CN106420891A - 杜仲叶总黄酮在制备治疗围绝经期综合征药物中的应用 - Google Patents
杜仲叶总黄酮在制备治疗围绝经期综合征药物中的应用 Download PDFInfo
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Abstract
本发明涉及杜仲叶总黄酮在制备治疗围绝经期综合征药物中的应用，可有效解决杜仲叶总黄酮作为唯一活性成分在制备围绝经期综合征药物中的应用问题，该杜仲叶总黄酮是将杜仲叶粉碎成粗粉，先用石油醚回流提取，脱脂，杜仲叶药渣挥干石油醚，加乙醇浸泡，回流提取，过滤，减压回收乙醇至无醇味，加蒸馏水分散，得药液；药液上AB‑8型大孔吸附树脂，先用蒸馏水洗脱，弃去水洗脱液；再用质量浓度10%乙醇洗脱，弃去洗脱液；再用质量浓度80%乙醇洗脱，收集80%乙醇洗脱液，减压回收乙醇至无醇味，冷冻干燥，粉碎，得杜仲叶总黄酮，本发明原料丰富，制备方法简单，易操作，生产效率高，有效用于制备治疗围绝经期综合征的药物，开拓了杜仲叶的新用途。
Description
技术领域
本发明涉及医药，特别是一种杜仲叶总黄酮在制备治疗围绝经期综合征药物中的应用。
背景技术
杜仲叶不仅与杜仲皮有相同的有效成分和药理作用，而且来源更丰富，成本更低。具有较好的开发利用前景，微辛，温。归肝、肾经，补肝肾；强筋骨；降血压，用于肝肾不足，头晕目眩，腰膝酸痛，筋骨痿软，腰背疼痛；足膝酸软乏力；高血压病。
围绝经期综合征是指妇女绝经前后的一段时期(从45岁左右开始至停经后12个月内的时期)，包括从接近绝经出现与绝经有关的内分泌、生物学和临床特征起至最后1次月经后1年，也就是卵巢功能衰退的征兆，一直持续到最后1次月经后1年，是正常的生理变化时期。但在围绝经期易发生不适，临床表现为月经改变，面部潮红、阵阵发热、出汗等血管舒张症状。情绪不稳定、激动易怒、抑郁多烦、记忆力减退、工作能力下降等，皮肤皱纹逐渐增多，有的出现瘙痒、毛发开始变白脱落。腹部和臀部脂肪增多，容易发胖，血压易波动，常出现高血压、心前区闷痛不适、心悸、气短，动脉硬化发生率增加，冠心病发病率也上升，骨质疏松，上述症状被称为围绝经期综合征。
现虽有治疗围绝经期综合征的多种药物，但至今未见有从杜仲叶中提取的杜仲叶总黄酮在制备治疗围绝经期综合征药物中的应用。
发明内容
针对上述情况，为克服现有技术之缺陷，本发明之目的就是提供一种杜仲叶总黄酮在制备治疗围绝经期综合征药物中的应用，可有效解决杜仲叶总黄酮作为唯一活性成分在制备围绝经期综合征药物中的应用问题。
本发明解决的技术方案是，杜仲叶总黄酮作为唯一活性部位(成分)在制备治疗围绝经期综合征药物中的应用，该杜仲叶总黄酮是将杜仲叶粉碎成粗粉，先用石油醚回流提取，脱脂，杜仲叶药渣挥干石油醚，加乙醇浸泡，回流提取，过滤，减压回收乙醇至无醇味，加蒸馏水分散，得药液；药液上AB-8型大孔吸附树脂，先用蒸馏水洗脱，弃去水洗脱液；再用质量浓度10％乙醇洗脱，弃去洗脱液；再用质量浓度80％乙醇洗脱，收集80％乙醇洗脱液，减压回收乙醇至无醇味，冷冻干燥，粉碎，得杜仲叶总黄酮。
本发明制备的杜仲叶总黄酮作为唯一活性成分在制备治疗围绝经期综合征药物中的应用，原料丰富，制备方法简单，易操作，生产效率高，有效用于制备治疗围绝经期综合征的药物，开拓了杜仲叶的新用途，是中药上的创新，有显著的经济和社会效益。
具体实施方式
以下结合具体情况对本发明的具体实施方式作详细说明。
本发明在具体实施中，杜仲叶总黄酮作为唯一活性部位(成分)在制备治疗围绝经期综合征药物中的应用，该杜仲叶总黄酮是将杜仲叶粉碎成过20-30目筛的粗粉，先加粗粉10倍重量的石油醚回流提取1h，脱脂，杜仲叶药渣挥干石油醚，加粗粉10倍重量、质量浓度80％乙醇浸泡0.5h，回流提取1h，过滤，得第一次滤液；药渣再加粗粉10倍重量、质量浓度80％乙醇回流提取1h，过滤，得第二次滤液，合并两次滤液，减压回收乙醇至无醇味，加蒸馏水分散至相当于含生药量0.3g/ml，得药液；药液上AB-8型大孔吸附树脂，先用2倍柱体积蒸馏水洗脱，弃去水洗脱液；再用4倍柱体积的质量浓度10％乙醇洗脱，弃去洗脱液；再用6倍柱体积的质量浓度80％乙醇洗脱，收集80％乙醇洗脱液，减压回收乙醇至无醇味，冷冻干燥，粉碎，得杜仲叶总黄酮。
本发明经试验，杜仲叶总黄酮作为唯一活性成分，可有效用于制备治疗围绝经期综合征的药物，有关试验资料如下：
1实验材料
1.1实验动物
KM小鼠，SPF级；性别：雌性；体重：23-25g，提供单位：山东鲁抗医药股份有限公司；动物合格证号：37005400000020；实验伦理编号：DWLL16020050；河南中医学院动物实验中心许可证号：SYXK(豫)2015-0005。
1.2实验药物
本发明杜仲叶总黄酮，含量>55％。更年安胶囊(地黄、熟地黄、泽泻、麦冬、玄参、牡丹皮、茯苓、珍珠母、仙茅、五味子、磁石、首乌藤、钩藤、浮小麦、制何首乌。辅料为空心胶囊。性状：本品为胶囊剂，内容物为黑褐色的颗粒；气微香，味微甜而后苦。功能主治：滋阴潜阳，除烦安神。用于更年期潮热汗出，眩晕耳鸣，烦躁失眠。规格：每粒装0.3g；用法用量：口服，一次3粒，一日3次,山西黄河中药有限公司生产。
1.3实验试剂
水合氯醛，天津市科密欧化学制剂开发中心；羧甲基纤维素钠，天津市恒兴化学试剂制造有限公司；注射用青霉素钠，华北制药股份有限公司；0.9％氯化钠注射液，河南科伦药业有限分司；甲醛溶液(分析纯)，烟台市双双化工有限公司；酒精：新乡市三伟消毒制剂有限公司；小鼠E2ELISA检测试剂盒，R&D公司；小鼠T ELISA检测试剂盒，R&D公司；小鼠LHELISA检测试剂盒，R&D公司；小鼠FSH ELISA检测试剂盒，R&D公司；小鼠IL-2ELISA 检测试剂盒，R&D公司。
1.4实验仪器
电子称，上海民桥医疗器械有限公司，型号JY601；电子分析天平，奥豪斯(上海)公司，型号AR1140/C；高速台式离心机，上海安亭科学仪器厂，型号TGL-168；电热恒温水浴锅，上海一恒科学仪器有限公司，型号HWS12；高速冷冻离心机，科大创新股份有限公司中佳分公司，型号KDC-160HR；可调式移液器，上海雷勃分析仪器有限公司；小鼠自主活动测试仪，成都泰盟科技有限公司，型号ZZ-6；小鼠避暗仪，成都泰盟科技有限公司，型号：BA-200；酶标仪，美国BIO-RAD公司，型号680；电动显微镜，日本OLYMPUS公司，型号BX61。
2实验方法
2.1造模与给药
造模方法：取体重23～25g雌性昆明小鼠84只，随机选出10只作为空白，做假手术处理，其余小鼠造围绝经期模型。小鼠称重后腹腔注射10％水合氯醛(0.03ml/10g)麻醉后腹位固定，然后从小鼠背部最末肋骨下，在腋中线和距脊柱外侧约1cm交叉处剪毛，消毒后切开皮肤和背肌约0.5～1cm，切口视野中可见一乳白色发亮的脂肪团，卵巢即包埋其中。用小镊子轻轻夹住脂肪团拉出切口外，分离脂肪团，即可见到一团细线状不规则呈黄红色的卵巢。剪取时先将卵巢下输卵管(包括脂肪)用细线结扎，再摘除卵巢，术后顺势将子宫角放回腹腔中，缝合肌肉及皮肤，同法摘除双侧卵巢。术后精心饲养，肌肉注射青霉素25万u/kg(每只0.02mL)以防感染，连续3d，每天1次。手术5d后开始逐只进行小鼠阴道涂片检查，每天1次，连续5d，以确定卵巢是否完全切除。涂片呈现动情反应的小鼠弃去不用，选择60只去势完全的小鼠随机均匀分为6组供实验用，分别为模型组，更年安胶囊组，大豆异黄酮软胶囊组，大、中、小剂量杜仲叶总黄酮组。
配药：0.5％CMC配制方法：称取羧甲基纤维素钠4g，用蒸馏水配成800ml。
大、中、小剂量杜仲叶总黄酮组给药剂量分别为400mg·kg-1、200mg·kg-1、100mg·kg-1(给药体积0.1ml/10g)。
更年安胶囊(675mg·kg-1，浓度67.5mg·mL-1，相当于临床用量的15倍)；配置方法：取更年安胶囊9粒，先混悬于少量的0.5％CMC溶液，然后再定容到40ml，混匀，即为所需更年安胶囊混悬液。
大豆异黄酮维E软胶囊(250mg·kg-1，浓度25mg·mL-1，相当于临床用量的15倍)；配置方法：取大豆异黄酮胶囊2粒，先混悬于少量的0.5％CMC溶液，然后再定容到40ml，混匀，即为所需大豆异黄酮混悬液。
储存方法：冷藏，用时隔水温热
给药方法：各组动物于手术后第10天给予相应药物。更年安胶囊组灌服更年安胶囊混悬液675mg·kg-1，大豆异黄酮软胶囊组灌服大豆异黄酮软胶囊混悬液250mg·kg-1，大、中、小剂量杜仲叶总黄酮组分别组别灌服大、中、小剂量杜仲叶总黄酮400mg·kg-1、200mg·kg-1、100mg·kg-1。空白组和模型组分别灌服同体积蒸馏水，每日灌胃给药1次，连续给药21d。
2.2观察项目及检测方法
各组小鼠于给药18d时测定5min内自主活动次数，于给药19～20d时测定小鼠首次进入暗室的潜伏期及5min内进入暗室遭电击的次数。于末次给药后2h(禁食不禁水12h)，小鼠称重后摘眼球取血，分离血清，测定血清中E2、T、LH、FSH、IL-2的含量；然后脱颈椎处死小鼠，解剖小鼠，摘除胸腺、脾脏、子宫组织，称量其湿重并计算脏器指数(脏器指数＝脏器湿重mg/小鼠体重g)，然后取脑；将胸腺、脾脏、子宫、脑组织固定于10％甲醛溶液中，石蜡包埋，切片，HE染色，光镜下观察各组的组织形态学变化。
2.2.1自主活动次数测试
将各组小鼠放入自主活动仪中，先适应环境1min，然后测定5min内自主活动次数。
2.2.2避暗法测试
将各组小鼠尾部对着进入暗室的小口放入测定盒中，进行训练，24h后重新测定小鼠首次进入暗室的潜伏期及5min内进入暗室遭电击的次数。
2.2.3试剂盒测定方法
小鼠雌二醇(E2)、睾酮(T)、黄体生成素(LH)、促卵泡素(FSH)、白细胞介素2(IL-2)ELISA检测试剂盒测定方法详见说明书。
2.3统计学处理方法
数据分析用SPSS17.0医用统计包进行数据资料的统计学处理，计量资料用平均数±标准差表示，各组间比较采用单因素方差分析，方差检验齐者用LSD法，方差不齐者用Games-Howell法检验，等级资料采用Ridit检验。
结果
1.对围绝经期模型小鼠自主活动的影响
杜仲叶总黄酮对围绝经期模型小鼠自主活动的影响，结果见表1。
表1杜仲叶总黄酮对围绝经期模型小鼠自主活动的影响
注：与模型组比较，*P<0.05，**P<0.01
由表1可看出，与空白组比，模型组小鼠的活动、站立次数均显著减少(P<0.01)，反映了围绝经期模型小鼠对新鲜环境的好奇程度降低。与模型组比，大豆异黄酮软胶囊组、更年安胶囊组、大、中剂量杜仲叶总黄酮组均可显著提高小鼠的活动次数(P<0.01)。
2.对围绝经期模型小鼠避暗法潜伏期和受电击次数的影响
杜仲叶总黄酮对围绝经期模型小鼠暗室潜伏期和受电击次数的影响，结果见表2
表2杜仲叶总黄酮对围绝经期模型小鼠暗室潜伏期和受电击次数的影响
注：与模型组比较，*P<0.05，**P<0.01
由表2可知，与空白组比，模型组小鼠的受电击次数显著增加(P<0.01)，反映了围绝经期模型小鼠记忆力降低。与模型组比，大豆异黄酮软胶囊组、更年安胶囊组、大、中、小剂量杜仲叶总黄酮组均可显著延长暗室潜伏期，减少小鼠受电击次数(P<0.01)。
3.对围绝经期模型小鼠脏器指数的影响
杜仲叶总黄酮对围绝经期模型小鼠胸腺、脾脏、子宫指数的影响，结果见表3。表3杜仲叶总黄酮对围绝经期模型小鼠胸腺、脾脏、子宫指数的影响
注：*P<0.05，**P<0.01，表示与模型组比较
由表3可知，与空白组比，模型组小鼠的子宫指数显著降低(P<0.01)，说明摘除卵巢导致围绝经期模型小鼠子宫萎缩。与模型组比，大豆异黄酮软胶囊组、更年安胶囊组、中剂量杜仲叶总黄酮组均可显著提高胸腺、脾脏、子宫指数(P<0.01)。
4.对围绝经期模型小鼠血清性激素含量的影响
杜仲叶总黄酮对围绝经期模型小鼠血清中E2、T含量的影响，结果见表4；杜仲叶总黄酮对围绝经期模型小鼠血清中FSH、LH含量的影响，结果见表5。
表4杜仲叶总黄酮对围绝经期模型小鼠血清中E2、T含量的影响
注：*P<0.05，**P<0.01，表示与模型组比较
表5杜仲叶总黄酮对围绝经期模型小鼠血清中FSH、LH含量的影响
注：*P<0.05，**P<0.01，表示与模型组比较
由表4、表5可以看出，与空白组比，模型组小鼠血清中E2、T水平显著下降(P<0.01)，LH、FSH水平均显著升高(P<0.01)，说明小鼠血清中的性激素水平紊乱，围绝经期小鼠模型复制成功。与模型组比，大剂量杜仲叶总黄酮组均可显著升高血清E2、T水平(P<0.01)，明显降低升高的LH、FSH水平(P<0.05)。
5.对围绝经期模型小鼠血清中IL-2含量的影响
杜仲叶总黄酮对围绝经期模型小鼠血清中IL-2含量的影响，结果见表6。
表6杜仲叶总黄酮对围绝经期模型小鼠血清中IL-2含量的影响
注:*P<0.05，**P<0.01，表示与模型组比较
由表6可知，与空白组比，模型组小鼠血清中IL-2水平显著下降(P<0.01)，说明完全摘除卵巢所致围绝经期小鼠模型机体免疫力有所降低。与模型组比，各给药组均可显著升高血清IL-2水平(P<0.01)。
6.对围绝经期模型小鼠器官组织形态的影响
杜仲叶总黄酮模型小鼠子宫、胸腺、脾脏、垂体的病理组织学观察，采用测微尺，测定实验各组每只小鼠子宫内膜的厚度。结果见表7；采用测微尺，对实验各组每只小鼠胸腺皮质最厚处和最窄处测定其厚度求得均数；然后用测微尺的基线测得压在基线上的胸腺皮质最厚处和最窄处的淋巴细胞数求得均数，结果见表8；采用测微尺的基线落在脾小结上，以中央动脉为中心分别测定两侧的脾小结厚度大小，求均数；同时计算落在基线上两侧的淋巴细胞数的个数，求均数；采用测微尺测量对角线上腺垂体的数量并求得平均值。
表7杜仲叶总黄酮对围绝经期模型小鼠子宫内膜厚度的影响
注:*P<0.05，**P<0.01，表示与模型组比较
由表7可知，与空白组比，模型组小鼠子宫内膜厚度显著下降(P<0.01)，说明完全摘除卵巢所致围绝经期小鼠模型显著子宫萎缩。与模型组比，大豆异黄酮软胶囊组、更年安胶囊组，大剂量杜仲叶总黄酮胶囊组均可显著升高子宫内膜的厚度(P<0.01)。
6.2对围绝经期模型小鼠胸腺、脾脏组织形态的影响
表8杜仲叶总黄酮对围绝经期模型小鼠胸腺的影响
注：*P<0.05，**P<0.01，表示与模型组比较
从表8可知，与空白组比，模型组小鼠胸腺皮质厚度、脾小结体积显著减少(P<0.01)，说明小鼠造围绝经期模型后胸腺、脾脏体积萎缩。与模型组比，大豆异黄酮胶囊、更年安胶囊、大、中剂量杜仲叶总黄酮组可显著增厚胸腺皮质和脾小结体积(P<0.01)。
6.3对小鼠围绝经期模型垂体组织形态的影响
表9杜仲叶总黄酮对围绝经期模型小鼠腺垂体数量的影响
注:*P<0.05，**P<0.01，表示与模型组比较
从表9可知，与空白组比，模型组腺垂体的数量显著减少(P<0.01)，说明小鼠造围绝经期模型后分泌促性激素的细胞源减少。与模型组比，大、中、小剂量杜仲叶总黄酮组可显著增加腺垂体的数量体积(P<0.01)。
应有本品制备用于治疗围绝经期综合征的药物(胶囊)，并经88例围绝经期综合征患者临床应用，取得了显著的治疗效果。患者每日服用3次，每次2粒，每粒含杜仲叶总黄酮0.5g，连服60天，除2人外，临床症状消失或临床症状有明显改善或改善者86人，有效率高达97.7％，效果之好是未曾料到的，是治疗围绝经期综合征药物上的创新，开拓了杜仲叶的新用途，经济和社会效益显著。
Claims (1)
1.一种杜仲叶总黄酮作为唯一活性部位在制备治疗围绝经期综合征药物中的应用，该杜仲叶总黄酮是将杜仲叶粉碎成过20-30目筛的粗粉，先加粗粉10倍重量的石油醚回流提取1h，脱脂，杜仲叶药渣挥干石油醚，加粗粉10倍重量、质量浓度80%乙醇浸泡0.5h，回流提取1h，过滤，得第一次滤液；药渣再加粗粉10倍重量、质量浓度80%乙醇回流提取1h，过滤，得第二次滤液，合并两次滤液，减压回收乙醇至无醇味，加蒸馏水分散至相当于含生药量0.3g/ml，得药液；药液上AB-8型大孔吸附树脂，先用2倍柱体积蒸馏水洗脱，弃去水洗脱液；再用4倍柱体积的质量浓度10%乙醇洗脱，弃去洗脱液；再用6倍柱体积的质量浓度80%乙醇洗脱，收集80%乙醇洗脱液，减压回收乙醇至无醇味，冷冻干燥，粉碎，得杜仲叶总黄酮。
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