CN116196364A - 番红花废弃物的提取物在制备预防和/或治疗肠道炎症疾病药物中的应用 - Google Patents
番红花废弃物的提取物在制备预防和/或治疗肠道炎症疾病药物中的应用 Download PDFInfo
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Abstract
本发明公开了番红花废弃物的提取物在制备预防和/或治疗肠道炎症疾病药物中的应用，涉及生物医药技术领域。本发明提供番红花废弃物的提取物在制备预防和/或治疗肠道炎症疾病药物中的应用，开辟了一种全新的番红花废弃物的提取物用于预防和/或治疗肠道炎症疾病药物的应用，并且对其作用机制进行了初步的研究，有助于将藏红花生药生产过程中的废弃物——番红花的花瓣和雄蕊、花茎实现进一步的开发利用。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及番红花废弃物的提取物在制备预防和/或治疗肠道炎症疾病药物中的应用。
背景技术
炎症性肠病(IBD)包括溃疡性结肠炎(UC)和克罗恩病(CD)两种独立的疾病，是一种累及回肠、直肠、结肠甚至全胃肠道的反复发作的慢性炎症性肠道疾病。由于IBD的确切发病机制尚未完全阐明，IBD患者仍无法治愈，需要终身维持治疗来预防复发。目前推荐用于治疗IBD的经典治疗药物包括氨基水杨酸盐、糖皮质激素、免疫抑制剂和生物制剂，但这些药物由于各种副作用而不能满足患者的需求。
最近，大量研究表明，食物来源的天然产物具有促进IBD缓解的巨大潜力，并提示开发来自药食同源草药的新辅助疗法用于IBD治疗具有前景。
藏红花是番红花(Crocus sativus L.)的干柱头，是一种价值极高的农业和药用香料，在中东地区广泛用于烹饪，使食物具有风味、颜色和香气。在中国，藏红花被广泛用作活血化瘀的传统中药，广泛用于治疗闭经、产后瘀血、毒性轻微、抑郁、心悸和癫狂。如今，藏红花在中国被广泛用作一种草药茶来促进健康。藏红花非常昂贵，因为每公斤番红花只有12克柱头。藏红花花瓣是番红花的核心成分，占番红花湿重的86.4％，干重的96.4％。在藏红花生药生产过程中，番红花的花瓣和雄蕊、花茎通常作为废弃物被丢弃。与生药藏红花相似，藏红花花瓣主要含有类黄酮、花青素和萜类化合物，具有有效的神经保护、保肝、抗氧化、降压和止咳活性。
目前，尚未有报道关于番红花废弃物的提取物对炎症性肠病的药效研究。
发明内容
本发明所要解决的技术问题是提供番红花废弃物的提取物尤其是番红花花瓣提取物在制备预防和/或治疗肠道炎症疾病药物中的应用，进一步开发番红花废弃物的利用价值。
为了解决上述问题，本发明提出以下技术方案：
第一方面，本发明提供番红花废弃物的提取物在制备预防和/或治疗肠道炎症疾病药物中的应用。
进一步地，所述番红花废弃物的提取物选自番红花花瓣提取物、番红花雄蕊提取物或番红花花茎提取物中的至少一种。
进一步地，所述番红花废弃物的提取物选自番红花花瓣提取物。
进一步地，所述肠道炎症疾病包括炎症性肠病、肠易激综合症。
本发明所述的肠道炎症疾病包括但不限于结肠炎，本发明实施例以小鼠结肠炎疾病模研究番红花废弃物的提取物对肠道炎症疾病的治疗效果。
进一步地，所述番红花废弃物的提取物的制备方法包括：将风干的番红花花瓣、雄蕊和花茎分别用8-12倍质量的有机溶剂回流提取2次，每次回流1.5-3h；然后将提取液浓缩并减压干燥，得到番红花花瓣SP提取物、番红花雄蕊SSA提取物及番红花花茎SF提取物。
进一步地，所述有机溶剂包括甲醇、乙醇。
本发明实施例以75％(w/v)的甲醇为例作为提取溶剂，制备番红花废弃物的提取物。
第二方面，本发明提供一种预防和/或治疗肠道炎症疾病的药物组合物，包括有效治疗剂量的番红花废弃物的提取物。
进一步地，所述番红花废弃物的提取物选自番红花花瓣提取物。
进一步地，所述药物组合物还包括药学上可接受的载体和/或辅剂。
进一步地，所述有效剂量为大于或等于200mg/Kg。
本发明所述的药物组合物，该药物组合物以所述的有效治疗剂量的番红花废弃物的提取物作为活性成分，尤其是以番红花花瓣提取物作为活性成份。
在本发明中，可将本发明的番红花废弃物的提取物、尤其是番红花花瓣提取物作为活性成分配制于无毒的、惰性的和药学上可接受的载体介质；配制好的药物可以通过常规途径进行给药，包括但并不限口服、肌内、腹膜内、静脉内、皮下、皮内、或局部给药。
当本发明的药物组合物的剂型为用于口服施用的药物时，其含有安全有效量的番红花废弃物的提取物、尤其是番红花花瓣提取物以及药学上可接受的载体和/或辅剂，口服施用的药物可制成片剂、丸剂、散剂、颗粒剂、胶囊剂、乳剂、糖浆剂、软膏剂、栓剂等常用剂型；本发明中，不对载体和/或辅剂做具体限定。
本发明的药物组合物还可以被制成注射剂，可以在无菌操作环境下与注射用水、生理盐水、葡萄糖水制成注射剂，上述注射剂可通过常规方法进行制备。
本发明研究发现，番红花废弃物的提取物尤其是番红花花瓣提取物SP可显著抑制lps刺激的RAW264.7细胞中的炎症反应，并发现番红花花瓣提取物SP对dss诱导的结肠炎小鼠具有有效的治疗作用，其机制与抑制巨噬细胞活化有关。
与现有技术相比，本发明所能达到的技术效果包括：
本发明提供番红花废弃物的提取物在制备预防和/或治疗肠道炎症疾病药物中的应用，开辟了一种全新的番红花废弃物的提取物用于预防和/或治疗肠道炎症疾病药物的应用，并且对其作用机制进行了初步的研究，有助于将藏红花生药生产过程中的废弃物——番红花的花瓣和雄蕊、花茎实现进一步的开发利用。
附图说明
图1为SP提取物对lps诱导的RAW246.7细胞的体外抗炎作用：(A)TNF-α和IL-1β的mRNA表达；(B)TNF-α和IL-1β的产生；(C)COX-2和iNOS蛋白表达。数据表示平均值±SD(n＝3)；###p<0.001vs对照组(Con)；与LPS组比较，*p<0.05或**p<0.01或***p<0.001。
图2为SP提取物对dss诱导的小鼠结肠炎的影响：(A)实验设计的示意图；(B)小鼠体重变化；(C)疾病活动指数(DAI)评分；(D)结肠代表图像；(E)结肠长度；(F)组织学评分和(G)结肠的he染色(放大，×100)。数据表示为平均值±SEM(n＝6)。###p<0.001vs对照组(Con)；与DSS组比较，*p<0.05或**p<0.01。
图3为SP提取物抑制dss处理小鼠结肠中巨噬细胞浸润，TNF-α，IL-1β和IL-6的转录和产生。(A)F4/80阳性巨噬细胞浸润；(B)TNF-α、IL-1β和IL-6的转录；(C)TNF-α，IL-1β和IL-6的产生。数据表示为平均值±SEM(n＝5-6)。与对照组(Con)比较，##p<0.01，###p<0.001；与DSS组比较，*p<0.05或**p<0.01。
图4为SP提取物对体内外PI3K/Akt和MAPKs信号通路中信号分子表达的影响。(A)SP提取物对lps诱导的RAW246.7细胞中PI3K/Akt和MAPKs信号通路中信号分子表达的影响；(B)SP提取物对dss处理小鼠结肠中PI3K/Akt和MAPKs信号通路中信号分子表达的影响。数据表示平均值±SD(细胞样本n＝3；动物样本n＝6)。###p<0.001vs对照组(Con)；与LPS或DSS组比较，*p<0.05或**p<0.01。
图5为不同浓度的SP、SSA、SF和SST对RAW264.7细胞存活率的影响。数据表示平均值±SD(n＝3)。***p<0.001vs对照组(Con)。
图6为不同浓度的SP、SSA、SF和SST对LPS诱导的RAW264.7细胞中NO释放的抑制率。数据表示平均值±SD(n＝3)。
具体实施方式
下面将结合本发明实施例中的附图，对实施例中的技术方案进行清楚、完整地描述。显然，以下将描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
(1)番红花花瓣提取物、番红花柱头提取物、番红花雄蕊提取物、番红花花茎提取物的制备过程：
将风干的藏红花花瓣、雄蕊、花茎和柱头分别用10倍75％甲醇(w/v)回流提取2次，每次回流2h。然后将提取液浓缩并减压干燥，得到藏红花花瓣(SP)、雄蕊(SSA)、花茎(SF)和柱头(SST)的粗提物用于生物测定。
(2)细胞实验及结果
细胞培养和活力测定：
从ATCC中获得RAW264.7细胞。将1.5×104个/孔细胞接种于96孔板过夜，然后用不同浓度的SP、SSA、SF和SST处理24h，采用CCK-8试剂盒检测细胞活力，结果如图5所示。
确定SP、SSA、SF和SST用于RAW264.7细胞的安全浓度后，进一步通过LPS刺激下的NO释放实验初步筛选其抗炎活性。
NO释放试验：
将5×104个细胞/孔接种于48孔培养板过夜，用0.4～250μg/mL的SP、SSA、SF和SST处理1h，然后用1μg/mL的LPS刺激24h。结果见图6，SP提取物在体外表现出有效的抗炎活性，结果如图6所示。
SP提取物和SSA提取物均表现出对NO释放的有效抑制作用，且呈剂量依赖性。由于SP提取物是番红花的主要副产物，其对NO释放的抑制活性优于其他副产物，我们进一步研究了SP提取物对lps刺激的RAW264.7细胞中其他炎症标志物的影响，结果见图1。
结果表明，SP提取物以剂量依赖性方式对TNF-α和IL-6的转录和蛋白生成具有有效的抑制作用(图1A和图1B)。COX-2和iNOS是两种炎症核心蛋白，分别产生PGE2和NO。Westernblot结果也显示，SP提取物可以显著下调COX-2和iNOS的表达(图1C)。综上所述，这些数据表明SP提取物具有有效的抗炎特性。
(3)动物研究实验及结果：
动物选择及模型建立：
参见图2A，选择68周龄、体质量18～22g的雄性C57BL/6J小鼠，小鼠取自北京维通河实验动物科技有限公司。适应1周后，用2％DSS饮水5d建立小鼠结肠炎模型，选取构建成功的结肠炎小鼠，然后将结肠炎小鼠随机分为DSS模型组、SASP组和两个SP提取物治疗组。同时设置不含DSS饮用水的溶剂对照组。在实验过程中，根据前期实验设定SP提取物的剂量分别为200和400mg/Kg，连续7天口服给小鼠。SASP组的给药剂量设定为200g/Kg。溶剂对照组和DSS模型组给予等体积的溶剂。体重和疾病活动指数每日测定。实验结束后对所有小鼠实施安乐死。
组织学检查和免疫荧光染色：
将结肠组织固定在4％多聚甲醛中24h，用石蜡包裹，用苏木精-伊红(H&E)染色，并盲法评估组织病理学损伤。免疫荧光染色：首先对石蜡包埋的结肠切片进行3％氢气和氧气处理、脱蜡和补液，然后用10％牛血清白蛋白(BSA)封闭。随后，将切片与1∶100小鼠抗f4/80抗体(Invitrogen)在4℃避光孵育过夜，然后用PBS洗涤，并与Alexa Fluor 594标记的山羊抗兔IgG H&L(1∶500)在室温避光孵育30min。用DAPI(4,6-二脒基-2-苯基吲哚)标记细胞核。
RNA提取和RT-PCR分析：
Trizol法提取RAW264.7细胞和结肠组织总RNA，逆转录酶扩增cDNA；采用SYBRGreen Master实时荧光定量PCR系统进行定量。用β-actin对靶基因的转录进行标准化，并用2-ΔΔCT方法分析数据。引物序列见表1。
表1引物序列
Westernblot和细胞因子检测：
使用含有蛋白酶和磷酸酶抑制剂的RIPA缓冲液从RAW 264.7细胞和结肠组织中提取蛋白质，并通过免疫印迹分析。使用的一抗是Akt、p-Akt、p38、p-p38、ERK、p-ERK、JNK、p-JNK和β-actin(Cell Signaling Technology,MA,USA)。使用Imagej测量条带的强度。与此同时，我们从结肠组织中提取总蛋白用于细胞因子检测；使用小鼠TNF-α，IL-1β和IL-6ELISA试剂盒检测结肠匀浆和lps处理的RAW 264.7细胞的上清液中TNF-α，IL-1β和IL-6的水平。
统计分析：
采用Graph Pad Prism Version 8(Graph Pad Software Company,USA)软件对数据进行单因素方差分析和Duncan多重距离检验。以P<0.05为差异有统计学意义。
动物实验结果表明：SP提取物可有效减轻dss诱导的小鼠结肠炎的结肠炎症。具体地：根据dss诱导的小鼠结肠炎实验结果，如图2B和2C所示，与对照组相比，dss治疗组小鼠的体重显著降低，存在严重的血便和腹泻，导致dss治疗组小鼠的DAI评分较高。经SP提取物治疗后，上述临床症状明显改善。此外，DSS治疗导致结肠长度缩短，结肠组织损伤广泛，炎症细胞浸润，病变形成，隐窝破坏，组织学评分高，而SP提取物可以逆转结肠缩短和损伤，具有有很大改善(图2D-2G)。
巨噬细胞浸润是结肠损伤的重要标志。根据免疫荧光染色结果显示，经SP提取物处理后，结肠中浸润F4/80阳性巨噬细胞的数量明显减少(图3A)。TNF-α、IL-1β、IL-6等促炎细胞因子可由活化的巨噬细胞释放，促进IBD的发生发展。RT-PCR和ELISA结果也表明，SP提取物处理显著降低了结肠炎小鼠结肠组织中TNF-α、IL-1β和IL-6的转录和产生(图3B和3C)。这些数据表明，SP提取物对dss诱导的结肠炎症具有有效的改善作用，可作为治疗IBD的有效补充和替代药物。
通过检测lps诱导的RAW 264.7细胞和dss诱导的结肠炎小鼠结肠组织中p-Akt、p-p38、p-ERK和p-JNK的蛋白表达。结果显示，SP提取物在体内外均可调节Akt和MAPK信号通路。如图4A所示，LPS暴露显著增加了RAW264.7细胞中Akt、p38、ERK和JNK的磷酸化，而SP提取物有效地抑制了这种上调。我们还发现，SP提取物显著下调了dss处理的结肠炎小鼠结肠组织中Akt、p38、ERK和JNK的磷酸化水平(图4B)。这些数据验证了SP提取物在体内和体外能够有效调节Akt和MAPK信号通路。
在上述实施例中，对各个实施例的描述都各有侧重，某个实施例中没有详细描述的部分，可以参见其他实施例的相关描述。
以上所述，为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到各种等效的修改或替换，这些修改或替换都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以权利要求的保护范围为准。
Claims (10)
1.番红花废弃物的提取物在制备预防和/或治疗肠道炎症疾病药物中的应用。
2.如权利要求1所述的应用，其特征在于，所述番红花废弃物的提取物选自番红花花瓣提取物、番红花雄蕊提取物或番红花花茎提取物中的至少一种。
3.如权利要求1所述的应用，其特征在于，所述番红花废弃物的提取物选自番红花花瓣提取物。
4.如权利要求1所述的应用，其特征在于，所述肠道炎症疾病包括炎症性肠病、肠易激综合症。
5.如权利要求1所述的应用，其特征在于，所述番红花废弃物的提取物的制备方法包括：将风干的番红花花瓣、雄蕊和花茎分别用8-12倍质量的有机溶剂回流提取2次，每次回流1.5-3h；然后将提取液浓缩并减压干燥，得到番红花花瓣提取物、番红花雄蕊提取物及番红花花茎提取物。
6.如权利要求5所述的应用，其特征在于，所述有机溶剂包括甲醇、乙醇。
7.一种预防和/或治疗肠道炎症疾病的药物组合物，其特征在于，包括有效治疗剂量的番红花废弃物的提取物。
8.如权利要求7所述的药物组合物，其特征在于，所述番红花废弃物的提取物选自番红花花瓣提取物。
9.如权利要求7所述的药物组合物，其特征在于，所述药物组合物还包括药学上可接受的载体和/或辅剂。
10.如权利要求7所述的药物组合物，其特征在于，所述有效剂量为大于或等于200mg/Kg。
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|---|---|---|---|---|
| CN102657747A (zh) * | 2012-05-14 | 2012-09-12 | 四川大学 | 藏红花副产物花瓣及雄蕊总黄酮提取纯化方法 |
| CN109303823A (zh) * | 2018-12-05 | 2019-02-05 | 浙江寿仙谷医药股份有限公司 | 一种藏红花花瓣精制醇提物在制备预防和治疗炎症的药物中的应用 |
| CN111228372A (zh) * | 2020-03-12 | 2020-06-05 | 厦门旗腾文化传媒有限公司 | 一种草本发酵平衡液及其制备方法 |
| WO2020182822A1 (de) * | 2019-03-12 | 2020-09-17 | Tagora Ip Ag | Safranknollenextrakt zur behandlung von entzündungen |
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102657747A (zh) * | 2012-05-14 | 2012-09-12 | 四川大学 | 藏红花副产物花瓣及雄蕊总黄酮提取纯化方法 |
| CN109303823A (zh) * | 2018-12-05 | 2019-02-05 | 浙江寿仙谷医药股份有限公司 | 一种藏红花花瓣精制醇提物在制备预防和治疗炎症的药物中的应用 |
| WO2020182822A1 (de) * | 2019-03-12 | 2020-09-17 | Tagora Ip Ag | Safranknollenextrakt zur behandlung von entzündungen |
| CN111228372A (zh) * | 2020-03-12 | 2020-06-05 | 厦门旗腾文化传媒有限公司 | 一种草本发酵平衡液及其制备方法 |
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