CN114796318A - 桑叶总生物碱在制备防治脑缺血损伤作用药物中的应用 - Google Patents
桑叶总生物碱在制备防治脑缺血损伤作用药物中的应用 Download PDFInfo
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Abstract
本发明涉及桑叶提取物的新用途,特别涉及桑叶总生物碱在制备防治脑缺血损伤作用药物中的应用。本发明所述的桑叶总生物碱可作为有效成分,应用于脑缺血性中风的防治药物中,拓展了桑叶的新用途。为防治脑缺血性中风的防治提供新选择;具体的说,桑叶总生物碱在防治脑缺血性中风药物中的应用;桑叶总生物碱在防治脑缺血性疾病药物中的应用;桑叶总生物碱在制备防治脑缺氧疲劳的药物中的应用;在制备防治心肌缺血疾病的药物中的应用。
Description
技术领域
本发明涉及桑叶提取物的新用途,特别涉及桑叶总生物碱在制备防治脑缺血损伤作用药物中的应用。
背景技术
桑叶,是桑科落叶乔木植物桑树Morus alba L.的叶,味苦、甘,性寒,归肺、肝经。具有疏散风寒,清肺润燥,平肝明目等功效。尽管桑叶作为中药使用已有悠久的历史,但未见桑叶及其总生物碱有效部位减轻脑缺血损伤药效的报道。我们前期研究证实,将桑叶提取后纯化获得总生物碱,通过整体动物实验证实桑叶总生物碱预防性给药,能有效减轻脑缺血损伤
发明内容
发明的目的:提供桑叶总生物碱有效部位的新用途。本发明提供的桑叶总生物碱是在制备用于防治脑缺血的药物、保健食品中的应用。
技术方案
桑叶总生物碱在制备防治脑缺血损伤作用药物中的应用。
所述的桑叶总生物碱由如下步骤制备获得:
桑叶按照物料比W/V=1:8加入ddH2O,加热回流提取,纱布过滤和抽滤,重复三次,合并滤液,浓缩,以2BV/h动态吸附上样LSI-010阳离子交换柱,10-15BV ddH2O洗脱直至流出液显中性后,7-8BV 0.5mol/L氨水洗脱,收集洗脱液并浓缩,抽滤,2BV/h动态吸附上样D941阴离子交换树脂,收集未吸附部分,浓缩,活性炭吸附以脱色,浓缩,冻干,雷氏盐比色法检测总生物碱含量,生物碱含量达到52%。
在防治脑缺血疾病时,以桑叶总生物碱为活性成分,使用制剂工艺,单独使用或与其他药物或提取物配合成在临床上可以使用的药物。如丸剂、散剂、胶囊剂,片剂,徽囊剂,软胶囊剂,膜剂,栓剂,注射剂,膏剂,酊剂,散剂,冲剂,气雾剂以及各种外用制剂等。本发明实验研究发现,桑叶总生物碱预给药7天,能有效改善脑缺血损伤小鼠的行为学评分,减小脑缺血再灌注小鼠的脑梗死体积,降低血清丙二醛MDA含量,
本发明的技术关键点在于:
1、桑叶总生物碱能改善脑缺血再灌注损伤小鼠的神经行为学评分;
2、桑叶总生物碱能减小脑缺血再灌注损伤小鼠的脑梗死体积;
3、桑叶总生物碱能减少脑缺血再灌注损伤小鼠血清中丙二醛(MDA)含量。
有益效果
1、本发明证实桑叶总生物碱高剂量(100mg/kg)对脑缺血的预防效果与银杏叶片(198mg/kg)相似,且总生物碱的剂量更小。证明桑叶总生物碱高剂量(100mg/kg)和总生物碱低剂量(50mg/kg)对脑缺血动物脑梗死体积的预防效果与银杏叶片(198mg/kg)相似,且剂量更小。银杏叶片(198mg/kg)和总生物碱高剂量(100mg/kg)、总黄酮低剂量(50mg/kg)均能减少血清中脂质过氧化产物量,且总生物碱的效应与银杏叶片相当,用量更小。
2、本发明所述的桑叶总生物碱可作为有效成分,应用于脑缺血性中风的防治药物中,拓展了桑叶的新用途。为防治脑缺血性中风的防治提供新选择;具体的说,桑叶总生物碱在防治脑缺血性中风药物中的应用;桑叶总生物碱在防治脑缺血性疾病药物中的应用;桑叶总生物碱在制备防治脑缺氧疲劳的药物中的应用;在制备防治心肌缺血疾病的药物中的应用。
附图说明
图1为桑叶总生物碱提取分离流程图;
图2为桑叶总生物碱的薄层色谱图;
图3为桑叶总生物碱的HPLC色谱图,其中A桑叶总生物碱的HPLC色谱图,B为标准品HPLC色谱图;
图4为桑叶总生物碱标准曲线;
图5桑叶总生物碱改善脑缺血损伤小鼠的行为学评分;
图6桑叶总生物碱减小脑缺血损伤小鼠的脑梗死体积;A小鼠大脑冠状切片后TTC染色图;B各组小鼠脑梗死体积统计图;
图7桑叶总生物碱降低脑缺血损伤小鼠血清中MDA含量;
图8桑叶总生物碱对脑缺血损伤小鼠脑缺血区域皮质时钟基因的调控作用;A-E依次为Per1,Per2,Clock,Bmal1,Rev-erbα;
图9桑叶总生物碱对脑缺血损伤小鼠脑缺血区域皮质时钟基因蛋白表达的调控作用;A为WB结果,B-E依次为Per1,Clock,Bmal1,Rev-erbα。
其中图中*P<0.05,**P<0.01 vs MCAO;#P<0.05,##P<0.01 vs MCAO;a P>0.05 vs阳性药组。
具体实施方式
1-脱氧野尻霉素(DNJ),购自成都埃法生物有限公司,批号AF20060303。
雷氏盐购自上海阿拉丁生化科技股份有限公司,I2024067BAW(正丁醇:醋酸:水=4:1:5)溶液:量取正丁醇40mL,醋酸10mL,ddH2O50mL,混匀,室温保存;
银杏叶片购自大连美罗中药厂有限公司。
实施例1
1、桑叶总生物碱的提取
如图1所示,按照物料比(W/V=1:8)加入ddH2O,加热回流提取,纱布过滤和抽滤,重复三次,合并滤液,浓缩,动态吸附上样(2BV/h)LSI-010阳离子交换柱,10-15BV ddH2O洗脱直至流出液显中性后,7-8BV 0.5mol/L氨水洗脱,收集洗脱液并浓缩,抽滤,动态吸附上样(2BV/h)D941阴离子交换树脂,收集未吸附部分,浓缩,活性炭吸附以脱色,浓缩,冻干,雷氏盐比色法检测总生物碱含量,生物碱含量达到52%。
2、桑叶总生物碱定性及定量检测
定性检测(薄层色谱法):以DNJ作为对照品,BAW溶液为展开剂,3%茚三酮为染色剂。
定性检测(高效液相色谱):以DNJ作为标准品,流动相:40%乙腈-水,色谱柱:C18,检测器:ELSD,流速:1.0mL/min,进样体积:10μL。
含量测定:按照如下表格加入0.04mol/L 4-羟基哌啶标准液和0.05mol/LHCl,各溶液中加入0.02g/mL雷氏盐3.0mL,混匀,冰浴2h,抽滤,沉淀附着于滤纸上。用冰浴后的乙酸乙酯冲洗滤纸直至其沉淀外其它地方变白,烘干滤纸,放入50mL离心管中,用10mL 70%丙酮溶解,吸200μL于λ=523nm处检测吸光值。以A523为纵坐标,浓度为横坐标绘制标准曲线并计算回归方程。
薄层色谱定性检测总生物碱见图2,左侧两个为样品,右侧两个为标准品DNJ,在Rf=0.3左右出现,说明样品中含有DNJ。HPLC色谱图见图3,保留时间在150-175s。含量检测所作标准曲线见图4,y=11.602x+0.0389(R2=0.9982)。
3桑叶总生物碱减轻脑缺血损伤的药效评价
将C57BL/6J小鼠随机分为假手术组、模型组、阳性药(银杏叶片)组、总生物碱(纯度53%)高剂量组、总生物碱低剂量组。银杏叶片组灌胃给药198mg/kg银杏叶片。总生物碱高剂量组、总生物碱低剂量组分别灌胃用200mg/kg和100mg/kg总生物碱,连续给药14天。假手术组和模型组分别灌胃相同剂量的溶媒。
给药14天后,除假手术组外,将其他各组小鼠麻醉,麻醉,仰卧位固定于手术台上,在颈部正中作一定长度的切口,采用0.25%的盐酸布比卡因浸润麻醉,轻柔地分离出右侧颈总动脉(common carotid artery,CCA)、颈内动脉(internal carotid artery,ICA)及颈外动脉(external carotid artery,ECA)。在靠近ECA分叉处约2mm作一切口,将备好的线栓插入ICA,当线栓进入距ECA和ICA分叉处约9-10mm时略感阻力即停止进栓,缺血1h后,拔出线栓实现再灌注。假手术组小鼠仅分离CCA、ICA和ECA而不插线栓。术后小鼠体温保持在37℃左右,自由进水进食,观察24h。再灌注24小时后,对各组小鼠做行为学评分[参照文献——张三利,盛明月,吴琪,陈逊,俞妍,寇俊萍,陈刚领.鲁斯可皂苷元对脑缺血再灌注损伤小鼠时钟基因的调控作用,中国药理学与毒理学杂志,2021,35(07):502-506.],取血测定丙二醛(MAD)含量,结果如下:
(1)桑叶总生物碱对脑缺血损伤小鼠神经功能评分的影响
再灌注24h后,对各组小鼠进行神经功能评分,评价小鼠神经功能受损情况。结果如图5所示,与Sham组相比,MCAO组小鼠行为学评分增加,差异具有非常显著性意义(P<0.01),表明模型小鼠神经功能受损;与MCAO组相比,阳性药组、总生物碱高剂量组,差异具有非常显著性意义(P<0.01),证明预防性给药能改善脑缺血小鼠的神经功能。阳性药组与总生物碱高剂量组相比,差异无显著性意义(P>0.05),证明桑叶总生物碱高剂量(100mg/kg)对脑缺血的预防效果与银杏叶片(198mg/kg)相似,且总生物碱的剂量更小。
(2)桑叶总生物碱对脑缺血损伤小鼠脑梗死体积的影响
行为学评分结束后,取小鼠大脑组织进行TTC染色。如图6所示,与Sham组相比,MCAO组小鼠脑梗死体积增加,差异具有非常显著性意义(P<0.01);与MCAO组相比,阳性药组、总生物碱高剂量组、总生物碱低剂量组小鼠的脑梗死体积减小,差异具有非常显著性意义(P<0.01)。证明银杏叶片、总生物碱高剂量、总生物碱低剂量均能有效减轻脑缺血动物的脑梗死。银杏叶片组与总黄酮高剂量相比,差异无显著性意义(P>0.05)。证明桑叶总生物碱高剂量(100mg/kg)和总生物碱低剂量(50mg/kg)对脑缺血动物脑梗死体积的预防效果与银杏叶片(198mg/kg)相似,且剂量更小。
(3)桑叶总生物碱对脑缺血损伤小鼠血清SOD活力和MDA含量的影响
见图7,分离各组小鼠血清测定MDA含量。与Sham组相比,MCAO组小鼠血清中MDA含量增多,差异具有显著性意义(P<0.05);与MCAO组相比,阳性药、总生物碱高剂量组和总生物碱低剂量组小鼠血清中MDA含量降低,差异具有显著性或显著性意义(P<0.05)。阳性药组与总生物碱高剂量、总生物碱低剂量组相比,差异无显著性意义(P>0.05),表明脑缺血后,模型小鼠血清中脂质过氧化产物增多。银杏叶片(198mg/kg)和总生物碱高剂量(100mg/kg)、总黄酮低剂量(50mg/kg)均能减少血清中脂质过氧化产物量,且总生物碱的效应与银杏叶片相当,用量更小。
(4)桑叶总生物碱对脑缺血损伤小鼠皮质时钟基因表达的影响
再灌注24h后,提取小鼠脑部皮层组织及进行q-PCR实验。如图8结果显示,与Sham组相比,MCAO组时钟基因Per 1、Per 2、Clock、Bmal1和Rev-erbαmRNA表达水平降低,差异具有非常显著性意义(P<0.01);与MCAO组相比,总生物碱高剂量组Per 1、Per 2、Clock、Bmal1和Rev-erbαmRNA表达水平增加,差异具有显著性意义(Per,Bmal1,P<0.05)或非常显著性意义(Per2,Clock,Rev-erbα,P<0.01),总生物碱低剂量组的Bmal1和Rev-erbαmRNA表达水平增加,差异具有非常显著性意义(Bmal1,P<0.01)或显著性意义(Rev-erbα,P<0.05)。前期研究表明,时钟基因表达的增高与药物的脑保护效应有相关性。
提取小鼠脑部皮层组织及进行Western blot实验。如图9结果显示,与Sham组相比,MCAO组中时钟基因Per 1、CLOCK、BMAL1和REV-ERBα蛋白表达水平减少,差异具有非常显著性意义(P<0.01);与MCAO组相比,总生物碱高剂量组Per1、Per2、Bmal1、CLOCK和REV-ERBα蛋白水平表达增加,差异具有非常显著性意义(Per1,CLOCK,Bmal1,P<0.01)或显著性意义(Per2,REV-ERBα,P<0.05),总生物碱低剂量组Per1、CLOCK、BMAL1和REV-ERBα蛋白表达水平增加,差异具有非常显著性意义(BMAL1,REV-ERBα,P<0.01)或显著性意义(Per1,CLOCK,P<0.05)。前期研究表明,时钟基因表达的增高与药物的脑保护效应有相关性。
Claims (3)
1.桑叶总生物碱在制备防治脑缺血损伤作用药物中的应用。
2.根据权利要求1所述的应用,其特征在于所述的桑叶总生物碱由如下步骤制备获得:
桑叶按照物料比W/V= 1: 8加入ddH2O,加热回流提取,纱布过滤和抽滤,重复三次,合并滤液,浓缩,以2 BV/h动态吸附上样LSI-010阳离子交换柱,10-15 BV ddH2O洗脱直至流出液显中性后,7-8 BV 0.5 mol/L氨水洗脱,收集洗脱液并浓缩,抽滤,2 BV/h动态吸附上样D941阴离子交换树脂,收集未吸附部分,浓缩,活性炭吸附以脱色,浓缩,冻干。
3.根据权利要求2所述的应用,其特征在于,以雷氏盐比色法检测总生物碱含量,生物碱含量为52%。
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李名洁等: "桑不同药用部位总生物碱对α-葡萄糖苷酶活性的抑制作用", 《中国现代中药》 * |
杨锐等: "脑白质衰老该表及其对卒中影响的研究进展", 《基础医学论坛》 * |
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