CN115093388B - 一种黄酮类化合物及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种黄酮类化合物及其制备方法和应用，涉及医药技术领域，解决现有非甾体抗炎药长期使用会产生较多副作用的技术问题，包括全新骨架的黄酮化合物6‑甲氧基‑4',5,7‑三羟基‑8‑(1”‑(3”'‑甲氧基‑4”'‑羟苯基)乙基)异黄酮，该黄酮类化合物是从川射干采用硅胶柱层析、SephadexLH‑20、ODS、高效液相色谱等方法提取分离出的全新化合物；本发明的黄酮化合物IT24具有较好的抗炎作用，且具有选择性抑制mPGES‑1的特性，并具有开发成新型抗炎药物的潜力，本发明为天然化合物的抗炎作用提供了一定的基础。
Description
技术领域
本发明涉及医药技术领域，更具体的是涉及一种黄酮类化合物与应用。
背景技术
中药川射干为鸢尾科植物鸢尾的干燥根茎，性味苦寒。有清热解毒，祛痰，利咽之功效，常用于热毒痰火郁结，咽喉肿痛，痰涎壅盛，咳嗽气喘。
炎症是机体对外界刺激或伤害作出的防御性反应，表现为红、肿、热和痛。一项全球疾病负担研究中心的调查显示，从1990至2017年，慢性炎症患者约有36.3亿。类风湿性关节炎(Rheumatoid arthritis,RA)，阿尔茨海默病(Alzheimer'sdisease,AD)，癌症和糖尿病等在内的多种炎症性疾病长期以来给患者的身心健康带来了巨大伤害。
巨噬细胞是先天性免疫细胞，对炎症具有重要作用。巨噬细胞可以被LPS、干扰素-γ(IFN-γ)、TNF-α、细胞外基质蛋白和其他化学介质激活，进而产生炎症介质如(NO、PGE2)和促炎症细胞因子如白介素-1β(Interleukin-1β,IL-1β)、IL-6和TNF-α。MAPK级联是细胞过程的一个关键途径，它包括增殖、分化和应激反应。众所周知，MAPK信号通路包括三个重要的亚家族，分别是细胞外信号调节激酶(the extracellular-signal-regulatedkinases,ERK/MAPK)、c-Jun氨基末端激酶/应激活化蛋白激酶(the c-jun N-terminalkinase/stress-activated protein kinase,JNK/SAPK)以及p38，均可以调控炎症反应。在LPS的刺激下，可以激活MAPK通路以分泌炎症介质和促炎因子。因此，MAPK信号通路可以作为治疗炎症性疾病的靶点。
目前临床治疗常使用非甾体抗炎药(non-steroidal anti-inflammatory drugs,NSAIDs)和糖皮质激素(Glucocorticoids,GCs)治疗炎症性疾病。NSAIDs通过抑制环氧合酶(如：COX-1/COX-2)从而抑制***素的产生。美国食品和药物管理局(Food and DrugAdministration,FDA)以及欧洲药品管理局都发布了有关NSAIDs的警告。因此，如果在不影响环氧合酶的前提下作用于其他靶点，可以减少副作用，并有望成为新颖且有潜力的疗法。多项研究表明，炎症反应与COX-2/mPGES-1/PGE2的异常磷脂代谢链有关。PGE2是炎症性疾病的一个关键介质。***素合酶(Prostaglandin synthase,PGES)，包括胞质***素E合酶(Cytosolic prostaglandin E synthase,cPGES)、mPGES-1和微粒体***素E合酶2(Microsomal prostaglandin E synthase-2,mPGES-2)，它们在PGE2的合成中发挥不同的作用。mPGES-1在炎症刺激下将PGH2异构化为病理性PGE2。与此同时，cPGES和mPGES-2产生生理性PGE2。这表明在不抑制上游COX-1/COX-2的情况下抑制mPGES-1对治疗炎症性疾病具有更高的安全性。
目前临床上常用NSAIDs治疗炎症性疾病。但长期使用会产生较多副作用，因此寻找副作用较小的替代性抗炎药物迫在眉睫。
发明内容
本发明的目的在于：为了解决上述非甾体抗炎药长期使用会产生较多副作用的技术问题，本发明提供一种黄酮类化合物及其制备方法和应用。
本发明为了实现上述目的具体采用以下技术方案：一种黄酮类化合物，所述化合物的结构式如下所示：
本申请的技术方案中：鸢尾科植物川射干是传统中药材，常用于治疗炎症性疾病和肝脏性疾病。研究表明，川射干中分离的鸢尾苷和鸢尾黄素对NO具有69.7％的抑制率，提取的獐牙菜素具有抗乙型肝炎病毒活性的作用。除此以外，川射干中的化合物还具有抗癌作用。通过细胞凋亡实验，研究者发现提取分离的化合物7-O-methylaromadendrin和Iritectol B可在体外诱导人肺癌细胞COR-L23的凋亡。通过质谱法鉴定口服川射干提取物Wistar大鼠体内胆汁、尿液和粪便样本中生物活性候选代谢物，通过网络药理学分析其相互作用，发现这些代谢物调控与癌症、心血管、泌尿生殖***和消化***疾病相关的重要蛋白质靶点。川射干的主要成分是异黄酮、醌类和三萜类。异黄酮成分被认为是川射干中含量最丰富，作用最关键的成分。近年来，天然植物中黄酮化合物的抗炎作用也受到广泛关注，例如木犀草素、黄芩素、槲皮素等。本申请的IT24即从川射干中分离出的具有全新骨架的黄酮化合物6-甲氧基-4',5,7-三羟基-8-(1”-(3”'-甲氧基-4”'-羟苯基)乙基)异黄酮[6-methoxy-4',5,7-trihydroxy-8-(1”-(3”'-methoxy-4”'-hydroxyphenyl)ethyl)isoflavone，IT24]，通过实验发现其具有较好的抗炎作用，且具有选择性抑制mPGES-1的特性，并具有开发成新型抗炎药物的潜力，本发明为天然化合物的抗炎作用提供了一定的基础。
本申请利用LPS诱导的巨噬细胞模型首次发现并证明了黄酮化合物IT24的抗炎作用，包括(1)抑制LPS诱导的巨噬细胞中炎症介质(NO和PGE2)和促炎因子(IL-6和TNF-α)的产生；(2)在LPS诱导的大鼠腹腔巨噬细胞中，IT24选择性抑制mPGES-1的蛋白和mRNA表达水平，对COX-1和COX-2蛋白无抑制作用；(3)IT24通过抑制LPS诱导的巨噬细胞中p38/JNK蛋白磷酸化进而抑制mPGES-1的表达发挥抗炎作用。因此，IT24可能成为具有更高安全性的mPGES-1选择性抑制剂。
一种黄酮类化合物的制备方法，包括如下步骤：
步骤1、将川射干用乙醇进行提取，提取液真空减压至无醇味，得到总浸膏；
步骤2、将总浸膏经过D101大孔树脂柱层析，乙醇水梯度洗脱，分别得到40％乙醇水、70％乙醇水、95％乙醇水部位；
步骤3、70％乙醇洗脱部位经过硅胶柱层析，用二氯甲烷-甲醇作流动相，分离得到6个部位，分别为馏分A、馏分B、馏分C、馏分D、馏分E及馏分F，馏分B由二氯甲烷-甲醇洗脱得到，再依次经过硅胶柱层析、ODS柱层析、Sephadex LH-20柱层析、高效液相色谱得到上述黄酮类化合物IT-24。
进一步的，步骤1中取干燥的川射干，粉碎至60目后，用70％乙醇水回流提取2次，每次2小时，川射干与70％乙醇水的质量体积比为1kg:7L。
进一步的，步骤2中，70％乙醇水的质量为2.1kg。
进一步的，馏分B的质量为580.3g。
进一步的，二氯甲烷与甲醇的体积比为99:1。
进一步的，高效液相色谱流动相为40％乙腈水洗脱，tR＝56～66min。
进一步的，一种药物组合物，其中含有所述的一种黄酮类化合物或其药学上可以接受的盐及药学上可接受的载体。
进一步的，所述的一种黄酮类化合物或其药学上可以接受的盐或所述的一种药物组合物在制备治疗类风湿性关节炎、骨关节炎、强直性脊柱炎、哮喘、动脉硬化、克罗恩病、阿尔茨海默病、帕金森病、肠癌、肝癌或冠心病药物中的应用。其中，类风湿性关节炎、骨关节炎、强直性脊柱炎、哮喘、动脉硬化和克罗恩病为炎症性疾病。
进一步的，所述的一种黄酮类化合物或其药学上可以接受的盐或所述的一种药物组合物在用作制备抑制LPS诱导的巨噬细胞中炎症介质和促炎因子，或LPS诱导的大鼠腹腔巨噬细胞中mPGES-1的蛋白和mRNA表达水平，或LPS诱导的巨噬细胞中p38/JNK蛋白磷酸化的药物中的应用，优选的，炎症介质包括NO和PGE2，促炎因子包括IL-6和TNF-α。
本发明的有益效果如下：
1、本申请的黄酮类化合物6-甲氧基-4',5,7-三羟基-8-(1”-(3”'-甲氧基-4”'-羟苯基)乙基)异黄酮[6-methoxy-4',5,7-trihydroxy-8-(1”-(3”'-methoxy-4”'-hydroxyphenyl)ethyl)isoflavone，IT24]是从川射干采用硅胶柱层析、SephadexLH-20、ODS、高效液相色谱等方法提取分离出的全新化合物；
2、本申请的黄酮化合物IT24显著抑制LPS诱导的RAW264.7巨噬细胞和大鼠腹腔巨噬细胞中NO和PGE2的表达水平，但不影响COX-1/COX-2的表达。此外，IT24选择性下调LPS诱导的大鼠腹膜巨噬细胞中的mPGES-1蛋白表达。在LPS诱导的RAW264.7细胞中，IT24无法抑制NF-κB通路关键蛋白的磷酸化与核转位，但可以抑制丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号通路中p38/JNK蛋白的磷酸化。IT24通过抑制MAPK通路中p38/JNK蛋白磷酸化从而抑制mPGES-1的表达，进而发挥抑制炎症的作用；
3、本申请IT24作为一种潜在的mPGES-1选择性抑制剂，可用于类风湿性关节炎、骨关节炎、强直性脊柱炎、哮喘、动脉硬化和克罗恩病等炎症性疾病、阿尔茨海默病、帕金森病、肠癌、肝癌、冠心病的治疗，具有进一步研究与药物开发的前景；
4、LPS可以与细胞膜上的toll样受体(TLR4)结合，通过激活MAPK信号通路导致巨噬细胞的激活。激活的MAPK信号通路会产生JNK、p38和ERK的磷酸化，导致炎症介质和促炎因子的后续转录。IT24抑制iNOS、TNF-α、IL-6和mPGES-1的表达。PGE2是由PGES合成，包括mPGES-1。因此，IT24可以通过减少mPGES-1的含量来抑制PGE2的生成。IT24通过抑制LPS诱导的iNOS和mPGES-1的表达来抑制NO和PGE2的产生，发挥抗炎作用；
5、本申请的IT24即从川射干中分离出的具有全新骨架的黄酮化合物，通过实验发现其具有较好的抗炎作用，且具有选择性抑制mPGES-1的特性，本发明为天然化合物的抗炎作用提供了一定的基础；
6、本申请利用LPS诱导的巨噬细胞模型首次发现并证明了黄酮化合物IT24的抗炎作用，包括(1)抑制LPS诱导的巨噬细胞中炎症介质(NO和PGE2)和促炎因子(IL-6和TNF-α)的产生；(2)在LPS诱导的大鼠腹腔巨噬细胞中，IT24选择性抑制mPGES-1的蛋白和mRNA表达水平，对COX-1和COX-2蛋白无抑制作用；(3)IT24通过抑制LPS诱导的巨噬细胞中p38/JNK蛋白磷酸化进而抑制mPGES-1的表达发挥抗炎作用。因此，IT24可能成为具有更高安全性的mPGES-1选择性抑制剂。
附图说明
图1是本发明IT24对RAW264.7细胞和大鼠腹膜巨噬细胞的毒性试验；
图2是本发明IT24和***素合成酶相互作用的对接结果；
图3是本发明IT24对LPS诱导的RAW264.7细胞和大鼠腹膜巨噬细胞中促炎因子、炎症介质、NO含量及IL-6和TNF-α蛋白表达水平和mRNA表达水平的影响；
图4是本发明IT24对LPS诱导的RAW264.7细胞中iNOS和COX-2蛋白表达水平和mRNA表达水平的影响；
图5是本发明IT24对LPS诱导的大鼠腹腔巨噬细胞中COX-1、COX-2和mPGES-1蛋白表达和mRNA表达水平的影响；
图6是本发明IT24对LPS诱导的RAW264.7细胞中p65、p-p65、IκBα、p-IκBα、IKKβ和p-IKKα/β蛋白表达的影响；
图7是本发明IT24对LPS诱导的RAW264.7细胞中NF-κB核转位的作用；
图8是本发明T24对LPS诱导的RAW264.7细胞中p38、p-p38、ERK、p-ERK、JNK和p-JNK蛋白表达的影响；
图9是本发明IT24抑制LPS诱导的巨噬细胞激活的分子机制；
图10是本发明化合物IT-24的质谱数据图；
图11是本发明化合物IT-24的红外数据图；
图12是本发明化合物IT-24的紫外数据图；
图13是本发明化合物IT-24的核磁共振氢谱；
图14是本发明化合物IT-24的核磁共振碳谱；
图15是本发明化合物IT-24的核磁共振DEPT-135谱；
图16是本发明化合物IT-24的1H-1H COSY图谱；
图17是本发明化合物IT-24的HSQC图谱；
图18是本发明化合物IT-24的HMBC图谱。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。
因此，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
本实施例提供一种黄酮类化合物的制备方法：
取干燥的川射干20.0kg，粉碎至60目后，用400L 70％乙醇水回流提取2次，每次2小时，真空减压至无醇味，得到总浸膏2.1kg，然后将总浸膏经过D101大孔树脂柱层析，乙醇水梯度洗脱，分别得到40％乙醇水、70％乙醇水(580.3g)、95％乙醇水部位。70％乙醇洗脱部位经过硅胶柱层析，用二氯甲烷-甲醇作流动相，分离得到6个部位(A-F)，馏分B(85.5g)由二氯甲烷-甲醇(99：1)洗脱得到，再依次经过硅胶柱层析、Sephadex LH-20柱层析、ODS柱层析、高效液相色谱(40％乙腈水洗脱，tR＝61min)得到化合物IT-24(10mg)。
试验例1
对实施例1制备的化合物IT-24，根据理化性质、紫外光谱、红外光谱、质谱、核磁共振谱以及旋光谱(图10-18)确定该化合物的结构。
IT-24为黄色无定型粉末，[α]25.8D+4.5(c 0.95CH3OH)，HR-ESI-MS m/z451.1393[M+H]+(calcd.for 451.1393)(如图10所示，化合物IT-24的质谱数据)，推断IT-24的分子式为C25H22O8。用石油醚和乙酸乙酯(1:2)作为展开剂，香草醛浓硫酸作为显色剂，TLC鉴定为蓝色。
UV谱(图12，化合物IT-24的紫外数据)显示(272nm)有吸收，IR谱(图11，化合物IT-24的红外数据)显示羟基吸收(3447cm-1)，羰基吸收(1644cm-1)。1H NMR谱(图13化合物IT-24的核磁共振氢谱)显示δH 8.36为一个尖锐的单峰信号，说明该化合物的母核为异黄酮；化学位移δH7.35(2H,J＝8.8Hz),δH6.80(2H,J＝8.8Hz)中，每组质子均为双重峰，表明该化合物存在2对苯环邻位耦合的情况，进一步说明该化合物为4′-氧取代异黄酮类化合物；δH13.24(1H,s)表明该化合物中存在5-OH取代；化学位移δH 6.89(1H,d,J＝1.6Hz),6.63(1H,d,J＝8.0Hz),6.68(1H,dd,J＝8.4,1.6Hz)为苯环上的ABX***，表明该化合物中含有一个1，3，4三取代苯环。结合13C NMR谱(图14化合物IT-24的核磁共振碳谱)和DEPT135谱(图15化合物IT-24的核磁共振DEPT-135谱)中含有3个甲基，9个次甲基，13个季碳。δC180.9、153.9、121.5为异黄酮母核中央三碳核的特征信号。δC60.2、55.7为两个甲氧基碳信号。1H-1H COSY谱(图16化合物IT-24的1H-1H COSY图谱)显示δH 4.71(1H,q,J＝7.6Hz,H-1″)与δH 1.68(3H,d,J＝7.6Hz,H-2″)有相关，提示该化合物存在-CH-CH3结构片段。将IT-24的核磁波谱数据与Compound 11(J Asian Nat Prod Res.2010Nov；12(11):978–84.)比较发现，差异比较大的数据集中在异黄酮母核的B环部分。在HMBC图谱中(图18化合物IT-24的HMBC图谱)，δH7.35(2H,d,H-2′,6′)、6.80(2H,d,H-3′,5′)均与δC157.4(C-4′)有远程相关，可知B环上C-4′被羟基化。δH 6.89(H-2′″)、6.80(H-5′″)均与δC144.5(C-4′″)，147.1(C-3′″)有远程相关，δH 6.68(H-6′″)与δC144.5(C-4′″)有远程相关，δH3.71(3H,s)与147.1(C-3′″)有远程相关，提示3′″位连有-OCH3，4′″被羟基化。通过HSQC谱(图17化合物IT-24的HSQC图谱)将化合物IT-24的核磁共振光谱数据进行归属，如表1所示。综上可知化合物IT-24的结构如下：
在Scifinder数据库中对化合物IT-24进行检索，未见相关报道，将其命名6-methoxy-4′,5,7-trihydroxy-8-(1″-(3″′-methoxy-4″′-hydroxyphenyl)ethyl)isoflavone。
表1.化合物IT-24的核磁共振光谱数据
δinppm；J in Hz；in DMSO
试验例2
细胞毒性试验
细胞培养和给药：RAW264.7细胞系购于美国模式培养物研究所(American TypeCulture Collection,ATCC)，并培养于含10％血清(FBS)的DMEM高糖培养基中。培养条件为恒温37℃且含5％CO2。处理条件为IT24预给药1h，然后给予LPS(100ng/mL)刺激。
原代巨噬细胞的分离与培养：大鼠腹腔巨噬细胞是从SD大鼠(200-250g)体内分离。分离方法参考文献(Biogerontology.2016Apr；17(2):359–71)。取得细胞后，立即进行离心，条件为1500rpm/10min，弃上清，培养于含10％FBS的DMEM培养基中。然后将细胞种于6孔板中，37℃，5％CO2，培养2h。给予不同浓度的IT24预处理1h，然后给予LPS(1μg/mL)刺激。
采用MTT法检测细胞活力，根据公式计算各组细胞存活率，并确定七组不同的浓度梯度，以进行后续实验。将RAW264.7细胞(1.2×104)和大鼠腹腔巨噬细胞(1.5×105)接种于96孔板中，培养24h。采用不同浓度的IT24预处理1h。采用浓度为100ng/mL和1μg/mL的LPS分别刺激细胞24h/18h。每孔中加入MTT溶液(5g/L)，37℃培养4h。每孔中再加入100μL 10％的SDS-HCl溶液，培养18h。使用紫外可见分光光度计在570nm处读取吸光度。对照组不做任何干预处理，认为其细胞活力为100％。细胞存活率(％)＝处理组OD值/对照组OD值×100％。
MTT结果显示，在LPS诱导的RAW264.7细胞中，当IT24<25μM时，对细胞没有明显毒性(图1中的A)。在LPS诱导的大鼠腹腔巨噬细胞中，当IT24<50μM时，对细胞没有明显毒性(图1中的B)。因此，分别选择25μM和50μM作为两类细胞的最高给药浓度。
由图1知：图1中的(A)IT24对RAW264.7细胞的毒性试验；IT24(6.25-100μM)预处理细胞1h，部分细胞采用LPS刺激18h，分析其细胞活力。图1中的(B)IT24对大鼠腹膜巨噬细胞的毒性试验；IT24(6.25-100μM)预处理细胞1h，部分细胞采用LPS刺激18h，所有数据均来自三个独立实验。*P<0.05,**P<0.01,***P<0.001,vs.正常细胞或受LPS刺激的细胞。
试验例3
分子对接试验
本实验使用Discovery Studio和PyMOL软件进行图形编辑。COX-1、COX-2和mPGES-1结构分别从PubChem数据库和PDB数据库中获得。
采用计算机模拟分子对接技术分析IT24潜在的作用靶点。根据表2中对接分数，IT24可能不直接与COX-1和COX-2的活性位点结合。相反，IT24与mPGES-1靶点具有较高的结合能。基于以上分析，IT24可能不直接与COX-1和COX-2结合，而是与mPGES-1具有结合作用。
表2.IT24与***素合成酶的相互作用预测
图2是IT24和***素合成酶相互作用的对接结果；本实验采用DiscoveryStudio软件进行分析，PyMOL软件进行图形编辑。
试验例4
ELISA与Griess实验：将RAW264.7细胞(4.5×105)和大鼠腹腔巨噬细胞(3×105)培养于6孔板中。用不同浓度的IT24预处理细胞1h，采用浓度为100ng/mL和1μg/mL的LPS分别刺激细胞24h/18h，收集上清液。采用ELISA试剂盒检测上清液中促炎细胞因子TNF-α和IL-6以及炎症介质PGE2、PGI2、PGD2和TXA2的水平。采用Griess试剂盒检测上清液中NO含量。采用实时荧光定量逆转录聚合酶链反应检测细胞的TNF-α和IL-6的基因表达水平。
采用Griess试验、ELISA试验、实时荧光定量逆转录聚合酶链反应测定IT24的抗炎作用。如图3所示，IT24显著抑制LPS诱导的RAW264.7细胞(图3中的A)和大鼠腹腔巨噬细胞(图3中的B)中NO和PGE2的表达水平。在图3中的C中，IT24显著抑制TXA2和PGD2的水平，但不影响LPS诱导的大鼠腹腔巨噬细胞中的PGI2的表达。同时，IT24还抑制LPS诱导的RAW264.7巨噬细胞中IL-6和TNF-α的蛋白(图3中的D)和基因(图3中的E)表达水平。基于以上结果，说明IT24对LPS诱导的巨噬细胞中炎症介质和促炎因子具有抑制作用。
图3.(A-B)IT24对LPS诱导的RAW264.7细胞和大鼠腹膜巨噬细胞中NO和PGE2含量的影响；用IT24和阳性药(DEX)预处理细胞1h，部分细胞采用LPS(100ng/mL/1μg/mL)刺激24h/18h，收集上清，使用Griess试验检测NO浓度，使用ELISA试剂盒测量PGE2的浓度。(C)IT24对LPS诱导的大鼠腹腔巨噬细胞中PGD2、PGI2和TXA2含量的影响；(D)IT24对LPS诱导的RAW264.7细胞中TNF-α和IL-6含量的影响；(E)IT24对LPS诱导的RAW264.7细胞中TNF-α和IL-6mRNA表达水平的影响；蛋白均由ELISA试剂盒或实时荧光定量逆转录聚合酶链反应测量。所有数据均来自三个独立实验。###P<0.001,vs.正常细胞或*P<0.05,**P<0.01,***P<0.001,vs.LPS刺激的细胞。
NO作为关键的信号分子，不仅会引起组织损伤，还会影响炎症环境中多种细胞和组织中的炎症信号的传导。NO作为参与炎症过程的最重要的炎症介质，可以将白细胞招募到特定组织。炎症性刺激可以激活iNOS，从而产生NO。在LPS诱导的巨噬细胞中，iNOS的表达显著增加，本申请的IT24处理后显示可以明显抑制iNOS的产生，因此，IT24通过抑制iNOS的表达来抑制NO的产生。
***素H2可以进一步转化为PGE2、PGI2、PGD2、TXA2以及***素F2α(prostaglandin F2α,PGF2α)等生物活性脂质，从而影响免疫、心血管、胃肠和中枢神经***。mPGES-1是人类末端限速酶，于1999年被首次表征。多项研究表明，mPGES-1的异常表达与多种疾病密切相关。例如mPGES-1在类风湿性关节炎和骨关节炎患者的滑膜组织样本与软骨细胞中过度表达。我们此前的研究曾发现，mPGES-1在胶原诱导的小鼠关节炎模型(Collagen-II-induced non-immune inflammatory arthritis,CIA)和角叉菜胶诱导的大鼠的炎症足组织中过量表达。临床研究发现，具有高胆固醇血症的骨关节炎患者体内髌下脂肪垫(Infrapatellar fat pad,IPFP)中mPGES-1的mRNA水平降低，但尚不清楚其下调后对骨关节炎患者的影响。阿尔茨海默病作为原发性退行性脑病，目前尚无特效治疗或逆转疾病进展的治疗药物。研究人员发现mPGES-1缺失可以减少老年斑周围小胶质细胞的积累，并减轻Tg2576小鼠的学***。结果表明，IT24可以抑制mPGES-1蛋白表达而不影响COX-1和COX-2的表达。截至目前，mPGES-1抑制剂GS-248已于2020年完成I期临床，但GS-248会显著引起头痛等不良反应。ISC 27864已于2015年完成I期临床。目前，ISC 27864在II期的安全性和有效性数据尚未公布。候选药物LY3031207和LY3023703在临床前实验中表现出对mPGES-1的选择性抑制作用，但也会引起严重的不良反应，并已在I期试验中终止。本申请的IT24作为一种更安全的潜在的mPGES-1抑制剂，为治疗类风湿性关节炎、骨关节炎、强直性脊柱炎、哮喘、动脉硬化和克罗恩病等炎症性疾病、阿尔茨海默病、帕金森病、肠癌、肝癌、冠心病提供候选药物分子，用于这些疾病的治疗。
试验例5
提取总蛋白与Western blot实验：蛋白浓度采用Bio-Rad试剂测定。首先采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)将蛋白分离，转移至NC膜上。将NC膜置于5％的脱脂牛奶中，封闭1h，加入TBST缓冲液，置于摇床上清洗三次，每次5min。使用TBST稀释一抗，置于4℃摇床中孵化过夜。回收一抗后，再次使用TBST缓冲液置于摇床上清洗三次，每次5min。加入二抗室温避光孵育1h。回收二抗，再次使用TBST缓冲液清洗条带三次，每次5min。使用Odyssey(Li-COR,Lincoln,NE,USA)软件检测抗原-抗体复合物条带，分析蛋白表达水平。
实时荧光定量逆转录聚合酶链反应：采用TransZol Up Plus(TransGen Biotech)RNA提取试剂盒分离RAW264.7和大鼠腹腔巨噬细胞中的总RNA。采用cDNA逆转录试剂盒(Roche,Nutley,New Jersey,USA)将RNA进行逆转录。通过qRT-PCR实验检测LPS诱导的RAW264.7细胞中IL-6、TNF-α、iNOS和COX-2以及LPS诱导的大鼠腹腔巨噬细胞中COX-2和mPGES-1的mRNA表达水平。使用FastStart Universal SYBR Green Master试剂盒(Roche,Nutley,New Jersey,USA)通过ViiATM 7实时PCR测量目标mRNA水平。引物设计分别如表3和表4所示。
表3.RAW264.7细胞引物
表4.大鼠腹腔巨噬细胞引物
IT24在LPS诱导的RAW264.7细胞中对iNOS和COX-2蛋白质与mRNA表达水平的作用：iNOS和COX-2是激活免疫***和炎症的关键介质。采用Western blot和qRT-PCR实验测定IT24对iNOS和COX-2的作用。如图4中的(A-C)所示，在LPS诱导的RAW264.7细胞中，IT24不抑制COX-2蛋白表达但显著抑制iNOS蛋白表达。此外，如图4中的(D-E)所示，IT24抑制LPS诱导的RAW264.7细胞中iNOS的mRNA表达水平而不影响COX-2的表达水平。说明，IT24在LPS诱导的RAW264.7细胞中，可以抑制iNOS蛋白和mRNA的表达而不抑制COX-2蛋白。
图4.(A-C)IT24对LPS诱导的RAW264.7细胞中iNOS和COX-2蛋白表达水平的影响；IT24和阳性药(DEX)预处理细胞1h，部分细胞用LPS(100ng/mL)刺激15min。提取细胞的总蛋白，用Western blot实验分析iNOS和COX-2的表达水平。(D-E)IT24对LPS诱导的RAW264.7细胞中iNOS和COX-2的mRNA表达水平的影响；所有数据均来自三个独立实验。###P<0.001,vs.正常细胞或**P<0.01,***P<0.001,vs.LPS刺激的细胞。
IT24在LPS诱导的大鼠腹腔巨噬细胞中对mPGES-1和COX-1/COX-2蛋白质与mRNA表达的作用：在mPGES-1酶的作用下可以生成PGE2。将下游mPGES-1作为新的靶点是一种更安全的治疗方法。采用Western blot和qRT-PCR实验检测IT24对LPS诱导的巨噬细胞中mPGES-1、COX-1和COX-2蛋白和mRNA表达水平的影响。如图5中的(A-B)所示，LPS刺激后，mPGES-1、COX-1和COX-2蛋白表达水平明显上调，IT24选择性下调mPGES-1蛋白表达水平，但不抑制COX-1和COX-2蛋白的表达。阳性药DEX也显著性下调mPGES-1和COX-2的表达(图5中的(A-B))。阳性药MK886抑制5-脂氧合酶激活蛋白(5-lipoxygenase-activating protein,FLAP)和COX-1活性，通常作为二两者的抑制剂。在本实验中，MK886在5μM/mL的浓度时，抑制mPGES-1蛋白质表达水平(图5中的(A-B))。根据qRT-PCR实验结果(图5中的C)，浓度为50μM/mL的IT24显著抑制LPS诱导的大鼠腹腔巨噬细胞中mPGES-1的mRNA表达。阳性药DEX对COX-2和mPGES-1的mRNA表达也具有相似的抑制作用。以上结果说明，IT24可以抑制LPS诱导的大鼠腹腔巨噬细胞中mPGES-1蛋白质和mRNA的表达而不影响COX-1和COX-2。
图5.(A)IT24对LPS诱导的大鼠腹腔巨噬细胞中COX-1、COX-2和mPGES-1蛋白表达的影响；IT24和阳性药(DEX)预处理细胞1h，部分细胞采用LPS(1μg/mL)刺激18h，收集细胞总蛋白，用Western blot实验分析COX-1、COX-2和mPGES-1的表达水平。所有数据均来自三个独立实验。###P<0.001,vs.正常细胞或*P<0.05,**P<0.01,***P<0.001,vs.LPS刺激的细胞。
试验例6
高内涵试验：将RAW 264.7细胞(1×104)接种于96孔板中，培养18h，不同浓度IT24预给药6h。其中部分给予LPS(200ng/mL)刺激15min。阳性药物采用Bay(1μM/mL)。采用高通量Incell 6000(GE Healthcare)仪器分析NF-κB通路的核转位情况。
IT24对NF-κB通路的作用：NF-κB是核转录因子，可以调控炎症性反应。该通路可诱导iNOS和COX-2等促炎介质以及TNF-α和IL-6等炎症因子。激活NF-κB通路有两个途径，分别是经典途径和非经典途径，其中经典途径最为常见，主要涉及IKKβ、IκBα和p65蛋白。采用Western blot实验测定IT24对LPS诱导的RAW264.7细胞中IKKβ、p-IKKα/β、IκBα、p-IκBα、p65和p-p65蛋白表达水平的影响。如图6中(A-C)所示，LPS诱导RAW264.7细胞后，IT24不抑制NF-κB通路中IKK-β、IκBα和p65蛋白的表达。根据上述结果，说明IT24无法抑制NF-κB通路的激活。
图6.(A-C)IT24对LPS诱导的RAW264.7细胞中p65、p-p65、IκBα、p-IκBα、IKKβ和p-IKKα/β蛋白表达的影响；IT24和阳性药(DEX)预处理细胞1h，部分细胞用LPS(100ng/mL)刺激细胞15min。收集细胞的总蛋白，用Western blot实验分析p65、p-p65、IκBα、p-IκBα、IKKβ和p-IKKα/β的表达水平。所有数据均来自三个独立实验。###P<0.001,vs.正常细胞或**P<0.01,vs.LPS刺激的细胞。
IT24对NF-κB通路核转位的作用：激活转录因子复合物NF-κB(p65/p50)后，Relp65/p50从IκBα释放出来并通过核孔转运，进一步影响炎性因子和炎症介质的表达。上述结果表明IT24不抑制NF-κB通路的激活。为了进一步验证，通过高内涵成像实验分析了IT24对LPS诱导的RAW264.7细胞中NF-κB核转位的影响。如图7所示，IT24在任何给药浓度下均不抑制NF-κB核转位，此结果与Western blot实验结果保持一致。
图7.IT24对LPS诱导的RAW264.7细胞中NF-κB核转位的作用；细胞培养于96孔中18h，用不同浓度的IT24预处理6h。部分细胞使用LPS(200ng/mL)刺激15min。使用Incell6000分析核转位情况。###P<0.001,vs.正常细胞或***P<0.001,vs.LPS刺激的细胞。
IT24在LPS诱导的RAW264.7细胞中对p38/JNK蛋白磷酸化表达的作用：MAPK信号通路具有三个重要的亚家族，包括JNK、ERK和p38，均可以调节炎症反应。采用Western blot实验探究IT24对LPS诱导的RAW264.7细胞中p38、p-p38、ERK、p-ERK、JNK和p-JNK蛋白表达水平的影响。结果表明，LPS刺激后显著增强磷酸化JNK、p38和ERK蛋白的表达。然而，我们发现IT24预给药后可以抑制磷酸化JNK和p38蛋白的表达，但不影响磷酸化ERK蛋白的表达。这说明，IT24抑制LPS诱导的RAW264.7细胞中p38/JNK蛋白的磷酸化表达。
图8.IT24对LPS诱导的RAW264.7细胞中p38、p-p38、ERK、p-ERK、JNK和p-JNK蛋白表达的影响；IT24和阳性药(DEX)预处理细胞1h，部分细胞用LPS(100ng/mL)刺激15min。收集细胞的总蛋白，用Western blot实验分析p38、p-p38、ERK、p-ERK、JNK和p-JNK的表达水平。###P<0.001,vs.正常细胞或*P<0.05,**P<0.01,***P<0.001,vs.LPS刺激的细胞。
促炎细胞因子(如TNF-α和IL-6)能激活中性粒细胞和淋巴细胞，增加血管内皮细胞通透性，调节其他组织代谢活性并进一步介导炎症过程。实验结果表明，IT24显著抑制LPS诱导的RAW264.7细胞中TNF-α和IL-6的蛋白表达水平。此外，由于NF-κB诱导调节免疫反应的细胞因子以及粘附分子，并招募白细胞到炎症部位。本申请进一步探究IT24对p65、IκBα和IKKβ蛋白表达的影响。结果表明，IT24无法抑制这些蛋白的磷酸化。同时，IT24对NF-κBp65的核转位也不具有抑制作用。说明IT24无法抑制NF-κB通路的激活。众所周知，MAPK在调节炎症反应方面也发挥重要作用。因此，本申请进一步探究IT24对p38、JNK和ERK蛋白的影响。如图8所示，IT24抑制MAPK通路中JNK和p38蛋白磷酸化表达，对ERK的表达无抑制作用。已有研究表明，MAPK信号通路中的亚家族可以相互调节mPGES-1，但机制较为复杂。因此，mPGES-1和MAPK信号通路的联合调控可能是治疗炎症性疾病的潜在方法，而IT24可能是一个有效的候选药物分子(图9)。
图9.IT24抑制LPS诱导的巨噬细胞激活的分子机制；LPS可以与细胞膜上的toll样受体(TLR4)结合，通过激活MAPK信号通路导致巨噬细胞的激活。激活的MAPK信号通路会产生JNK、p38和ERK的磷酸化，导致炎症介质和促炎因子的后续转录。IT24抑制iNOS、TNF-α、IL-6和mPGES-1的表达。PGE2是由PGES合成，包括mPGES-1。因此，IT24可以通过减少mPGES-1的含量来抑制PGE2的生成。IT24通过抑制LPS诱导的iNOS和mPGES-1的表达来抑制NO和PGE2的产生，发挥抗炎作用。
上述试验例中，脂多糖(LPS)，***(Dexamethasone,DEX)，Bay117085和MK886均购自Sigma公司。COX-1、COX-2、iNOS、p65、p-p65、IκBα、p-IκBα、IKKβ、p-IKKβ、p38、p-p38、ERK、p-ERK、JNK和p-JNK抗体购自美国CST(Cell Signaling Technology)公司。β-actin和mPGES-1抗体购自美国Santa(Santa Cruz)公司。P65(D14E12)Rabbit mAb(Alexa488Conjugate)抗体购于CST公司。酶联免疫试剂盒购于美国Cayman公司。Griess试剂购于美国Promega公司。
上述试验例中，所有数据均来自三个独立实验，使用Graph Prism 9进行统计分析。组间差异通过单因素方差分析(ANOVA)来确定。p<0.05时差异具有统计学意义。
Claims (11)
1.一种黄酮类化合物或其药学上可接受的盐，其特征在于，所述化合物的结构式如下所示：
。
2.根据权利要求1所述的一种黄酮类化合物的制备方法，其特征在于，包括如下步骤：
步骤1、将川射干用乙醇进行提取，提取液真空减压至无醇味，得到总浸膏；
步骤2、将总浸膏经过D101大孔树脂柱层析，乙醇水梯度洗脱，分别得到40%乙醇水、70%乙醇水、95%乙醇水部位；
步骤3、70%乙醇洗脱部位经过硅胶柱层析，用二氯甲烷-甲醇作流动相，分离得到6个部位，分别为馏分A、馏分B、馏分C、馏分D、馏分E及馏分F，馏分B由二氯甲烷-甲醇洗脱得到，再依次经过硅胶柱层析、ODS柱层析、Sephadex LH-20柱层析、高效液相色谱得到上述黄酮类化合物IT-24；二氯甲烷与甲醇的体积比为99~1；高效液相色谱流动相为40%乙腈水洗脱，tR= 56~66 min。
3.根据权利要求2所述的制备方法，其特征在于，步骤1中取干燥的川射干，粉碎至60目后，用70%乙醇水回流提取2次，每次2小时，川射干与70%乙醇水的质量体积比为1 kg:7 L。
4.根据权利要求2所述的制备方法，其特征在于，步骤2中，70%乙醇水的质量为2.1 kg。
5.根据权利要求2所述的制备方法，其特征在于，馏分B的质量为580.3 g。
6.一种药物组合物，其中含有权利要求1的一种黄酮类化合物或其药学上可接受的盐及药学上可接受的载体。
7.如权利要求1所述的一种黄酮类化合物或其药学上可接受的盐或权利要求6所述的一种药物组合物在制备抑制LPS诱导的巨噬细胞中炎症介质和促炎因子的药物中的应用。
8.如权利要求1所述的一种黄酮类化合物或其药学上可接受的盐或权利要求6所述的一种药物组合物在制备LPS诱导的大鼠腹腔巨噬细胞中mPGES-1的蛋白和mRNA表达水平的药物中的应用。
9.如权利要求1所述的一种黄酮类化合物或其药学上可接受的盐或权利要求6所述的一种药物组合物在制备LPS诱导的巨噬细胞中p38/JNK蛋白磷酸化的药物中的应用。
10.根据权利要求7所述的应用，炎症介质为NO和PGE2，促炎因子为IL-6和TNF-α。
11.如权利要求1所述的一种黄酮类化合物或其药学上可接受的盐或权利要求6所述的一种药物组合物在制备治疗类风湿性关节炎、骨关节炎、强直性脊柱炎、哮喘、动脉硬化、克罗恩病、阿尔茨海默病、帕金森病、肠癌、肝癌或冠心病药物中的应用。
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| CN1594308A (zh) * | 2003-05-15 | 2005-03-16 | 成都迪康药物研究所 | 鸢尾苷元的异黄酮类衍生物及其制备方法和以其为有效药用成分的抗病毒药物 |
| CN101171241A (zh) * | 2005-03-11 | 2008-04-30 | 密执安州立大学董事会 | 抗细胞凋亡的bcl-2家族成员的色烯-4-酮抑制剂及其应用 |
| CN101664404A (zh) * | 2009-09-11 | 2010-03-10 | 吉林省中医药科学院 | 玉竹二氢高异黄酮提取物及其制备方法和用途 |
| CN101723926A (zh) * | 2008-10-10 | 2010-06-09 | 四川省中医药科学院 | 一种异黄酮衍生物及其制备方法和用途 |
| CN106963853A (zh) * | 2017-04-11 | 2017-07-21 | 四川省中医药科学院 | 一种川射干总黄酮苷元提取物及其制备方法和用途 |
| CN106995425A (zh) * | 2017-04-20 | 2017-08-01 | 四川省中医药科学院 | 一种川射干黄酮苷元的提取工艺及其分离纯化方法 |
| CN109748941A (zh) * | 2019-01-31 | 2019-05-14 | 王伟明 | 桄榔子镇痛化合物及其制备方法和应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1594308A (zh) * | 2003-05-15 | 2005-03-16 | 成都迪康药物研究所 | 鸢尾苷元的异黄酮类衍生物及其制备方法和以其为有效药用成分的抗病毒药物 |
| CN101171241A (zh) * | 2005-03-11 | 2008-04-30 | 密执安州立大学董事会 | 抗细胞凋亡的bcl-2家族成员的色烯-4-酮抑制剂及其应用 |
| CN101723926A (zh) * | 2008-10-10 | 2010-06-09 | 四川省中医药科学院 | 一种异黄酮衍生物及其制备方法和用途 |
| CN101664404A (zh) * | 2009-09-11 | 2010-03-10 | 吉林省中医药科学院 | 玉竹二氢高异黄酮提取物及其制备方法和用途 |
| CN106963853A (zh) * | 2017-04-11 | 2017-07-21 | 四川省中医药科学院 | 一种川射干总黄酮苷元提取物及其制备方法和用途 |
| CN106995425A (zh) * | 2017-04-20 | 2017-08-01 | 四川省中医药科学院 | 一种川射干黄酮苷元的提取工艺及其分离纯化方法 |
| CN109748941A (zh) * | 2019-01-31 | 2019-05-14 | 王伟明 | 桄榔子镇痛化合物及其制备方法和应用 |
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