CN108159042B - 辛可耐因Ib在制备治疗炎症性肠病的药物的应用 - Google Patents

辛可耐因Ib在制备治疗炎症性肠病的药物的应用 Download PDF

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CN108159042B
CN108159042B CN201711306968.7A CN201711306968A CN108159042B CN 108159042 B CN108159042 B CN 108159042B CN 201711306968 A CN201711306968 A CN 201711306968A CN 108159042 B CN108159042 B CN 108159042B
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刘成航
刘岱琳
付山
贺凌霜
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Abstract

本发明公开了辛可耐因Ib在制备治疗炎症性肠病的药物的应用。实验证明：辛可耐因Ib具有治疗炎症性肠病的作用，且疗效确切。本发明挖掘出天然产物辛可耐因Ib的新用途。

Description

辛可耐因Ib在制备治疗炎症性肠病的药物的应用
技术领域
本发明涉及一种天然产物的新用途，特别涉及一种辛可耐因Ib在制备治疗炎症性肠病的药物的应用。
背景技术
黄酮木脂素是一类新型的具C-9取代基的黄酮类化合物，即以黄酮和苯丙素类衍生物缩合而成的化合物，该类化合物具有很强的抗氧化、保肝、抗肿瘤、杀菌等生物活性[1]。辛可耐因Ib就是一种黄酮木脂素类化合物，来源于楝科鹧鸪花属巴西海木，蓼科菊三七属血三七，报春花科珍珠菜属珍珠菜，红树科红树属红海榄根、秋茄属秋茄，百合科菝葜属菝葜，蔷薇科枇杷属枇杷、苹果属苹果等6个科8个属植物中根、茎、果实、种子为原料。目前研究显示辛可耐因Ib具有较强的抗病毒[2]、抗氧化活性[3]，能够促进胰岛素的分泌[4]，但是其他的活性研究报道相对较少。
炎症性肠病(inflammatoryboweldisease；IBD)是一种慢性、复发性的肠道炎症疾病。近年来在发达国家，IBD的发病率已达到了1/1000[5]。到目前为止，IBD的病因还没有完全研究清楚，被认为是基因，环境，微生物和机体免疫系统的相互作用造成的[6]。由于确切的病因不清，常规的治疗策略主要是抗炎和非特异性的抑制免疫反应[7]。但大剂量的非甾体抗炎药物的使用会产生高热、消化不良、腹泻、转氨酶升高、脂酶水平升高等不良反应，皮质激素和免疫抑制剂的长期使用也会产生剂量依赖性。近年来，学者开始关注中药及天然产物治疗炎症性肠病，研究表明中药及天然产物的疗效稳定，且具有综合性双向调节作用，既可调节免疫功能又能抵御病原微生物的侵袭，并可减轻或缓解症状等优点。本发明通过实验证明辛可耐因Ib可以对抗炎症性肠病的发生，增加了辛可耐因Ib的新用途。
参考文献：
[1]Botany.Screening of biflavonoid compounds and British Columbianbryophytes for antiviral activity against potato virus X[D].The University ofBritish Columbia,2000.
[2]Takara K,Kuniyoshi A,Wada K,et al.Antioxidative flavan-3-olglycosides from stems of Rhizophora stylosa[J].Bioscience,biotechnology,andbiochemistry,2008,72(8):2191-4.
[3]Qa’dan F,Verspohl EJ,Nahrstedt A,et al.Cinchonain Ib isolated fromEriobotrya japonica induces insulin secretion in vitro and in vivo[J].Journalof ethnopharmacology,2009,124(2):224-7.
[4]Xavier R.J.Podolsky DKUnravelling the pathogenesis of inflammatorybowel disease[J],Nature,2007,448:427-434.
[5]Liehtenstein GR,Hanauer SB,Sandborn WJ.Management of Crohn’sdisease in adults[J],Am J.Gastroenterol,2009,104:46-483.
[6]Sandborn WJ,Feagan BG.Review artiele:mild to moderate Crohn’sdisease-defining the basis for a new treatment algorithm[J],AlimentPharmaeol,2003,18:263-277。
发明内容
本发明的目的是克服现有技术的不足，提供辛可耐因Ib在制备治疗炎症性肠病的药物的应用。
本发明的技术方案概述如下：
辛可耐因Ib在制备治疗炎症性肠病的药物的应用。
所述辛可耐因Ib的剂型优选为硬胶囊剂、软胶囊剂、散剂、颗粒剂、片剂、丸剂、蜜膏剂、口服液、栓剂、酒剂或注射剂。
我们通过实验观察到辛可耐因Ib能够明显抑制葡萄酸葡聚糖硫酸钠致小鼠实验性结肠病，并且能够提升机体的自身免疫能力，对抗炎症性肠病的发生。辛可耐因Ib高效无毒，安全性极高，本发明挖掘出辛可耐因Ib的新用途。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1实施例1中本发明中化合物的1H，13C-NMR数据；
图2实施例2中各实验组DAI评分和组织学评分比较(n＝8，x±s)；
图3实施例2中辛可耐因Ib调节NF-kB炎症通路中相关蛋白的表达；
图4实施例2中结肠组织IL-4、TNF-a和PGE2的含量比较(n＝8，x±s)。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
实施例1
辛可耐因Ib的制备方法
(1)提取：200g苹果多酚(天津市尖峰天然产物研究研究开发有限公司提供，多酚含量80％)中加入2000mL的水常温搅拌溶解，备用；
(2)大孔树脂吸附分离：将(1)中苹果多酚溶液经HP20型大孔树脂吸附，用水、体积百分比浓度为10％、30％、50％、70％、95％的乙醇水溶液梯度洗脱，每半个柱床体积作为一个接收体积，通过薄层层析方法并结合分析型HPLC检测，收集富含辛可耐因Ib的组分，将其合并，在温度≤60℃，真空度为0.06～0.08MPa减压浓缩，糖度为40，得辛可耐因Ib的富集物；
(3)硅胶柱分离：大孔树脂吸附分离后得到的辛可耐因Ib的富集物用等倍量的甲醇溶解以1.5--2.5倍量比例加入60-100目硅胶减压拌样至干，干法上样，用10-20倍量的硅胶柱色谱分离，用二氯甲烷-甲醇体积比为95:5，93:7，92:8，91:10梯度洗脱，半个柱床体积作为一个接收体积，通过薄层层析方法并结合分析型HPLC检测，收集富含辛可耐因Ib的组分，将其合并，在温度≤60℃，真空度为0.06～0.08MPa减压浓缩至干，得辛可耐因Ib的富集物，含量为32.1％；
(4)Toyopearl HW-40分离：将辛可耐因Ib富集物用2倍水溶解，经Toyopearl HW-40柱色谱分离，用甲醇-水的体积比为10％，15％，20％，25％梯度洗脱，每四分之一个柱床体积作为一个接收体积，通过薄层层析方法并结合分析型HPLC检测，收集富含辛可耐因Ib的组分，将其合并，在温度≤60℃，真空度为0.06～0.08MPa减压浓缩，糖度为20，得辛可耐因Ib的富集物，含量为67.2％；
(5)制备液相色谱精制：将(4)中辛可耐因Ib的富集物利用制备液相色谱精制，Cosmosil ODS柱(5u，10×250mm)254nm检测，50％甲醇水分离，收集辛可耐因Ib的色谱峰，并将其在温度≤60℃，真空度为0.06～0.08MPa减压浓缩，糖度为16，冷冻干燥得到辛可耐因Ib(460mg)。
利用ESI-MS、1H-NMR和13C-NMR等光谱手段并于文献数据进行对照，鉴定了根据上述方法分离得到的辛可耐因Ib的结构。在此基础上利用HPLC的面积归一化进行标定，辛可耐因Ib含量为97％。辛可耐因Ib淡黄色粉末，聚酰胺薄膜以正丁醇-醋酸-水(4:1:3)展开，Rf为0.4，5％三氯化铁乙醇溶液显单一蓝色斑点。HR ESI-MS：(negative)m/z：451.0986[M-H]-，计算值(451.1035)，可以确定该化合物的分子量为452。结合氢谱碳谱给出的信息，确定其分子式为C24H20O9，计算其不饱和度为15。其1H-NMR(300MHz，inDMSO)中信号交叠较严重，其13C-NMR(75MHz，in DMSO)中信号清晰，与文献相对照进行了全归属，鉴定为辛可耐因Ib，结果见附图1
实施例2
辛可耐因Ib对葡聚糖硫酸钠致小鼠实验性结肠病作用。
实验方法：
(1)葡聚糖硫酸钠(dextran sulfate sodium，DSS)溶于蒸馏水配制成3％DSS(W/V)水溶液，即配即用。40只c57小鼠随机分为6组：正常对照组、模型组、辛可耐因Ib(实施例2制备)高、中、低剂量组(200、100、50mg/kg)和阳性药物组(柳氮磺嘧啶组，220mg/kg)，每组各8只。除正常对照组外，上述各组小鼠均给予3％DSS溶液自由饮用，造模开始分别按上述剂量灌胃给药，正常组和模型组给予等量体积生理盐水。记录每天每只小鼠DSS的应用量，确保各组DSS饮用量大致相等。每日观察小鼠一般状态和体重变化，观察粪便性状改变及便血情况，根据Cooper HS评分标准，进行疾病活动指数(Disease activity index，DAI)评分，评估小鼠结肠粘膜炎症损伤情况。评分标准：体质量不下降者记0分，下降1％～5％为1分，下降5％～10％为2分，下降10％～15％为3分，下降>15％为4分；大便正常为0分，松散为2分，稀便为4分；大便潜血正常为0分，弱阳性为1分，阳性为2分，强阳性为3分，肉眼血便为4分。实验进行8d后，处死小鼠，取下结肠，4％中性福尔马林固定，石蜡包埋切片后进行HE染色，镜下观察肠黏膜损伤情况，根据Cooper HS评分标准，观察15个放大100倍视野，取平均值，观察组织学损伤评分。评分标准：正常结肠黏膜者为0分；结肠隐窝腺体丢失三分之一者为1分；隐窝腺体丢失三分之二者为2分；若隐窝腺体全部丢失，黏膜上皮完整，伴有轻度炎性细胞浸润者为3分；黏膜上皮糜烂、破坏，伴有明显炎性细胞浸润者为4分。
实验结果：
正常对照组小鼠活动自如，反应灵敏，大便正常，毛色光泽，体重增加；模型组小鼠饮用3％DSS 8d后出现稀便、血便或脓血便、大便潜血实验阳性，体重减轻，活动进食均减少；各给予辛可耐因Ib组和阳性药物组小鼠状况好于模型组，其腹泻、便血症状较模型组明显减轻。DAI评分显示模型组组小鼠DAI升高，与正常对照组相比，具有统计学意义(P<0.05)，阳性药物组和辛可耐因Ib各组DAI评分明显降低，与模型组相比，差异具有统计学意义(P<0.05)。
光镜下观察正常组小鼠结肠黏膜完整连续，腺体排列规则整齐，无炎细胞浸润和溃疡形成。而模型组小鼠结肠充血水肿，粘膜丧失，糜烂、溃疡、出血，腺体排列紊乱，可见大量炎细胞浸润，说明造模成功。阳性药物组和辛可耐因Ib各组症状改善明显，结肠黏膜较完整，有轻度充血水肿和少量炎细胞浸润，肠腺体排列基本规则，组织学评分示，各治疗组组织学评分显著低于模型组，差异具有统计学意义(P<0.05)。上述实验结果说明辛可耐因Ib能够明显抑制葡萄酸葡聚糖硫酸钠致小鼠实验性结肠病。
各实验组DAI评分和组织学评分比较(n＝8，x±s)如附图2
注：与正常组比较，
Figure GDA0001640072930000041
为P<0.05；与模型组比较，*为P<0.05
(2)免疫组化法检测相应蛋白的活性
常规梯度脱水，石蜡包埋切片，脱蜡水化，过氧化氢灭活内源性酶，PBS冲洗，枸橼酸盐缓冲液热抗原修复，PBS冲洗，加一抗，稀释度为1:100，PBS作为阴性对照，4℃湿盒过夜。PBS洗涤后，加入生物素化羊抗兔IgG二抗，37℃，30min，PBS洗涤，DAB显色，常规脱水、封片。实验结果见附图3。正常组小鼠结肠粘膜仅微弱表达NF-kB p65。而模型组中NF-kBp65显著增加，其阳性药物组NF-kBp65的表达收到明显抑制，二者相比，差异具有统计意义(P<0.05)。各给予辛可耐因Ib的治疗组NF-kBp65表达均减少，可耐因Ib高、中剂量组降低最明显，与模型组相比，差异具有统计学意义(P<0.05)。上述结果说明辛可耐因Ib能够有效降低炎症通路中NF-kB和Ikkα/β的表达，说明辛可耐因Ib能够调节炎症通路中相关蛋白的表达而发挥抗炎作用。
(3)ELISA法检测TNF-a、PGE2和IL-4的表达水平
摘取结肠组织匀浆离心后，采用酶联免疫吸附法，严格按照试剂盒说明，检测细胞因子TNF-a、PGE2和IL-4的水平。实验结果见附图4。
实验结果：
模型组TNF-a和PGE2水平升高，与正常组相比有统计学差异(P<0.05)。阳性药物组和辛可耐因Ib(实施例2制备)各组TNF-a和PGE2水平与模型组相比显著下降(P<0.05)，其中TNF-a水平随着给药剂量的增加逐渐降低，呈剂量效应关系。各治疗组IL-4水平略有升高趋势。结果见附图4。
结肠组织IL-4、TNF-a和PGE2的含量比较(n＝8，x±s)见附图4
注：与正常组比较，
Figure GDA0001640072930000051
为P<0.05；与模型组比较，*为P<0.05
上述实验结果说明辛可耐因Ib能够明显抑制葡萄酸葡聚糖硫酸钠致小鼠实验性结肠病，并且能够提供机体的自身免疫能力，对抗炎症性肠病的发生。
实验证明：辛可耐因Ib可以对抗炎症性肠病的发生。
按常规方法将辛可耐因Ib成硬胶囊剂、软胶囊剂、散剂、颗粒剂、片剂、丸剂、蜜膏剂、口服液、栓剂、酒剂或注射剂的剂型，可以方便药物的使用。

Claims (2)

1.辛可耐因Ib作为单一活性成分在制备治疗炎症性肠病的药物中的应用。
2.根据权利要求1所述的应用，其特征是所述药物的剂型为硬胶囊剂、软胶囊剂、散剂、颗粒剂、片剂、丸剂、蜜膏剂、口服液、栓剂、酒剂或注射剂。
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