CN110251524A - 化合物党参炔苷(Lobetyolin)的制备方法及在药物和保健品中的新用途 - Google Patents
化合物党参炔苷(Lobetyolin)的制备方法及在药物和保健品中的新用途 Download PDFInfo
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- CN110251524A CN110251524A CN201910571315.4A CN201910571315A CN110251524A CN 110251524 A CN110251524 A CN 110251524A CN 201910571315 A CN201910571315 A CN 201910571315A CN 110251524 A CN110251524 A CN 110251524A
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Abstract
本发明公开了化合物党参炔苷（Lobetyolin，C20H28O8）的制备方法及在制备拮抗顺铂（CDDP）诱导的急性肾损伤药物中的应用，属于化合物制备分离及医药用途开发领域。经体内外研究表明：党参炔苷可以有效减轻顺铂导致的小鼠急性肾损伤，包括减轻小鼠血清BUN和CRE水平，减少血清中TNF‑α和IL‑1β水平，抑制肾组织中促凋亡蛋白Bax和Casapase的表达水平。经体外实验表明：党参炔苷能够减轻HEK293肾细胞活力降低和细胞毒性，提高细胞中GSH水平同时降低MDA含量。因此，可以把该化合物及含有该化合物的有效组分制备成预防急性肾损伤的药物、保健品、食品添加剂或复方制剂等。其药物或保健品可以是现有的任何剂型，如片剂，胶囊剂，粉针剂，注射剂，丸剂，软胶囊，颗粒剂和贴剂等。
Description
技术领域
本发明涉及化合物党参炔苷（Lobetyolin）在制备预防急性肾损伤药物及保健品中的新用途，属于中药化合物的制备方法及其医药用途技术领域。
背景技术
顺铂(CDDP)，全名顺式-1,2-二氯二氨合铂，是临床使用的最有效和最常见的抗肿瘤药物之一，其在有效剂量范围内广泛应用于多种骨肉瘤和实体瘤的治疗，包括卵巢癌，宫颈癌，头颈癌以及非小细胞肺癌等。然而，高剂量顺铂产生的副作用限制了其在临床中的应用。临床试验报道，顺铂常见的副作用包括肾毒性，神经毒性，耳毒性，骨髓毒性和电解质紊乱等，其中肾毒性是最常见的不良反应。研究结果显示，肾小管上皮细胞中的顺铂浓度是血液中的5倍，毒性效应主要发生在近端小管，特别是在肾小管上皮细胞的S3区段，肾小球和远端小管随后受到影响。
一般而言，顺铂诱导的肾小管损伤涉及了相互关联的因素，例如DNA损伤，线粒体功能障碍，氧化应激，炎症反应及凋亡。因此，寻找一种能够有效抑制顺铂累计所致肾毒性的药物仍是当前研究的重点。氧化应激在顺铂诱发的肾毒性的病理生理学中起着重要的作用。先前的报道显示，顺铂进入机体后，通过抑制细胞还原和清除活性氧簇(ROS)的能力，使细胞中ROS水平升高，破坏了机体内氧化和抗氧化能力的平衡，使机体发生氧化应激，最终导致机体组织器官的损伤。此外，顺铂也可能损害抗氧化剂防御机制如谷胱甘肽（GSH），超氧化物歧化酶（SOD），使它们降解或失活，从而引发氧化应激损伤。同时，顺铂在水合代谢过程中会产生大量的氧自由基可引发生物膜中多价不饱和脂肪的过氧化反应，形成脂质过氧化物MDA等，破坏膜脂质的完整性，导致肾细胞功能紊乱甚至死亡。MDA是氧自由基与生物膜不饱和脂肪酸发生脂质过氧化反应的稳定代谢产物，其含量变化能直接反映细胞内酶系统或非酶系统产生的氧自由基，触发细胞膜发生脂质过氧化损伤。
最近有关于顺铂肾毒性机制的研究聚焦于肾小管上皮细胞的凋亡，已经证实在一些凋亡途径参与了顺铂所致肾小管上皮细胞损伤，包括通过死亡受体激活的外源性途径，如肿瘤坏死因子受体或Fas途径，内源性线粒体途径及内质网应激途径。各种外界因素可以通过不同的信号传递系统传递凋亡信号，引起细胞凋亡，Fas是一种跨膜蛋白，属于肿瘤坏死因子受体超家族成员，它与FasL结合可以启动凋亡信号的转导引起细胞凋亡。此外，顺铂可激活凋亡的线粒体途径，应用顺铂处理肾小管上皮细胞，可导致Bax向线粒体移位，活化caspase-2，线粒体释放细胞色素C、凋亡诱导因子及第二个线粒体来源的胱氨酸酶激活物(SMAC/Diablo)，并激活caspase-9，体内及体外试验均证实，caspase家族在顺铂致肾小管上皮细胞凋亡的执行阶段起重要作用。
综上所述，顺铂的副作用，在一定程度上限制了其在临床治疗上的应用，在天然药物中寻求有效预防急性肾损伤的成分，得到越来越多人们的普遍关注。目前，有研究报道，从中草药中获得提取物及活性成分对急性肾损伤具有一定的保护作用（参见：ZhangW.Z.et.al Platycodon grandiflorum Saponins Ameliorate Cisplatin-Induced AcuteNephrotoxicity through the NF-κB-Mediated Inflammation and PI3K/Akt/ApoptosisSignaling Pathways. Nutrients，2018；刘新文，等.三七总皂苷通过线粒体途径抑制顺铂诱导的大鼠肾细胞凋亡，中国药理学通报，2015）。
目前，大量研究表明，从中草药中获得的提取物及活性成分对急性肾损伤具有一定的保护作用。党参为桔梗科植物党参（Codonopsis pilosula (Franch.) Nannf.）的干燥根,是我国常用的一种传统补益类中药,具有调节血糖、促进造血机能、抗缺氧、抗应激、抗疲劳、增强机体免疫力、延缓衰老等多种药理作用。据报道，党参中含有皂苷、挥发油、微量生物碱蛋白质、多糖、维生素及多种氨基酸。其中党参炔苷对乙醇所致的胃黏膜损伤有很好的保护作用，与党参补益脾胃的传统功效相符，是党参保护胃黏膜的活性成分之一。此外，党参炔苷还具有较强的抗氧化活性。（参见：蒋小飞，等.党参总皂苷的提取分离和纯化[J].安徽农业科学,2011; 杨亚蕾.党参炔苷与浸出物含量的相关性研究[J].中国中医药资讯,2010; M. U. Dumlu, et al,. Chemical composition and antioxidant activity ofCampanula alliariifolia. Nat Prod Res.）。
到目前为止，未见有关党参炔苷对顺铂（CDDP）所致急性肾损伤保护作用的报道，本发明通过体内和体外实验创造性的提出并证明了该成分的急性肾损伤保护作用，对该化合物药用价值进行了充分的开发利用，为临床上治疗肾损伤提供了理论基础支撑。
发明内容
本发明提供了党参炔苷在制备预防顺铂（CDDP）诱导的急性肾损伤药物及保健品中的应用。
所述的党参炔苷可通过以下步骤获得：（1）取干燥的党参属样品，粉碎过200目筛，经适当的溶剂和提取方法，得到提取液，减压浓缩得浸膏；（2）用水稀释浸膏，上大孔树脂吸附柱色谱，依次用水和70%以上乙醇进行洗脱，收集醇洗脱部分，减压浓缩收集液，冷冻干燥得党参炔苷粗提物；（3）将所得样品上ODS柱，并用5%~85%甲醇的水溶液进行不同浓度洗脱，分段收集不同流出液，减压浓缩流出液，最后经重结晶，可得到纯度为95~99.99%的党参炔苷。
前述的党参属样品，可包括党参、桔梗等植物，其优选党参，提取方法可包括浸泡提取、回流提取、超声提取、超临界萃取等的任何一种；提取溶剂选自水，甲醇，乙醇任何一种及不同比例混合物；用于后续分离纯化的大孔树脂及ODS柱色谱，可包括药学上能够接受的如D101、NKA12等常规树脂及其他柱色谱。
前述的党参炔苷，主要对药源性肾损伤具有预防和治疗作用，能够在日常生活中使用，以缓解和或改善顺铂引起的肾损伤，同样该用途也可以应用到其他致病源导致的肾脏损伤。
当本发明用于上述用途时，其口服或胃肠外给药，均是安全的，在口服情况下，其可以以任何常规的形式给药，如片剂，胶囊剂，粉针剂，注射剂，丸剂，软胶囊，颗粒剂和贴剂等。
本发明制备预防和制备药源性肾损伤的党参炔苷可以与药用载体或食品载体混合，这里使用的固体或液体的赋形剂在本领域是总所周知的，下面举几个例子，散剂是内服的粉末剂，它的赋形剂有乳糖，淀粉，糊精，碳酸钙，合成或天然硫酸铝，氧化镁，硬脂酸镁，碳酸氢钠，干燥酵母等；溶液剂的赋形剂有水，甘油，1，2-丙二醇，单糖浆，乙醇，乙二醇，聚乙二醇，山梨糖醇等；软膏剂的赋形剂可以使用脂油，含水羊毛脂、凡士林、甘油、蜂蜡、木蜡、液体石蜡等组合成的疏水剂或亲水剂。
附图说明
图1：党参炔苷结构。
具体实施方式
实验例1. 桔梗中党参炔苷的制备分离
制备分离过程步骤如下：（1）取干燥的桔梗根或茎叶样品，粉碎过200目筛，经甲醇或乙醇提取，得到提取液，减压浓缩得浸膏；（2）用水稀释上述浸膏，上D101大孔树脂吸附柱色谱，依次用水和70%以上乙醇进行洗脱，收集70%醇洗脱部分，减压浓缩收集液，冷冻干燥得富含党参炔苷的粗提物；（3）将所得样品过反相ODS柱，并用5%~85%甲醇的水溶液进行不同浓度洗脱，分段收集不同流出液，减压浓缩流出液，最后经重结晶，可得到纯度为95.5%的党参炔苷。
茎叶做提取试材时候优选乙醇做提取溶剂，可以有效避免叶绿素等杂质的干扰。
实验例2. 轮叶党参中党参炔苷的制备分离
制备分离过程步骤如下：（1）取干燥的轮叶党参根或茎叶样品，粉碎过200目筛，经甲醇或乙醇提取，得到提取液，减压浓缩得浸膏；（2）用水稀释上述浸膏，上D101大孔树脂吸附柱色谱，依次用水和70%以上乙醇进行洗脱，收集70%醇洗脱部分，减压浓缩收集液，冷冻干燥得富含党参炔苷的粗提物；（3）将所得样品过反相ODS柱，并用5%~85%甲醇的水溶液进行不同浓度洗脱，分段收集不同流出液，减压浓缩流出液，最后经重结晶，可得到纯度为97.2%的党参炔苷。
茎叶做提取试材时候优选乙醇做提取溶剂，可以有效避免叶绿素等杂质的干扰。
实验例3. 党参中党参炔苷的制备分离
制备分离过程步骤如下：（1）取干燥的党参干燥根样品，粉碎过200目筛，经甲醇或乙醇提取，得到提取液，减压浓缩得浸膏；（2）用水稀释上述浸膏，上D101大孔树脂吸附柱色谱，依次用水和70%以上乙醇进行洗脱，收集70%醇洗脱部分，减压浓缩收集液，冷冻干燥得富含党参炔苷的粗提物；（3）将所得样品过反相ODS柱，并用5%~85%甲醇的水溶液进行不同浓度洗脱，分段收集不同流出液，减压浓缩流出液，最后经重结晶，可得到纯度为96.1%的党参炔苷。
实验例4. 党参炔苷对顺铂(DDDP)诱导的小鼠肾损伤的保护作用
实验方法主要参考文献（徐颀, 等. 毒理学杂志, 2016, 30, 260-264），简述如下：
4.1 动物分组及给药
将ICR小鼠随机分成空白对照组(Normal)、肾损伤模型组(Cisplatin)、党参炔苷低剂量给药组(10 mg/kg)、党参炔苷高剂量给药组(20 mg/kg)剂量组，每组10只。每天各给药组按体重10mL/Kg连续灌胃10天，每天1次，空白组和模型组均灌胃同体积生理盐水。于第7天给药后，禁食不禁水12h，肾损伤模型组和各给药组一次性腹腔注射20mg/Kg顺铂生理盐水溶液，继续给药3天，末次给药后禁食不禁水12h后眼眶静脉丛取血，离心制备血清，脱臼处死小鼠，迅速取出肾组织。将左肾置于生理盐水中制成匀浆，3000 rmp/min离心5 min，取上清液，试剂盒检测相关生化指标；右肾10%甲醛固定后进行病理学及免疫组织化学分析。
血清中CRE, BUN及肾组织中MDA的测定
取离心后的血清，分别采用肌氨酸氧化酶法和脲酶法试剂盒测定血清中CRE和BUN水平；取肾组织匀浆上清液，采用TBA法测试盒测定MDA的含量，按照试剂盒说明书进行操作，酶标仪相应波长处比色测定OD值，计算含量。
血清中TNF-α和IL-1β炎症因子的测定
采用ELISA法测定血清中炎症因子TNF-α和IL-1β的水平，根据说明书进行操作，酶标仪450nm波长处比色法测定OD值，计算含量。
病理组织学染色
取小鼠肾脏中部做横切面取材，常规石蜡包埋、切成5μm的切片，每组随机选取3张组织切片，进行脱蜡，梯度过酒精，苏木素(Hematoxylin)染细胞核，自来水冲洗返蓝，之后伊红(Eosin)染细胞质，常规脱水，透明，中性树胶封片，光学显微镜下观察肾小管病变。
统计学方法
所有数据应用SPSS 21.0软件进行分析处理。各项数据以Mean ± SD表示，P<0.01或P<0.05具有统计学意义。
结果如下（数据略）：
1、与正常对照组小鼠相比，一次性顺铂注射3天后，小鼠状态萎靡，毛色灰暗，体重略有减轻，肾指数和脾指数显著升高（P<0.05），表明代谢器官受损严重，出现了免疫抑制反应；党参炔苷2个给药组，小鼠状态明显好转，体重下降得到缓解，可以明显改善顺铂所致的肾指数下降（P<0.05），改善率大于65%，但对肝和脾指数几乎不影响。
、与模型组相比，党参炔苷2个剂量组均能不同程度的降低肾组织中MDA的含量，高剂量具有极显著性差异（P<0.01）
3、与模型组相比，党参炔苷2个剂量给药组均能够降低小鼠血清中TNF-α和IL-1β的水平，且具有显著性差异（P < 0.05）。表明党参炔苷能够部分通过抑制炎症因子的表达而发挥肾保护作用。
4、与模型组相比，党参炔苷2个剂量给药组均能够降低小鼠肾组织中Bax和caspase 3的水平，且具有显著性差异（P < 0.05）。表明党参炔苷能够部分通过抗凋亡作用而发挥肾保护作用。
实验例5. 党参炔苷对顺铂致HEK293细胞损伤的保护作用
1 材料和方法
1.1 材料、试剂及仪器
党参炔苷，纯度>98.5%（HPLC）；顺铂，(CDDP, 纯度>99%，Pt, 65%，CAS NO. 15663-27-1),二甲基亚砜（DMSO）等均购自美国Sigma化学品有限公司；青霉素和链霉素各10000U/mL的DMEM培养基、胎牛血清（FBS）等均购自美国Gibco公司；3-(4, 5-二甲基噻唑-2)-2, 5-二苯基四氮唑溴盐（MTT），北京生物技术有限公司；还原型谷胱甘肽（GSH）和丙二醛（MDA）等均购自南京建成生物工程研究所；活性氧试剂盒购自沈阳万类生物科技有限公司；Hoechst33258染色液购自上海碧云天生物技术有限公司；司；HC-2517高速离心机，安徽中科中佳科学仪器有限公司；显微镜Leica DM500，德国徕卡；SPECTRO star Nano全波长扫描式酶标仪，德国BMG LABTECH公司。
细胞的培养
从ATCC细胞库获得的HEK293细胞，细胞在进行细胞培养前在液氮中保存。将HEK293细胞置于含有10％FBS以及链霉素和青霉素的DMEM培养基中，并在环境为95％空气和5％CO2的培养箱中培养。为了细胞能够良好的传代，培养基每2天更换一次。当细胞充满于培养瓶80-90％时。用1mL胰蛋白酶-EDTA溶液进行消化，已备进行下一步实验。
细胞活力测试（MTT法）
使用MTT定量比色法测定细胞活力。将细胞接种到96孔板中并在37℃下培养。经过适当的培养时间后，用不同浓度的党参炔苷进行预给药，24h后，给予顺铂（20 μM）进行处理，造模满24 h后向每孔滴加20μL MTT溶液（5mg/mL）并在37 ℃下温育3.5 h。然后，向每孔中滴加150μL DMSO，并在平板振荡器中振摇5 min。最后，使用酶标仪(Nano，德国）在490nm处测量吸光度，产生的颜色强度与HEK 293细胞的存活数量成正比。
细胞中GSH和MDA含量及活性氧（ROS）的测定
细胞中GSH含量采用二硫代对硝基苯法和钼酸铵法测定，MDA水平采用硫代巴比妥酸反应物质（TBARS）法检测，使用荧光测定法（DCFH-DA）测定细胞内活性氧的相对水平，GSH、MDA测定方法均按照试剂盒说明书进行操作，酶标仪相应波长处比色测定OD值，计算其含量。
将HEK293细胞接种到6孔板中并在37℃下培养。经过适当的培养时间后，用不同浓度的党参炔苷进行预给药。24h后，给予顺铂（20 μM）进行处理，造模满24 h后用10μM DCFH-DA探针在37℃，5％CO2条件下在黑暗中孵育30 min。弃去培养基，用PBS洗涤细胞两次，然后在荧光显微镜（Leica TCS SP8，德国）下通过荧光强度测定产生的ROS。
数据处理
实验数据均用均数±标准差( ±s)表示，用SPSS 22.0统计软件进行分析，组间采用单因素方差分析比较差异。P < 0.05为明显差异。
结果
2.1 党参炔苷对顺铂诱导的HEK 293细胞活力降低的缓解作用
从表1可以看出，顺铂所产生的细胞毒性能够降低HEK293的细胞活力并呈剂量和时间依赖性。我们选择20 μM作为模型组剂量。党参炔苷在（5-20 μg/mL）的剂量以内并不影响HEK293细胞活力。与正常对照组相比，用20 μM的顺铂处理后，模型组的细胞活力显著降低。而党参炔苷（5-20 μg/mL）组对顺铂致药物损伤具有显著的保护作用并呈剂量依赖性，这些结果表明党参炔苷对顺铂所致的HEK293细胞活力降低具有明显改善作用，结果如下表。
表1 党参炔苷对顺铂所致的HEK293细胞损伤的保护作用
2.2 党参炔苷对顺铂诱导的HEK293细胞脂质过氧化的影响
从表2可以看出，与正常对照组相比，模型组细胞中GSH水平显著下降(P<0.05)，而MDA水平明显升高(P<0.05)，具有统计学意义，提示顺铂诱导急性肾细胞损伤模型成功建立，细胞间脂质过氧化产物累积，抗氧化代谢水平降低；与模型组相比，党参炔苷各给药剂量组细胞中的MDA含量随着给药剂量的增加而逐渐降低，并有显著性差异（P<0.05）；而GSH的含量随着给予党参炔苷剂量的增加均有所提升并呈一定的剂量关系(P<0.05)，表明党参炔苷可以在一定程度上缓解顺铂引起的脂质过氧化，有效改善细胞中的氧化应激损伤，结果如下表。
表2 党参炔苷对顺铂致HEK 293细胞损伤中GSH和MDA水平影响
2.3 党参炔苷对顺铂诱导的HEK293细胞氧化应激的影响
如表3所示，结果表明：正常HEK293细胞内存在少量的ROS，而与正常对照组相比，模型组经过20 μM顺铂处理24 h之后，细胞内ROS水平明显增多，而经党参炔苷给药组预处理后能明显抑制顺铂诱导的HEK293细胞氧化应激，图略。
表3 党参炔苷对顺铂致HEK 293细胞损伤中ROS和凋亡的影响
2.4 党参炔苷对顺铂诱导的HEK293细胞凋亡的影响
为判断党参炔苷能否减轻顺铂所引起的HEK293细胞凋亡，我们采用了Hoechst 33258染色法检测了细胞的凋亡程度。如表3所示，结果表明：正常对照组相比，顺铂模型组的细胞核致密浓染，荧光强度表达较高。然而，党参炔苷给药后，与模型组比较，荧光强度明显降低且细胞核轮廓规则，以上结果表明，党参炔苷对顺铂诱导的HEK293细胞的凋亡发挥了显著地抑制作用。
结论
党参炔苷在5~20 µM剂量范围内，可以显著改善顺铂诱导的HEK293细胞活力降低和细胞损伤，主要通过改善急性氧化应激水平及抑制凋亡反应等，在一定程度上为顺铂肾毒性防护措施的研究开辟了新的思路。
实施例 药物的实施例
制备药剂的实施例一
胶囊剂的制备 100g党参炔苷，药用淀粉适量，两者充分混匀，装胶囊，制成1000粒胶囊，每粒重0.25g，每粒含党参炔苷100mg。口服，每次4粒，每日三次。
制备药剂的实施例二
片剂的制备 100g党参炔苷，药用淀粉适量，两者充分混匀，用乙醇做粘合剂制湿颗粒，干燥，过120目筛整粒，装胶囊，每粒100 mg，每次口服1-2粒，每日2次。
制备药剂的实施例三
滴丸剂的制备 聚乙二醇4000 300g，在水浴上熔化，加入原料党参炔苷100g，搅拌均匀，倾入保温管中，调节恒温装置，使药液在80-90℃下滴入冷却过的液体石蜡中（温度±4℃），滴完后，将药丸倾入滤纸上吸干石蜡油，再加入少量滑石粉，混匀，得三七皂苷ST-4滴丸1000粒。口服，一次4粒，每日三次，饭后服用。
上述实施方式对本发明的党参炔苷在制备预防急性肾损伤药物及保健品方面的应用进行了简要说明，也可将党参炔苷与其他药物及活性成分组合使用，所以本发明并不局限于上述实施方式，在不脱离其要旨的范围内，可以对本发明技术方案的细节和形式进行修改或替换，在各种方式中实施本发明，其这些修改技术方案也应当在其保护范围之内，在此不再一一叙述。
Claims (4)
1.党参炔苷（Lobetyolin，C20H28O8）在制备预防急性肾损伤药物中的应用，所述的肾损伤疾病为顺铂（CDDP）所致的急性肾损伤，表现为明显改善了顺铂所致的小鼠急性肾毒性和HEK293细胞毒性，包括抑制凋亡、减少炎症反应和氧化应激。
2.按权利要求1所述的应用，其所述的党参炔苷分离可通过如下步骤获得：（1）取干燥的党参属（桔梗、轮叶党参和党参）样品，粉碎过200目筛，经适当的溶剂和提取方法，得到提取液，减压浓缩得浸膏；（2）用水稀释浸膏，上大孔树脂吸附柱色谱，依次用水和70%以上乙醇进行洗脱，收集醇洗脱部分，减压浓缩收集液，冷冻干燥得含党参炔苷的粗提物；（3）将所得样品过ODS柱，并用5%~85%甲醇的水溶液进行不同浓度洗脱，分段收集不同流出液，减压浓缩流出液，富集含党参炔苷样品；（4）上述样品经乙醇重结晶，可得到高纯度的党参炔苷。
3.按权利要求2所述的分离方法，其特征在于：所获得的党参炔苷纯度为95~99.99%。
4.根据权利要求1-3所述的应用，其特征在于：将党参炔苷作为唯一活性成分或与其它药物组合后使用，可按常规的制药方法和工艺要求，制成用以治疗或预防急性肾损伤的药物及保健品，并可以制作成任意剂型，如片剂，胶囊剂，粉针剂，注射剂，丸剂，软胶囊，颗粒剂和贴剂等。
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| CN114931645B (zh) * | 2022-06-29 | 2024-03-22 | 广州中医药大学第一附属医院 | 抑制骨桥蛋白表达的试剂在治疗尿毒症心衰中的应用 |
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