-SMA)。因而本实验通过研究黄柏酮对肺肌成纤维细胞α-SMA表达的影响，为黄柏酮在肺纤维化病的防治中的应用提供资料依据。
免疫组织化学染色步骤：
1)石蜡切片放入60℃电热恒温培养箱中烤片30min；
2)二甲苯脱蜡(Ⅰ、Ⅱ各8min)，下行梯度酒精水化(各级8min)，水洗；
3)抗原修复，PBS洗片，3次×3min；
4)滴加A液(S-P试剂盒)，室温阻断10min，PBS洗片，3次×3min；
5)滴加B液(S-P试剂盒)封闭，室温10min，吸干多余液体；
6)滴加一抗，置湿盒4℃过夜，PBS洗片，3次×3min；
7)滴加C液(S-P试剂盒)，室温10min，PBS洗片，3次×3min；
8)滴加D液(S-P试剂盒)，室温10min，PBS洗片，3次×3min；
9)DAB显色，水洗；
10)复染，滴加苏木素，室温1min，水洗；0.5％盐酸酒精分化，PBS返蓝，水洗；
11)梯度酒精脱水，二甲苯透明，封片。
结果表明，Bleomycin小鼠肺组织中α-SMA的表达量显著高于正常组，主要出现于肺泡上皮细胞、细支气管上皮细胞、巨噬细胞及血管内皮细胞。而给予黄柏酮后，α-SMA的表达与Bleomycin组相比较明显减弱(图3)，这表明黄柏酮明显抑制了Bleomycin诱导的肺纤维化进程。
实验四：黄柏酮对小鼠肝肾损伤实验
1.实验材料
1.1实验动物 小鼠动物8周龄的C57BL/6小鼠18只，体重18～22g，由广州中医药大学实验动物中心提供，合格证号44007200023897。
1.2药材、试剂和仪器黄柏酮，购于天津士兰科技有限公司。玉米油(广东长兴食品贸易有限公司)；DMSO(Sigma公司)；磷酸盐缓冲液(PBS，Hyclone开发公司)；HE染液(谷 歌生物)；无水乙醇(大茂)；二甲苯(大茂)。倒置相差显微镜(CKX41奥林巴斯)；自动细胞计数仪(Countstar IC-100)；生物安全柜(BSC-1000IIAC)；全自动染色封片一体化工作站(LEICA ST5020),组织包埋机(HisTOSTAR)，全自动封闭式组织脱水机(SHANDON PATACENTRE)，石蜡切片机(LEICA RM2245)。
2.实验方法与结果
8周龄B6小鼠玉米油或10mg/kg黄柏酮，共注射六次，每周两次。最后一次注射后7天，分离肝、肾组织，固定，制成切片，HE染色(图4)，过程如下所述。
苏木素-伊红(Hematoxylin-eosin，HE)染色：用石蜡切片机进行石蜡样本的切片，厚度3.5μm，连续切片，染色程序为烤片干燥20分钟，二甲苯2次×10分钟，无水乙醇2次×2分钟，95％乙醇1分钟，80％乙醇1分钟，70％乙醇1分钟，水洗1分钟，苏木素8分钟，苏木素10分钟，水洗2次×1分钟，0.5％盐酸酒精10秒，水洗10分钟，伊红2分钟，水洗1分钟，80％乙醇5秒，85％乙醇5秒，90％乙醇5秒，95％乙醇1分钟，无水乙醇2次×2分钟，无水乙醇3分钟，二甲苯2次×2分钟，结束染色后直接用中性树胶封片。
结果如图4所示，黄柏酮对小鼠肝肾无明显影响。其中，肝脏切片可见肝窦、肝索形态结构完整，肝脏未见细胞核水肿，细胞质无病变；肾脏切片可见肾小球结构完整，且数量与对照组一致，细胞质、细胞核均未见病变形态。
综上研究结果表明：黄柏酮在抑制肺部炎症和纤维化过程中有显著的作用，并且无明显毒副作用，从而表现出良好的抵抗肺损伤和肺纤维化的作用。
二、药剂制备例
例1(注射剂)
1、处方：
Figure PCTCN2017075179-appb-000002
2、制备方法
将黄柏酮浸润在大豆油和油酸中作为油相，不断搅拌加热至70℃，将聚乙二醇15-羟基 硬脂酸酯、大豆磷脂、甘油、植酸六钠分散于水中作为水相，不断搅拌加热至70℃，将油相缓慢加入水相，用高速分散器高速搅拌后8分钟(转速2000转/分)制得初乳，将初乳用高压均质机均化(压力65MPa，温度75℃)7次后，注射用水稀释至1000ml，用0.45μm微孔滤膜初过滤后充氮分装熔封，热压灭菌(121℃，15min)，即得。每支1ml，肌肉注射，每天1次，每次2支，10～20天为一疗程，可连续使用2～3疗程。
例2(片剂)
1、处方：
Figure PCTCN2017075179-appb-000003
2、制备方法：
将黄柏酮与羟丙基纤维素SSL溶解在乙醇800mL中，将上述溶液加入到液体石蜡中，减压干燥除去乙醇，黄柏酮与羟丙基纤维素形成微粒析出，将析出的微粒50℃干燥；干燥后的微粒与过100目筛的交联羧甲基纤维素钠和硬脂酸镁混合均匀，压制成每片含黄柏酮30mg的片剂10000片，每片净重0.13g。口服每次2片，一日1次，10～20天为一疗程，可连续使用2～3疗程。
例3(胶囊剂)
1、处方：
Figure PCTCN2017075179-appb-000004
2、制备方法：
称取处方量的十二烷基硫酸钠和聚维酮K30，加水溶解，配制成10％聚维酮K30的溶液，制成粘合剂；称取处方量黄柏酮原料药置混合机中，加入粘合剂制软材，再用制粒机制粒； 湿颗粒在40℃～45℃下干燥1～3小时后，整粒；将上述颗粒置混合机中，加入过60目筛的低取代羟丙基纤维素、交联聚维酮和硬脂酸镁，混合30分钟，取样测定含量和干燥失重；按颗粒含量测定结果计算出平均装量，用4#胶囊充填，制成胶囊1000粒，每粒填充0.1g颗粒，含黄柏酮20mg，口服每次3粒，一日1次，10～20天为一疗程，可连续使用。

Claims (6)

  1. 黄柏酮在制备防治肺损伤和肺纤维化的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述的药物由黄柏酮和医学上可接受的辅料和添加剂组成，其中，黄柏酮在药物中的质量百分含量为3-25％。
  3. 根据权利要求1或2所述的应用，其特征在于，所述的药物是注射剂、片剂或胶囊剂。
  4. 根据权利要求3所述的应用，其特征在于，所述的注射剂每1000mL由以下原料组成：
    黄柏酮30g，注射级玉米油270g，聚乙二醇15-羟基硬脂酸酯18g，注射级大豆磷脂2g，注射级甘油2g，油酸5g，植酸六钠0.5g，其余为水。
  5. 根据权利要求3所述的应用，其特征在于，所述的片剂每10000片由以下原料组成：
    黄柏酮300g，羟丙基纤维素SSL 80g，液体石蜡1000mL，交联羧甲基纤维素钠20g，硬脂酸镁2g。
  6. 根据权利要求3所述的应用，其特征在于，所述的胶囊剂每1000粒的内容物由以下原料组成：
    黄柏酮20g，低取代羟丙基纤维素50g，交联聚维酮5g，十二烷基硫酸钠2g，聚维酮K305g，硬脂酸镁3g。
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