CN111393492B - 一种炔烯对苯二酚糖苷及其制备方法和在制备肝x受体激动剂中的应用 - Google Patents
一种炔烯对苯二酚糖苷及其制备方法和在制备肝x受体激动剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种炔烯对苯二酚糖苷及其制备方法和在制备肝X受体激动剂中的应用。炔烯对苯二酚糖苷，或其药用盐，其结构式如式I所示。本发明从Pestalotiopsis neglecta SCSIO41403的发酵培养物中分离得到了具有激发了LXRα的转录活性，致使下游基因如ABCA1进行相应调控的炔烯对苯二酚糖苷，能够将其用于作为肝X受体激动剂的。
Description
技术领域：
本发明属天然产物应用技术领域，具体涉及一种炔烯对苯二酚糖苷化合物及其制备方法和在制备肝X受体激动剂中的应用。
背景技术：
肝X受体(1iver X receptor，LXR)属于核受体超家族成员，包含LXRα和LXRβ两种亚型，其中，LXRα主要分布在肝脏，而LXRβ则遍布全身组。LXR在动脉粥样硬化的发生和发展中起关键性作用，以LXRs为靶点寻找抗动脉粥样硬化的药物,对心血管疾病的治疗具有重要意义。LXRs作为胆固醇感受器,可以调节包括胆固醇流出、转运、排泄等大量与胆固醇代谢有关的基因的表达，LXR又被发现是脂代谢和糖代谢的关键转录调节因子。另外，LXR在巨噬细胞和系统水平均有抗炎作用，LXR激动剂在脑和中枢神经系统中对免疫和胆固醇稳态也有作用。因此，LXR有望作为治疗动脉粥样硬化、高胆固醇血症、Ⅱ型糖尿病等的药物靶点。
发明内容：
本发明的第一个目的是提供一种具有激发了LXRα的转录活性，致使下游基因如ABCA1进行相应调控的炔烯对苯二酚糖苷。
本发明的炔烯对苯二酚糖苷，或其药用盐，其结构式如式I所示：
本发明发现，炔烯对苯二酚糖苷可以显著上调LXR靶基因ABCA1(图4)，证实了PQC是LXRα激动剂。因此，炔烯对苯二酚糖苷衍生物PQC是通过与LXRα受体结合，激发了LXRα的转录活性，致使下游基因如ABCA1进行相应调控。
因此，本发明的第二个目的是提供上述炔烯对苯二酚糖苷，或其药用盐在制备肝X受体激动剂中的应用。
本发明的第三个目的是提供一种肝X受体激动剂，其包括炔烯对苯二酚糖苷，或其药用盐作为活性成分的。
本发明的第四个目的是提供一种炔烯对苯二酚糖苷的制备方法，包括以下步骤：
制备Pestalotiopsis neglecta SCSIO41403的发酵培养液，发酵液用乙酸乙酯萃取，乙酸乙酯萃取液经浓缩去除乙酸乙酯后得到浸膏，浸膏经硅胶柱层析，石油醚/乙酸乙酯从体积比20:1,10:1,5:1,2:1,1:1,和0:1梯度洗脱，收集石油醚/乙酸乙酯体积比1:1洗脱馏分，再次经过硅胶柱层析，用石油醚/乙酸乙酯从4:1～1:1梯度洗脱，收集石油醚/乙酸乙酯体积比2:1洗脱馏分，经纯化后得到pestalotioquinoside C。
所述的纯化是半制备HPLC纯化，采用SunFireTM C18色谱柱，柱温40℃，CH3CN/H2O梯度洗脱，0-3min，25％CH3CN；3-20min，25％-55％CH3CN；20-23min，55％-100％CH3CN；23-28min，100％CH3CN；28-30min，100％-5％CH3CN；30-33min，5％CH3CN；2mL/min，保留时间为13.5min时分离得到纯化合物pestalotioquinoside C。
所述的制备Pestalotiopsis neglecta SCSIO41403的发酵培养液是将Pestalotiopsis neglecta SCSIO41403接种到发酵培养基中，28℃，180转/每分，培养7天，获得发酵液，所述的发酵培养基是每升含有甘露醇20g，大豆蛋白胨10g，磷酸氢二钾0.35g，碳酸钙0.5g，豆油0.5ml，余量为水，pH 7.0。
本发明的第五个目的是提供Pestalotiopsis neglecta SCSIO41403在制备炔烯对苯二酚糖苷中的应用。
本发明的第六个目的是提供Pestalotiopsis neglecta SCSIO41403，其保藏号为：GDMCC No：60987。
本发明从Pestalotiopsis neglecta SCSIO41403的发酵培养物中分离得到了具有激发了LXRα的转录活性，致使下游基因如ABCA1进行相应调控的炔烯对苯二酚糖苷，能够将其用于作为肝X受体激动剂的。
本发明的真菌Pestalotiopsis neglecta SCSIO41403，于2020年3月27日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，其保藏编号为GDMCC No：60987。
附图说明：
图1为炔烯对苯二酚糖苷衍生物pestalotioquinoside C(PQC)的化学结构和重要的二维核磁相关信息(COSY and HMBC)图。
图2为pestalotioquinoside C(PQC)与肝X受体α配体结合域(T090复合物)的分子对接分析。(a)PQC与LXRα配体结合口袋的三维结构；(b)PQC与LXRα配体结合模式的二维推测结构。计算机分析结果显示PQC与肝X受体α结合较好。
图3为pestalotioquinoside C(PQC)与LXRα受体的蛋白-小分子相互作用实验(SPR)，实验结果显示PQC与LXRα受体结合较好。
图4为pestalotioquinoside C(PQC)共培养后的ACHN细胞中LXRα和ABCA1的mRNA水平。结果以平均值±SD表示(n＝9,ns＝不显著,****p<0.0001)。实验结果显示PQC可以显著上调LXR靶基因ABCA1，证实PQC是LXRα激动剂。
具体实施方式：
为了使本发明的目的、技术方案和有益技术效果更加清晰，以下结合实施例，对本发明进行进一步详细说明。应当理解的是，本说明书中描述的实施例仅仅是为了解释本发明，并非为了限定本发明，实施例的参数、比例等可因地制宜做出选择而对结果并无实质性影响。
实施例1真菌Pestalotiopsis neglecta SCSIO41403发酵和炔烯对苯二酚糖苷的分离
将种子培养基各组分(麦芽糖6.25g，麦芽提取物6.25g，酵母提取物1g，蛋白胨6.25g，磷酸二氢钾1.25g)溶解于1L蒸馏水中，1M NaOH溶液调节pH至7.0。配制4L体积后将其分装于500ml三角瓶(200ml/瓶，20瓶)，每瓶加入10-15颗玻璃珠以避免菌丝振摇成团，棉塞封口包扎后放入高压灭菌锅121℃灭菌30min。待灭菌培养基冷却后，从保存斜面挑取真菌Pestalotiopsis neglecta SCSIO41403菌丝体接种入瓶中，25℃下摇床振摇(180转/每分)。
种子发酵液培养3天后，按10％v/v的接种量接种种子发酵液于5L发酵罐中进行大量发酵。发酵培养基配方(甘露醇20g，大豆蛋白胨10g，磷酸氢二钾0.35g，碳酸钙0.5g，豆油0.5ml，蒸馏水1L，pH 7.0，其配制方法是将各成分混合，1M NaOH溶液调节pH至7.0，灭菌制得)，除分装体积不同，且不添加玻璃珠外，其它配置步骤与种子发酵液一致，发酵体积共计40L(1L/罐，40罐)，培养条件为28℃，180转/每分，培养7天，获得发酵液。
发酵液用乙酸乙酯萃取3次，减压回收溶剂得到提取物浸膏。提取物浸膏(10g)以少量甲醇溶解后硅胶拌样，进行中压硅胶柱层析(200-300目)，洗脱液为石油醚/乙酸乙酯从体积比20:1,10:1,5:1,2:1,1:1,和0:1梯度洗脱，顺序得到6个洗脱部位Fr.a–Fr.f。Fr.e(石油醚/乙酸乙酯体积比1:1洗脱馏分)再次经过硅胶柱层析(用石油醚/乙酸乙酯从4:1～1:1梯度洗脱)分离得到3个组分Fr.e1-Fr.e3，其中Fr.e2(石油醚/乙酸乙酯体积比2:1洗脱馏分)经半制备HPLC纯化，采用SunFireTM C18色谱柱(5μm，10×250mm)，柱温40℃，CH3CN/H2O梯度洗脱(0-3min，25％CH3CN；3-20min，25％-55％CH3CN；20-23min，55％-100％CH3CN；23-28min，100％CH3CN；28-30min，100％-5％CH3CN；30-33min，5％CH3CN；2mL/min)，保留时间为13.5min时分离得到纯化合物2.87mg，将其命名为pestalotioquinoside C(PQC)。
实施例2 Pestalotioquinoside C(PQC)的结构鉴定
实施例1中分离获得的炔烯对苯二酚类天然产物PQC的化学结构如图1所示。结构推导过程如下：
PQC为黄色油状物，HR-ESIMS显示m/z 387.1825[M-H]-，分子式为C22H28O6。该化合物的1H NMR(表1)显示3个甲基[δH 1.69(3H,s,H3-8′),1.58(3H,s,H3-10′),1.25(3H,d,J＝6.2,H3-6″)]，2个sp3杂化亚甲基[δH 2.26(2H,t,J＝7.2Hz,H2-4′),2.31(2H,dt,J＝7.0,7.2Hz,H2-5′)]，5个sp3杂化次甲基[δH 5.27(H,d,J＝1.7Hz,H-1″),3.97(H,dd,J＝3.4,1.7Hz,H-2″),3.81(H,dd,J＝9.5,3.4Hz,H-3″),3.45(H,t,J＝9.5Hz,H-4″),3.68(H,dq,J＝9.5,6.2Hz,H-5″)]，1个烯烃亚甲基[δH 5.38(1H,d,J＝1.8Hz,H2-9′),5.27(1H,d,J＝1.8Hz,H2-9′)]，4个烯烃次甲基[δH 6.99(1H,d,J＝2.9Hz,H-3),6.90(1H,dd,J＝8.9,2.9Hz,H-5),6.76(1H,d,J＝8.9Hz,H-6),5.17(1H,t,J＝7.0Hz,H-6′)]。13C NMR谱(表1)显示22个碳信号，其中包括3个甲基，2个sp3杂化亚甲基，5个sp3杂化次甲基，1个sp2杂化亚甲基，4个sp2杂化次甲基，5个sp2杂化季碳，2个sp杂化碳。综合1H和13C NMR数据分析，PQC中存在单取代对苯二酚结构。根据C-1′(δC 86.5)和C-2′(δC 94.6)的化学位移以及不饱和度分析，2个sp杂化碳确定为炔键；HMBC谱中，H-4′与C-2′和C-9′(δC 121.2)相关，H2-9′和C-2′相关，H-3和C-1′相关(图1)，表明存在炔烯片段连接在苯环C-2位置。1H-1H COSY谱中H2-4′/H2-5′/H-6′相关，HMBC谱中H3-8′与C-6′(δC 124.6)，C-7′(δC 136.8)以及C-10′(δC 17.9)相关，H2-4′与C-5′(δC 28.0)和C-6′(δC 124.6)相关(图1)，说明化合物中存在异戊烯基结构片段与C-4位相连。
1H NMR中的端基质子信号[δH 5.26(d,J＝1.7Hz,H-1″)]提示PQC为糖苷衍生物，1H-1H COSY谱中H-1″/H-2″/H-3″/H-4″/H-5″/H3-6″存在相关信号，HMBC谱中H-1″与C-2″，C-3″存在相关，H-3″与C-4″，C-5″存在相关，H-5″与C-4″，C-6″存在相关(图1)，进而验证了PQC结构中存在脱氧己糖片段，H-1″与C-4的HMBC相关则表明该糖基片段连接在C-4位上。通过对PQC进行酸水解，将反应产物的1H NMR和旋光
与标准鼠李糖测得数据进行对比，证实PQC中存在L-鼠李糖基团。氢-氢耦合常数3JH 1″,H 2″(1.7Hz)以及碳-氢耦合常数1JC 1″,H 1″(170Hz)则证实鼠李糖基团的相对构型为α构型。因此，PQC被确定为新的炔烯对苯二酚鼠李糖苷衍生物，命名为pestalotioquinoside C(炔烯对苯二酚糖苷)，其结构式如式I所示：
表1.PQC的1H NMR和13C NMR数据(氘代甲醇)
实施例3 Pestalotioquinoside C(PQC)作用肝X受体α活性测定
为了寻找潜在的LXR激动剂，我们通过高性能计算机的分子对接分析对多个炔烯对苯二酚类天然产物进行筛选。使用
软件进行对接，从可用的晶体结构(PDB ID：1UHL)中检索出与T090形成复合物的LXRα配体结合域，按照Maestro软件包中的Protein Prepare Wizard工作流程进行构建。使用Grid生成程序选择键合位点，然后再使用默认参数下的Glide(XP模式)将制备的配体灵活对接到受体上。研究分析LXRα配体结合域与化合物PQC之间的分子相互作用。结果发现，PQC可与LXRα受体结合域进行结合(图2)，结合自由能值(S值)为-11.132。在二维结合模型中，对苯二酚结构基团中的羟基与LXRα的活性位点残基THR302或HID421形成氢键相互作用，糖基片段则与LXRα受体结合域形成另外的氢键。由此推测糖基具有促进炔烯对苯二酚类化合物与LXRα受体蛋白结合的作用。
体外表面等离子体共振(SPR)分析用于测量LXRα受体和配体的亲和相互作用。将LXRα蛋白固定于CM5芯片上。以50μL/min的流速注入不同稀释浓度的受试化合物，持续180秒后用50mM NaOH作为清洗液使芯片再生。用GT Biacore T100在25℃采集数据，使用Biacore Evaluation Software 2.0计算蛋白与小分子之间的亲和力KD值。用LXR强激动剂GW3965作为阳性对照。PQC与LXRα受体结合作用较佳，呈剂量依赖型。PQC动力学曲线与LXR强效激动剂GW3965的动力学曲线相似，在SPR分析中与LXRα结合的解离平衡常数(KD)为50.0μM(图3)。
为了进一步研究PQC对LXRα的作用，通过定量聚合酶链反应(qPCR)方法检测PQC对细胞中的LXRα及其靶基因ABCA1的mRNA水平的影响。体外培养ACHN细胞，选择PQC作用LXRα受体的KD值作为最佳浓度，ACHN细胞培养液中加入PQC化合物，使其终浓度为50μM，共培养48小时。使用RNAprep纯组织试剂盒(中国天根生物科技有限公司)，提取PQC作用和未作用的ACHN细胞裂解物中的总RNA。使用PrimeScript RT试剂盒(日本Takara公司)和SYBRGreen PCR预混液(美国Promega公司)进行qPCR检测，按试剂盒说明书操作。β-肌动蛋白作为对照基因，以评估LXRα和ABCA1的相对mRNA水平。结果显示，PQC可以显著上调LXR靶基因ABCA1(图4)，证实了PQC是LXRα激动剂。因此，炔烯对苯二酚糖苷衍生物PQC是通过与LXRα受体结合，激发了LXRα的转录活性，致使下游基因如ABCA1进行相应调控。
Claims (6)
1.炔烯对苯二酚糖苷，或其药用盐，其结构式如式I所示：
2.权利要求1所述的炔烯对苯二酚糖苷，或其药用盐在制备肝X受体激动剂中的应用。
3.一种肝X受体激动剂，其特征在于，包括权利要求1所述的炔烯对苯二酚糖苷，或其药用盐作为活性成分的。
4.一种权利要求1所述的炔烯对苯二酚糖苷的制备方法，其特征在于，包括以下步骤：
制备Pestalotiopsis neglecta SCSIO41403的发酵培养液，发酵液用乙酸乙酯萃取，乙酸乙酯萃取液经浓缩去除乙酸乙酯后得到浸膏，浸膏经硅胶柱层析，石油醚/乙酸乙酯从体积比20:1,10:1,5:1,2:1,1:1,和0:1梯度洗脱，收集石油醚/乙酸乙酯体积比1:1洗脱馏分，再次经过硅胶柱层析，用石油醚/乙酸乙酯从4:1～1:1梯度洗脱，收集石油醚/乙酸乙酯体积比2:1洗脱馏分，经纯化后得到炔烯对苯二酚糖苷。
5.根据权利要求4所述的制备方法，其特征在于，所述的纯化是半制备HPLC纯化，采用SunFireTMC18色谱柱，柱温40℃，CH3CN/H2O梯度洗脱，0-3min，25％CH3CN；3-20min，25％-55％CH3CN；20-23min，55％-100％CH3CN；23-28min，100％CH3CN；28-30min，100％-5％CH3CN；30-33min，5％CH3CN；2mL/min，保留时间为13.5min时分离得到纯化合物炔烯对苯二酚糖苷。
6.根据权利要求4所述的制备方法，其特征在于，所述的制备Pestalotiopsisneglecta SCSIO41403的发酵培养液是将Pestalotiopsis neglecta SCSIO41403接种到发酵培养基中，28℃，180转/每分，培养7天，获得发酵液，所述的发酵培养基是每升含有甘露醇20g，大豆蛋白胨10g，磷酸氢二钾0.35g，碳酸钙0.5g，豆油0.5ml，余量为水，pH 7.0。
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