CN103819437B - 螺环赤芝素类化合物及其药物组合物和其应用 - Google Patents
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Abstract
提供灵芝中获得的7个结构新颖的螺环杂萜化合物,及其药物组合物,它们的制备方法,及其在制备治疗糖尿病性肾病或慢性肾病的药物或保健食品中的应用。本发明的化合物具有显著的抑制高糖诱导的大鼠肾系膜细胞株产生活性氧作用,由于氧化应激在慢性肾病中普遍存在,因此本发明提示该类化合物在慢性肾病药物中的应用前景。
Description
技术领域:
本发明属于药物技术领域,具体地,涉及灵芝中结构新颖的螺环杂萜衍生物,及其药物组合物,它们的制备方法,及其在制备治疗糖尿病性肾病或慢性肾病的药物或保健食品中的应用。
背景技术:
糖尿病肾病或慢性肾病的危害、病理机制及药物研究的方法和原理,本申请人在已公开的专利公布号:CN102153630A中均有明确的描述。众所周知,现代饮食起居生活方式变化,加之环境污染,药物滥用等因素,导致全球范围内糖尿病并发症及慢性肾病如肾纤维化多发、高发,但目前这些疾病的临床有效治疗药物还比较缺乏。
现代研究表明糖尿病肾病和肾病的发生发展是活性氧自由基密切相关的。正常情况下,自由基在机体内扮演信号分子,维持生理需要,但病理状态下,机体活性自由基产生过量,产生危害,自由基还可激活NF-kB通路,引发机体炎症。糖尿病肾病发病过程中,高糖易诱发活性氧过量产生,活性氧过量和炎症也是其它慢性肾病的主要病理特征。因此抑制活性氧,改善机体氧化应激状态将有助于肾病干预。
灵芝是传统中药,在东亚及美国被承认和广泛应用。目前其已被收录于美国草药药典中。我国关于灵芝的保健品众多,表明其功能备受关注。深入研究过程中,本发明从灵芝(Ganoderma lucidum)中获得7个结构新颖的杂萜类成分,并发现其具有明显地抑制高糖诱导的肾系膜细胞产生活性氧作用。该发明是对中医药理论进行有效运用揭示灵芝科学内涵的又一例证。现有技术中未见有本发明的化合物及其具有糖尿病肾病或慢性肾病价值治疗前景的活性的相关报道。
发明内容:
本发明的目的在于提供灵芝中获得的7个结构新颖的螺环杂萜化合物,及其药物组合物,它们的制备方法,及其在制备治疗糖尿病性肾病或慢性肾病的药物或保健食品中的应用。
本发明的上述目的是由下述的技术方案得以实现的:
具有下述结构式的灵芝杂萜类化合物,
制备上述化合物的方法,取赤芝,粉碎,用95%乙醇回流提取2h,合并提取液并减压回收溶剂得粗提取物,将粗提取物混悬于水中,然后用等体积乙酸乙酯萃取三次,合并萃取液,减压浓缩得乙酸乙酯萃取物,该萃取物经硅胶200-300目柱层析,氯仿-甲醇系统梯度99:1,98:2,97:3,96:4,95:5,94:6,93:7,92:8,90:10,85:15,80:20,50:50洗脱,每种溶剂梯度为1.5倍柱体积,按照每份500mL收集得7个合并组份,其中组份7经MCI gel CHP20P柱层析,甲醇-水(20–100%)洗脱得9个亚组份,其中组份7.2经Sephadex LH-20(MeOH)柱层析及反相RP-18柱层析(甲醇-水,35-50%),最后经半制备HPLC(甲醇-水,40%),依次得到化合物4,1,2,组分7.3经反相RP-18柱层析(甲醇-水,40-50%)得到化合物3,化合物1-3均为消旋体,它们经手型材料HPLC拆分获得光学纯对映体,条件分别如下:化合物1:固定相:Daicel Chiralpak(IC);流动相:正己烷-乙醇,85:15;流速:1mL/min)拆分为(+)-1and(-)-1.;化合物2:固定相:Daicel Chiralpak(IC);流动相:正己烷-乙醇,80:20;流速:1mL/min)拆分为(+)-2and(-)-2.;化合物3:固定相:Daicel Chiralpak(AD-H);流动相:正己烷-二氧六环,90:10;流速:1mL/min)拆分为(+)-3and(-)-3。
治疗糖尿病肾病或慢性肾病的药物组合物,含有治疗有效量的所述的化合物和药学上可接受的载体。
所述的化合物在制备治疗糖尿病肾病或慢性肾病的药物或保健食品中的应用。
更具体地,本发明制备上述化合物的方法为:取赤芝(Ganodermalucidum)80kg,粉碎,用95%乙醇回流提取(2×360L×2h),合并提取液并减压回收溶剂得粗提取物,将粗提取物混悬于水中,然后用等体积乙酸乙酯萃取三次。合并萃取液,减压浓缩得乙酸乙酯萃取物1.1kg。该萃取物经硅胶柱层析(硅胶200-300目,7kg),氯仿-甲醇系统梯度洗脱(99:1,98:2,97:3,96:4,95:5,94:6,93:7,92:8,90:10,85:15,80:20,50:50),每种溶剂梯度为1.5倍柱体积,按照每份500mL收集)得7个合并组份。组份7(36g)经MCI gel CHP20P柱层析,甲醇-水(20–100%)洗脱得9个亚组份,其中组份7.2(1.3g)经Sephadex LH-20(MeOH)柱层析及反相RP-18柱层析(甲醇-水,35-50%),最后经半制备HPLC(甲醇-水,40%),依次得到化合物4(46mg),1(12mg),2(14mg)。组分7.3(930mg)经反相RP-18柱层析(甲醇-水,40-50%)得到化合物3(12mg)。化合物1-3均为消旋体,它们经手型材料HPLC拆分获得光学纯对映体。条件分别如下:化合物1:固定相:Daicel Chiralpak(IC);流动相:正己烷-乙醇,85:15;流速:1mL/min)拆分为(+)-1(4.7mg)and(-)-1(5.2mg).化合物2:固定相:Daicel Chiralpak(IC);流动相:正己烷-乙醇,80:20;流速:1mL/min)拆分为(+)-2(5.8mg)and(-)-2(6.1mg).化合物3:固定相:Daicel Chiralpak(AD-H);流动相:正己烷-二氧六环,90:10;流速:1mL/min)拆分为(+)-3(5.5mg)and(-)-3(5.5mg)。
本发明化合物可以单独直接应用或组合应用,也可以与其它药物包括植物提取物组成复方的形式使用,可以使用不同的药用辅料,制成多种固体制剂和液体制剂。将本发明的药物组合物以单位体重服用量的形式使用。本发明的药物可经口服和注射两种形式给药。使用量可根据给药途径、患者的年龄、体重、所治疗疾病的类型和严重程度等变化进行一次或多次使用。
附图说明:
图1为本发明7个结构新颖的螺环杂萜化合物的结构式;
图2为本发明化合物的绝对构型示意图;
图3表示本发明化合物抑制高糖诱导的肾系膜细胞活性氧产生:*P<0.05vs正常糖;#P<0.05vs高糖。
具体实施方式:
下面用本发明的实施例来进一步说明本发明的实质性内容,但并不以此来限定本发明。根据本发明的实质对本发明进行的简单改进都属于本发明的范围。
实施例1:
化合物1-4的分离纯化:
取赤芝(Ganoderma lucidum)80kg,粉碎,用95%乙醇回流提取(2×360L×2h),合并提取液并减压回收溶剂得粗提取物,将粗提取物混悬于水中,然后用等体积乙酸乙酯萃取三次。合并萃取液,减压浓缩得乙酸乙酯萃取物1.1kg。该萃取物经硅胶柱层析(硅胶200-300目,7kg),氯仿-甲醇系统梯度洗脱(99:1,98:2,97:3,96:4,95:5,94:6,93:7,92:8,90:10,85:15,80:20,50:50),每种溶剂梯度为1.5倍柱体积,按照每份500mL收集)得7个合并组份。组份7(36g)经MCI gel CHP20P柱层析,甲醇-水(20–100%)洗脱得9个亚组份,其中组份7.2(1.3g)经Sephadex LH-20(MeOH)柱层析及反相RP-18柱层析(甲醇-水,35-50%),最后经半制备HPLC(甲醇-水,40%),依次得到化合物4(46mg),1(12mg),2(14mg)。组分7.3(930mg)经反相RP-18柱层析(甲醇-水,40-50%)得到化合物3(12mg)。化合物1-3均为消旋体,它们经手型材料HPLC拆分获得光学纯对映体。条件分别如下:化合物1:固定相:Daicel Chiralpak(IC);流动相:正己烷-乙醇,85:15;流速:1mL/min)拆分为(+)-1(4.7mg)and(-)-1(5.2mg).化合物2:固定相:Daicel Chiralpak(IC);流动相:正己烷-乙醇,80:20;流速:1mL/min)拆分为(+)-2(5.8mg)and(-)-2(6.1mg).化合物3:固定相:Daicel Chiralpak(AD-H);流动相:正己烷-二氧六环,90:10;流速:1mL/min)拆分为(+)-3(5.5mg)and(-)-3(5.5mg)。
化合物的结构确证:
化合物的结构式如下所示:
化合物Spirolingzhines A-D(螺环灵芝素A-D)的结构鉴定数据:
Table1.The1H NMR data(δin ppm).
Table2.The13C NMR data.
Spirolingzhine A(1).yellowish gum;{[α]D 24+27.8(c3.31,MeOH);UV(MeOH)λmax(logε)370(3.60),254(3.86),220(4.20)nm;CD(MeOH)Δε234+5.51,Δε327+0.73,Δε369–2.25;(+)-spirolingzhine A};{[α]D 24–31.2(c2.63,MeOH);UV(MeOH)λmax(logε)371(3.60),254(3.87),221(4.20)nm;CD(MeOH)Δε234–5.78,Δε327–1.27,Δε369+1.71;(–)-spirolingzhine A};EI-MS m/z306[M]+;HREI-MS m/z306.1105[M]+(calcd for C16H18O6,306.1103).1H and13C NMR data,see Table1and Table2.
Spirolingzhine B(2).yellowish gum;{[α]D 24+37.4(c0.65,MeOH);UV(MeOH)λmax(logε)371(3.52),254(3.79),222(4.11),196(3.88)nm;CD(MeOH)Δε232+5.94,Δε321–2.16,Δε366+1.62;(+)-spirolingzhineB};{[α]D 24–43.2(c0.27,MeOH);UV(MeOH)λmax(logε)371(3.50),254(3.77),220(4.10),196(3.88)nm;CD(MeOH)Δε232–5.01,Δε321+1.47,Δε366–1.78;(–)-spirolingzhine B};ESI-MS m/z329[M+Na]+;HRESI-MS m/z329.0993[M+Na]+(calcd for C16H18NaO6,329.1001);1H and13C NMR data,see Table1and Table2.
Spirolingzhine C(3).yellowish gum;{[α]D 27+22.9(c0.27,MeOH);UV(MeOH)λmax(logε)369(3.59),254(3.87),219(4.21)nm;CD(MeOH)Δε229+7.46,Δε284–2.49;(+)-spirolingzhine C};{[α]D 27–29.5(c0.25,MeOH);UV(MeOH)λmax(logε)369(3.62),254(3.91),219(4.23)nm;CD(MeOH)Δε229–6.71,Δε284+1.90;(–)-spirolingzhine C};ESI-MS m/z289[M–H]-;HRESI-MS m/z289.0718[M–H]-(calcd forC15H13O6,289.0712);1H and13C NMR data,see Table1and Table2.
Spirolingzhine D(4).yellowish gum;[α]D 27+165.9(c2.36,MeOH);UV(MeOH)λmax(logε)372(3.62),254(3.90),217(4.22)nm;CD(MeOH)Δε234+7.83,Δε321–3.45,Δε367+3.79;EI-MS m/z304[M]+;HREI-MS m/z304.0956[M]+(calcd for C16H16O6,304.0947);1H and13CNMR data,see Table1and Table2.
化合物的结构经X-ray单晶衍射得到进一步确证,其数据如下:
Crystal data of(-)-1:C16H18O6,M=306.30,orthorhombic, α=90.00°,β=90.00°,γ=90.00°,T=100(2)K,space groupP212121,Z=4,μ(CuKα)=0.941mm-1,7105reflections measured,2393independent reflections(Rint=0.0571).The final R1values were0.0491(I>2σ(I)).The final wR(F2)values were0.1261(I>2σ(I)).The final R1values were0.0496(all data).The final wR(F2)values were0.1265(alldata).The goodness of fit on F2was1.100.Flack parameter=0.0(2).TheHooft parameter is0.11(10)for916Bijvoet pairs.The deposition numberCCDC963799for(-)-1can be obtained free of charge from TheCambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
化合物1-4的绝对构型均参照下述文献方法,采取下述参考文献[1]-[5]中量子化学计算的方法得以确证,如图2所示。
[1]P.J.Stephens,N.Harada,Chirality2010,22,229-233.
[2]T.Bruhn,A.Y.Hemberger,G.Bringmann,SpecDisversion1.53.Germany:University of2012
[3]M.J.Frisch,G.W.Trucks,H.B.Schlegel,G.E.Scuseria,M.A.Robb,J.R.Cheeseman,G.Scalmani,V.Barone,B.Mennucci,G.A.Petersson,H.Nakatsuji,M.Caricato,X.Li,H.P.Hratchian,A.F.Izmaylov,J.Bloino,G.Zheng,J.L.Sonnenberg,M.Hada,M.Ehara,K.Toyota,R.Fukuda,J.Hasegawa,M.Ishida,T.Nakajima,Y.Honda,O.Kitao,H.Nakai,T.Vreven,J.A.Montgomery,Jr.J.E.Peralta,F.Ogliaro,M.Bearpark,J.J.Heyd,E.Brothers,K.N.Kudin,V.N.Staroverov,T.Keith,R.Kobayashi,J.Normand,K.Raghavachari,A.Rendell,J.C.Burant,S.S.Iyengar,J.Tomasi,M.Cossi,N.Rega,J.M.Millam,M.Klene,J.E.Knox,J.B.Cross,V.Bakken,C.Adamo,J.Jaramillo,R.Gomperts,R.E.Stratmann,O.Yazyev,A.J.Austin,R.Cammi,C.Pomelli,J.W.Ochterski,R.L.Martin,K.Morokuma,V.G.Zakrzewski,G.A.Voth,P.Salvador,J.J.Dannenberg,S.Dapprich,A.D.Daniels,O.Farkas,J.B.Foresman,J.V.Ortiz,J.Cioslowski,D.J.Fox,Gaussian09,Revision C.01;Gaussian,Inc.,Wallingford CT:2010.
[4]T.Lu,F.Chen,J.Comput.Chem.2012,33,580-592.
[5]M.Humphrey,A.Dalke,K.Schulten,J.Mol.Graph.1996,14,33-38
实施例2:
实施例1中化合物中的任一种,按常规法加注射用水,精滤,灌封灭菌后可制成注射液。
实施例3:
实施例1中化合物中的任一种,将其溶于无菌注射用水中,用无菌漏斗过滤,分装,低温冷冻干燥后无菌熔封即得粉针剂。
实施例4:
实施例1中化合物中的任一种,按常规法配以各种药用辅料可制成片剂。
使用实施例1中化合物中的任一种作为药物活性成分,使用几种赋形剂作为制备组合药物片剂的辅料成分,按照一定比例配比制成每片含有药物成分1-100mg的片剂样品,表1给出普通片剂的配方比例。
将一定数量实施例1中化合物中的任一种原料与赋形剂辅料制备成不同剂量片剂制剂(如表1):将几种赋形剂辅料与原料药均匀混合,加入1%羟甲基纤维素钠溶液适量制成软料,过筛制粒,湿粒烘干并过筛整粒,加入硬脂酸镁和滑石粉混合均匀后压片即得。
表1实施例1中化合物中的任一种组合药物片剂的原料药和辅料配方
实施例5:
实施例1中化合物中的任一种按常规法配以各种药用辅料可制成胶囊剂:
含有实施例1中化合物中的任一种作为有效成分的药物组合胶囊制剂的制备,使用实施例1中化合物中的任一种作为药物活性成分、使用几种赋形剂作为制备组合药物胶囊剂的辅料成分,按照一定比例配比制成每粒胶囊中含有化合物成分1-100mg的胶囊制剂。
实施例6:
取1份实施例1制得的化合物任一种,分别与20份聚合度为300的醋酸乙烯酯树脂,2份邻苯二甲酸丁酯,3份巴西棕蜡和20份麦芽糖,在捏机中于50℃混合3分钟,再加入50份砂糖,1份薄荷,混炼均匀后在50℃恒温下,从挤出机中挤出口香糖,切割成规定厚度,作为功能食品。
为了更好地理解本发明,下面结合本发明化合物与药用辅料或赋形剂组成的药物组合物的药理作用作为具体试验例进行说明,但并不以此来限定本发明。
实施例7:
本发明化合物及其与药用辅料组成的药物组合物的抗糖尿病肾病或慢性肾病的药理作用。
本发明化合物抗肾系膜细胞活性氧实验:
参照文献方法(Wei XF,Zhou QG,Hou FF et al.Advanced oxidationprotein products induce mesangial cell perturbation throughPKC-dependent activation of NADPH oxidase.Am J Physiol RenalPhysiol2009;296:F427-F437),大鼠系膜细胞株(HBZY-1,Life-Science Academy of Wuhan University,Wuhan,China)在37℃条件下,培养在pH值为7.4的DMEM(Invitrogen,Carlsbad,CA)培养液中,培养液中加入10%胎牛血清(Invitrogen,Carlsbad,CA),2mM谷氨酰胺,100U/ml青霉素和100μg/ml链霉素。细胞融合达到80%时,采用含有0.5%胎牛血清的培养液饥饿24小时,使细胞同步化于G0期,用于后续实验。
为了检测化合物的作用,系膜细胞首先与不同浓度的化合物预孵育1小时,然后以5.6mM(normal glucose,NG)或25mM(highglucose,HG)的D-葡萄糖(Xia L,Wang H,Munk S et al.Reactiveoxygen species,PKC-β1,and PKC-ζmediate high-glucose-inducedvascular endothelial growth factor expression in mesangial cells.Am JPhysiol Endocrinol Metab2007;293:E1280-E1288)刺激,观察各项指标。
细胞内活性氧(reactive oxygen species,ROS)含量测定:
细胞内ROS含量采用荧光探针carboxymethyl-H2-dichlorofluorescein diacetate(CM-H2-DCF-DA,Sigma Chemical Co.,St.Louis,MO)染色,流式细胞仪检测(Xia L,Wang H,Goldberg HJ et al.Mesangial cell NADPH oxidase upregulationin high glucose is protein kinase C dependent and required for collagenIV expression.Am J Physiol Renal Physiol2006;290:F345-F356)。消化搜集细胞,与CM-H2-DCF-DA(1μM)孵育30分钟。以流式细胞仪(BD FACSCalibur system,Franklin Lakes,NJ)测定荧光强度(激发波长λ=488nm,发射波长λ=515nm)。每组ROS含量用各组细胞荧光强度与正常糖浓度培养的细胞荧光强度的比值表示,结果以细胞总蛋白含量进行校正(Rygiel TP,Mertens AE,Strumane K et al.TheRac activator Tiam1prevents keratinocyte apoptosis by controllingROS-mediated ERK phosphorylation.J Cell Sci2008;121:1183-1192)。
统计学分析:
所有实验重复三次。连续变量表示为均数±标准差,采用单因素方差分析进行比较,SPSS13.0进行统计学分析。方差齐时采用Student-Newman-Keuls法进行比较,方差不齐时采用Dunnett's T3法进行比较。双尾检验P值小于0.05认为具有统计学差异。结果见图3。
以上结果说明本发明的化合物1、2和4对高糖诱导的肾系膜细胞株产生的活性氧具有明显的抑制作用。由于氧化应激是糖尿病肾病和慢性肾病的主要发病机制之一,因此本结果提示螺环灵芝素A-D对于糖尿病肾病或慢性肾病具有重要的应用价值。
Claims (5)
1.具有下述结构式所示的灵芝杂萜类化合物,
2.制备权利要求1中的化合物的方法,取赤芝,粉碎,用95%乙醇回流提取2h,合并提取液并减压回收溶剂得粗提取物,将粗提取物混悬于水中,然后用等体积乙酸乙酯萃取三次,合并萃取液,减压浓缩得乙酸乙酯萃取物,该萃取物经硅胶200‐300目柱层析,氯仿-甲醇系统梯度99:1,98:2,97:3,96:4,95:5,94:6,93:7,92:8,90:10,85:15,80:20,50:50洗脱,每种溶剂梯度为1.5倍柱体积,按照每份500mL收集得7个合并组份,其中组份7经MCI gel CHP 20P柱层析,20–100%的甲醇‐水洗脱得9个亚组份,其中组份7.2经Sephadex LH‐20MeOH柱层析及反相RP‐18柱层析35‐50%的甲醇‐水,最后经半制备HPLC40%的甲醇‐水,依次得到化合物4,1,2,组分7.3经反相RP‐18柱层析40‐50%的甲醇‐水得到化合物3,化合物1-3均为消旋体,它们经手性材料HPLC拆分获得光学纯对映体,条件分别如下:化合物1:固定相:Daicel ChiralpakIC;流动相:正己烷‐乙醇,85:15;流速:1mL/min拆分为(+)‐1和(-)‐1.;化合物2:固定相:Daicel ChiralpakIC;流动相:正己烷‐乙醇,80:20;流速:1mL/min拆分为(+)‐2和(-)‐2.;化合物3:固定相:Daicel ChiralpakAD‐H;流动相:正己烷‐二氧六环,90:10;流速:1mL/min拆分为(+)‐3和(-)‐3。
3.治疗糖尿病肾病或慢性肾病的药物组合物,含有治疗有效量的权利要求1所述的化合物和药学上可接受的载体。
4.权利要求1所述的化合物在制备治疗糖尿病肾病或慢性肾病的药物中的应用。
5.权利要求1所述的化合物在制备治疗糖尿病肾病或慢性肾病的保健食品中的应用。
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