CN116966259A - 一种用于预防或治疗多囊卵巢综合征的中药组合物 - Google Patents
一种用于预防或治疗多囊卵巢综合征的中药组合物 Download PDFInfo
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Abstract
本发明提供一种中药组合物,以重量分数计包括以下成分:柴胡3‑5份、白花蒿1‑3份,黄芩1‑3份、甘草1‑3份、党参1‑3份、白姜1‑3份、大枣1‑3份、桂枝1‑3份、当归1‑3份、川芎1‑3份、白芍1‑3份、枸杞子3‑5份、仙茅3‑5份、菟丝子3‑5份。本发明提供的中药组合物对多囊卵巢综合征有很好的治疗效果。
Description
技术领域
本发明属于中药领域,具体涉及一种用于预防或治疗多囊卵巢综合征的中药组合物。
背景技术
多囊卵巢综合征(polycystic ovary syndrome,PCOS)是一种常见的女性生殖、内分泌和代谢疾病,在年轻女性中的发病率为5%-10%,其临床表现多种多样,如闭经、肥胖和不孕等。由于其临床表现的高度异质性,其发病机制尚不清楚,诊断标准仍存在争议。PCOS治疗包括生活方式调节和代谢紊乱、高雄激素血症和生殖异常的改善。PCOS目前还没有有效的治疗方法,已经严重威胁到青少年和育龄妇女的健康。因此,迫切需要探索治疗PCOS的新治疗策略。
中医药在治疗PCOS方面积累了丰富的临床经验,显示出巨大的治疗价值和科研潜力。
中国专利202110619720.6中公开了一种治疗多囊卵巢综合征的中药组合物及其制备方法和用途。提供一种中药组合物,包括肉桂、淫羊藿、附子、吴茱萸、土茯苓、土贝母、莪术、刘寄奴、红花、丹参、王不留行和木香。该中药组合物通过上述12味中药在上述穴位上进行隔药灸,即通过借助灸火的热力来促进穴位皮肤组织对这些药物有效成分的吸收,从而达到温阳益肾、化痰利湿、活血化瘀、消肿散结之功效,能够有效治疗肾虚痰湿型和肾虚痰瘀型多囊卵巢综合征。但该方法施行需要借助灸火,对医师技术要求较高。
中国专利202010380045.1中公开了治疗肾虚痰湿型多囊卵巢综合征不孕症的中药汤,可有效解决治疗肾虚痰湿型多囊卵巢综合征不孕症的用药问题,由杜仲15-20g、菟丝子20-30g、炒苍术10-15g、白术10-15g、清半夏10-15g、茯苓10-20g、香附10-15g、陈皮10-15g和桂枝3-6g制成,其中,上述药物加水400-600mL,在18-25℃下,浸泡15-25分钟,然后武火烧开,再转文火,煎煮20-30min,过滤,得滤液,备用;滤渣加水300-400mL,武火烧开后,转文火,煎煮10-15min,过滤,弃去滤渣,得滤液,合并两次滤液,浓缩至两次加水量的1/2-1/3,即可,该发明原料丰富,制备方法简单,成本低,易服用,效果好,具有温肾助孕、理气化痰之功效,是治疗肾虚痰湿型多囊卵巢综合征不孕症上创新。但其有效率衡量标准较低,药效有待验证。
目前仍需要一种更好的中药组合物来治疗多囊卵巢综合征。
发明内容
为了解决上述问题,本发明提供了一种中药组合物,通过科学配伍,使其对PCOS具有了更好地治疗效果。
一方面,本发明提供了一种中药组合物。
所述的中药组合物以重量分数计包括以下成分:
柴胡3-5份、白花蒿1-3份,黄芩1-3份、甘草1-3份、党参1-3份、白姜1-3份、大枣1-3份、桂枝1-3份、当归1-3份、川芎1-3份、白芍1-3份、枸杞子3-5份,仙茅3-5份,菟丝子3-5份。
优选地,所述的中药组合物以重量分数计包括以下成分:柴胡3-5份、白花蒿2份,黄芩1-3份、甘草1-3份、党参1-3份、白姜1-3份、大枣1-3份、桂枝2份、当归1-3份、川芎1-3份、白芍1-3份、枸杞子3-5份,仙茅4份,菟丝子3-5份。
进一步优选地,所述的中药组合物以重量分数计包括以下成分:柴胡4份、白花蒿2份、黄芩2份、甘草2份、党参2份、白姜2份、大枣2份、桂枝2份、当归2份、川芎2份、白芍2份、枸杞子4份、仙茅4份、菟丝子4份。
所述的柴胡中药名为柴胡、白花蒿中药名为半夏、黄芩中药名为黄芩、甘草中药名为甘草、党参中药名为党参、白姜中药名为生姜、大枣中药名为大枣、桂枝中药名为桂枝、当归中药名为当归、川芎中药名为川芎、白芍中药名为白芍、枸杞子中药名为蛇床子、仙茅中药名为小毛、菟丝子中药名为土司子。
另一方面,本发明提供了前述的中药组合物在制备用于预防或治疗多囊卵巢综合征的药物中的应用。
所述的药物中包括前述的中药组合物或其提取物。
再一方面,本发明提供了一种治疗多囊卵巢综合征的药物的制备方法。
所述的制备方法利用前述的中药组合物为原料进行制备。
所述的制备方法中包括将前述的中药组合物进行熬煮后制备。
优选地,熬煮后得到药液20g。
所述的制备方法中可能还包括其他常用的制药技术。
又一方面,本发明提供了一种用于预防或治疗多囊卵巢综合征的药物。
所述的药物中包括前述的中药组合物或其提取物。
所述的提取物包括但不限于水提取物。
所述的药物的剂型包括但不限于:汤剂、片剂、糖浆剂、颗粒剂、口服液、丸剂。
所述的药物中还包括其他药学上可接受的载体或赋形剂。
附图说明
图1为实施例1中糖耐量实验结果。
图2为实施例1中AKT mRNA表达量检测结果。
图3为实施例1中PI3K mRNA表达量检测结果。
图4为实施例1治疗组卵巢形态HE染色光学放大100倍照片。
图5为实施例1对照组卵巢形态HE染色光学放大40倍照片。
图6为实施例1对照组卵巢形态HE染色光学放大100倍照片。
图7为实施例1差异基因图(A为差异基因热图;B为差异基因火山图)。
图8为实施例1全转录组测序差异miRNA分析。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例中:
从广西医科大学(中国南宁)实验动物中心获得24天大的SPF雌性Sprague-Dawley大鼠,体重250-300g。动物生产许可证号:SCXK(桂)2020-0003。所有大鼠均饲养于广西中医药大学实验室SPF级动物房,使用标准水、鼠粮和标准环境(温度25±2℃,相对湿度60±5%,12h明暗循环)。
原药均购自北京同仁堂健康药业有限公司(中国北京)。汤剂加中药浓缩冲剂共20克。
左炔诺孕酮胶囊,规格:75mg,购自上海大华药业有限公司,批号YZ20190103;
绒毛膜促性腺激素购自丽珠制药厂,批准号200805;
逆转录试剂盒购自诺和蛋白科技有限公司,批号0519281;
荧光定量PCR试剂盒(SYBR Green Master Mix)购自卓伊生物科技有限公司,批号A25742;
引物由上海生工生物工程有限公司合成;
电动组织研磨机购自天根生化科技有限公司(北京,型号:OSE-Y30);
离心机型号:Stratos,Thermo Fisher Scientific;
PCR基因扩增仪,型号:3200,珠海海马医疗器械有限公司;
掌上离心机:海门凯莉贝尔实验室仪器有限公司,型号:LX-100;
实时荧光定量PCR(qRT-PCR),型号:7500,Applied Biosystems;
血糖仪和血糖试纸购自One Touch UltraEasy。
实施例1
本实施例中中药组合物配方如下:
柴胡(中药名:柴胡)20克、白花蒿(中药名:半夏)10克,黄芩(中药名:黄芩)10克、甘草(中药名:甘草)10克、党参(中药名:党参)10克、白姜(中药名:生姜)5克、大枣(中药名:大枣)5克、桂枝(中药名:桂枝)10克、当归(中药名:当归)10克、川芎(中药名:川芎)10克、白芍(中药名:白芍)10克、枸杞子(中药名:蛇床子)20克,仙茅(中药名:小毛)20克,菟丝子(中药名:土司子)20克。
以上组合物通过加水熬煮30-60分钟制成中药汤剂20g。
适应性喂养3天后,将30只大鼠随机分为对照组(n=6)、PCOS组(n=12)和治疗组(n=12)。黄体酮联合HCG用于建立大鼠多囊卵巢综合征模型。PCOS组和治疗组大鼠分别皮下植入左炔诺孕酮棒(3-4mm),3天后注射绒毛膜促性腺激素(3U加入0.1mL生理盐水中)连续9天建立PCOS模型。假手术组注射等量生理盐水。治疗组接受4200mg/kg/d的中药汤剂,而对照组和PCOS模型组接受等量的蒸馏水。
(1)血糖检测:
OGTT(口服葡萄糖耐量试验)在三组大鼠禁食12小时后进行。大鼠可以自由饮水。采集尾血,使用OneTouch UltraEasy血糖仪和血糖试纸检测血清葡萄糖水平。使用口服管饲法,大鼠接受50%的葡萄糖(2g葡萄糖/kg)。然后,在30分钟、60分钟和120分钟后测量血清葡萄糖水平。
ITT(胰岛素耐受试验)将三组大鼠禁食4小时,自由饮水。使用OneTouchUltraEasy血糖仪和血糖试纸确定从尾部采集的血液中的葡萄糖水平。然后以0.5/kg给大鼠腹腔注射胰岛素,并在30分钟、60分钟、90分钟和120分钟后在尾部测量血糖水平。
(2)HE染色
包括以下步骤:卵巢组织予4%福尔马林溶液浸泡固定送检,置于组织包埋托上,组织块周围用OCT胶包埋后使用切片机切成4μm左右厚度,置于载玻片上,苏木素染色,蓝化后加伊红染色,10倍目镜,10-100倍光镜下观察。
(3)基因表达量的检测
采用TRIzol法从小鼠卵巢组织中提取总RNA。使用逆转录cDNA试剂盒,反应条件为:42℃,5分钟;50℃,15分钟;75℃,5分钟。大鼠GAPDH用作内部对照基因。加入引物。qRT-PCR采用以下扩增条件:94℃,1分钟;94℃,20秒,57℃,20秒,72℃,60秒/1kb,30个循环;72℃,5分钟。确定qPCR荧光曲线的Ct值。ΔCt=目标基因Ct值-GAPDH Ct值。
定量PCR引物序列见下图:
(4)全转录组测序
使用TRIzol试剂从大鼠卵巢组织中提取RNA,DNaseI进行DNA消化。Qubit3.0 withQubitTM RNABroad Range Assay试剂盒进行RNA检测。使用Ribo-off rRNADepletion Kit和KCTM Stranded mRNALibrary Prep Kit将2μg总RNA用于RNA测序文库制备。对200-500bp对应的文库产物进行富集、定量,最后在DNBSEQ-T7测序仪用PE150模式进行测序。
RNA-Seq数据分析原始测序数据首先由Trimmomatic(0.36版本)过滤,使用STRA软件(版本2.5.3a)的默认参数将Clean Reads映射到大鼠的参考基因组,通过featureCounts(Subread-1.5.1;Bioconductor)对映射到每个基因的外显子区域的读数进行计数,然后计算RPKM。使用edgeR包(版本3.12.1)鉴定组间差异表达的基因。p-valuecutoff 0.05和fold-change cutoff 2判断为基因差异表达具有统计学意义。差异表达基因的GO分析和KEGG富集分析均由KOBAS软件(版本:2.1.1)执行,p值阈值为0.05,以判断具有统计学意义的富集。使用rMATS(版本3.2.5)检测可变剪接事件,FDR值截止值为0.05,Δψ绝对值为0.05。
LncRNA分析,通过lncRNA分析预测了新的lncRNA。通过featureCounts(Subread-1.5.1;Bioconductor)对新的lncRNA和基因组注释的已知lncRNA的表达进行计数,然后计算RPKM。使用edgeR包(版本3.12.1)鉴定组间差异表达的LncRNA。P值cutoff 0.05和fold-change cutoff 2用于判断lncRNA表达差异的统计学意义。预测差异表达lncRNA的靶基因。差异表达基因的GO分析和KEGG富集分析均由KOBAS软件(版本:2.1.1)执行,p值阈值为0.05,以判断具有统计学意义的富集。
(5)miRNA测序及数据分析
使用TRIzol试剂从大鼠卵巢组织中提取RNA,使用KC-DigitalTM small RNALibrary Prep Kit for Illumina试剂盒进行miRNA文库构建,使用唯一分子标识符(UMI)标记预扩增的miRNA分子。洗脱的cDNA文库使用6%PAGE凝胶电泳进行分离。大小约为160bp的条带被分离出来后,经过Qubit 3.0分离、纯化和定量后,最终使用Hiseq X-10测序仪(Illumina)测序仪通过PE150模板进行测序。
原始测序数据首先通过fastx_toolkit(版本:0.0.13.2)进行初筛,使用bowtie软件(版本:1.1.2)将共有序列映射到大鼠的参考基因组。包用于将读数映射到miRBase数据库中已知的初级miRNA并预测新的miRNA。使用edgeR包(版本:3.12.1)确定组间表达的miRNA差异。P<0.05和|Log2Fold-change|>1的阈值用于判断miRNA表达差异是否具有统计学意义。miRanda v3.3a软件用于预测差异表达miRNA的靶mRNA。使用KOBAS软件(版本:2.1.1)对预测到的靶mRNA进行GO和KEGG富集分析,P<0.05判定为具有统计学意义。miRNA-mRNA共表达网络由WGCNA(1.61版)构建,miRNA和mRNA之间的相关阈值>0.8。
结果及分析:
(1)胰岛素耐量试验中,对照组和治疗组的血糖显著低于PCOS组(P<0.05);然而,对照组和治疗组之间的血清葡萄糖差异不显着。糖耐量试验中,三组血糖水平无显著差异(P>0.05),如图1所示。
(2)与PCOS大鼠模型相比,通过本申请药物的干预,PCOS治疗组的血糖水平得到显著改善,表明药物可能通过调节糖代谢对PCOS产生治疗作用。
(3)卵巢形态HE染色显示,在药物干预后,PCOS大鼠发现多个扩张的卵泡与多个发育阶段的卵泡共存,各级卵泡结构正常,卵母细胞、卵丘、放射冠等毛囊内可见结构,黄体存在。相比之下,对照组卵巢包膜增厚,卵巢内可见多个囊性扩张的卵泡,PCOS大鼠模型中卵母细胞疏松层数和冠状颗粒细胞丢失减少。见图4-6。图4为治疗组卵巢形态HE染色光学放大100倍,镜下见各个发育阶段的卵泡,成熟卵母细胞及黄体。颗粒细胞层排列规则。图5为对照组40倍下见多个同一发育阶段的卵泡,卵泡呈囊性扩张。图6为对照组100倍下见卵泡颗粒细胞层排列疏松。
(3)AKT mRNA表达量结果如图2所示,结果表明以正常大鼠的AKT基因表达量为基准,多囊卵巢大鼠的表达量为1.05,中药组多囊卵巢大鼠的表达量为1.04,无显著性差异。
(4)PI3K mRNA表达量结果如图3所示,结果表明:以正常大鼠的PI3K基因表达量为基准,多囊卵巢大鼠的表达量为1.43,中药组多囊卵巢大鼠的表达量为1.19,较对照组多囊卵巢大鼠下降。
(5)卵巢组织全转录组测序进行差异基因分析(图7),结果表明:中药组与PCOS模型组大鼠间差异表达基因189个,其中上调基因48个,下调基因141个,GO生物过程分析表明下调基因富集于有机羟基化合物的代谢过程、对激素的反应、酒精代谢过程、脂质转运等,上调基因富集于细胞对有机物质的反应、对有机物质的反应、对含氧化合物的反应等,KEGG分析表明上调基因富集于PI3K-Akt、Metabolic、Jak-STAT等信号通路,下调基因富集于Metabolic、PPAR等信号通路。GO分析和KEGG信号通路富集分析结果表明PI3K/Akt信号通路中,本实施例中药组合物在干预PCOS中起重要作用。本实施例中药组合物干预PCOS与PI3K/Akt信号通路调控有关。
(6)差异miRNA分析:治疗组与PCOS组大鼠间差异miRNA,上调miRNA4个,下调miRNA4个。与治疗组干预有关的miR-206-3p等做为ceRNA参与网络调控有关(图8中的A),全转录组测序分析miRNA热图分析(图8中的B)。结果表明miR-206-3p在治疗组干预PCOS中发挥重要作用,且已有研究表明miR-206-3p可能在PCOS发病中起重要作用。
对比例
参照实施例1的治疗组设置对比例,用药组分如下:
与实施例1治疗组的用药差别 | |
对比例1 | 不添加仙茅 |
对比例2 | 不添加桂枝 |
对比例3 | 不添加白花蒿 |
对比例4 | 不添加仙茅和桂枝 |
对比例5 | 不添加仙茅和白花蒿 |
对比例6 | 不添加桂枝和白花蒿 |
检测PI3K mRNA表达量,以正常大鼠的PI3K mRNA表达量为基准,结果如下:
序列表
<110> 北京三医智慧科技有限公司
<120> 一种用于预防或治疗多囊卵巢综合征的中药组合物
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gacatgccgc ctggagaaac 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
agcccaggat gccctttagt 20
<210> 3
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctacctcgcc gttcctgtg 19
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ttgacctgac ccatctgacc t 21
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aggcatccct tccttacagc 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cagcccgaag tccgttatct 20
Claims (10)
1.一种中药组合物,其特征在于,以重量分数计包括以下成分:
柴胡3-5份、白花蒿1-3份,黄芩1-3份、甘草1-3份、党参1-3份、白姜1-3份、大枣1-3份、桂枝1-3份、当归1-3份、川芎1-3份、白芍1-3份、枸杞子3-5份,仙茅3-5份,菟丝子3-5份。
2.根据权利要求1所述的中药组合物,其特征在于,以重量分数计包括以下成分:柴胡3-5份、白花蒿2份,黄芩1-3份、甘草1-3份、党参1-3份、白姜1-3份、大枣1-3份、桂枝2份、当归1-3份、川芎1-3份、白芍1-3份、枸杞子3-5份,仙茅4份,菟丝子3-5份。
3.根据权利要求1所述的中药组合物,其特征在于,以重量分数计包括以下成分:柴胡4份、白花蒿2份,黄芩2份、甘草2份、党参2份、白姜2份、大枣2份、桂枝2份、当归2份、川芎2份、白芍2份、枸杞子4份,仙茅4份,菟丝子4份。
4.权利要求1-3任一项所述的中药组合物在制备用于预防或治疗多囊卵巢综合征的药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述的药物中包括权利要求1-3任一项所述的中药组合物或其提取物。
6.一种治疗多囊卵巢综合征的药物的制备方法,其特征在于,利用权利要求1所述的中药组合物为原料进行制备。
7.根据权利要求6所述的制备方法,其特征在于,包括:将权利要求1-3任一项所述的中药组合物进行熬煮后制备。
8.根据权利要求7所述的制备方法,其特征在于,熬煮后得到药液20g。
9.一种用于预防或治疗多囊卵巢综合征的药物,其特征在于,所述的药物中包括权利要求1-3任一项所述的中药组合物或其提取物。
10.根据权利要求9所述的药物,其特征在于,所述的药物中还包括其他药学上可接受的载体或赋形剂。
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