CN101801907B - 从牛樟芝中分离的化合物及其应用 - Google Patents
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Abstract
本发明涉及来自牛樟芝的新颖化合物及其应用。
Description
技术领域
本发明涉及一种化合物,其来自从牛樟芝(Antrodia cinnamomea)中分离的Antrodin C的代谢物。
背景技术
牛樟芝(Antrodia cinnamomea,多孔菌科,非摺菌目)在台湾是一种有名的传统中药。其仅生长于台湾的地区性常绿牛樟树(Cinnamomunkanehirai,樟科)的内心材。其用作治疗食材来治疗中毒、腹泻、腹痛、高血压、皮肤瘙痒和肝癌(Tsai ZT等.1982 Sheng-Yun出版社,台中市,台湾省,pp 116-117)。类固醇酸化合物(Chen CH等.1995天然产物化学杂志58:1655-1661.;Yang SW等.1996植物化学41:1389-1392)、三萜类(Cherng IH等,1995天然产物化学杂志58:365-371;Cherng IW等.1996植物化学41:263-267)、双萜烯(Chen CC等.2006天然产物化学杂志69:689-691)、倍半萜烯内酯(Chiang HC等.1995植物化学,39,613-616)及苯和联苯(Chiang HC等.1995植物化学,39,613-616;Huang KF等.2001中华药学杂志53:327-331)从牛樟芝的子实体中分离而来,其具有细胞毒素活性、神经保护活性、抗炎活性、细胞凋亡活性。此外,菌丝体,牛樟芝的另一部分,具有抗氧化作用(Hsiao G.等.2003农业与食品化学杂志51:3302-3308;Song TY等.2003农业与食品化学杂志51:1571-1577)、护肝作用(Han HF等.2006 Chem Pharm Bull 54:496-500)、抗炎作用(ShenYC等.2004 Planta Medica 70:310-314;Hseu YC,et al.2005 IhtImmunopharmacol 5:1914-192)、抗HBV(Lee IH等.2002 FEMS MicrobiolLett 209:63-67)、血管舒张作用(Wang G.J,等.2003生命科学73:2769-2783)和细胞凋亡作用(Song TY等.2005农业与食品化学杂志53: 5559-5564)。
发明内容
本发明提供一种化合物,其具有以下通式
其中,R1是C1-10羧酸或C1-10酯;R2是C1-10羧酸或C1-10酯;R3是H、C1-10烷基、C2-10烯基或C2-10炔基;及R4是H、C1-10烷基、C2-10烯基或C2-10炔基。
本发明还提供一种组合物,其包含具有以下通式的化合物
其中,R1是C1-10羧酸或C1-10酯;R2是C1-10羧酸或C1-10酯;R3是H、C1-10烷基、C2-10烯基或C2-10炔基;及R4是H、C1-10烷基、C2-10烯基或C2-10炔基。
附图说明
图1表示M1的HMBC(a)和NOE(b)的相互关系。
图2表示M2和M3的HMBC的相互关系。
图3是口服50mg/kg剂量的Antrodin C后的粪便(a)、胆汁(b)和血浆(c)样品的TIC。
图4是粪便、胆汁和血浆样品中的M1-M5和Antrodin Cd MS谱(负离子模式)。
图5是Antrodin C及其代谢物的结构。
图6是静脉注射(I.V.)10mg/kg剂量的Antrocin C(a)和口服(P.O.)50mg/kg剂量的Antrocin C(b)后,胆汁样品中的M1的浓度-时间曲线。
图7是在大鼠服用Antrodin C后的胆汁和血浆的TIC及粪便的UV谱。
图8是静脉注射M1后,空白血浆样品和血浆样品的UV谱。
具体实施方式
在本发明中,首先从牛樟芝的菌丝体中分离三种马来酸衍生物和两种琥珀酸衍生物(Antrodin A-E),验证了Antrodin C和B的抗LLC细胞的细胞毒素活性(Nakamura N等.2004天然产物化学杂志7:46-48)。进一步地,在菌丝体中含有大量的Antrodin C,在LPS诱导的模式中其具有抗肝炎的保护作用。但是,关于牛樟芝中的化合物的新陈代谢研究尚未见报导。在本发明中,对大鼠胆汁和粪便样品中的Antrodin C的代谢物通过电喷雾电离(electrospary ionization,ESI)的LC/MS-MS鉴定,在口服50mg/kgAntrodin C和静脉注射10mg/kg Antrodin C后,通过PAD-HPLC对大鼠胆汁中的M1进行药代动力学研究。
本发明提供一种化合物,其具有以下通式
其中,R1是C1-10羧酸或C1-10酯;R2是C1-10羧酸或C1-10酯;R3是H、C1-10烷基、C2-10烯基或C2-10炔基;及R4是H、C1-10烷基、C2-10烯基或C2-10炔基。
该化合物的R1或R2是C1-6羧酸。在一优选实施例中,R1或R2是COOH,R3是C1-6烷基,且R4是异丁基。在更优选的实施例中,该化合物是(2顺)-2-异丁基-3-{4-[(3-甲基-2-丁烯-1-氧]苯}-2-丁烯二酸((2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid)、(2顺)-2-异丁基-3-{4-[(3-甲基-2-丁烯-1-氧]苯}-2-丁烯二酸-4-甲酯 ((2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioic acid 4-methyl ester)或者(2顺)-2-异丁基-3-{4-[(3-甲基-2-丁烯-1-氧]苯}-2-丁烯二酸-1-甲酯((2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioic acid 1-methyl easter)。
上述化合物是大鼠中的Antrodin C的代谢物,且Antrodin C从牛樟芝菌丝体中分离获得。
本发明提供一种组合物,其包含具有以下通式的化合物
其中,R1是C1-10羧酸或C1-10酯;R2是C1-10羧酸或C1-10酯;R3是H、C1-10烷基、C2-10烯基或C2-10炔基;及R4是H、C1-10烷基、C2-10烯基或C2-10炔基。
在一优选实施例中,该化合物是(2顺)-2-异丁基-3-{4-[(3-甲基-2-丁烯-1-氧]苯}-2-丁烯二酸((2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioic acid)、(2顺)-2-异丁基-3-{4-[(3-甲基-2-丁烯-1-氧]苯}-2-丁烯二酸-4-甲酯((2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioic acid 4-methyl ester)或者(2顺)-2-异丁基-3-{4-[(3-甲基-2-丁烯-1-氧]苯}-2-丁烯二酸-1-甲酯((2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioicacid 1-methyl easter)。
本发明提供的化合物具有抗氧化作用、抗微生物、抗菌作用,止痛活性、止痉挛或血管舒张活性。
本发明的化合物可以降低心脏收缩血压或增加高密度脂蛋白。另外,该化合物具有中枢类胆碱激动作用、肝保护作用、抗炎或抗肿瘤活性。特别地,本发明的化合物可以抑制选自由肝、肺、肠、骨、血、淋巴和乳房组成的群组中的细胞或组织的肿瘤。接受本发明的混合物的对象包括但不 限于人类、哺乳动物、小鼠、大鼠、马、猪、鸡、鸭、狗和猫。
本发明还提供一种组合物,其包含本发明的化合物。本发明的组合物可以降低心脏收缩血压或增加高密度脂蛋白。另外,该化合物具有中枢类胆碱激动作用、肝保护作用、抗炎或抗癌活性。特别地,本发明的组合物可以抑制选自由肝、肺、肠、骨、血、淋巴和乳房组成的群组中的细胞或组织的肿瘤。接受本发明的混合物的对象包括但不限于人类、哺乳动物、小鼠、大鼠、马、猪、鸡、鸭、狗和猫。
实施例
化学药品和试剂
GAM肉汤购自Nissui公司(日本东京)。用于开管柱层析的液体色谱级溶剂、三乙胺、4-二甲氨基吡啶(4-DMAP)、硅胶BW-820MH(FujiSilysia)、ODS DM 1020T(Fuji Silysia),用于TLC分析的Merck预涂膜硅胶60F254(0.25mm)和Merck RP-18F254(0.25mm)购自和光纯药公司(Wako Pure Chemical Industries Ltd.,日本大阪)。
仪器
以四甲基硅烷作为内标用Unity Plus 500(varian)NMR分光计分析 1H-及13C-NMR和2D NMR,化学位移表示为δ值。肠道细菌应用EAN-140(Tabai公司,日本大阪)厌氧孵育。HPLC仪器是Agilent 1100系统(Agilent技术公司,德国瓦尔德布龙),其包含具有光电二极管阵列检测器(PAD)的Agilent1100系列二元泵,和具有20μl回路(loop)的7725i注射器系列。通过化学工作站(ChemStation)获取和整合数据。HPLC系统连接到装有ESI源的Esquire 3000plus质谱仪(Bruker Daltonik GmbH,德国不莱梅)。所有LC/MS-MS数据通过Esquire Control软件获取,通过Bruker Daltonics公司的软件进行分析。
实施例1
M1-M3的合成
将50mg Antrodin C溶于5ml水中,加入0.5ml的1N KOH,搅拌分钟。用1N HCl调整pH至8。过滤后,溶液保持在室温下过夜。再次过滤,冻干上清液,并用一些甲醇移行,然后过滤并在真空中浓缩,获得13mg(26%)产量的M1。
M1的1H and 13C-NMR谱(表1)与Antrodin C的1H and 13C-NMR谱非常相似,这表示存在异丁基、3-甲基-2-丁烯氧基、对位取代的苯环,这通过1H-1H COSY、HMQC试验得到支持。但是异丁基的羰基碳(δ178.9:1)、亚甲基碳(δ39.7:1’)、质子(δ1.87:1’)和亚甲基质子(δ1.56:2’),苯碳共轭的烯烃(δ131.6:1”)及与其相邻的苯碳(δ7.11:2”,6”)与Antrodin C的不同,M1的这些所有的碳比Antrodin C中的所有碳向低场偏移,M1的所有质子比Antrodin C中的所有质子向高场偏移。在HMBC试验中,观察到长程相关性,其示于图1(a)中。发明人确定M1的烯烃连接(2-C和3-C)是顺式的,因为在M1的NOESY谱(图1(b))中,在1’-H和3’4’-H或6”-H之间观察到NOE。根据这些结果,M1确定为(2顺)-2-异丁基-3-{4-[(3-甲基-2-丁烯-1-氧]苯}-2-丁烯二酸((2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]-phenyl}but-2-enedioic acid)。酐M4(Antrodin A)和二羧酸M1通过酸、碱条件互相转化。
将500mg Antrodin A溶于1ml甲醇中,在溶液中加入0.2ml的1.6mmol的三乙胺和13.4mg的0.11mmol的4-二甲氨基吡啶(4-DMAP),置于25℃20小时。然后将混合物通过开管ODS柱用甲醇和水(30∶70→100∶0)洗脱,将包含M2和M3的组分在真空中浓缩,然后通过NMR和LC/MS分析。M1-M3的1H-NMR和13C-NMR数据示于表2中。
除了甲氧基外,M2和M3的1H和13C-NMR谱与M1的1H and 13C-NMR谱也相似,这表示存在异丁基、3-甲基-2-丁烯氧基、对位取代的苯环,在HMBC试验中,M2的异丁基(δ2.16:1’)的亚甲基质子和羰基碳(δ174.0:1)显示出了长程相关性,M3的异丁基的亚甲基质子(δ2.14:1’)和羰基碳(δ171.4:1)显示出了长程相关性(图2)。M2和M3的结构分别确定为(2顺)-2-异丁基-3-{4-[(3-甲基-2-丁烯-1-氧]苯}-2-丁烯二酸-4-甲酯((2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-ene dioic acid 4-methyl ester)和(2顺)-2-异丁基-3-{4-[(3- 甲基-2-丁烯-1-氧]苯}-2-丁烯二酸-1-甲酯((2Z)-2-isobutyl-3-{4-[(3-methylbut-2-en-1-yl)oxy]phenyl}but-2-enedioic acid 1-methyl ester)。
表1M1(D2O),M2和M3(CD3OD)的1H-NMR谱数据((δppm,J=Hz))
表2M1(D2O),M2和M3(CD3OD)的13C-NMR谱数据(δppm)
实施例2
动物治疗
将购自SLC公司(日本滨松)的雄性Wistar大鼠(9周龄)用标准的实验室普通饲料喂养一周,在给予药物前禁食一夜但允许自由饮水。在大鼠呆在隔离的代谢笼中时,收集尿和粪便样品。在外科手术过程中用乙醚对动物进行轻微麻醉。在口服50mg/kg Antrodin C和静脉注射10mg/kgAntrodin C后,通过将聚乙烯管(PE-10)插入大鼠胆管间隔0,0.25,0.5,1,2,4,8,12,24,36和48小时收集胆汁样品(n=5)。给予药物后,沿中线剖腹,待腹腔露出时,用肝素注射器从下腔静脉收集血样。在8000g下离心血浆样品15分钟,以分离血浆,然后将所有样品储存在-20℃下用于后 续分析。
分析用样品制备
将解冻的尿和胆汁样品(0.5ml)溶解于3倍体积的乙腈中,然后在8000g下离心15分钟。上清液通过0.45μm的Millipore针头式过滤器(Nihon Millipore,日本东京)后用于LC/MS-MS分析。血浆样品通过已用3ml乙腈洗脱并用6ml的水平衡的固相萃取柱(Waters公司,美国米尔福德)。用2-3ml的乙腈从柱中洗脱组分,然后在35℃在氮气流下浓缩洗脱液,将残渣溶解于100μl的乙腈中,用于LC/MS-MS分析。用于药代动力学研究的胆汁样品包含M2,M3和M4,其用相同体积的水稀释,然后在37℃水浴下孵育12小时,M2-M4会完全转化为M1。按照如上所述处理完胆汁样品后,M1的量通过PAD-HPLC计算。
大鼠粪便、胆汁和血浆中的代谢物的鉴定
用LC/MS-MS分析粪便、胆汁和血浆中的代谢物。LC/MS-MS仪器包含具有TSK gel ODS-80Ts的柱(离子大小,5μm;4.6×150mm直径.,Tosoh公司,日本东京)。样品用0.1%的甲醇∶乙腈(35∶65)在30℃以1ml/min的流速通过柱洗脱。标准的负离子模式在下述条件下筛选:全扫描范围,50-800m/z;扫描分辨率,13000m/z/sec;;喷雾器,50.0psi;干燥气体,10.01/min;干燥温度,360℃。在50到600m/z范围内全扫描,在TLC中Antrodin C和代谢物与其空白样品相比成多个峰(图3)。MS谱显示,M1,M2,M3,M4,M5和Antrodin C分别在m/z 331,345,345,313,312和328[M-H]-有强烈的离子峰(图4,表3)。在粪便中,代谢物是M1-M3,M5,具有最初的Antrodin C;在胆汁中是M2-4;在血浆中是M1,具有另一未知峰。在尿样品中,既无代谢物也无Antrodin C。与标准品和合成的化合物相比,MS图中m/z 328[M-H]-,保留时间(tR)=8.2分钟的峰型源自Antrodin C(MW 329),M1(m/z 331[M-H]-,tR=4.4分钟)是Antrodin C水解解的二羧酸;M2和M3(m/z 345[M-H]-,tR=7.7和8.4分钟)比M1的大14,其是两种M1的单甲酯;M4(m/z 313[M-H]-,tR=21.6分钟)是Antrodin A,M5(m/z 312[M-H]-,,tR=11.5分钟)是Antrodin B。 Antrodin C及其代谢物的结构示于图5中。
表3Antrodin C及其代谢物的保留时间(tR)和MS谱
肠道细菌在体外对Antrodin C和代谢物的代谢
将按照Biol Pharm Bull(Xie LH等.2003 Biol Pharm Bull 51:378-384)制备大鼠(RIB)或人(HIB)的肠道细菌各5g,与溶解于0.5ml吐温(Tween)20中的5mg的Antrodin C、溶解于1.0ml水中的5mg的M1、或者与在口服Antrodin C后收集的具有代谢物M2-M4的大鼠胆汁样品(10ml)一起加入50mlGAM肉汤中,在37℃下厌氧孵育3天。用3倍体积的乙腈萃取孵育混合物,然后通过0.45μm过滤器。之后,Antrodin C转化为M5(Antrodin B)。而且,在大鼠口服Antrodin C后收集的胆汁样品中的M2-M4代谢物,可以在30分钟的孵育后完全转化为M1。而即使将孵育时间延长到3天,M1也不能被肠道菌群代谢。
通过PAD-HPLC确定M1
线性(Linearity):将M1溶解于大鼠空白胆汁中,制备7级稀释的标准溶液。每个水平下注射三次确定7个浓度的响应线性。在信噪比(S/N)为5时确定该方法对每个组分的检测极限(LOD)。
准确度(Accuracy):一天五次分别分析高、中和低标准浓度的大鼠胆汁确定日内和日间变异系数,持续5天。
回收率(Recovery):在口服具有已知量的M1的Antrodin C后,将2个标准浓度溶液与大鼠胆汁样品混合,计算加样回收率。
稳定性(Stability):将制备用于PAD-HPL分析的3个浓度的胆汁样 品置于室温下12小时,或者在4℃冰箱放置1、3和5天。计算样品中的组分的平均峰面积和相对标准偏差(RSD)。
PAD-HPLC定量的验证
大鼠胆汁样品中的M1的回归方程是Y=610.22X-3.94;γ=0.9998;线性范围是0.05-2.0μg/ml。大鼠胆汁样品中的M1的日内和日间(n=5)的变异系数示于表4。CV没有超过6%,准确率都在85-110%之内。回收率的CV值示于表5,其在回收率为93.4和99.6%的低和高浓度下少于10%。稳定性测试显示,在所有情况下,相对标准偏差保持在5%内;因此,样品在测试期间是稳定的。因此,胆汁样品中的准确度、回收率和稳定性测试满足定量测定的标准。
表4大鼠胆汁中的M1的日内和日间(n=5)变异系数
表5大鼠胆汁中的M1的回收率
大鼠胆汁中M1的药代动力学
大鼠胆汁中的浓度-时间数据(n=5)使用中国药理学会数学药理学专业委员会编辑的药代动力学3p97程序处理。得到下述的药代动力学参数:在口服50mg/kg剂量的Antrodin C后,胆汁样品中的吸收半减期(t1/2(Kα))和消除半减期(t1/2(Kβ))。通过非房室药代动力学分析的统计矩方法计算浓度-时间曲线下面积(AUC(i.v.)和AUC(p.o.))。通过下述方程式:Clm,b(ml/h ·kg)=剂量(i.v.)/AUC(i.v.)和Fm,b(%)=AUC(p.o.)·剂量(i.v.)/[AUC(i.v.)·剂量(p.o.)]计算清除率(Clm,b)和绝对生物利用度(Fm,b)。数据以每组的均值和标准差(S.D.)表示。
在口服(P.O.)50mg/kg Antrodin C和静脉注射(I.V.)10mg/kg AntrodinC后,计算胆汁样品中的M1的浓度。M1的浓度-时间曲线示于图6中。药代动力学参数示于表6中。口服后,t1/2(kα)和t1/2(kβ)分别为0.95小时和12.64小时。AUC0-lim为1.61(P.O.)和1.68h mg/ml(I.V.),Clm.b.为5.96ml/h·kg,Fm.b.为19.43(%)。Antrodin C的累积排泄率为5.46±1.62%(P.O.)和56.85±13.40%(I.V.)。因此,Antrodin C不仅从胃肠很快吸收,而且在肝中快速代谢。大鼠中主要的排泄为胆汁-粪便路径。
表6口服(P.O.)和静脉注射(I.V.)Antrodin C后大鼠胆汁样品中的M1的药代动力学参数
实施例3
重复实施例2,结果示于图7和图8。
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