CN114246873B - 源自牛樟芝的化合物及提取物作为fgf21激动剂及相关疾病治疗剂或预防剂的用途 - Google Patents
源自牛樟芝的化合物及提取物作为fgf21激动剂及相关疾病治疗剂或预防剂的用途 Download PDFInfo
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Abstract
本发明涉及源自天然菌类牛樟芝的化合物及提取物用于制备FGF21激动剂以及非酒精性脂肪性肝炎等相关疾病的治疗剂或预防剂的用途。
Description
技术领域
本发明涉及源自天然菌类牛樟芝的化合物及提取物用于制备FGF21激动剂以及非酒精性脂肪性肝炎等相关疾病的治疗剂或预防剂的用途。
背景技术
成纤维细胞生长因子21(FGF21)是成纤维细胞生长因子(FGF)家族的一员，具有内分泌因子的功能，其生物活性包括促细胞增殖、机体发育、血管增生、创伤修复等，参与机体物质代谢，维持机体脂肪和糖代谢平衡。FGF21在胰脏、肝脏、白色脂肪组织中大量表达，主要经过PPAR和胰岛素/AKT1通路调控，在肝脏内PPARα和脂肪内PPARγ的调控下，通过细胞表面受体FGFR1c与共受体β-Klotho的相互作用，影响代谢，参与葡萄糖及脂质的代谢调节，促进葡萄糖异生、增加游离脂肪酸的氧化、生酮作用和葡萄糖的摄取，增加能量的产生和利用等(Fisher FM等,Annu Rev Physiol.2016；78:223-241)。研究发现FGF21水平在所有与胰岛素抵抗有关的疾病中均会升高，推测FGF21升高可能是机体防御糖毒性和脂毒性的保护机制，是机体的代偿性反应(Bonakdaran S等,Acta Endocrinol-Buch.2017；13:278-281)。
作为一种代谢调控因子，FGF21在肥胖、糖尿病(例如2型糖尿病)等代谢性疾病的治疗领域有非常重要的价值(Tezze C等,Front Physiol.2019；10:419)，可能成为治疗代谢性疾病的新治疗靶点，对心血管疾病、慢性肾病、糖尿病并发症的治疗也有重要意义。而且，FGF21是FGF家族中目前发现的唯一没有促有丝分裂作用的蛋白，从而大大降低了临床用药的风险。
并且，有研究表明，FGF-21通过与FGFR1c和β-Klotho的结合，激活MAPK/ERK、PI3K-Akt及PPARγ等重要的细胞信号通路，激活细胞核内葡萄糖转运体-1(GLUT-1)基因的转录水平，增加脂肪细胞对葡萄糖的摄取和利用，减轻胰岛素抵抗，有效调节血糖、胰岛素、胰高糖素及甘油三酯的水平，同时改善血脂紊乱，降低低密度脂蛋白(LDL)水平、增高高密度脂蛋白(HDL)水平(Zarei M等,Trends Pharmacol Sci.2020；41:199-208)。此外，内质网应激可诱导FGF21在肝脏的表达和合成增加，而给予外源性FGF21可以抑制由内质网应激诱发的肝脏脂肪变性，表明FGF21可以改善内质网应激，进而可减少非酒精性脂肪性肝炎(NASH)造成的损伤(Inagaki T等,Front Endocrinol.2015；6:147)。
目前，临床上作用于FGF21新靶点的药物多是FGF21类似物，如LY2405319、PF-05231023、BMS-986036等大分子或多肽类物质，将其用于改善肥胖和2型糖尿病患者的血脂异常。例如，已发现FGF21类似物BMS-986036在动物实验中能改善胰岛素敏感性，目前正在开展相应的II期临床试验(Mu J等，Diabetes.2012,61,505-512)。然而，上述FGF21类似物的有效剂量过高，可能与代谢性疾病患者体内FGF21抵抗有关(Gaich G等,CellMetab.2013；18:333-340)。并且，作为多肽，上述FGF21类似物均需要肠胃外给药，无法口服给药，由于高肾清除率和溶蛋白性裂解，血液循环半衰期较低(0.5-2h)，药代动力学性质较差(Lee JH等,Am J Transl Res.2016；8:4750-4763)。临床前研究还发现，FGF21类似物会导致啮齿动物大量的骨质流失，造成骨质疏松(Wei W等,Proc Natl Acad Sci U SA.2012；109:3143-3148)；FGF21类似物还会抑制肝脏生长激素-胰岛素样生长因子轴，影响机体生长，可能不适用于儿童和青少年(Inagaki T等,Cell Metab.2008；8:77-83)。
此外，目前也还没有发现任何具有良好效果的小分子FGF21激动剂。目前仅有研究发现奥贝胆酸、二甲双胍、talabostat等药物能通过FXR、PPAR、eIF2α等通路间接上调FGF21的表达。其中，据报道，奥贝胆酸能够增强FGF21的上调作用(Hu Y等,JHEP reports.2020；2:100093)，但其上调作用比较弱，并且在临床试验中也表现出了较大的副作用，包括严重的瘙痒反应等。
因此，为了更好地治疗可通过由于调控FGF21而带来治疗益处的疾病，本领域仍然需要找到更强效的FGF21激动剂。本发明的发明人意外地发现天然菌类牛樟芝提取物及特定的化学成分具有FGF21激动作用。牛樟芝又名樟芝，来源非常珍贵，将牛樟芝入药可用于治疗腹痛、抗过敏、解酒保肝等。目前还没有将牛樟芝或其中的任何成分用作FGF家族、特别是FGF21的激动剂的报道。例如，有文献报道了牛樟芝乙醇提取物中的成分DEA(结构如下)能够用于防治非酒精性脂肪性肝炎，但作用较弱，也没有提到其具有FGF21激动作用，因此DEA也不足以满足相关的临床需求。
发明内容
本发明的发明人发现，牛樟芝中的某些化学成分、其衍生物、及含有所述成分的特定提取物具有FGF21激动作用，由此完成了本发明。
具体地，本发明涉及以下内容。
本发明的一个方面涉及式(I)化合物或其盐或异构体在制备FGF21激动剂中的用途：
其中，R1和R2选自-H、-OH、C1-6烷基，或R1和R2共同形成＝O；R3选自-H、-OH、C1-6烷基；R4选自-H、-OH、糖基(优选氧-葡萄糖基)。
在一个优选的实施方案中，R1和R2选自H和-OH，或R1和R2共同形成＝O；R3选自-H和-OH；R4为-OH或氧-葡萄糖基。
在一个进一步优选的实施方案中，所述式(I)化合物为Antcin K、Antcin C、AK-GLU(即Antcin K-7-O-葡萄糖苷)或其盐或异构体：
本发明的另一个方面涉及一种牛樟芝提取物，该提取物满足以下一或多项：
(1)含有Antcin K的质量分数为5％以上、优选为10％以上，更优选为15％以上；
(2)含有所述Antcin C的质量分数为3％以上、优选为5％以上，更优选为8％以上；
(3)含有所述Antcin K和Antcin C的质量分数总计为8％以上、优选为15％以上，更优选为20％以上；
(4)含有DEA的质量分数为1％以下、优选为0.5％以下，更优选为0.1％以下。
本发明的另一个方面涉及前述牛樟芝提取物的提取方法，其包括以下步骤：
(1)将牛樟芝粉碎；
(2)用甲醇水溶液进行提取(例如通过回流或超声提取)，所述水溶液中的甲醇浓度为20-80％、优选为30-70％、更优选为40-60％(例如约50％)。
本发明的另一个方面涉及一种药物组合物，其中含有前述提取物。
本发明的另一个方面涉及前述提取物或药物组合物在制备FGF21激动剂中的用途。
本发明的另一个方面涉及前述任一项所述的化合物或其盐或异构体、提取物或药物组合物在制备药物中的用途，所述药物用于治疗或预防肥胖、糖尿病(例如2型糖尿病)、心血管疾病、非酒精性脂肪性肝炎等疾病，优选非酒精性脂肪性肝炎。
本发明的优势在于提供了前述式(I)化合物、特别是Antcin K、Antcin C和AK-GLU在用于制备FGF激动剂或相关疾病的治疗剂或预防剂中的用途，所述化合物的FGF激动活性活性显著优于现有技术中已知的FGF21激动剂奥贝胆酸以及文献中报道的牛樟芝成分DEA，在非酒精性脂肪性肝炎的治疗效果方面也取得了非常显著的提高。本发明还提供了能够富集前述化合物的牛樟芝提取物，其提取方法简便高效，可以直接将富集活性成分的提取物制成FGF21激动剂或相关疾病(如非酒精性脂肪性肝炎)的治疗剂或预防剂，从而大大降低药品的成本。
附图说明
图1：Antcin K的1H-NMR图谱(吡啶-d5，400MHz)。
图2：Antcin K的13C-NMR图谱(吡啶-d5，100MHz)。
图3：Antcin C的1H-NMR图谱(吡啶-d5，400MHz)。
图4：Antcin C的13C-NMR图谱(吡啶-d5，100MHz)。
图5：实施例3中牛樟芝不同提取物及标准品的HPLC图谱。
图6：实施例4中对蛋氨酸胆碱缺乏饮食造成的小鼠(MCD小鼠)的肝脏FGF21基因表达影响的实验结果。
图7：实施例5中对MCD小鼠的肝脏FGF21蛋白表达影响的实验结果。
图8：实施例6中对MCD小鼠血清肝功能的血生化指标检测结果。
图9：实施例6中对MCD小鼠肝脏进行病理切片染色的结果。
图10：实施例7中对MCD小鼠的血清FGF21蛋白表达影响的实验结果。
具体实施方案
本说明书上下文中所用的术语“约”表示在相应数值上下浮动10％的范围。例如，若某种成分的浓度为约5mM，表明其浓度为4.5-5.5mM；若某种成分的浓度范围为约5-10mM，表明其浓度范围为4.5-11mM。本说明书所用其它术语均具有本领域通用的含义。
本说明书上下文中所述的“式(I)化合物”及具体化合物Antcin K、Antcin C和AK-GLU的定义中包括其盐和异构体。所述盐优选为该化合物与酸或碱形成的药学上可接受的盐；所述“异构体”包括本领域技术人员公知的互变异构体、顺反异构体、非对映异构体(差向异构体)等，及其混合物。特别是，本发明的通式化合物和具体化合物中25位的碳原子(即羧基所连接的碳原子)为手性碳，因此本说明书所述的“异构体”包括25R/25S-差向异构体，以及其任意比例(优选等比例)的混合物。此外，对于R构型和S构型的差向异构体，在本说明书中可分别用“25R”和“25S”表示，例如用“25R-Antcin K”表示25位的碳原子呈R构型的Antcin K。
本说明书上下文中所用的术语“糖基”是指当苷元化合物与糖形成糖苷后由糖部分构成的基团。优选地，本说明书中所述的糖基为氧-葡萄糖基，其结构如下所示：
由于认识到了Antcin K、Antcin C这两种天然存在于牛樟芝中的成分具有显著优于现有技术的FGF21激动作用、特别是治疗或预防非酒精性脂肪性肝炎的作用，申请人设计了如前文所述的以甲醇水溶液对牛樟芝进行提取的方法，其能够简便高效地得到富集Antcin K、Antcin C且基本上不含DEA等杂质的提取物，即如前文所述的本发明的牛樟芝提取物。例如，在实施例4的实验条件下，本发明的牛樟芝提取物在以约80mg/kg给药时可以使MCD小鼠的肝脏FGF21基因表达水平提高至正常小鼠肝脏FGF21表达水平的约150倍以上或200倍以上；在实施例5的实验条件下，本发明的牛樟芝提取物在以约80mg/kg给药时可以使MCD小鼠的肝脏FGF21蛋白表达水平提高至正常小鼠肝脏FGF21蛋白表达水平的约100％以上或120％以上；在实施例6的实验条件下，本发明的牛樟芝提取物在以约80mg/kg给药时可以使MCD小鼠的血清ALT水平降低至120IU/L或100IU/L以下；在实施例7的实验条件下，本发明的牛樟芝提取物在以约80mg/kg给药时可以使MCD小鼠的血清FGF21水平升高至200pg/ml或300pg/ml以上。
本发明的化合物或提取物可以使用能够产生所需结果的任何方便的手段给药于治疗对象，例如人类患者，例如可以将所述化合物制成如前文所述的药物组合物，和/或制成已知的或新开发的剂型(如片剂、胶囊剂、注射剂等)中。本发明的化合物或提取物可以与其它治疗药物组合使用，当组合使用时，本发明的化合物或提取物可以与其它治疗药物制成同一剂型，或分别制成单独的剂型。前述药物组合物或剂型中可以加入一种或多种药学上可接受的载体或辅料，包括但不限于稀释剂、填充剂、粘合剂、润湿剂、崩解剂、润滑剂等。
本发明的具体的实施方案将通过以下实施例进行例示性解释说明，但应认识到所述实施例并非意在限制本发明的范围。实施例中所用的原料、试剂等，均为本领域技术人员所公知并且可通过市售或文献方法获得的物质；所用的试验或表征方法也是本领域技术人员公知的方法。
实施例1：Antcin K、Antcin C的提取分离
本实施例中提及的所有化学试剂均购自北京化工厂。
首先，称取干燥皿培牛樟芝25kg，粉碎。加入10倍量95％乙醇，加热回流2～3h，抽滤。滤渣用95％乙醇重复提取5次。合并提取液，减压浓缩回收溶剂，得总浸膏，即为牛樟芝乙醇提取物。可获得总浸膏约4.8kg，得率达19.2％。
为从乙醇提取物中分离Antcin K、Antcin C，取乙醇提取物1.2kg溶解于50％乙醇中，分4次上样于9.6kg大孔吸附树脂开放柱(AB-8)，以乙醇-水(50:50、70:30、85:15、95:5)作为流动相梯度洗脱，根据TLC和HPLC分析结果合并成6个流份(A-F)。
流份B(80.5g)通过硅胶柱分离，以二氯甲烷-甲醇(15:1-1:1，v/v)为流动相，梯度洗脱，得到4个流份(BA-BD)及化合物Antcin K(25R/S，10g)。采用半制备液相色谱法(乙腈-水，25:75，v/v)进一步纯化获得化合物25S-Antcin K(500mg)和25R-Antcin K(400mg)。
流份D(90.5g)溶解于适量甲醇，超声，过滤，获得滤液DA(12.6g)和固体DB(70.8g)。滤液DA经硅胶柱分离，以二氯甲烷-甲醇(10:1-1:1，v/v)作为洗脱剂，经TLC和HPLC检测结果合并成4个流份DAA至DAD。流份DAB(2.6g)通过LH-20凝胶色谱分离获得2个流份(DABA和DABB)，其中流份DABA(309.1mg)经半制备液相色谱法(乙腈-水，65:36，v/v)得到化合物25R-Antcin C(50.3mg)和25S-Antcin C(60.4mg)。
结构鉴定(详见图1至图4)：
Antcin K(25R,25S):1H NMR(400MHz,吡啶-d5)δ:2.10,3.16(2H,m,H-1),1.95,2.77(2H,m,H-2),4.09(1H,brs,H-3),4.64(1H,t,J＝8.2Hz,H-7),2.46,2.99(2H,d,J＝13.4Hz,H-12),0.92(3H,s,H-18),2.08(3H,s,H-19),0.91(3H,d,J＝6.6Hz,H-21),1.52(3H,d,J＝7.0Hz,H-27),5.09(1H,s,H-28a),5.23(1H,s,H-28b),1.75(3H,s,H-29)。13C NMR(100MHz,吡啶-d5)δ:30.0(C-1),27.1(C-2),211.7(C-3),75.1(C-4),43.9(C-5),30.5(C-6),71.2(C-7),144.4(C-8),154.6(C-9),39.1(C-10),201.8(C-11),59.2(C-12),48.3(C-13),54.1(C-14),25.8(C-15),28.6(C-16),55.2(C-17),12.9(C-18),21.3(C-19),36.6(C-20),19.0(C-21),34.8(C-22),32.3(C-23),150.7(C-24),46.9(C-25),177.3(C-26),17.4(C-27),110.8(C-28),28.4(C-29)。
Antcin C(25R,25S):1H NMR(400MHz,吡啶-d5)δ:1.25,2.90(2H,m,H-1),2.38(2H,m,H-2),4.53(1H,t,J＝8.6Hz,H-7),2.47(1H,d,J＝13.8Hz,H-12a),3.00(1H,d,J＝13.8Hz,H-12b),0.90(3H,s,H-18),1.61(3H,s,H-19),0.92(3H,d,J＝5.1Hz,H-21),1.53(3H,d,J＝7.0Hz,H-27),5.10(1H,s,H-28a),5.25(1H,s,H-28b),1.13(3H,d,J＝6.5Hz,H-29)。13CNMR(100MHz,吡啶-d5)δ:36.6(C-1),38.5(C-2),211.8(C-3),44.5(C-4),49.0(C-5),33.9(C-6),69.7(C-7),141.3(C-8),156.2(C-9),37.8(C-10),201.7(C-11),58.9(C-12),48.3(C-13),54.0(C-14),25.8(C-15),28.6(C-16),55.1(C-17),12.9(C-18),18.1(C-19),36.5(C-20),19.0(C-21),34.8(C-22),32.3(C-23),150.3(C-24),46.9(C-25),176.8(C-26),17.4(C-27),110.9(C-28),12.3(C-29)。
实施例2：AK-GLU的合成
AK-GLU是将牛樟芝中天然存在的Antcin K进行糖基化所得到的合成产物。其合成方法例示如下。
分别称取适量25R-Antcin K(9.5mg，0.02mM)和25S-Antcin K(9.9mg，0.02mM)并将其溶解于120mL 50mM的NaH2PO4-Na2HPO4缓冲液(pH 8.0)，加入两倍摩尔当量的UDP-Glc(25.5mg，0.04mM)和YjiC1纯酶液(240μg)，于37℃、200rpm的摇床中反应8h，加入甲醇(2倍体积)终止反应，用2倍体积的乙酸乙酯萃取3次，对乙酸乙酯相进行减压浓缩蒸干，用甲醇复溶。
采用半制备液相色谱YMC Pack ODS-A柱(10×250mm,5mm)，色谱条件：0-35min,15％-80％B，35-45min,80％-100％B；检测波长：254nm，流速：2mL/min，得到化合物25R-Antcin K-7-O-葡萄糖苷(11.1mg，得率85％，白色固体)和25S-Antcin K-7-O-葡萄糖苷(11.3mg，得率87％，白色固体)，并采用NMR和HRESIMS确定了其结构。
25R-Antcin K-7-O-葡萄糖苷，得率：85％，11.1mg。HRESIMS:m/z 649.3582([M-H]-,C35H53O11计算值：649.3588)。1H NMR(400MHz,吡啶-d5):δ:1.25,3.13(2H,m,H-1),1.97,2.78(2H,m,H-2),4.09(1H,brs,H-3),2.22(1H,m,H-5),4.63(1H,t,J＝8.4Hz,H-7),2.44(1H,d,J＝13.2Hz,H-12a),2.92(1H,d,J＝13.3Hz,H-12b),2.72(1H,m,H-14),0.82(3H,s,H-18),2.08(3H,s,H-19),0.85(3H,d,J＝6.0Hz,H-21),3.47(3H,q,J＝6.9Hz,H-25),1.51(3H,d,J＝7.0Hz,H-27),5.06(1H，重叠，H-28a),5.23(1H,s,H-28b),1.84(3H,s,H-29),5.05(1H，重叠，H-1'),3.99(1H,m,H-2'),4.04(1H,m,H-3'),4.19(1H,m,H-4'),4.26(1H,m,H-5'),4.30,4.55(2H,m,H-6')。13C NMR(100MHz,吡啶-d5)δ:30.0(C-1),27.0(C-2),74.9(C-3),74.4(C-4),43.7(C-5),29.6(C-6),79.7(C-7),151.4(C-8),146.7(C-9),38.6(C-10),201.9(C-11),59.0(C-12),48.5(C-13),54.3(C-14),25.1(C-15),28.5(C-16),55.3(C-17),12.7(C-18),21.2(C-19),36.6(C-20),18.9(C-21),34.9(C-22),32.1(C-23),150.8(C-24),47.1(C-25),177.2(C-26),17.5(C-27),110.8(C-28),28.3(C-29),105.6(C-1'),76.0(C-2'),78.5(C-3'),72.8(C-4'),79.0(C-5'),63.7(C-6')。
25S-Antcin K-7-O-葡萄糖苷，得率：87％，11.3mg。HRESIMS:m/z 649.3594([M-H]-,C35H53O11计算值：649.3588)。1H NMR(400MHz,吡啶-d5):δ:1.25,3.13(2H,m,H-1),1.97,2.78(2H,m,H-2),4.09(1H,brs,H-3),2.22(1H,m,H-5),4.63(1H,t,J＝8.4Hz,H-7),2.44(1H,d,J＝13.2Hz,H-12a),2.92(1H,d,J＝13.3Hz,H-12b),2.72(1H,m,H-14),0.82(3H,s,H-18),2.08(3H,s,H-19),0.85(3H,d,J＝6.0Hz,H-21),3.47(3H,q,J＝6.9Hz,H-25),1.51(3H,d,J＝7.0Hz,H-27),5.06(1H，重叠，H-28a),5.23(1H,s,H-28b),1.84(3H,s,H-29),5.05(1H，重叠，H-1'),3.99(1H,m,H-2'),4.04(1H,m,H-3'),4.19(1H,m,H-4'),4.26(1H,m,H-5'),4.30,4.55(2H,m,H-6')。13C NMR(100MHz,吡啶-d5)δ:30.0(C-1),27.0(C-2),74.9(C-3),74.4(C-4),43.7(C-5),29.6(C-6),79.7(C-7),151.4(C-8),146.7(C-9),38.6(C-10),201.9(C-11),59.0(C-12),48.5(C-13),54.3(C-14),25.1(C-15),28.5(C-16),55.3(C-17),12.7(C-18),21.2(C-19),36.6(C-20),18.9(C-21),34.9(C-22),32.1(C-23),150.8(C-24),47.1(C-25),177.2(C-26),17.5(C-27),110.8(C-28),28.3(C-29),105.6(C-1'),76.0(C-2'),78.5(C-3'),72.8(C-4'),79.0(C-5'),63.7(C-6')。
实施例3：牛樟芝提取方法的研究
与现有技术以及实施例1中所用的乙醇提取相比，采用甲醇水溶液提取所得的提取物中的Antcin K与Antcin C的含量更高，并且其中不存在DEA，表明能够更好地富集Antcin K与Antcin C这两种活性成分。以下实验验证了这一结论。
将皿培牛樟芝粉碎，分别用10倍体积的95％乙醇和50％甲醇超声提取30分钟。提取后减压浓缩回收溶剂，分别得到干浸膏。通过HPLC方法，将Antcin K、Antcin C和DEA的标准品的连续稀释液进样至HPLC仪器中，绘制峰面积-浓度曲线，并对两种提取物干浸膏中的Antcin K、Antcin C和DEA的含量进行测定。HPLC条件为：
-仪器：Agilent 1260高效液相色谱仪；
-色谱柱：YMC-C18柱(5μm,4.6×250mm)，Zorbax SB-C18预柱(5μm,4.6×12.5mm)；
-流动相：乙腈(A)-0.1％甲酸(B)；
-洗脱程序：0-15min,40-47％A；15-45min,47-67％A；45-50min,67-100％A；50-60min,100％A。
-检测波长：254nm。
各提取物和标准品的HPLC图谱如图5所示，测定结果如表1所示。
表1：提取物中的成分含量测定结果
实施例4：对MCD小鼠肝脏FGF21基因表达的影响
Tranzol(全式金，北京)，氯仿、乙醇、异丙醇(北京化工厂，北京)，无RNA酶(RNase-free)双蒸水、反转录试剂盒(全式金，北京)，SYBR Green I荧光染料法定量PCR体系(全式金，北京)。
4周龄C57BL/6J雄性小鼠购自北京大学医学部实验动物中心，随机分组，每组10只。给予蛋氨酸胆碱缺乏饲料(MCD)诱发非酒精性脂肪性肝炎(NASH)，得到MCD模型小鼠。对该模型小鼠灌胃给药，给药4周，末次给药后禁食过夜，处死小鼠，血清、肝脏组织保存于-80℃，并与未给药的MCD小鼠(“MCD”)和正常小鼠(“Nor”或“Normal”)进行对照。其中各组的给药量如下：低剂量Antcin C(“AC-L”)：20mg/kg；高剂量Antcin C(“AC-H”)：40mg/kg；DEA：20mg/kg；AK-Glu：26.6mg/kg。
总RNA提取：剪取小鼠肝脏组织10-20mg，剪碎后加入1ml的Tranzol，放置在冰上于匀浆器中碾磨，然后转移到无DNA酶和RNA酶的EP管中。每管加入0.2ml氯仿，剧烈振荡15s，室温放置3min，然后于4℃，12000rpm条件下离心15min。将上层水相转移到新的EP管中，在得到的水相中加入等体积的异丙醇，颠倒混匀，室温放置20-30min，在4℃，12000rpm条件下离心15min，管底可见白色透明胶状物质。弃上清，加入1ml 75％的乙醇，涡旋，在4℃，12000rpm条件下离心5min，弃上清，室温下放置晾干。加入20～50μL的无RNA酶双蒸水，轻轻反复吹打直至充分溶解RNA，用分光光度计检测A260/280，并进行RNA定量。
反转：将RNA浓度调整为一致，PCR体系中加入RNA 2μg、用无RNA酶双蒸水补足至15μL、All-in-one Mix 4μL、gDNA remover 1μL。PCR程序见表2。
表2：RNA反转PCR反应程序
实时定量PCR：qPCR引物信息如表3，按表4配置反应体系后按表5的程序进行定量PCR实验，并收集处理数据。
表3：qPCR引物信息
表4：qPCR反应体系
表5：qPCR反应程序
如图6所示，与文献中报道的成分DEA相比，Antcin C、AK-GLU均显著激活了体内FGF21的基因表达水平。Antcin C、AK-GLU可以使蛋氨酸胆碱缺乏饮食造成的小鼠(MCD小鼠)的肝脏FGF21表达水平提高至正常小鼠肝脏FGF21表达水平的300倍以上。
实施例5：对MCD小鼠肝脏FGF21蛋白表达的影响
本实施例所用实验材料包括：蛋氨酸胆碱缺乏饲料(Research Diets，美国)、RIPA裂解液、BSA蛋白检测试剂盒(碧云天，上海)，FGF21抗体、GAPDH抗体(Bioss，北京)，山羊抗小鼠二抗、山羊抗兔二抗(柏奥易杰，北京)。
MCD小鼠的造模和操作方式同实施例4。将各给药组与未给药的MCD小鼠(“MCD”)和正常小鼠(“Normal”)进行对照，其中各给药组的给药量如下：阳性药奥贝胆酸(“OCA”)：10mg/kg；Antcin K(“AK”)：20mg/kg；低剂量Antcin C(“AC-L”)：20mg/kg；高剂量Antcin C(“AC-H”)：40mg/kg；DEA：20mg/kg。
取肝脏组织15-20mg，剪碎后加入RIPA裂解液，在冰上充分研磨，在4℃、13000rpm条件下离心30min，将中层液体转移至新离心管，通过BCA法测定蛋白浓度。每个样品取20μg蛋白经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离，然后按免疫印迹方法，Bio-Rad的标准湿式转膜装置，设定电流200mA，转膜时间60分钟。转膜完毕后，按蛋白分子量剪下条带，依次用0.5％BSA室温封闭60分钟，一抗4℃孵育14小时，TBST室温洗涤3次，每次5分钟，二抗室温孵育1小时，TBST室温洗涤3次，每次10分钟，ECL试剂检测。
如图7所示，与文献中报道的成分DEA相比，Antcin K和不同浓度的Antcin C均显著上调了FGF21蛋白表达水平。
实施例6：对MCD小鼠非酒精性脂肪性肝炎的治疗作用
MCD小鼠造模给药方式同实施例4，末次给药后处死小鼠，肝脏于4％多聚甲醛固定过夜，石蜡切片，苏木精-伊红染色。具体染色步骤，切片依次浸泡于以下溶液：二甲苯(I)15分钟，二甲苯(II)15分钟，50％二甲苯-无水乙醇2分钟，无水乙醇(I)5分钟，无水乙醇(II)5分钟，80％乙醇5分钟，蒸馏水5分钟，苏木精染色液5分钟，流水冲洗5分钟，1％盐酸乙醇30秒，水洗30秒，蒸馏水过洗5秒，0.5％伊红染色液2分钟，蒸馏水30秒，80％乙醇30秒，95％乙醇(I)1分钟，95％乙醇(II)1分钟，无水乙醇(I)3分钟，无水乙醇(II)3分钟，二甲苯(I)3分钟，二甲苯(II)3分钟，中性树胶封固。
将各给药组与未给药的MCD小鼠(“MCD”)和正常小鼠(“Normal”)进行对照，其中各给药组的给药量如下：阳性药奥贝胆酸(“OCA”)：10mg/kg；Antcin K(“AK”)：20mg/kg；低剂量Antcin C(“AC-L”)：20mg/kg；高剂量Antcin C(“AC-H”)：40mg/kg；DEA：20mg/kg；AK-Glu：26.6mg/kg。
末次给药后，禁食过夜，小鼠麻醉，眼底静脉取血，全血静置2小时，6000rpm离心10分钟，取上清，用ALT试剂盒(博泰斯，北京)检测小鼠血清谷丙转氨酶(ALT)水平，结果如图8所示。该结果表明，Antcin K、Antcin C、AK-Glu均能显著降低谷丙转氨酶(ALT)的水平，展现出良好的肝脏保护活性，其效果均显著优于阳性药奥贝胆酸以及文献中报道的成分DEA。
另外，图9为病理切片染色结果，该结果显示，4周MCD模型小鼠肝组织中明显可见脂肪变性、气球样变等退行性改变，且病变弥漫，NAS评分≥4，诊断为NASH病变。经AntcinK、Antcin C、AK-Glu治疗后，明显可见小鼠肝脏肪变性、气球样变程度减轻，病变缓解，且其改善效果均显著优于阳性药奥贝胆酸以及文献中报道的成分DEA。
实施例7：对MCD小鼠血清FGF21蛋白表达的影响
MCD小鼠的造模和操作方式同实施例4。将各给药组与未给药的MCD小鼠(“MCD”)和正常小鼠(“Nor”)进行对照，其中各给药组的给药量如下：阳性药奥贝胆酸(“OCA”)：10mg/kg；Antcin K(“AK”)：20mg/kg；低剂量Antcin C(“AC-L”)：20mg/kg；高剂量Antcin C(“AC-H”)：40mg/kg；DEA：20mg/kg；AK-Glu：26.6mg/kg。
小鼠血清取样方式同实施例6。使用ELISA试剂盒(碧云天)进行测试，操作过程如下：微孔板条室温平衡20分钟，每孔加入血清样品或不同浓度的标准品50μL，加入辣根过氧化氢酶标记的检测抗体100μL，封板膜封住反应孔，37℃孵育60分钟，弃去液体，吸水纸上拍干，每孔加入350μL洗涤液，静置1分钟，弃去液体，吸水纸上拍干，重复洗板5次，每孔加入底物A、B各50μL，37℃避光孵育15分钟，每孔加入终止液50μL，在15分钟内检测450nm下吸光度。以标准品的吸光度为横坐标，标准品浓度为横坐标，绘制标准曲线，血清样品吸光度带入标准曲线，计算各样品中FGF21浓度。
如图10所示，与文献中报道的成分DEA相比，Antcin K、AK-Glu及不同浓度的Antcin C均显著上调了小鼠血清FGF21蛋白表达水平。
Claims (8)
1.化合物Antcin K、Antcin C、AK-GLU或其盐在制备FGF21激动剂中的用途
2.一种牛樟芝提取物在制备FGF21激动剂中的用途，所述牛樟芝提取物满足以下一或多项：
(1)含有Antcin K的质量分数为5％以上；
(2)含有Antcin C的质量分数为3％以上；
(3)含有Antcin K和Antcin C的质量分数总计为8％以上；
(4)含有DEA的质量分数为1％以下
3.根据权利要求2所述的用途，其中所述牛樟芝提取物满足以下一或多项：
(1)含有Antcin K的质量分数为10％以上；
(2)含有Antcin C的质量分数为5％以上；
(3)含有Antcin K和Antcin C的质量分数总计为15％以上；
(4)含有DEA的质量分数为0.5％以下。
4.根据权利要求2所述的用途，其中所述牛樟芝提取物满足以下一或多项：
(1)含有Antcin K的质量分数为15％以上；
(2)含有Antcin C的质量分数为8％以上；
(3)含有Antcin K和Antcin C的质量分数总计为20％以上；
(4)含有DEA的质量分数为0.1％以下。
5.化合物Antcin K、Antcin C、AK-GLU或其盐在制备药物中的用途，所述药物用于治疗或预防非酒精性脂肪性肝炎
6.一种牛樟芝提取物在制备药物中的用途，所述药物用于治疗或预防非酒精性脂肪性肝炎，所述牛樟芝提取物满足以下一或多项：
(1)含有Antcin K的质量分数为5％以上；
(2)含有Antcin C的质量分数为3％以上；
(3)含有Antcin K和Antcin C的质量分数总计为8％以上；
(4)含有DEA的质量分数为1％以下
7.根据权利要求6所述的用途，其中所述牛樟芝提取物满足以下一或多项：
(1)含有Antcin K的质量分数为10％以上；
(2)含有Antcin C的质量分数为5％以上；
(3)含有Antcin K和Antcin C的质量分数总计为15％以上；
(4)含有DEA的质量分数为0.5％以下。
8.根据权利要求6所述的用途，其中所述牛樟芝提取物满足以下一或多项：
(1)含有Antcin K的质量分数为15％以上；
(2)含有Antcin C的质量分数为8％以上；
(3)含有Antcin K和Antcin C的质量分数总计为20％以上；
(4)含有DEA的质量分数为0.1％以下。
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