CN110123851B - 沙棘籽油在治疗多囊卵巢综合征药物中的应用 - Google Patents
沙棘籽油在治疗多囊卵巢综合征药物中的应用 Download PDFInfo
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Abstract
本申请公开了沙棘籽油的新用途，即沙棘籽油在制备用于治疗多囊卵巢综合征(PCOS)的药物中的应用和沙棘籽油在制备用于治疗多囊卵巢综合征的保健食品中的应用。实验结果表明沙棘籽油对多囊卵巢综合征有治疗作用。沙棘籽油不仅可以改善PCOS小鼠卵巢多囊样病变，还可以使紊乱的动情周期正常化，增加卵巢储备，而且能有效降低PCOS小鼠的高血糖症状、减轻肥胖状态等。本申请对于直接利用沙棘籽油治疗多囊卵巢综合征提供了科学依据。
Description
技术领域
本申请涉及沙棘籽油用途技术领域，特别涉及沙棘籽油在治疗多囊卵巢综合征药物中的应用。
背景技术
多囊卵巢综合征(polycystic ovary syndrome，PCOS)是一种以胰岛素抵抗、雄激素增高和卵巢多囊为主要临床特征的内分泌疾病。PCOS所表现的高雄激素血症和卵巢功能障碍，常伴有糖脂代谢紊乱如高胰岛素血症和血脂异常，最终导致肥胖、2型糖尿病和心血管疾病的发生，PCOS患者患糖尿病的风险是健康人群的4倍。肥胖的PCOS患者则表现出更为严重的胰岛素抵抗、腹部脂肪沉积和高雄激素血症，超过60％的PCOS患者超重或肥胖，约70％存在胰岛素抵抗。
PCOS是育龄女性最常见的生殖内分泌紊乱性疾病之一，在我国的发病率为12.5％～15％，可导致女性不孕，严重危害女性健康。PCOS的病因尚不明确，其核心病理机制是胰岛素抵抗和高雄激素血症。主要临床表现为胰岛素抵抗、高雄激素血症、高脂血症、肥胖和慢性炎症等，并伴随异常月经、卵巢多囊样病变。因此，PCOS治疗的重点在于降低体重、改善胰岛素抵抗及高雄激素血症，恢复卵巢功能。
目前对于PCOS的治疗主要包括手术治疗和药物治疗。手术有卵巢打孔术，但因为其疗效差、并发症多，且会影响卵巢储备，目前已很少采用；药物治疗主要分为四方面的对症治疗：①高雄激素血症治疗——达英-35；②调整月经周期——口服避孕药，孕激素；③胰岛素抵抗治疗——二甲双胍；④促排卵治疗——枸橼酸氯米芬。但由于每种药物只是对症治疗，疗效欠佳，且大多为激素类药物，并发症较多，所以目前对于PCOS的治疗缺少有效的综合性治疗药物。
中药沙棘在我国具有悠久的药用历史，《四部医典》记载了沙棘可用于止咳、平喘、活血化瘀等作用。沙棘的活性成份有400多种，主要包括：维生素类、黄酮类化合物、三萜、甾体类化合物、油和脂肪酸、氨基酸和蛋白质、酚类及有机酸类、挥发油类、微量元素、磷脂类、5-羟色胺、糖类等物质。沙棘籽油由沙棘籽经过超临界萃取或亚临界低温萃取得到的棕黄色到棕红色透明油状液体，内含黄酮、有机酸、生物碱、甾醇类、三萜烯类、SOD及各种维生素(VC、VE等)和微量元素(锌、铬、锰、硒、镁、钙)等140多种生物活性成分。沙棘籽油作为保健食品原料，已在抗氧化、抗疲劳、护肝、降血脂等方面得到广泛运用；沙棘籽油作为药用原料，具有明显生物作用，其抗感染强、促进愈合快、被广泛用于治疗烧伤、烫伤、冻伤、刀伤等方面。沙棘籽油对扁桃体炎、口腔炎、结膜炎、角膜炎、妇科宫颈炎等都有良好而稳定的疗效。迄今为止，尚没有报道沙棘籽油对多囊卵巢综合征(生殖领域)有治疗作用。
发明内容
本申请公开沙棘籽油的新用途，即沙棘籽油在治疗多囊卵巢综合征药物中的应用，解决目前对于PCOS治疗缺少有效综合性治疗药物的问题。
根据本申请的实施例，提供沙棘籽油在制备用于治疗多囊卵巢综合征的药物中的应用；
根据本申请的实施例，提供沙棘籽油在制备用于治疗多囊卵巢综合征的保健食品中的应用。
进一步地，所述药物的剂型包括软胶囊、滴丸、乳剂、片剂、洗剂、搽剂、软膏剂或栓剂。
进一步地，所述保健食品的剂型包括软胶囊、滴丸、乳剂、片剂、洗剂、搽剂、软膏剂或栓剂。
本申请首次证实沙棘籽油具有完全不同于以上所述的新功能：沙棘籽油对多囊卵巢综合征有治疗作用。沙棘籽油不仅可以改善PCOS小鼠卵巢多囊样病变，还可以使紊乱的动情周期正常化，在内分泌代谢方面，还可以有效降低血糖、减轻肥胖状态等。该申请对于直接利用沙棘籽油治疗多囊卵巢综合征提供基础。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本申请的技术路线图；
图2为H&E染色镜下卵巢形态观察结果；
图3为显微镜下小鼠阴道分泌物观察结果；
图4为各组小鼠形态观察结果；
图5为各组小鼠体重变化数据图；
图6为各组小鼠体重增加比柱状图；
图7为各组小鼠血糖水平数据图；
图8为各组小鼠血糖水平AUC数据图；
图9为各组小鼠AMH表达结果。
其中，1-正常对照组，2-模型组(sham组)，3-阳性对照组，4-沙棘籽油组。
具体实施方式
本申请提供沙棘籽油在制备用于治疗多囊卵巢综合征的药物中的应用；
本申请提供沙棘籽油在制备用于治疗多囊卵巢综合征的保健食品中的应用。
进一步地，所述药物的剂型包括软胶囊、滴丸、乳剂、片剂、洗剂、搽剂、软膏剂或栓剂。
进一步地，所述保健食品的剂型包括软胶囊、滴丸、乳剂、片剂、洗剂、搽剂、软膏剂或栓剂。
本申请首次证实沙棘籽油具有完全不同于以上所述的新功能：沙棘籽油对多囊卵巢综合征有治疗作用。沙棘籽油增加卵巢储备功能的作用。沙棘籽油不仅可以改善PCOS小鼠卵巢多囊样病变，还可以使紊乱的动情周期正常化，在内分泌代谢方面，还可以有效降低血糖、减轻肥胖状态等。该申请对于直接利用沙棘籽油治疗多囊卵巢综合征提供基础。
为了证实沙棘籽油具有上述新功能，做了如下实验。
1.技术方案
(1)建立小鼠PCOS动物模型：
来曲唑是芳香化酶抑制剂，可以抑制卵巢雄激素向雌激素的转化，从而造成高雄激素血症。高脂喂养可以造成胰岛素抵抗。对ICR小鼠给予高脂喂养加灌胃给来曲唑，200ug/只/天，给予21天可以造成胰岛素抵抗及高雄激素血症的PCOS模型，动情周期紊乱，出现持续间期，血糖升高(此为业界公认的PCOS标准造模手段)。同时设置正常饲养的ICR小鼠一组，作为正常对照组。
(2)对小鼠PCOS动物模型给予干预：
本实验分为正常对照组、模型组(sham组)、二甲双胍(metformine)阳性对照组、沙棘籽油(Oil组)。在造模后第21天，确定造模成功后，正常对照组和模型组每日灌胃给予等量盐水，二甲双胍(metformine)阳性对照组每日灌胃给予50mg/kg(0.1ml)二甲双胍，沙棘籽油(Oil组)每日灌胃给予2.5ml/kg(0.1ml)沙棘籽油，共持续28天。
(3)实验结果监测及数据收集
每2天检测并记录各组小鼠体重、摄食及动情周期变化。
每7日检测禁食后血糖，并在给药处理后第27天给予葡萄糖耐量OGTT实验。
给药处理后第28天，取材，通过石蜡包埋剂切片染色技术，观察卵巢形态学改变。通过免疫组化技术检测AMH改变。
2.技术路线图
技术路线图如图1所示。
3.主要技术
1)建立小鼠PCOS动物模型
来曲唑是芳香化酶抑制剂，可以抑制卵巢雄激素向雌激素的转化，从而造成高雄激素血症。高脂喂养可以造成胰岛素抵抗。高脂喂养加灌胃给来曲唑，200ug/只/天，给予21天可以造成胰岛素抵抗及高雄激素血症的PCOS模型。
2)口服糖耐量实验(OGTT)
小鼠禁食16-18h，灌胃葡萄糖3g/kg体重，测定记录0min，15min，30min，60min，90min，120min时间点血糖。
3)阴道脱落细胞冲洗液图片法检测动情周期
用滴管吸取适量生理盐水，每隔2天，在同一时间点冲洗、吸取阴道分泌物于载玻片上，迅速于显微镜下观察细胞形态，判断动情周期。
4.实验结果
4.1沙棘籽油能治疗多囊卵巢综合征小鼠卵巢多囊样病变，改善卵巢形态
卵泡发育障碍及无排卵是PCOS患者生殖功能障碍的核心特征，PCOS患者卵泡发育障碍基本表现是卵泡募集数量过多，卵泡选择及优势化受阻，卵泡发育停滞，导致无排卵。病检结果显示卵巢呈多囊样改变，黄体数目显著减少，白膜增厚，白膜下见许多不同程度的卵泡及闭锁卵泡。卵巢白膜明显胶原化，呈板层状包绕卵巢，最终形成空泡样改变，导致排卵异常。
本申请发现沙棘籽油可以显著治疗PCOS小鼠卵巢多囊样病变，改善卵巢形态。
大体肉眼观察：正常对照组小鼠的卵巢形态正常，色泽红润；Sham组小鼠的卵巢偏大，表面苍白，卵巢表面可见单个或较多的囊性扩张卵泡。
H&E染色镜下形态观察结果如图2所示，正常对照组小鼠的卵巢在HE染色下可见多个不同发育阶段的卵泡及黄体，颗粒细胞形态完整且排列整齐，多为8～9层；Sham组小鼠的卵巢闭锁增多并且呈囊状扩张，可见多个初级卵泡及少量次级卵泡，成熟期的卵泡内未见卵细胞，可见颗粒细胞层数变薄，排列松散，部分脱落，卵泡膜细胞增生，卵泡内卵母细胞消失，黄体形成较少；沙棘籽油组和二甲双胍阳性对照组小鼠卵巢内均可见黄体和发育不同阶段的卵泡，接近成熟期的卵泡可见卵细胞及放射冠，颗粒细胞排列较紧密，层数增多，卵泡膜细胞层较薄，形态接近正常对照组。
4.2沙棘籽油可以显著恢复PCOS小鼠的动情周期
月经失调是多囊卵巢综合征患者最典型的临床表现之一，表现为月经稀发、月经量少甚至闭经。小鼠没有月经周期，表现为动情周期紊乱，出现持续动情间期。
本申请发现正常对照组保持规律的动情周期，模型组出现持续动情间期，不再出现动情期、动情前期和动情后期，而在给予二甲双胍的阳性干预对照组以及沙棘籽油组，在干预后6天开始逐渐恢复，直至干预后20天左右，均恢复动情周期，出现明显及规律的动情前期、动情期、动情后期表现。
本试验直观地证明沙棘籽油可有效改善PCOS，恢复小鼠正常动情周期。
如图3所示，从显微镜下观察小鼠阴道分泌物细胞组成可见：正常对照组(动情前期)可见在白细胞中分布梭形上皮细胞，即将进入动情器。模型组(sham组)(动情间期)可见大量白细胞，无其他细胞存在；阳性对照组(动情后期)可见多种细胞类型，白细胞、无核的上皮细胞及梭形上皮细胞；动情期(沙棘籽油组)见大量鳞状上皮细胞，形状大而不规则，无核，白细胞消失。
经过造模后每隔2天的连续动情周期观察发现：正常组对照组动情周期均基本正常，表现为规律的4～5d动情周期的变化。与正常组比较，其余三组均有显著性差异：模型组(sham组)小鼠从实验第9天始出现动情周期紊乱，阴道涂片示持续处于动情间期；阳性对照(二甲双胍组)之后出现动情周期延长，在实验第20天再次出现动情期，但延长至6～7日，动情周期虽有恢复，但并不稳定；沙棘籽油组在给药开始，少部分小鼠出现动情周期紊乱的现象，随着持续给药，小鼠均恢复正常的动情周期，并且沙棘籽油组在给药17天恢复正常的动情周期，为4～5天，分为动情前期(20h)，动情期(8～16h)，动情后期(25～35h)，动情间期(40～50h)，实验期间动情周期规律，共出现动情期3～5次。
4.3沙棘籽油可以减轻PCOS小鼠的肥胖状态
在PCOS患者中约50％-70％伴有超重或肥胖，临床研究表明，PCOS合并肥胖的患者，胰岛素抵抗、高性激素血症等症状更严重，因此减轻体重是缓解PCOS症状的一个有效途径。本研究中我们发现，沙棘籽油可以显著减轻PCOS小鼠的体重。如图4所示：
小鼠形态肉眼观：模型组(sham组)体型明显大于正常对照组，而给予二甲双胍的阳性对照组及给予沙棘籽油的实验组，体型则接近正常对照组。
如图5和图6所示，给予高脂喂养及来曲唑后，相对于正常对照组，小鼠体重明显增加，但在给予二甲双胍及沙棘籽油后，体重上升趋势有所改善，尤其沙棘籽油，减缓体重上升趋势的作用更优于二甲双胍。根据取材前一天最终体重亦可见，给予沙棘籽油治疗后的小鼠，体重与正常组接近，与模型组体重有显著性差异。
4.4沙棘果浆和沙棘果油可以改善PCOS小鼠的高血糖状态，减轻胰岛素抵抗
糖耐量受损是PCOS最主要的表现之一，在此项研究中，我们发现沙棘籽油有很好的改善糖耐量受损、降低血糖的作用。
如图7和图8所示，各组小鼠禁食16小时后，给予高糖，实行口服葡萄糖耐量实验(OGTT)。给予高脂喂养及来曲唑的模型组后，小鼠糖耐量明显恶化，远高于正常对照组。但在给予二甲双胍及沙棘籽油后，糖耐量有明显改善。沙棘籽油对血糖的改善与传统降糖药二甲双胍作用几乎没有差别。证明沙棘籽油具有非常好的改善PCOS小鼠糖耐量受损的作用。
4.5沙棘籽油可增加卵巢的储备功能，增加卵巢组织中AMH含量
多囊卵巢综合征(PCOS)的患者疾病改善的特征之一是卵巢中有大量的生长期卵泡，意味着有丰富的卵巢储备。AMH(Anti-Mullerian Hormone，抗穆勒氏激素)由窦前卵泡和小窦卵泡的颗粒细胞分泌，抑制卵泡的生长，防止卵泡过快过早的消耗，保存卵巢的储备，是卵巢功能储备的重要指标。在自然月经周期中,募集的卵泡少，AMH水平在卵泡期仅发生轻微的改变,而在超排卵过程中,大量始基卵泡被募集进入窦前卵泡期时,AMH表达则显著增加。AMH在卵巢不同反应人群中表达不同，AMH与卵巢反应、临床妊娠率、胚胎的种植率等呈正相关，在卵泡的发育和功能上起作用。近年的临床研究发现月经周期第3天血清中AMH水平随着年龄的增加而进行性的下降，在绝经期后无法检测到。因此AMH是用于评估卵巢储备功能最有效直接的因子之一。在本研究中，我们发现沙棘籽油明显提高PCOS小鼠卵巢中AMH的表达，证明沙棘籽油可以增加卵巢储备功能。
这可能是沙棘籽油改善PCOS的重要机制。
如图9所示，AMH蛋白主要表达于卵巢组织切片颗粒细胞的胞浆、胞膜和胞核，呈棕黄色。通过免疫组化实验可以证实：较正常对照组相比，沙棘果浆组中AMH的表达明显增加，主要分布于颗粒细胞所在的区域；模型(Sham)组的表达较正常组有所减少。这表明沙棘籽油可以增加卵巢组织中AMH的含量，进而增加卵巢的储备功能。
本申请发现，相比二甲双胍，沙棘籽油对PCOS有更好的治疗作用。它不仅可以治疗胰岛素抵抗，还有有效降低PCOS鼠的体重、恢复规律的动情周期、改善卵巢状态等，可谓治疗PCOS有效的综合性药物。
由以上技术方案可知，沙棘籽油对多囊卵巢综合征有治疗作用。沙棘籽油不仅可以改善PCOS小鼠卵巢多囊样病变，还可以使紊乱的动情周期正常化，在内分泌代谢方面，还可以有效降低血糖、减轻肥胖状态等。本申请对于直接利用沙棘籽油治疗多囊卵巢综合征提供了基础。
本领域技术人员在考虑说明书及实践这里公开的申请后，将容易想到本申请的其它实施方案。本申请旨在涵盖本申请的任何变型、用途或者适应性变化，这些变型、用途或者适应性变化遵循本申请的一般性原理并包括本申请未公开的本技术领域中的公知常识或惯用技术手段。说明书和实施例仅被视为示例性的，本申请的真正范围和精神由下面的权利要求指出。
应当理解的是，本申请并不局限于上面已经描述内容，并且可以在不脱离其范围进行各种修改和改变。本申请的范围仅由所附的权利要求来限制。
Claims (1)
1.沙棘籽油作为单一活性成分用于制备治疗多囊卵巢综合征的药物中的应用。
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