CN113041336A - 治疗卵巢早衰的中药组合物及其制备方法、应用 - Google Patents
治疗卵巢早衰的中药组合物及其制备方法、应用 Download PDFInfo
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Abstract
本发明属于中药技术领域,具体公开一种治疗卵巢早衰的中药组合物,其按重量份数计由以下原料制成:附子6‑10份、肉桂6‑10份、鹿角胶3‑8份、熟地8‑24份、山药12‑30份、山茱萸6‑10份、枸杞子10‑15份、杜仲10‑15份、菟丝子12‑25份、当归9‑15份、续断10‑15份、桑寄生10‑15份、炮姜6‑10份、川芎10‑15份、淫羊藿6‑10份、巴戟天6‑10份、香附6‑10份、炙甘草6‑10份。本发明提供的治疗卵巢早衰的中药组合物,能够有效抑制颗粒细胞的凋亡,促进卵泡发育成熟,提升卵巢储备力,且不良反应小。
Description
技术领域
本发明属于中药技术领域,具体公开一种治疗卵巢早衰的中药组合物及其制备方法、应用。
背景技术
卵巢早衰(POF)指女性在40岁以前因卵巢卵泡耗竭引起的闭经、不孕、雌激素过少和促性腺激素水平升高等临床症状。目前POF发病率为1%~3%,呈逐年升高且低龄化趋势,甚至在<20岁女性中的发病率高达0.1%,严重影响女性生殖健康及身心健康。随着国家二胎政策实施,临床上有二胎意愿的女性骤增,许多年轻女性虽然处于生育年龄,但多种原因引起卵巢储备功能低下或卵巢早衰,导致女性生殖功能低于同龄正常水平。因此,保护和恢复女性生育年龄应有的生殖功能是临床亟待解决的难题。
现代医学治疗卵巢早衰主要采用激素替代疗法,虽然有效,但无法根治,停药后往往不能重新恢复卵巢功能,且使患乳腺癌、子宫内膜癌、血栓性疾病的风险增高。研究证实,中医药治疗卵巢早衰在改善临床症状、调节激素水平及机体细胞免疫作用等方面有明显优势。
发明内容
本发明提供的治疗卵巢早衰的中药组合物,不良反应小,且能够从根本上改善患者的体质,恢复卵巢功能,在临床治疗卵巢早衰方面取得良好效果;且在实验中已证明,该中药组合物能够有效抑制颗粒细胞的凋亡,促进卵泡发育成熟,提升卵巢储备力,为临床上治疗卵巢早衰提供治疗、理论依据以及新的治疗思路。
本发明提供的治疗卵巢早衰的中药组合物,其按重量份数计由以下原料制成:附子6-10份、肉桂6-10份、鹿角胶3-8份、熟地8-24份、山药12-30份、山茱萸6-10份、枸杞子10-15份、杜仲10-15份、菟丝子12-25份、当归9-15份、续断10-15份、桑寄生10-15份、炮姜6-10份、川芎10-15份、淫羊藿6-10份、巴戟天6-10份、香附6-10份、炙甘草6-10份。
优选地,其按重量份数计由以下原料制成:附子6份、肉桂6份、鹿角胶6份、熟地24份、山药12份、山茱萸9份、枸杞子12份、杜仲12份、菟丝子12份、当归9份、续断12份、桑寄生12份、炮姜10份、川芎10份、淫羊藿10份、巴戟天10份、香附10份、炙甘草6份。
本发明还提供一种上述中药组合物的制备方法,包括以下步骤:称取附子、肉桂、熟地、山药、山茱萸、枸杞子、杜仲、菟丝子、当归、续断、桑寄生、炮姜、川芎、淫羊藿、巴戟天、香附、炙甘草,混合,加水煎煮,收集煎煮液,加入烊化的鹿角胶,即得所述中药组合物。
优选地,所述加水煎煮的具体过程为:取附子加入相当于其重量4-6倍量的沸水先煎30-60min,其他药材用相当于药材总重量10-12倍量的水浸泡30-60min后,与附子煎煮液合并煎煮1-2h,过滤,得第一次滤液;滤渣再加入6-8倍量的水煎煮40-60min,过滤,得第二次滤液,合并两次滤液,即得所述煎煮液。
优选地,所述加水煎煮的具体过程为:取附子加入相当于其重量6倍量的沸水先煎40min,其他药材用相当于药材总重量10倍量的水浸泡30min后,与附子煎煮液合并煎煮1h,过滤,得第一次滤液;滤渣再加入8倍量的水煎煮40min,过滤,得第二次滤液,合并两次滤液,即得所述煎煮液。
上述中药组合物能够进一步被制备成药学上可接受的中药制剂,如颗粒剂、胶囊剂、片剂、糖浆剂等口服制剂,或经静脉、肌肉、腹腔给药的注射剂等。
本发明还提供一种上述组合物在制备治疗肾阳虚型卵巢早衰的药物中的应用。
卵巢早衰属于中医“经水早断”、“血枯经闭”、“不孕”等范畴,基于“肾主生殖”理论,临床认为卵巢早衰的基本病机多属肾虚,常见肾阴虚证、肾阳虚证,而肾阳是肾中阳气,为一身阳气之本,来源于先天,滋养充盛于后天。肾阳鼓动体内气机,滋养五脏阳气,温养阴血、肾精,具有温煦胞宫、温通胞脉,鼓动成熟卵子排出的作用,是推动生殖之精发育的原动力。因此,补肾助阳对月经周期的发生发展起着重要的促进作用。
本发明提供的中药组方适用于治疗肾阳不足、命门火衰引起的卵巢早衰,患者表现为月经后期、量少甚至闭经,婚久不孕,伴有腰膝痠冷、精神不振、怯寒畏冷、大便溏薄、尿频而清。功效为温补肾阳、填精助孕。右归丸为温补肾阳的基础方,但对于肾阳不足、命门火衰引起的卵巢早衰,补肾气和温补力量不足,而且没有补而不滞的理血之药。本发明提供的中药组方中加入了补肾促生殖的续断、桑寄生,以促进卵巢生殖功能的尽快恢复;加入淫羊藿、巴戟天,加大了促排卵、暖宫助孕的作用;肾阳虚多伴有脾阳虚,后天气血的化生有赖脾胃运化,故又加入了炮姜助温补脾胃之力;加入川芎、香附理气行气,使气血通畅,冲任满溢;加入炙甘草加强健脾益气,又为使药,起到调合诸药的作用。
本发明提供的中药组方中肉桂、附子辛甘大热,善补火助阳、引火归元;鹿角胶甘咸性温,血肉有情,善壮肾阳、益精血,三药相合,既温补肾阳,又填精益髓故共为君药;杜仲、续断、桑寄生,善补肝肾、强腰膝;菟丝子、淫羊藿、巴戟天,补肾助阳;山茱萸酸甘微温,温补肝肾;熟地黄甘补质润微温,善滋阴填精益髓;枸杞子甘平质润,善滋阴补肾、兼助肾阳,九药合用,阴阳双补,辅助君药温肾填精、固精止遗,故共为臣药;当归、川芎、香附辛散甘补温通,善补血行血活血,以求精血互生,气血流畅;山药、炮姜,能益气养阴、健脾补肾。五药相合,助君臣药补阴血,理气血、温脾胃,故共为佐药;炙甘草加强健脾益气,又为使药,起到调合诸药的作用。全方配伍,共奏温补肾阳、填精助孕之功,善治肾阳不足、命门火衰所致的腰膝痠冷、精神不振、怯寒畏冷、阳痿遗精、大便溏薄、尿频而清等,及月经后期、量少甚至闭经,婚久不孕。
附图说明
图1是空白对照组大鼠卵巢组织形态图;
图2是模型对照组大鼠卵巢组织形态图;
图3是高剂量组大鼠卵巢组织形态图;
图4是中剂量组大鼠卵巢组织形态图;
图5是低剂量组大鼠卵巢组织形态图;
图6是右归丸组大鼠卵巢组织形态图;
图7是空白对照组大鼠子宫组织形态图;
图8是模型对照组大鼠子宫组织形态图;
图9是高剂量组大鼠子宫组织形态图;
图10是中剂量组大鼠子宫组织形态图;
图11是低剂量组大鼠子宫组织形态图;
图12是右归丸组大鼠子宫组织形态图。
具体实施方式
下面通过实施例进一步描述本发明,但是本发明不受这些实施例的限制。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
下述实施例中1g表示一个重量份。
实施例1
一种治疗卵巢早衰的中药组合物,其由以下原料制成:附子6g、肉桂6g、鹿角胶6g、熟地24g、山药12g、山茱萸9g、枸杞子12g、杜仲12g、菟丝子12g、当归9g、续断12g、桑寄生12g、炮姜10g、川芎10g、淫羊藿10g、巴戟天10g、香附10g、炙甘草6g。
实施例2
一种治疗卵巢早衰的中药组合物,其由以下原料制成:附子6g、肉桂6g、鹿角胶3g、熟地8g、山药12g、山茱萸6g、枸杞子10g、杜仲10g、菟丝子12g、当归9g、续断10g、桑寄生10g、炮姜6g、川芎10g、淫羊藿6g、巴戟天6g、香附6g、炙甘草6g。
实施例3
一种治疗卵巢早衰的中药组合物,其由以下原料制成:附子10g、肉桂10g、鹿角胶6g、熟地24g、山药30g、山茱萸10g、枸杞子15g、杜仲15g、菟丝子25g、当归15g、续断15g、桑寄生15g、炮姜10g、川芎15g、淫羊藿10g、巴戟天10g、香附10g、炙甘草10g。
实施例4
一种治疗卵巢早衰的中药组合物,其由以下原料制成:附子10g、肉桂6g、鹿角胶8g、熟地10g、山药25g、山茱萸8g、枸杞子15g、杜仲15g、菟丝子20g、当归15g、续断12g、桑寄生15g、炮姜8g、川芎12g、淫羊藿10g、巴戟天8g、香附10g、炙甘草10g。
实施例5
一种治疗卵巢早衰的中药组合物,其由以下原料制成:附子8g、肉桂6g、鹿角胶8g、熟地8g、山药25g、山茱萸10g、枸杞子12g、杜仲15g、菟丝子20g、当归15g、续断12g、桑寄生12g、炮姜8g、川芎12g、淫羊藿10g、巴戟天8g、香附8g、炙甘草10g。
实施例6
一种治疗卵巢早衰的中药组合物,其由以下原料制成:附子10g、肉桂6g、鹿角胶6g、熟地8g、山药25g、山茱萸10g、枸杞子12g、杜仲10g、菟丝子20g、当归12g、续断12g、桑寄生12g、炮姜6g、川芎12g、淫羊藿6g、巴戟天10g、香附8g、炙甘草8g。
上述各实施例提供的中药组合物的制备方法,包括以下步骤:称取附子、肉桂、鹿角胶、熟地、山药、山茱萸、枸杞子、杜仲、菟丝子、当归、续断、桑寄生、炮姜、川芎、淫羊藿、巴戟天、香附、炙甘草,先取附子加入相当于其重量6倍量的沸水先煎60min,其他除鹿角胶之外的药材加入相当于药材总重量10倍量的水浸泡30min后,与附子煎煮液合并煎煮1h,过滤,得第一次煎煮液;滤渣再加入8倍量的水煎煮40min,过滤,得第二次煎煮液,合并两次煎煮液,加入烊化的鹿角胶,即得该中药组合物。
实施例7
一种治疗卵巢早衰的中药组合物,与实施例1的不同在于制备方法不同,具体为:称取附子、肉桂、鹿角胶、熟地、山药、山茱萸、枸杞子、杜仲、菟丝子、当归、续断、桑寄生、炮姜、川芎、淫羊藿、巴戟天、香附、炙甘草,先取附子加入相当于其重量4倍量的沸水先煎30min,其他除鹿角胶之外的药材加入相当于药材总重量12倍量的水浸泡40min后,与附子煎煮液合并煎煮2h,过滤,得第一次煎煮液;滤渣再加入6倍量的水煎煮60min,过滤,得第二次煎煮液,合并两次煎煮液,加入烊化的鹿角胶,即得该中药组合物。
由于上述实施例1-7提供的中药组合物的治疗效果基本相同,故以下仅以实施例1制备得到的中药组合物为例进行效果说明。
实验方案
1.1主要试剂
雌激素试剂盒、促卵泡刺激素试剂盒、孕酮放免试剂盒、促黄体生成激素放免试剂盒、睾酮放免试剂盒、10%的水合氯醛、4%多聚甲醛、10%中性福尔马林固定液、0.9%生理盐水、苏木素伊红染色液等。
1.2主要测试仪器
精密轮转切片机(RM2135)、DP70数码显微照相系统、BX-51数码显微镜、Image-ProPlus5.1型图像分析系统、GIS-120D数码凝胶成像分析系统、标准规格酶标仪、生物组织包埋机、电子天平(MP-120-2)、37℃恒温箱、超低温冰箱MDF-382EN型、高速冷冻离心机TGL-16G-A型、Sysmex XE-2100全自动血液分析仪和配套试剂。
1.3、动物来源
选用SPF级健康雌性12周龄SD大鼠60只,体重(250±20)g;实验动物由西安交通大学实验动物中心提供。
1.4、动物管理
实验在陕西中医药大学医学实验中心进行。饲养动物前,笼具、笼架、饮水瓶均用质量分数2%NaOH浸泡消毒并清洗,鼠舍关闭口窗,用甲醛和高锰酸钾混合进行熏蒸消毒。饲养环境温度控制在20-25℃,相对湿度为50-70%,光照、黑暗各12h(8:00-20:00)。所有大鼠适应性正常喂养7天,此时采用足量饲料喂食,自由饮水。每日08时阴道脱落细胞涂片,连续观察10天,给每只大鼠建立动情周期档案。每日10时称体重,每天观察大鼠精神和健康状态,隔天清洗托粪盘,发现异常隔离观察并记录。
1.5、SD大鼠卵巢早衰造模
入组标准:动物购入后适应性正常喂养7天,连续10天做阴道脱落细胞涂片,建立动情周期档案,选择动情周期规律的SD雌性大鼠60只纳入实验。
造模方法:每日09时腹腔注射环磷酰胺(CTX),首次负荷剂量50mg/kg,继以8mg/kg的剂量连续腹腔注射14天,造模共15天。
1.6、实验分组
实验用大鼠共计60只,分为6组。
选取正常健康SD大鼠10只,记为空白对照组;
选取造模成功的SD卵巢早衰大鼠50只,随机分成5组,每组10只,即模型对照组、高剂量组、中剂量组、低剂量组、阳性对照组。
1.7、给药剂量及灌胃方法
1.7.1实验药物来源
将实施例1制备得到的中药组合物,常压浓缩至不同的浓度,其中高剂量组浓度为5g/mL、中剂量组浓度为2.5g/mL、低剂量组浓度为1.25g/mL。
阳性对照组:使用右归丸,每丸重9g,丸剂,北京同仁堂股份有限公司,国药准字Z11021040。根据人和大鼠体表面积换算的等效剂量比率表计算,右归丸药物剂量:27g/60kg×6=2.7g/kg/d,以质量分数0.9%生理盐水配成混悬液备用。
1.7.2给药方法
1.7.2.1本发明实施例1提供的中药组合物高、中、低剂量组
根据《试验动物学》,一般大鼠灌胃的容积0.2-1ml/100g。因中药复方固形物含量高,太浓灌胃针不容易抽取,故给予大鼠给药容积:1ml/100g。
高剂量组:造模结束后24h,灌胃,1ml/100g/d,连续灌服20天;
中剂量组:造模结束后24h,灌胃,1ml/100g/d,连续灌服20天;
低剂量组:造模结束后24h,灌胃,1ml/100g/d,连续灌服20天。
1.7.2.2阳性对照组
造模结束后24h,右归丸2.7g/kg/d灌胃,连续灌服20天。
1.7.2.3模型对照组
造模结束后24h,生理盐水1ml/100g/d灌胃,连续灌服20天。
1.7.2.4空白对照组
生理盐水1ml/100g/d灌胃,连续灌服20天。
1.8、标本采集与处理
于末次给药后禁食过夜,次日上午9:00给予质量分数10%水合氯醛腹腔注射麻醉(剂量为0.3ml/100g),掐尾确定麻醉完全。用手术器械打开腹腔,将脏器拨到一边,轻轻撕开腹后膜,然后用棉球朝同一方向轻轻擦开脂肪及结缔组织,充分暴露腹主动脉。取一次性采血针,针尖朝下,沿血管方向平放刺入约1cm后固定不动以防戳破血管,由其他组员协助将采血针另一端插入采血管取血。采得血样室温下静置1h,然后3000rpm、4℃离心管离心15min,分装上层血清,封口膜封口,置-20℃冰箱保存待测。另取双侧卵巢及子宫(注意除净输卵管及器官周围的结缔组织),将左侧子宫及卵巢放入质量分数4%多聚甲醛固定液中固定,其余样本置于-80℃超低温冰箱保存。
1.9、实验观察指标
1.9.1一般状态
每日10时详细观察并记录每只大鼠体重、毛色光泽、精神状态、活动度、饮食状况、大小便等情况。
1.9.2阴道上皮脱落细胞角化指数的观察
实验期间每日8时行大鼠阴道上皮脱落细胞涂片观察。
具体方法:制作纤细棉签,用生理盐水浸湿,插入大鼠阴道顺时针方向旋转一周,取脱落细胞,将采样棉签在清洁载玻片上反时针旋转一周涂片,涂片立即放入质量分数95%乙醇液中固定,15分钟后作伊红染色1小时,清水漂洗10分钟后自然晾干,在普通光学显微镜下观察各细胞形态及数目以确定大鼠所处的动情周期阶段:动情前期——大量上皮细胞,胞浆呈粒状,少量角化(无核)细胞,无白细胞;动情间期——大量多核白细胞,少量上皮细胞;动情期——大量角化细胞,形状大而不规则,尚有少量上皮细胞;动情后期——大量白细胞,尚有融合的角化细胞。光镜下观察成熟角化细胞百分比,以每200个脱落细胞中成熟角化细胞的数目所占百分比计算角化细胞指数,比较同一动情时期的角化指数。
1.9.3大鼠血清性激素检测
采用双抗体夹心酶联免疫吸附技术(ELISA),于末次给药后禁食过夜,次日上午九点大鼠称重后,以质量分数10%水合氯醛(剂量为0.3ml/100g)腹腔注射麻醉,从腹主动脉取血5ml于含有促凝集的真空生化管中,以3000r/min速度离心20min,分离上层血清,置于-20℃以下保存待测。检测步骤严格按照放射免疫试剂盒说明进行,详细记录其数值。具体步骤:
(1)将所需各试剂及样本放置室温,平衡至少30min以上再进行操作;
(2)取60×12mm聚苯乙烯管并分别编号,所有的检测指标都做双管重复;
(3)取每一个浓度的的标准品、质控血清和样本200ul加入相应的编号试管中;
(4)各管分别添加200ul标记物工作液;
(5)T管和NSB管除外,其余各管均加入200ul抗体工作液,并充分混匀;
(6)在16-25℃的室温下放置过夜12-16小时;
(7)每管各加入500u1分离剂,然后充分混匀;
(8)室温放置5-l0min后,以3500r/min离心20min,吸去上清液;
(9)用y一计数器测定各管沉淀物放射性计数CPM,检测时间:1min;
(10)在标准曲线上读取样本及质控血清的浓度。
1.9.4大鼠血清AMH检测:
末次给药后禁食24h,所有大鼠称量体重,仍按体重计算麻醉药物(质量分数10%的水合氯醛)剂量给予麻醉,然后经腹主动脉采血,用试管收集每只大鼠血液约8~10ml,静置,待血液凝固后以3000r/min速度离心20min,随后用移液枪分装血清,并放置冰箱内(-20℃)保存备检,用于酶联免疫吸附法(ELISA法)检测血清AMH水平。
(1)实验前从冰箱中取出ELISA试剂盒及大鼠血清,在室温(20-25℃)的环境下放置约20min,待用。
(2)试剂准备:
a.用双蒸水稀释浓缩洗涤液(25:1)。使用时将洗涤液放置室温,并当日使用,未用完的放回4℃冰箱保存;
b.标准品:将标准品稀释液1.0ml滴入冻干标准品中,盖紧管帽,并反复颠倒,静置8~10min,待冻干标准品充分溶解,此时浓度为10ng/ml。然后根据实验需要将其倍比稀释,标准品稀释液可作为空白孔0ng/ml,余孔溶度分别为:10、5、2.5、1.25、0.625、0.313、0.156ng/ml;
c.生物素化抗体工作液:实验开始前20min,按1:100的比例稀释浓缩生物素化抗体;
d.酶结合物工作液:实验开始前20min,按1:100的比例稀释浓缩辣根过氧化物酶(HRP)结合物。
(3)操作步骤:
a.加样:将标准品稀释液100μl滴入空白孔,余孔各滴入标准品或待测样品100μl,注意不要产生气泡,轻轻晃动,混合均匀。贴上酶标板覆膜,放入37℃孵育90min;
b.倒掉酶标板内液体,并甩干,无需洗涤。向每个孔内滴加生物素化抗体工作液100μl,37℃温育约1小时;
c.温育1小时完成后洗板3次,方法:甩尽孔中液体,并在干净的吸水纸上轻轻拍干,每孔滴入洗涤液350μl,静置1-2min后,再次甩掉酶标板中的液体;
d.每孔滴入酶结合物工作液100μl,37℃温育约30min后,洗板5次;
e.每孔滴入底物溶液(TMB)100μl,37℃避光孵育0min左右(可根据试验时的实际显色状况酌情缩短或延长孵育时间,但不能超过30min。当标准孔显现出较为明显的色差时,终止孵育);
f.滴加终止液50μl,此时蓝色转为黄色;
g.用酶标仪在450nm波长测定每孔的光密度(OD值)。
1.9.5脏器指数
大鼠采血处死后,立即寻找子宫、卵巢,分别剥离周围脂肪组织,于宫颈上方完全剪下子宫体,剪离双侧卵巢。滤纸稍拭后,电子分析天平称出子宫湿重及双侧卵巢湿重,子宫重量法将子宫湿重换算为子宫指数,子宫指数=子宫湿重(mg)/大鼠体重(g)×100%。卵巢重量法将卵巢湿重换算为卵巢指数,卵巢指数=卵巢湿重(mg)/大鼠体重(g)×100%。称重后将子宫、卵巢用质量分数4%的多聚甲醛固定液固定。
1.9.6大鼠卵巢组织病理学观察
大鼠麻醉后,固定头部及四肢,下腹部剃毛,常规消毒,迅速摘取左侧卵巢及子宫,分离卵巢子宫及其周围脂肪和其他组织,左侧卵巢经精密电子称湿重、记录数据,常规固定、脱水、石蜡包埋、切片、HE染色,在400倍光镜下观察卵巢闭锁卵泡的形态,各级卵泡(原始卵泡、初级卵泡、次级卵泡、成熟卵泡)形态、数目、大小,颗粒细胞数目,黄体及间质等的组织学变化。具体步骤如下:
(1)固定:大鼠卵巢称重后,置于4%多聚甲醛溶液中固定,6~8h取出;
(2)酒精梯度脱水:70%乙醇60min→80%乙醇60min→95%乙醇(Ⅰ)60min→95%乙醇(Ⅱ)60min→无水乙醇(Ⅰ)60min→无水乙醇(Ⅱ)60min;
(3)透明:(50%无水乙醇+50%二甲苯)60min→二甲苯(Ⅰ)60min→二甲苯(Ⅱ)60min;
(4)渗透:(50%二甲苯+50%石蜡)90min→石蜡(Ⅰ)120min→石蜡(Ⅱ)120min;
(5)包埋:将组织放入包埋框的中央,用加热后的液体石蜡进行包埋,包埋后置于冷却台上冷却;
(6)切片:将完全凝固的石蜡块置于切片机中,连续切取5μm左右厚度切片,取最大卵巢切面者贴片;
(7)贴片:切片放56℃水中烫平,用防脱载玻片捞取,编号,在45℃恒温箱中烘干。
(8)脱蜡、水洗:二甲苯(Ⅰ)10min→二甲苯(Ⅱ)10min→(50%二甲苯+50%无水乙醇)2min→无水乙醇(Ⅰ)5min→无水乙醇(Ⅱ)5min→95%乙醇5min→80%乙醇5min→75%乙醇5min→蒸馏水5min;
(9)HE染色:苏木素5min→自来水冲洗30s→1%盐酸分化5s→自来水冲洗30s→1%伊红1min→自来水冲洗30s;
(10)脱水:75%乙醇5min→80%乙醇5min→95%乙醇(Ⅰ)5min→95%乙醇(Ⅱ)5min→无水乙醇(Ⅰ)5min→无水乙醇(Ⅱ)5min;
(11)透明:二甲苯(Ⅰ)2min→二甲苯(Ⅱ)2min;
(12)封片:将每个载玻片上滴加中性树胶,用盖玻片封片,置通风橱使其自然干燥;
(13)在带有医学图像分析系统的光学显微镜下观察大鼠卵巢组织切片,照相,并计录结果。
1.9.7大鼠子宫病理组织学观察
大鼠麻醉后取左侧子宫,取材、称重、固定、脱水、透明具体方法步骤同左侧卵巢,切取左侧子宫中段0.5cm长度子宫组织进行石蜡包埋、切片、HE染色,具体方法亦同左侧卵巢,在400倍光镜下观察大鼠子宫壁厚度、内膜、肌层及浆膜层结构、腺体及腺腔大小、腔内分泌物、间质细胞与蜕膜样细胞分布等。
1.10实验数据的统计处理
1.11结果分析
1.11.1一般状态
空白对照组:毛发浓密有光泽,行动自如,反应灵敏,大小便正常。
模型对照组:大鼠眼眶凹陷,精神萎靡,皮毛干枯,稀疏变硬,无光泽感,活动明显减少,反应迟钝,烦躁不安,拱背呲牙,竖毛怕人,嗜睡易惊,饮食减少,大便或干结色黑,或大便稀溏,小便色黄等。
高、中、低剂量组与右归丸组:大鼠一般状态较模型对照组有不同程度的改善,其中以高、中剂量组症状缓解最明显,其次为右归丸组,低剂量组症状缓解不明显。
以上结果表明:POF严重影响大鼠的整体机能,各治疗组通过药物干预后可在不同程度上改善大鼠卵巢早衰症状。
1.11.2大鼠体重变化
结果见表1。
组别 | n | 体重(g) |
空白对照组 | 10 | 250.9±8.7 |
模型对照组 | 10 | 222.2±8.3<sup>△△</sup> |
低剂量组 | 9 | 233.1±13.1<sup>★</sup> |
中剂量组 | 8 | 243.3±10.5<sup>★★</sup> |
高剂量组 | 10 | 240.1±15.2<sup>★★</sup> |
右归丸组 | 8 | 237.4±14.5<sup>★</sup> |
注:n代表总数。大鼠在造模及药物干预过程中,因大鼠对抗性增加,同组之间撕咬明显及CTX对大鼠机能的损伤,共有5只大鼠自然耗损。与空白组比:△△P<0.01;与模型组比:★P<0.05,★★P<0.01.
通过SPSS 22分析,由表1可知:与空白组比,模型组大鼠体重下降显著(P<0.01)。与模型组比,低剂量组与右归丸组大鼠体重有所上升((P<0.05);高、中剂量组大鼠体重增加显著(P<0.01);各药物干预组之间无相关性(P>0.05)。由此可见,CTX致POF严重影响大鼠体重水平,药物治疗后大鼠体重的逐步增加,可为临床治疗POF提供依据。
1.11.3大鼠动情周期变化
通过电子显微镜结果可见:实验前各组大鼠动情周期规律,动情周期4-5日;造模后各组大鼠动情周期全部紊乱,可见持续的动情间期或动情周期无明显的规律性。结果见表2。
组别 | n | N | n/N(%) |
空白对照组 | 10 | 10 | 100% |
模型对照组 | 0 | 10 | 0%<sup>△△</sup> |
低剂量组 | 2 | 9 | 22% |
中剂量组 | 4 | 8 | 50%<sup>★</sup> |
高剂量组 | 5 | 10 | 50%<sup>★★</sup> |
右归丸组 | 4 | 8 | 50%<sup>★</sup> |
注:n为动情周期规律的数量;N代表总数。与空白组比:△△P<0.01;与模型组比:★P<0.05,★★P<0.01.
通过SPSS 22分析,运用卡方检验方法,由表2可知:与空白组比,模型组恢复动情周期大鼠的数量有显著统计学意义(P<0.01)。与模型组比,低剂量组无意义(P>0.05);中剂量组与右归丸组恢复动情周期大鼠的数量有统计学意义(P<0.05);高剂量组恢复动情周期大鼠的数量有显著统计学意义(P<0.01);各药物干预组之间无差异性(P>0.05)。由此可见,使用化疗药物环磷酰胺致动情周期紊乱严重影响卵巢储备功能;给予本发明药物治疗后可逐步恢复动情周期,提高大鼠卵巢储备能力,改善POF大鼠临床症状。
1.11.4大鼠血清FSH、LH、E2、P、T、PRL水平变化
通过CTX进行POF大鼠造模,造模成功后,大鼠血清FSH、LH、T值水平明显升高,血清E2、P值水平显著降低,与临床POF患者出现高促性腺激素和低雌激素的表现相一致,提示POF大鼠造模成功,证明了腹腔注射环磷酰胺进行卵巢早衰造模的科学性。结果见表3-5。
组别 | n | FSH(IU/L) | LH(mIU/ml) |
空白对照组 | 10 | 15.18±4.25 | 40.85±12.58 |
模型对照组 | 10 | 27.55±3.39<sup>△△</sup> | 55.86±11.22<sup>△△</sup> |
低剂量组 | 9 | 23.04±4.51<sup>★</sup> | 48.87±10.42 |
中剂量组 | 8 | 21.18±3.77<sup>★★</sup> | 44.02±10.60<sup>★</sup> |
高剂量组 | 10 | 19.22±3.98<sup>★★</sup> | 42.11±10.53<sup>★★</sup> |
右归丸组 | 8 | 22.74±4.43<sup>★</sup> | 42.67±11.51<sup>★</sup> |
注:n代表总数。与空白组比:△△P<0.01;与模型组比:★P<0.05,★★P<0.01.
通过SPSS 22分析,由表3可知:与空白组比,模型组FSH、LH水平升高显著(P<0.01)。与模型组比,低剂量组与右归丸组FSH水平降低(P<0.05);中、高剂量组FSH水平显著下降(P<0.01);高剂量组LH水平显著下降(P<0.01);中剂量组与右归丸组LH水平下降(P<0.05);低剂量组LH水平稍有下降,但不具有统计学意义(P>0.05)。
组别 | n | E<sub>2</sub>(pmol/l) | P(ng/ml) |
空白对照组 | 10 | 82.73±11.53 | 15.22±4.36 |
模型对照组 | 10 | 62.61±12.62<sup>△△</sup> | 11.04±1.29<sup>△△</sup> |
低剂量组 | 9 | 73.66±9.00 | 14.11±2.92<sup>★</sup> |
中剂量组 | 8 | 77.90±17.64<sup>★</sup> | 15.15±2.65<sup>★★</sup> |
高剂量组 | 10 | 80.18±15.39<sup>★★</sup> | 15.08±3.11<sup>★★</sup> |
右归丸组 | 8 | 72.26±12.09 | 15.04±3.86<sup>★</sup> |
注:n代表总数。与空白组比:△△P<0.01;与模型组比:★P<0.05,★★P<0.01.
通过SPSS 22分析,由表4可知:与空白组比,模型组E2、P水平均显著下降(P<0.01)。与模型组比,低剂量组与右归丸组E2水平均稍有增加,但不具有统计学意义(P>0.05);中剂量组E2水平增加(P<0.05);高剂量组E2、P水平及中剂量组P值均显著上升(P<0.01);低剂量组与右归丸组大鼠血清P值上升(P<0.05)。
组别 | n | T(pg/ml) | PRL(ng/ml) |
空白对照组 | 10 | 143.94±24.68 | 19.28±3.86 |
模型对照组 | 10 | 165.45±22.17<sup>△△</sup> | 19.11±3.44 |
低剂量组 | 9 | 148.48±22.74 | 19.07±5.12 |
中剂量组 | 8 | 148.46±21.07 | 19.40±3.38 |
高剂量组 | 10 | 144.11±21.83<sup>★</sup> | 19.30±4.65 |
右归丸组 | 8 | 149.08±26.36 | 19.23±4.14 |
注:n代表总数。与空白组比:△△P<0.01;与模型组比:★P<0.05,★★P<0.01.
通过SPSS 22分析,由表5可知:与空白组比,模型组T水平升高显著(P<0.01);模型组PRL水平无明显差异性(P>0.05)。与模型组比,高剂量组T水平下降(P<0.05);其余各组T值均无明显差异性(P>0.05);高、中、低剂量组与右归丸组PRL水平无差异性,不具有统计学意义(P>0.05)。
综合表3-5可知:①大鼠血清FSH、LH水平均有所下降,血清FSH水平变化最为显著,其中,高剂量组大鼠血清FSH、LH水平下降最为显著,其次为中剂量组与阳性对照组,低剂量组大鼠血清FSH水平下降较好,血清LH水平下降较差;②血清E2水平以高剂量组上升最为显著,其次为中剂量组,低剂量组与阳性对照组虽有所上升,但上升效果不显著;血清P值以中、高剂量组上升最为显著,其次为阳性对照组及低剂量组;③高剂量组可降低大鼠血清T值,其余各组虽有部分降低,但降低效果不明显,无明显相关性;④大鼠血清PRL水平未见明显差异性或规律性,故可见卵巢早衰与血清PRL无明显相关性。
腹腔注射环磷酰胺造模大鼠性激素水平变化显著,同样,性激素水平的不同程度亦可反应卵巢生殖功能。造模后各治疗组给予药物干预后,大鼠性激素水平均有一定程度调节与改善,提示本发明药物可提高大鼠卵巢储备能力,改善卵巢生殖功能。
1.11.5大鼠血清AMH水平变化
AMH由卵巢卵泡颗粒细胞产生,是卵巢功能评估的准确标志物。实验结果表明:腹腔注射CTX进行大鼠POF造模可显著降低血清AMH水平,提示卵巢生殖功能降低。结果如表6所示。
注:n代表总数。与空白组比:△△P<0.01;与模型组比:★P<0.05,★★P<0.01.
通过SPSS 22分析,由表7可知:与空白组比,模型组AMH水平显著降低(P<0.01)。与模型组比,低剂量组AMH水平稍有上升,但无差异性(P>0.05);中剂量组AMH水平显著上升(P<0.01);高剂量组及右归丸组AMH水平升高(P<0.05)。由此可见,本发明提供的中药组合物可通过提高大鼠AMH水平,改善卵巢功能,提高卵巢生殖能力。
1.11.6大鼠脏器指数变化
结果见表7。
组别 | n | 卵巢指数 | 子宫指数 |
空白对照组 | 10 | 35.39±1.27 | 2.15±0.24 |
模型对照组 | 10 | 31.40±1.95<sup>△△</sup> | 1.83±0.21<sup>△△</sup> |
低剂量组 | 9 | 33.46±3.03 | 1.95±0.19 |
中剂量组 | 8 | 34.25±3.43<sup>★</sup> | 2.03±0.14<sup>★</sup> |
高剂量组 | 10 | 33.87±2.98 | 2.03±0.20<sup>★</sup> |
右归丸组 | 8 | 34.30±3.68<sup>★</sup> | 1.99±0.22 |
注:n代表总数。与空白组比:△△P<0.01;与模型组比:★P<0.05.
通过SPSS 22分析,由表7可知:与空白组比,模型组大鼠卵巢指数与子宫指数均显著下降(P<0.01)。与模型组比,中剂量组与右归丸组大鼠卵巢指数升高(P<0.05);低剂量组与高剂量组卵巢指数稍有升高,但无差异性(P>0.05);中、高剂量组子宫指数升高(P<0.05);低剂量组与右归丸组子宫指数稍有升高,但无差异性(P>0.05)。提示本发明提供的中药组合物可在一定程度上增加卵巢湿重与子宫湿重,为临床运用本发明组方治疗POF提供科学依据。
1.11.7大鼠卵巢组织病理学观察
通过电子光镜下观察,参考图1-6。结果显示:造模完成后,卵巢体积较空白组明显缩小,发育成熟卵泡少见,闭锁卵泡增加,颗粒细胞层排列紊乱疏松,且卵泡数目明显减少,黄体数量较少,问质可见炎细胞浸润及纤维化。各治疗组给予药物干预后,可见卵巢体积不同程度的增加,卵巢组织中各级卵泡数量增加,偶见成熟卵泡,闭锁卵泡及发育不良卵泡有所减少,黄体数量增加,颗粒细胞层排列逐渐有序趋于正常形态。具体为:
空白对照组:卵巢内皮质、髓质结构清晰,原始卵泡、初级卵泡和次级卵泡均同时存在,可见成熟卵泡,且成熟卵泡有丰富的卵泡液,颗粒细胞排列整齐,卵母细胞完整,透明带清晰。可见较多黄体,间质腺少见,各级血管丰富通畅。
模型对照组:卵巢体积明显缩小,卵泡数目明显较少,大部分卵泡有炎细胞浸润,少见发育成熟卵泡,闭锁卵泡增加,颗粒细胞层排列紊乱疏松,黄体数量较少,问质可见炎细胞浸润及纤维化。
低剂量组:卵巢体积小,可见少量各级卵泡,少数发育不良,偶见黄体,部分黄体可见脂肪变性,间质轻度增生,可见炎细胞浸润,血管细少迂曲。
中剂量组:卵巢体积略小,可见各级卵泡,数量较少,多数形态良好,部分卵泡发育不良,黄体数稍多,偶有间质轻度增生,血管尚丰富。
高剂量组:卵巢体积接近正常,可见各级卵泡,偶见卵泡发育不良,黄体数目较多,少数间质可见炎细胞浸润,血管形态及分布接近正常。
右归丸组:卵巢体积缩小,可见各级卵泡,多见闭锁卵泡,成熟卵泡少见,可见部分卵泡发育不良,黄体数量少,间质轻度纤维化及炎细胞浸润。
以上结果表明化疗药环磷酰胺破坏卵巢卵泡发育,POF后卵巢功能严重下降;本发明提供的中药组合物能一定程度上改善卵巢血流,促进卵泡发育,组织卵泡闭锁,提高卵巢生殖功能,为临床补肾促生殖治疗POF提供理论依据。
1.11.8大鼠子宫组织病理学观察
通过电子光镜下观察,参考图7-12。结果显示:与空白组相比,模型组大鼠子宫组织病理切片可见子宫内膜菲薄,腺体减少且腺腔变小,内膜上皮细胞排列紊乱无规则,基质稀疏水肿,可见嗜酸性粒细胞浸润。各治疗组给予药物干预后,大鼠子宫内膜出现不同程度的增厚,内膜上皮细胞较模型对照组排列规则,形态趋于正常,腺体及血管组织增多,结缔组织较疏松,水肿及嗜酸性粒细胞明显减少。具体为:
空白对照组:大鼠子宫内膜较厚,腺体数量多,上皮细胞复层化,细胞呈柱状、无异型性,可见分泌泡,结缔组织疏松。
模型对照组:大鼠子宫内膜菲薄,腺体减少,且腺腔变小,内膜上皮细胞排列紊乱无规则,基质紧致,可见嗜酸性粒细胞浸润。
低剂量组:大鼠子宫内膜薄,腺体数量较少,内膜上皮细胞结构紊乱,结缔组织较疏松,组织水肿明显,可见部分嗜酸性粒细胞浸润。
中剂量组:大鼠子宫内膜趋于正常,可见部分腺体及血管,内膜上皮细胞排列尚有序,结缔组织较疏松。
高剂量组:大鼠子宫内膜增厚,可见腺体及血管组织,内膜上皮细胞排列规则,结缔组织较疏松。
右归丸组:大鼠子宫内膜较薄,可见少量腺体组织,内膜上皮细胞排列稍有紊乱,结缔组织疏松水肿。
以上结果表明腹腔注射环磷酰胺严重损伤卵巢、子宫组织;本发明提供的中药组合物能在一定程度上丰富子宫血供,增加卵巢内卵泡对促性腺激素的反应性,提高卵泡质量,促进卵泡发育,改善子宫内膜状态,提高卵巢储备功能,改善卵巢组织损伤,促进大鼠恢复动情周期,从而达到治疗肾阳虚型卵巢早衰的作用。
以上公开的仅为本发明的具体实施例,但是,本发明实施例并非局限于此,任何本领域的技术人员能思之的变化都应落入本发明的保护范围。
Claims (7)
1.治疗卵巢早衰的中药组合物,其特征在于,其按重量份数计由以下原料制成:附子6-10份、肉桂6-10份、鹿角胶3-8份、熟地8-24份、山药12-30份、山茱萸6-10份、枸杞子10-15份、杜仲10-15份、菟丝子12-25份、当归9-15份、续断10-15份、桑寄生10-15份、炮姜6-10份、川芎10-15份、淫羊藿6-10份、巴戟天6-10份、香附6-10份、炙甘草6-10份。
2.根据权利要求1所述的治疗卵巢早衰的中药组合物,其特征在于,其按重量份数计由以下原料制成:附子6份、肉桂6份、鹿角胶6份、熟地24份、山药12份、山茱萸9份、枸杞子12份、杜仲12份、菟丝子12份、当归9份、续断12份、桑寄生12份、炮姜10份、川芎10份、淫羊藿10份、巴戟天10份、香附10份、炙甘草6份。
3.根据权利要求1所述的治疗卵巢早衰的中药组合物的制备方法,其特征在于,包括以下步骤:称取附子、肉桂、熟地、山药、山茱萸、枸杞子、杜仲、菟丝子、当归、续断、桑寄生、炮姜、川芎、淫羊藿、巴戟天、香附、炙甘草,混合,加水煎煮,收集煎煮液,加入烊化的鹿角胶,即得所述中药组合物。
4.根据权利要求3所述的治疗卵巢早衰的中药组合物的制备方法,其特征在于,所述加水煎煮的具体过程为:取附子加入相当于其重量4-6倍量的沸水先煎30-60min,其他药材用相当于药材总重量10-12倍量的水浸泡30-60min后,与附子煎煮液合并煎煮1-2h,过滤,得第一次滤液;滤渣再加入6-8倍量的水煎煮40-60min,过滤,得第二次滤液,合并两次滤液,即得所述煎煮液。
5.根据权利要求4所述的治疗卵巢早衰的中药组合物的制备方法,其特征在于,所述加水煎煮的具体过程为:取附子加入相当于其重量6倍量的沸水先煎40min,其他药材用相当于药材总重量10倍量的水浸泡30min后,与附子煎煮液合并煎煮1h,过滤,得第一次滤液;滤渣再加入8倍量的水煎煮40min,过滤,得第二次滤液,合并两次滤液,即得所述煎煮液。
6.根据权利要求3-5任一项所述的治疗卵巢早衰的中药组合物的制备方法,其特征在于,所述中药组合物能够进一步制备成药学上可接受的中药制剂。
7.根据权利要求1或2所述的中药组合物在制备治疗肾阳虚型卵巢早衰的药物中的应用。
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