CN106266476B - 葱籽总黄酮在制备治疗绝经前综合征药物中的应用 - Google Patents
葱籽总黄酮在制备治疗绝经前综合征药物中的应用 Download PDFInfo
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Abstract
本发明涉及葱籽总黄酮在制备治疗绝经前综合征药物中的应用，可有效解决葱籽总黄酮作为唯一活性成分在制备绝经前综合征药物中的应用问题，该葱籽总黄酮是将葱籽粉碎成粗粉，先用石油醚回流提取，脱脂，葱籽药渣挥干石油醚，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，得葱籽总黄酮，本发明制备方法简单，易操作，生产效率高，有效用于制备治疗绝经前综合征的药物，开拓了葱籽的新用途，是中药上的创新，有显著的经济和社会效益。
Description
技术领域
本发明涉及医药，特别是一种葱籽总黄酮在制备治疗绝经前综合征药物中的应用。
背景技术
葱子(Fistular Onion Seed)为炎百合科植物葱Allium fistulosum L.的种子，性温，味辛，补肾明目。用于肾虚、阳痿、目眩。
绝经前综合征是指妇女绝经前出现性激素波动或减少所致的一系列躯体及精神心理症状。绝经(menopause)分为自然绝经和人工绝经。自然绝经指卵巢内卵泡生理性耗竭所致的绝经；人工绝经指两侧卵巢经手术切除或受放射治疗所致的绝经。人工绝经患者更易发生绝经前综合征，临床症状表现为：月经紊乱，常出现如心悸、眩晕、头痛、失眠、耳鸣等自主神经失调症状，注意力不易集中，并且情绪波动大。表现为激动易怒、焦虑不安或情绪低落、抑郁、不能自我控制等情绪症状，记忆力减退也较常见，影响着患者的身体健康和生活质量。
现虽有治疗绝经前综合征的多种药物，但至今未见有从葱籽中提取的葱籽总黄酮在制备治疗绝经前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葱籽总黄酮在制备治疗绝经前综合征药物中的应用，可有效解决葱籽总黄酮作为唯一活性成分在制备绝经前综合征药物中的应用问题。
本发明解决的技术方案是，葱籽总黄酮作为唯一活性部位(成分)在制备治疗绝经前综合征药物中的应用，该葱籽总黄酮是将葱籽粉碎成粗粉，先用石油醚回流提取，脱脂，葱籽药渣挥干石油醚，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，得葱籽总黄酮。
本发明制备的葱籽总黄酮作为唯一活性成分在制备治疗绝经前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，有效用于制备治疗绝经前综合征的药物，开拓了葱籽的新用途，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，葱籽总黄酮作为唯一活性部位(成分)在制备治疗绝经前综合征药物中的应用，该葱籽总黄酮是将葱籽粉碎成过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，葱籽药渣挥干石油醚，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，得葱籽总黄酮。
本发明经试验，葱籽总黄酮作为唯一活性成分，可有效用于制备治疗绝经前综合征的药物，有关试验资料如下：
1实验材料
1.1药品试剂
本发明葱籽总黄酮，含量>52％。更年安胶囊，山西黄河中药有限公司(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)，滋阴潜阳，除烦安神；用于围绝经期潮热汗出，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。大鼠E2检测试剂盒，R&D公司；注射用青霉素钠，华北制药股份有限公司，规格：400万单位；甲醛溶液(分析纯)，烟台市双双化工有限公司；0.9％氯化钠注射液，河南科伦药业有限公司l；水合氯醛：天津市光复精密化工研究所；酒精：新乡市三伟消毒制剂有限公司。
1.3实验仪器
TDZ4-WS台式低速离心机，湖南湘仪实验室仪器开发有限公司；BIORAD-680酶标仪，680Microplate Reader，Bio-Rad Laboratories；BX61电动显微镜，日本OLYMPUS公司；手动移液器，美国赛默飞世尔科技有限公司，型号：JZ19600；HHS-21-8型电热恒温水浴锅，上海博迅实业有限公司医疗设备厂；BCD-248T型冰箱，新飞电器有限公司；JM 1000电子秤，余姚记铭称重校验设备有限公司；AL204电子天平，梅特勒-托利多仪器(上海)有限公司。
1.4实验动物
清洁级小鼠，昆明种，雌性，18－22g，山东鲁抗医药股份有限公司,动物合格证号37005400000020；实验室合格证号SYXK(豫)2010-001。
2实验方法
2.1造模与给药
造模方法：取昆明种小鼠70只，雌性，22～25g，随机取60只小鼠，腹腔注射戊巴比妥钠50mg/kg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。另取10只小鼠为空白对照组，按上法做手术，但不切除卵巢。术后精心饲养，肌肉注射青霉素预防感染，连续3d。手术5d后开始做手术小鼠逐只进行大鼠阴道涂片检查，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动物弃去不用，选择60只去势完全的动物随机均匀分为5组。分别为模型组，更年安胶囊组，大、小剂量葱籽总黄酮组，单做手术不切除卵巢的为空白对照组。
给药方法：将CMC-Na溶解于煮沸的蒸馏水中，制成0.5％的溶液做为溶媒，将以下各药物混悬其中。取葱籽总黄酮大、小剂量：30mg·mL-1、15mg·mL-1，按600mg·kg-1、300mg·kg-1灌胃；更年安胶囊：33.75mg·mL-1，按675mg·kg-1灌胃(相当于临床用量的15倍)灌胃；模型组和空白组给予同体积的CMC-Na溶液，给药体积为0.2mL/10g，每天1次，连续3周。
2.2观察项目及检测方法
各组小鼠末次灌胃后2h(禁食15小时)，眶静脉取血，分离血清，采用放射免疫法测定血清中E2、LH、FSH的含量；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/大鼠体重g)；将子宫固定于10％福尔马林溶液中，石蜡包埋，切片，HE染色，光镜下观察各组的组织形态学变化。
雌二醇的测定方法
测定原理：本实验采用放射免疫分析方法。先将校准品或样品、标记物和抗体按程序规定依次加入试管中，使标记和非标记抗原与有限量抗体互相竞争结合，待反应平衡后，加入分离剂，离心沉淀，使游离抗原与抗原抗体复合物分离，测量沉淀物的放射性强度。随E2含量增加，其放射性强度则相应降低。
校准试剂：125I-E2，1瓶(红色液体)；
抗体，1瓶(蓝色液体)；
校准品，7瓶(液体)，浓度为0、10、50、100、250、750、2000pg/mL；
分离剂，1瓶(悬浮液)用前摇匀；
质控血清，2瓶(液体)，含低、高两个剂量。
操作程序：将圆底聚苯乙烯试管编号(按复管操作)，按下表程序操作
加样测定程序表 单位：μL
数据处理：以γ计数仪预先编制的程序直接给出有关参数、校准曲线及样品浓度。
实验结果
3.1对围绝经期模型小鼠脏器指数的影响
葱籽总黄酮对围绝经前综合征模型小鼠胸腺、脾脏、子宫指数的影响见表1。
表1葱籽总黄酮对围绝经前综合征模型小鼠胸腺、脾脏、子宫指数的影响
注：与模型组比较，*P<0.05，**P<0.01
从上表可看出，与空白对照组比，模型组小鼠的胸腺指数、脾脏指数和子宫指数均显著降低(P<0.01)，说明摘除卵巢导致围绝经期模型小鼠胸腺、脾脏及子宫的萎缩。与模型组比，葱籽总黄酮大、小剂量组和更年安胶囊组均可显著提高模型小鼠胸腺指数(P<0.01)、可显著提高模型小鼠脾脏指数(P<0.01)，葱籽总黄酮大剂量组和更年安胶囊组均可显著提高模型小鼠子宫指数(P<0.01)。
3.2对围绝经期模型小鼠E2水平的影响
葱籽总黄酮对围绝经前综合征模型小鼠血清E2水平的影响见表2。
表2葱籽总黄酮对围绝经前综合征模型小鼠血清E2水平的影响
注：与模型组比较，*P<0.05，**P<0.01
从上表可看出，与空白对照组比，模型组小鼠血清中E2水平显著下降(P<0.01)，说明摘除卵巢导致围绝经期模型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，葱籽总黄酮大剂量组和更年安胶囊组均可显著提高模型小鼠血清E2水平(P<0.01)。
3.3对围绝经期模型小鼠子宫组织形态的影响
空白组小鼠子宫黏膜层、肌层、浆膜层正常，黏膜层中上皮细胞、粘膜腺均正常。模型组小鼠子宫黏膜层、肌层、浆膜层明显萎缩，黏膜层中上皮细胞、粘膜腺均出现萎缩并减少。更年安胶囊组小鼠子宫黏膜层、肌层、浆膜层得到一定程度的恢复，黏膜层中上皮细胞、粘膜腺也得到一定恢复。葱籽总黄酮大剂量组小鼠子宫黏膜层、肌层及浆膜层均明显得到恢复，黏膜层中上皮细胞、粘膜腺明显得到恢复。葱籽总黄酮小剂量组小鼠子宫黏膜层、肌层及浆膜层恢复不明显，黏膜层中上皮细胞及粘膜腺恢复不明显。
采用测微尺测定子宫内膜和肌层厚度的最宽处及最窄处，求均数，用计数方法，计算内膜中腺体数量，求均数，测的结果见表3。
表3葱籽总黄酮对围绝经前综合征模型小鼠子宫内膜、肌层和内膜中腺体的影响
注：与模型组比较，*P<0.05，**P<0.01
从上表可看出，与空白对照组比，模型组小鼠子宫内膜厚度、肌层厚度显著减少(P<0.01)，腺体个数显著降低(P<0.01)，说明小鼠造围绝经期模型后子宫萎缩，腺体减少。与模型组比，葱籽总黄酮大剂量组和更年安胶囊组均可显著增加模型小鼠子宫内膜厚度(P<0.01)；葱籽总黄酮大、小剂量组有增加模型小鼠子宫肌层厚度的趋势(P>0.05)，更年安胶囊组均可显著增加模型小鼠子宫肌层厚度(P<0.01)；葱籽总黄酮大剂量组可明显增加模型小鼠子宫内膜中腺体数量(P<0.05)，更年安胶囊组均可显著增加模型小鼠可子宫内膜中腺体数量(P<0.01)。
应用葱籽总黄酮用于治疗绝经前综合征的药物(胶囊)，经78例绝经前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，2-3粒，每粒含葱籽总黄酮0.5g，连服60天。除1人外，临床症状消失或临床症状有明显改善或改善者77人，有效率高达98.7％，效果之好是未曾料到的，是治疗绝经前综合征药物上的创新，开拓了葱籽的新用途，经济和社会效益显著。
Claims (1)
1.一种葱籽总黄酮作为唯一活性部位在制备治疗绝经前综合征药物中的应用，该葱籽总黄酮是将葱籽粉碎成过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，葱籽药渣挥干石油醚，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，得葱籽总黄酮。
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