WO2021179902A1 - 岩黄连及其制剂在制备治疗非酒精性脂肪性肝病的药物中的应用 - Google Patents

岩黄连及其制剂在制备治疗非酒精性脂肪性肝病的药物中的应用 Download PDF

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WO2021179902A1
WO2021179902A1 PCT/CN2021/077410 CN2021077410W WO2021179902A1 WO 2021179902 A1 WO2021179902 A1 WO 2021179902A1 CN 2021077410 W CN2021077410 W CN 2021077410W WO 2021179902 A1 WO2021179902 A1 WO 2021179902A1
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preparation
treating non
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total alkali
alcoholic fatty
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成俊
刁和芳
黄芳
郑艳萍
赵开军
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南京中山制药有限公司
江苏弘典中药产业研究院有限公司
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Priority to US17/797,305 priority Critical patent/US20230057861A1/en
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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)
    • A61K36/66—Papaveraceae (Poppy family), e.g. bloodroot
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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)
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    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

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  • the present invention relates to a new use of siberian huanglian and its total alkali capsules of siberian huanglian, in particular to the application of siberian huanglian and its total alkali capsules in the treatment of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, belonging to Medical technology field.
  • Non-alcoholic fatty liver disease is defined as liver steatosis confirmed by imaging and liver histology, and other causes of liver steatosis are excluded, such as heavy drinking, long-term use of fat-forming drugs or Single-gene genetic disorders, etc., based on their clinical manifestations, mainly include liver steatosis, non-alcoholic steato-hepatitis (NASH) and liver cirrhosis.
  • NASH non-alcoholic steato-hepatitis
  • liver cirrhosis The pathogenesis of non-alcoholic fatty liver disease is very complicated, and there is currently no recognized effective drug treatment. Therefore, it is still necessary to continue efforts to develop effective drugs based on the research on the pathogenesis.
  • Drug-induced liver disease also known as drug-induced liver injury (DILI)
  • DILI drug-induced liver injury
  • the main component of Scutellaria sibiricum is the total alkali of Scutellaria sibiricus.
  • the medicinal material of Corydalis saxicola has the effects of clearing away heat, detoxifying, removing dampness, relieving pain and stopping bleeding. It is commonly used for hepatitis, tongue erosion, fire eyes, eye cloud, dysentery, diarrhea, abdominal pain, hemorrhoids and bleeding.
  • Yanhuanglian Capsule for Nanjing Zhongshan Pharmaceutical Co., Ltd. has completed clinical phase 2 and is currently undergoing clinical phase 3.
  • the objective of the present invention is to overcome the shortcomings of the prior art, and through a large number of experimental screenings, on the basis of the existing effects of Yanhuanglian and Yanhuanglian total alkali capsules, develop its new clinical efficacy and expand its clinical indications .
  • Siberian rhizome in the preparation of medicines for treating non-alcoholic steatohepatitis.
  • Siberian rhizome in the preparation of a medicine for treating non-alcoholic fatty liver disease combined with drug-induced liver injury disease.
  • Siberian rhizome in the preparation of a medicine for treating non-alcoholic fatty liver disease combined with chemical liver injury disease.
  • the preparation method of the total alkali of Rhizoma Scutellariae in the present invention is:
  • the present invention has been screened through a large number of experiments, and developed its new clinical efficacy on the basis of the existing effects of S. huanglian and the total alkali capsules of S. huanglian.
  • Reduce the synthesis of fatty acids in cells reduce the levels of TG, TC, LDL cholesterol, HDL cholesterol and free fatty acids induced by HFD, and anatomical experiments show that the total alkali of Scutellaria sibiricum can reduce the weight of the liver.
  • the pathological section shows that the liver tissue is carried out very well. protect.
  • it was found that the total alkalis of Rhizoma Scutellariae can significantly improve the biochemical indicators and imaging evaluation results of fatty liver patients.
  • the present invention shows through the experimental results of drug-induced liver injury that the Yanhuanglian total alkali capsules can significantly improve drug-induced liver injury and have the effect of preventing liver injury.
  • the present invention shows through the experimental results of drug-induced liver injury that the Yanhuanglian total alkali capsule can significantly improve the chemical and drug-induced liver injury, and has the effect of preventing liver injury.
  • the experiment of the present invention can expand the clinical indications of the total alkali of Rhizoma Scutellariae, and provide an effective basis for its clinical promotion.
  • Figure 1 is a bar graph of the cytotoxicity test.
  • Figure 2 is a histogram of the liver weight index of each group of mice.
  • Figure 3 is a test chart of changes in blood glucose of mice in each group.
  • Figure 4 is a test chart of the lipid distribution of each group of mice.
  • Figure 5 shows the result of HE staining.
  • Figure 6 shows the result of oil red staining.
  • C represents the model group
  • B represents the blank group
  • L represents the total alkalis of Scutellaria scoparia (25mg/kg)
  • H represents the total alkalis of Scutellaria scoparia (100mg/kg)
  • MET indicates metformin (200mg/kg).
  • 75g of total alkaloids of Scutellaria sibiricum can be taken, added with 125g of soluble starch, mixed uniformly, granulated by adding 85% ethanol in an appropriate amount, dried, sized, and filled into capsules to obtain S. chinensis capsules.
  • Test drug The total alkali of Scutellariae (CSBTA) prepared in Example 1.
  • MTT cell proliferation and cytotoxicity detection kit (article number: KGA321, Jiangsu KGI Biotechnology Co., Ltd.); the total alkalis of Scutellariae prepared in Example 1.
  • Dilute 5 ⁇ MTT with Dilution Buffer to 1 ⁇ MTT add 50 ⁇ L 1 ⁇ MTT to each well, incubate at 37°C for 4 hours to reduce MTT to formazan, aspirate the supernatant, add 150 ⁇ L DMSO to each well to dissolve formazan, use Shake well on a plate shaker, and detect the optical density of each well with a microplate reader at a wavelength of 490nm.
  • mice After the administration in the above paragraph 1.4, an OGTT experiment was performed on mice to understand the function of pancreatic islet ⁇ -cells and the body's ability to regulate blood sugar. Fasting for 12 hours after 8 o'clock in the evening, the fasting blood glucose concentration FBG was measured the next day, and glucose solution was given to each group of mice by gavage (2g/kg). At 15min, 30min, 60min, and 120min after glucose gavage, the blood glucose concentration of mice was measured respectively. Calculate AUC (area under the curve).
  • TC determination kit (A111-1-1, Nanjing Jiancheng Biotechnology Co., Ltd.), TG determination kit (A110-1-1, Nanjing Jiancheng Biotechnology Co., Ltd.), HDL-C determination kit (A112- 1-1, Nanjing Jiancheng Biotechnology Co., Ltd.), LDL-C Assay Kit (A113-1-1, Nanjing Jiancheng Biotechnology Co., Ltd.), NEFA: Assay Kit (A042-2-1, Nanjing Jiancheng Biotechnology Co., Ltd.) company).
  • mice were fasted overnight, blood samples were collected from the eye veins, and centrifuged at 3000 rpm at 4°C for 10 minutes. Collect plasma and store it at -80°C until use.
  • the kit is determined in accordance with the manufacturer's instructions.

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Abstract

岩黄连及岩黄连总碱胶囊在制备治疗非酒精性脂肪性肝病、非酒精性脂肪性肝炎、药物性肝损伤、化学性肝损伤的药物中的应用。

Description

岩黄连及其制剂在制备治疗非酒精性脂肪性肝病的药物中的应用 技术领域
本发明涉及一种岩黄连及其岩黄连总碱胶囊的新用途，特别涉及岩黄连及其岩黄连总碱胶囊在治疗非酒精性脂肪性肝病和非酒精性脂肪性肝炎疾病中的应用，属于医药技术领域。
背景技术
非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)定义为影像学和肝组织学证实肝脂肪变，并除外导致肝脂肪变的其它原因，如大量饮酒、长期应用促脂肪形成药物或单基因遗传紊乱等，根据其临床表现主要包括肝脏脂肪变性、非酒精性脂肪性肝炎(non-alcoholic steato-hepatitis，NASH)和肝硬化。非酒精性脂肪性肝病的发病机制非常复杂，目前也没有公认的有效药物治疗，因此目前在研究发病机制基础之上开发有效的药物仍需要继续努力。
另外随着我国人民生活水平的提高和中药、保健品使用的日益增多，药物中毒性肝损伤的发病率有上升趋势。药物性肝病又称药物性肝损伤(drug induced liver injury,DILI)，是我国非感染性肝病的第二大病种，药物引起的肝毒性现已成为急性肝功能衰竭的主要原因，过去数十年间因为肝毒性从市场撤除的药物造成的损失达数十亿美金。
岩黄连主要成分是岩黄连总碱。岩黄连药材具有清热解毒，利湿，止痛止血之功效。常用于肝炎，口舌糜烂，火眼，目翳，痢疾，腹泻，腹痛，痔疮出血。岩黄连胶囊为南京中山制药有限公司的已经做完临床2期，目前正在做临床3期。
发明内容
发明目的：本发明的目的是为了克服现有技术的不足，通过大量实验筛选，在岩黄连及其岩黄连总碱胶囊现有功效的基础上，开发其新的临床功效，扩大其临床适应症。
技术方案：为了实现以上目的，本发明采取的技术方案为：
岩黄连在制备治疗非酒精性脂肪性肝病的药物中的应用。
岩黄连在制备治疗非酒精性脂肪性肝炎的药物中的应用。
岩黄连在制备治疗非酒精性脂肪性肝病合并药物性肝损伤疾病的药物中的应用。
岩黄连在制备治疗非酒精性脂肪性肝病合并化学性肝损伤疾病的药物中的应用。
岩黄连总碱胶囊在制备治疗非酒精性脂肪性肝病的药物中的应用。
岩黄连总碱胶囊在制备治疗非酒精性脂肪性肝炎的药物中的应用。
岩黄连总碱胶囊在制备治疗非酒精性脂肪性肝病合并药物性肝损伤疾病的药物中的应用。
岩黄连总碱胶囊在制备治疗非酒精性脂肪性肝病合并化学性肝损伤疾病的药物中的应用。
本发明所述的岩黄连总碱制备方法为：
(1)取岩黄连饮片30kg，加10倍量体积浓度75％乙醇浸泡1h；
(2)然后加热回流提取三次，每次2小时，合并提取液；
(3)回收尽乙醇并浓缩至1：1.3(岩黄连饮片W：浓缩液V)得到浓缩液；
(4)取上述浓缩液加4倍于原料量(岩黄连饮片)的1％盐酸，置耐酸多功能提取罐加热回流提取1小时；
(5)提取液用40％氢氧化钠调节并保持pH值至8，然后浓缩至相对密度为1.06～1.08(60℃)，浓缩液冷藏，静置48小时，滤过；滤过所得沉淀用16倍于沉淀量(W/V)的1％盐酸加热回流提取1小时，趁热滤过；
(6)滤液用40％氢氧化钠调节并保持pH值至6-8，浓缩至相对密度为1.06～1.08(60℃)，冷藏，静置48小时，滤过；
(7)滤过沉淀物进行减压干燥(60℃，0.08MPa)，即得岩黄连总碱提取物。
本发明的技术效果：
本发明通过大量实验筛选，在岩黄连及其制备得到的岩黄连总碱胶囊现有功效的基础上，开发其新的临床功效，通过实验发现，岩黄连和岩黄连总碱胶囊具有很好的降低脂肪酸细胞内的合成、克降低HFD诱导的TG，TC，LDL胆固醇，HDL胆固醇以及游离脂肪酸水平，并且解剖实验表明岩黄连总碱可降低肝脏的重量，病理切片显示对肝脏组织进行了很好保护。并且通过临床的实验发现，岩黄连总碱能明显改善脂肪肝患者的生化指标和影像学评价结果。另外本发明通过 药物性肝损伤实验结果表明，岩黄连总碱胶囊可显著改善药物性肝损伤，具有防止肝损伤的作用。
另外本发明通过药物性肝损伤实验结果表明，岩黄连总碱胶囊可显著改善化学性和药物性肝损伤，具有防止肝损伤的作用。通过本发明的实验可以扩大岩黄连总碱的临床适应症，为其临床上的推广提供有效基础。
附图说明
图1为细胞毒性作用测试的柱状图。
图2为各组小鼠肝重指数的柱状图。
图3为各组小鼠血糖变化的测试图。
图4为各组小鼠脂质分布的测试图。
图5为HE染色的结果图。
图6为油红染色的结果图。
以上各图中，C表示模型组，B表示空白组，L表示岩黄连总碱(25mg/kg),H表示岩黄连总碱(100mg/kg)，MET表示二甲双胍(200mg/kg)。*表示与模型组相比具有统计学意义P＜0.05。****表示与模型组相比具有统计学意义P＜0.01。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。在阅读了本发明之后，本领域技术人员对本发明的各种非实质性的修改均落于本申请所附权利要求所限定的范围。
实施例1岩黄连总碱制备
(1)取岩黄连饮片30kg，加10倍量体积浓度75％乙醇浸泡1h；
(2)然后加热回流提取三次，每次2小时，合并提取液；
(3)回收尽乙醇并浓缩至1：1.3(岩黄连饮片W：浓缩液V)得到浓缩液；
(4)取上述浓缩液加4倍于原料量(岩黄连饮片)的1％盐酸，置耐酸多功能提取罐加热回流提取1小时；
(5)提取液用40％氢氧化钠调节并保持pH值至8，然后浓缩至相对密度为1.06～1.08(60℃)，浓缩液冷藏，静置48小时，滤过；滤过所得沉淀用16倍于沉淀量(W/V)的1％盐酸加热回流提取1小时，趁热滤过；
(6)滤液用40％氢氧化钠调节并保持pH值至6-8，浓缩至相对密度为1.06～ 1.08(60℃)，冷藏，静置48小时，滤过；
(7)滤过沉淀物进行减压干燥(60℃，0.08MPa)，即得岩黄连总碱提取物。
根据临床需要，可取岩黄连总生物碱75g，加125g可溶性淀粉，混合均匀，加85％乙醇适量制粒，干燥，整粒，装入胶囊，即得岩黄连胶囊。
实施例2岩黄连总碱胶囊对细胞实验的疗效观察
1、试验用药：实施例1制备得到的岩黄连总碱(CSBTA)。
2、细胞毒性实验
1.1实验仪器：MULTSKAN Sky全波长酶标仪(Thermo Scientific，美国)。
1.2试剂：MTT细胞增殖及细胞毒性检测试剂盒(货号：KGA321，江苏凯基生物技术股份有限公司)；实施例1制备得到的岩黄连总碱。
1.3实验方法：在96孔板加入细胞100μL/孔(约1×104)，置37℃、5％CO2细胞培养箱培养24小时。吸出所有孔的培养基，使用未加血清的空白培养基配置CSTBA(100ug/mL)溶液，按两倍梯度依次稀释至50、25、12.5等七个浓度岩黄连总碱，将空白培养基与各浓度培养基加入96孔板中，将其置于37℃，含5％CO2空气及100％湿度的细胞培养箱中孵育24h。将5×MTT用Dilution Buffer稀释成1×MTT,每孔加50μL 1×MTT，在37℃孵育4小时，使MTT还原为甲臜,吸出上清液，每孔加150μLDMSO使甲臜溶解，用平板摇床摇匀,酶标仪在490nm波长处检测每孔的光密度。
1.4实验结果：为了确定岩黄连总碱胶囊的最适的给药剂量，分七个梯度测定了其对细胞的毒性作用，如图1测试结果，发现其对细胞无明显的毒害作用。
实施例3岩黄连总碱对高脂血症模型大鼠降血脂作用的疗效观察
1.实验造模
1.1实验动物：雄性C57BL/6小黑鼠，4周龄，体重19g左右，购自扬州大学比较医学中心。
1.2饲料：60％高脂饲料，果糖，正常饲料，购自南通特洛菲饲料科技有限公司。
1.3试剂：实施例1制备得到的岩黄连总碱。
1.4实验方法：取35只雄性C57BL/6小黑鼠，4周龄，适应性喂养一周后，将小鼠随机分为2个初始组，喂以正常饲料或高脂(60％脂肪)高糖饮食(20％ 果糖)。喂养10周后，将小鼠随机分为模型组，岩黄连总碱(25mg/kg),岩黄连总碱(100mg/kg)，二甲双胍(200mg/kg)(n＝7)，空白组[使用等体积的0.5％甲基纤维素(中国国家药典，北京)]。造模过程中，小鼠自由摄食，饮水，动物实验室内室温23～25℃，温度适宜，且保持12h昼夜循环。在11周开始各组灌胃给药持续5周，期间每周测定一次体重。
1.5、实验结果
小鼠肝重指数
如图2所示，模型组的肝脏的重量增加，肿胀并呈现棕黄色，此现象在给药组和阳性药组得到明显改善。
2.小鼠血糖变化
2.1实验仪器：鱼跃血糖仪，血糖试纸
2.2试剂：葡萄糖(sigma，美国)
2.3实验方法：上述1.4在给药结束后，对小鼠进行OGTT实验，了解胰岛β细胞功能和机体对血糖的调节能力。晚8点后进行禁食12h，次日测定空腹血糖浓度FBG，给各组小鼠进行葡萄糖溶液灌胃(2g/kg)。在葡萄糖灌胃后15min,30min,60min,120min时，分别测定小鼠的血糖浓度。计算AUC(曲线下面积)。
2.4实验结果：图3曲线下面积可看出，与模型组小鼠相比，岩黄连总碱(CSBTA)治疗对糖耐量产生了有益的作用，此外，HFD引起的空腹血糖(FBG)浓度受损也通过岩黄连总碱治疗得到了很大的恢复。
3.小鼠血脂变化
3.1实验仪器：MULTSKAN Sky全波长酶标仪(Thermo Scientific，美国)
3.2试剂：TC测定试剂盒(A111-1-1,南京建成生物科技有限公司)，TG测定试剂盒(A110-1-1,南京建成生物科技有限公司)，HDL-C测定试剂盒(A112-1-1,南京建成生物科技有限公司)，LDL-C测定试剂盒(A113-1-1,南京建成生物科技有限公司),NEFA：测定试剂盒(A042-2-1,南京建成生物科技有限公司)。
3.3实验方法：小鼠禁食过夜，从眼静脉收集血样，并在4℃下以3000rpm离心10分钟。收集血浆并将其保存在-80℃直至使用。试剂盒按照制造商说明测定。
3.4实验结果：如图4所示，岩黄连总碱对HFD喂养小鼠的代谢特征的影响，测量了脂质分布，包括TG，TC，LDL胆固醇,HDL胆固醇以及游离脂肪酸的含量。岩黄连总碱可显著降低HFD诱导的TG，TC，LDL胆固醇，HDL胆固醇以及游离脂肪酸水平。
4.组织病理学检测
4.1 HE染色
4.1.1实验方法：收集新鲜的肝脏组织，并在4％中性福尔马林缓冲液中固定24小时，然后脱水。然后将块修整，加工并包埋在石蜡中。将组织玻片切成5μm的厚度，并用HE染色进行处理。
4.1.2实验结果：如图5中B所示正常肝组织中央静脉轮廓清晰，肝索沿着中央静脉放射状排列，肝细胞胞浆疏松，黄色框内是中央静脉，组织未见明显炎症细胞浸润。如C组所示，用HE染色在HFD小鼠肝脏中进行的组织病理学检查显示肝细胞膨胀，脂肪变性增加，肝细胞质稀疏和中间丝束成束。在给于岩黄连总碱(25mg/kg),岩黄连总碱(100mg/kg)，二甲双胍后均有明显改善。
4.2油红染色
4.2.1实验方法：对于油红O染色，将肝脏的冰冻切片(6μm厚)在室温下与10％福尔马林孵育30分钟，然后用新鲜的油红O工作溶液染色20分钟。用水洗涤后，将切片用苏木精染料复染1分钟，并置于显微镜下观察脂质沉积。
4.2.2实验结果：如图6中C所示，细胞周围出现明显的脂滴聚集，此现象在岩黄连总碱低剂量给药组(25mg/kg)中已有明显改善，在岩黄连总碱高剂量组(100mg/kg)改善更加明显，在阳性药组中也有明显改善。
4.3 Masson染色
4.3.1实验方法：根据制造商说明书染色
4.3.2实验结果：正常组血管壁周围可见少量胶原纤维，属正常范围。模型组肝组织内大量胶原纤维沉积，呈蓝色，自汇管区周围向外延伸，纤维条索较粗且染色着色较深，表明胶原纤维较多，包裹。此现象在岩黄连总碱低剂量给药组(25mg/kg)中已有明显改善，在岩黄连总碱高剂量组(100mg/kg)与阳性药组中几乎消失。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技 术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (9)

  1. 岩黄连在制备治疗非酒精性脂肪性肝病的药物中的应用。
  2. 岩黄连在制备治疗非酒精性脂肪性肝炎的药物中的应用。
  3. 岩黄连在制备治疗非酒精性脂肪性肝病合并药物性肝损伤疾病的药物中的应用。
  4. 岩黄连在制备治疗非酒精性脂肪性肝病合并化学性肝损伤疾病的药物中的应用。
  5. 岩黄连总碱胶囊在制备治疗非酒精性脂肪性肝病的药物中的应用。
  6. 岩黄连总碱胶囊在制备治疗非酒精性脂肪性肝炎的药物中的应用。
  7. 岩黄连总碱胶囊在制备治疗非酒精性脂肪性肝病合并药物性肝损伤疾病的药物中的应用。
  8. 岩黄连总碱胶囊在制备治疗非酒精性脂肪性肝病合并化学性肝损伤疾病的药物中的应用。
  9. 根据权利要求5至8任一项所述的应用，其特征在于，所述的黄连总碱胶囊的制备方法为：
    取岩黄连饮片，加8`10倍量体积浓度50～75％乙醇浸泡0.5～1h；
    (2)然后加热回流提取1～3次，每次0.5～2小时，合并提取液；
    (3)回收尽乙醇并浓缩，得到浓缩液；
    (4)取上述浓缩液加入盐酸，置耐酸多功能提取罐加热回流提取；
    (5)提取液用氢氧化钠调节并保持pH值，然后浓缩，浓缩液冷藏，静置，滤过；滤过所得沉淀用盐酸加热回流提取，趁热滤过；
    (6)滤液用氢氧化钠调节并保持pH值至6-8，浓缩，冷藏，静置，滤过；
    (7)滤过沉淀物进行减压干燥，即得岩黄连总碱提取物，与药学上可接受的载体制备成胶囊剂。
PCT/CN2021/077410 2020-03-13 2021-02-23 岩黄连及其制剂在制备治疗非酒精性脂肪性肝病的药物中的应用 WO2021179902A1 (zh)

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