CN103788038A - 赤芝内酯类化合物及其药物组合物和其制备方法与应用 - Google Patents
赤芝内酯类化合物及其药物组合物和其制备方法与应用 Download PDFInfo
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Abstract
提供从中药赤芝中纯化得到的六个杂萜类新化合物,以它们为活性成分的药物组合物,它们的制备方法,及其它们在制备治疗糖尿病肾病或慢性肾病的药物和保健食品中的应用。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分泌IL-6,纤粘连蛋白(Fibronectin),IV型胶原蛋白和活性氧作用。显示该类化合物在制备糖尿病肾病和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灵芝杂萜衍生物,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与细胞因子如IL-6,MCP-1等以及细胞外基质如I型、Ⅳ型胶原、纤粘蛋白等的过度分泌密切相关。因此观察药物对高糖诱导的肾系膜细胞的细胞因子和胶原蛋白的影响,已经成为目前研究糖尿病肾病或肾纤维化药物的常用方法。现代研究已经证明糖尿病肾病或慢性肾病与氧化应激相关,氧化应激会激活NF-kB信号通路,导致炎症发生,因此氧化应激与炎症是慢性肾病的主要特征。机体产生过度的活性氧与机体抗氧化系统受到损伤有关,其中Nuclear factor erythroid2-relatedfactor2(Nrf2)的激活有利于机体激活一系列抗氧化基因和酶,从而清除过多的自由基,发挥抗慢性肾病作用。因此Nrf2激活剂或活性氧抑制剂对慢性肾病有益。
灵芝被称为仙草,中药之王,我国有近百种。灵芝自古为名贵中药,在我国已有2000多年应用历史,据称灵芝可治疗百病,其不仅在我国,且在东亚和美国都有应用,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,本申请人从灵芝(Ganoderma lucidum)具有扶正固本功能推测其可能含有抗糖尿病肾病或慢性肾病活性物质,灵芝的扶正固本一方面可能与多糖相关,是大家通常理解的多糖具有调节免疫力作用,但本申请人认为中医药理论的深度对于认识中医药内涵是有益的。现有文献报道赤芝可治疗多种疾病,其为什么能够广泛地治疗临床疾病,可能与其“以不变应万变”有关。“不变”意味着一方面其可以通过改善免疫作用,解决很多疾病面临的共同问题,本申请人认为灵芝的“不变”可能还与其可能含有抗氧化应激活性成分有关,氧化应激广泛存在多种疾病中,通过清除活性氧可以减轻活性氧引起的一系列病理产物,这也是一种另外意义上的扶正固本。本发明即基于此中医药理论的灵活运用。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具有抗糖尿病肾病或慢性肾病价值的化合物1-3,该发明的化合物在制备抗糖尿病性肾病或/和慢性肾病的药物中的应用,以本发明化合物为活性成分的药物组合物,以及它们的制备方法。
本发明的上述目的是由下述的技术方案得以实现的:
具有下述结构式所示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99:1,98:2,97:3,96:4,95:5,94:6,93:7,92:8,90:10,85:15,80:20,50:50梯度洗脱,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,组份3经MCI gel CHP20P柱层析,甲醇-水(20–100%)洗脱得11个亚组份,其中组份3.10经Sephadex LH-20(MeOH)柱层析,TLC检测,合并相同斑点组份后经反相RP-18柱层析,甲醇-水(40-60%)洗脱,三氯化铁试剂阳性斑点合并,然后经制备薄层层析(CHCl3/Me2CO,12:1),得化合物3及组份3.10.1,该组份经半制备反相RP-18HPLC(MeCN/H2O,35:65)纯化获得化合物1,组份3.8经MCI gel CHP20P柱层析(MeOH/H2O,40-100%)得五个亚组份,组份3.8.1经Sephadex LH-20(MeOH)纯化,三氯化铁试剂显色跟踪,合并组份再经真空柱层析(petroleum ether/Me2CO,10:1-1:1)纯化得化合物2,化合物1-3均为消旋体,化合物1经手性材料半制备HPLC(n-hexane/dioxane,70:30,流速:1mL/min)纯化得化合物(+)-1和(-)-1;化合物2同样经手性材料半制备HPLC(n-hexane/dioxane,60:40,流速:1mL/min)获得(+)-2和(-)-2;化合物3经手性材料半制备HPLC(n-hexane/dioxane,88:12,流速:1mL/min)获得(+)-3和(-)-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中的应用。
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。
本发明化合物更具体的制备方法为: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3(51g)经MCI gel CHP20P柱层析,甲醇-水(20–100%)洗脱得11个亚组份,其中组份3.10(1.1g)经Sephadex LH-20(MeOH)柱层析,TLC检测,合并相同斑点组份后经反相RP-18柱层析,甲醇-水(40-60%)洗脱,三氯化铁试剂阳性斑点合并,然后经制备薄层层析(CHCl3/Me2CO,12:1),得化合物3(3.5mg)及组份3.10.1,该组份经半制备反相RP-18HPLC(MeCN/H2O,35:65)纯化获得化合物1(8mg)。组份3.8(3g)经MCI gel CHP20P柱层析(MeOH/H2O,40-100%)得五个亚组份。组份3.8.1(300mg)经Sephadex LH-20(MeOH)纯化,三氯化铁试剂显色跟踪,合并组份再经真空柱层析(petroleum ether/Me2CO,10:1-1:1)纯化得化合物2(150mg)。化合物1-3均为消旋体,化合物1经手性材料半制备HPLC(n-hexane/dioxane,70:30,流速:1mL/min)纯化得化合物(+)-1(3.8mg)和(-)-1(3.6mg);化合物2同样经手性材料半制备HPLC(n-hexane/dioxane,60:40,流速:1mL/min)获得(+)-2(1.8mg)和(-)-2(1.7mg);化合物3经手性材料半制备HPLC(n-hexane/dioxane,88:12,流速:1mL/min)获得(+)-3(1.3mg)和(-)-3(1.5mg)。
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表示化合物抑制高糖诱导的肾系膜细胞促炎症因子及细胞外基质测定实验(量效关系):采用USCK公司的ELISA试剂盒检测大鼠系膜细胞培养上清IL-6,Collagen IV和Fibronectin的表达;图1A-1C分别为化合物1对白细胞介素-6、纤粘连蛋白(fibronectin)、IV型胶原的抑制作用。*P<0.05vs正常糖;#P<0.05vs高糖。
图2表示化合物抑制高糖诱导的肾系膜细胞促炎症因子及细胞外基质测定实验(时效关系):采用USCK公司的ELISA试剂盒检测大鼠系膜细胞培养上清IL-6,Collagen IV和Fibronectin的表达;图2A-2C分别为化合物1对白细胞介素-6、纤粘连蛋白(fibronectin)、IV型胶原的抑制作用。*P<0.05vs正常糖;#P<0.05vs高糖。
图3表示化合物抑制高糖诱导的肾系膜细胞促炎症因子及细胞外基质测定实验(量效关系):采用USCK公司的ELISA试剂盒检测大鼠系膜细胞培养上清IL-6,Collagen IV和Fibronectin的表达;图3A-3C分别为化合物2对白细胞介素-6、纤粘连蛋白(fibronectin)、IV型胶原的抑制作用。*P<0.05vs正常糖;#P<0.05vs高糖。
图4表示化合物抑制高糖诱导的肾系膜细胞促炎症因子及细胞外基质测定实验(时效关系):采用USCK公司的ELISA试剂盒检测大鼠系膜细胞培养上清IL-6,Collagen IV和Fibronectin的表达;图4A-4C分别为化合物2对白细胞介素-6、纤粘连蛋白(fibronectin)、IV型胶原的抑制作用。*P<0.05vs正常糖;#P<0.05vs高糖。
图5表示化合物2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。
图6表示本发明化合物的结构示意图。
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1-3的分离纯化:
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,将粗提取物混悬于适量1%氢氧化钠水中(PH=13),然后用等体积乙酸乙酯萃取三次。碱水层用稀盐酸中和,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(主要为酚性物质)。该酚性物质经硅胶柱层析(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3(51g)经MCI gel CHP20P柱层析,甲醇-水(20–100%)洗脱得11个亚组份,其中组份3.10(1.1g)经Sephadex LH-20(MeOH)柱层析,三氯化铁试剂显色跟踪合并,合并组份经反相RP-18柱层析,甲醇-水(40-60%)洗脱,三氯化铁试剂阳性斑点合并,然后经制备薄层层析(CHCl3/Me2CO,12:1),得化合物3(3.5mg)及组份3.10.1,该组份经半制备反相RP-18HPLC(MeCN/H2O,35:65)纯化获得化合物1(8mg)。组份3.8(3g)经MCI gelCHP20P柱层析(MeOH/H2O,40-100%)得五个亚组份。组份3.8.1(300mg)经Sephadex LH-20(MeOH)纯化,三氯化铁试剂显色跟踪,合并组份再经真空柱层析(petroleum ether/Me2CO,10:1-1:1)纯化得化合物2(150mg)。化合物1-3均为消旋体,化合物1经手性材料半制备HPLC(n-hexane/dioxane,70:30,流速:1mL/min)纯化得化合物(+)-1(3.8mg)和(-)-1(3.6mg);化合物2同样经手性材料半制备HPLC(n-hexane/dioxane,60:40,流速:1mL/min)获得(+)-2(1.8mg)和(-)-2(1.7mg);化合物3经手性材料半制备HPLC(n-hexane/dioxane,88:12,流速:1mL/min)获得(+)-3(1.3mg)和(-)-3(1.5mg)。
化合物1-3的结构确证:
化合物1-3的结构式如下所示:
化合物1-3的结构鉴定数据:
Linzhilactone A(1):pale yellow amorphous solid;{+84.5(c0.15,CHCl3);UV(CHCl3)λmax(logε)366(3.59),261(3.93),241(3.95)nm;CD(CHCl3)Δε221–2.22,Δε369+1.19;ESIMS(negative)347[M–H]-;HRESIMS(negative)347.1136[M–H]-(calcd for C18H19O7,347.1136);(+)-linzhilactone A};{–78.8(c0.05,CHCl3);UV(CHCl3)λmax(logε)366(3.53),261(3.88),241(3.92)nm;CD(CHCl3)Δε221+2.24,Δε369–1.08;ESIMS(negative)347[M–H]-;HRESIMS(negative)347.1136[M–H]-(calcd for C18H19O7,347.1136);(–)-linzhilactone A}.
Linzhilactone B(2):pale yellow amorphous solid;{+57.1(c0.07,MeOH);UV(MeOH)λmax(logε)367(3.59),258(3.86),227(4.12)nm;CD(MeOH)Δε225–1.02,Δε369+0.37;ESIMS(negative)319[M–H]-;HRESIMS(negative)319.0823[M–H]-(calcd for C16H15O7,319.0823);(+)-linzhilactone B};{–71.4(c0.05,MeOH);UV(MeOH)λmax(logε)367(3.59),258(3.86),227(4.12)nm;CD(MeOH)Δε226+1.69,Δε369–1.10;ESIMS(negative)319[M–H]-;HRESIMS(negative)319.0827[M–H]-(calcd for C16H15O7,319.0823);(–)-linzhilactone B}.
Linzhilactone C(3):pale yellow amorphous solid;{+68.2(c0.02,MeOH);UV(CHCl3)λmax(logε)367(3.54),259(3.84),241(3.78)nm;CD(CHCl3)Δε229–1.48,Δε369+0.78;ESIMS(negative)393[M–H]-;HRESIMS(negative)393.1544[M–H]-(calcd for C20H25O8,393.1555);(+)-linzhilactone C};{–88.9(c0.03,MeOH);UV(CHCl3)λmax(logε)366(3.47),259(3.79),241(3.73)nm;CD(CHCl3)Δε221+1.40,Δε369–0.91;ESIMS(negative)393[M–H]-;HRESIMS(negative)393.1541[M–H]-(calcd for C20H25O8,393.1555);(–)-linzhilactone C}.
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。
化合物1和2抑制肾系膜细胞株炎症因子和细胞外基质测定实验(见图1所示):
采用Uscn公司的ELISA试剂盒检测大鼠系膜细胞培养上清IL-6,Collagen IV和Fibronectin表达。图1A为IL-6的化合物剂量依赖性表达;图1B为Fibronectin的化合物剂量依赖性表达;图1C为Collagen IV的化合物剂量依赖性表达。图2A为IL-6的化合物时效关系;图2B为Fibronectin的化合物时效关系;图2C为Collagen IV的化合物时效关系。
实验原理:
采用双抗体夹心法测定标本中大鼠系膜细胞培养上清IL-6、Fibronectin及Collagen IV表达水平。用纯化的抗IL-6、Fibronectin及Collagen IV抗体包被微孔板,制成固相抗体,向包被单抗的微孔中依次加入含表达IL-6、Fibronectin及Collagen IV的待测样品,再与HRP标记的抗体结合,形成抗体-抗原-酶标抗体复合物,经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的IL-6、Fibronectin及Collagen IV的表达呈正相关。按照文献(Journal ofNatural Products,2011,74,1392-1400;Organic Letters,2014,16,532-535)方法,用酶标仪测定吸光度(OD值),通过标准曲线计算样品中大鼠IL-6、Fibronectin及Collagen IV浓度。
样本处理:
1)细胞培养上清:无菌管收集,2,000rpm/min,离心20min。仔细收集上清。分装冻存于-80℃。
2)刮取底壁细胞,总蛋白裂解液裂解,12,000rpm/min,4℃离心10min,收集上清,Bradford法测量总蛋白含量。
ELISA检测:
操作按试剂盒说明进行:
1)标准品的稀释与加样:酶标包被板设标准品孔,倍比稀释(稀释后各孔加样量都为50μl)加入IL-6、Fibronectin及Collagen IV的标准品。
2)加样:分别设空白孔(空白对照孔不加样品和酶标试剂,其余各骤操作相同)、待测样品孔(样品稀释度为5倍)。轻轻晃动混匀。
3)温育:37℃,30min。
4)洗涤:弃去板中液体,甩干,洗液洗5次,30秒/次。吸水纸拍干。
5)加酶:每孔加入50μl酶标记液,空白孔除外。
6)温育:37℃,30min。
7)洗涤:弃去板中液体,甩干,洗液洗5次,30秒/次。吸水纸拍干。
8)显色:每孔加入底物A、B液各50μl/孔。37℃,15min。避光。
9)终止:每孔加入终止液各50μl/孔。
10)测定:以空白孔调零,酶标仪450nm读吸光度(OD值)。
11)实验重复3次。
结果计算:利用标准品定量曲线分析计算样品浓度并以细胞总蛋白含量校正(IL-6:pg/mg cell protein;Fibronectin&Collagen IV:ng/mgcell protein)。
实施例7:
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5。
以上结果说明本发明的化合物1和2对高糖诱导的肾系膜细胞株炎症因子IL-6、Fibronectin及Collagen IV均有剂量和时效依赖性的抑制作用。另外,这些化合物对活性氧具有明显的抑制作用。由于化合物3是由化合物2的醛基发生缩酮反应而来,该反应在酸性条件下可逆,因此化合物3在胃酸中将生成化合物2并与2形成平衡体,因此可推理化合物3在体内的有效性。鉴于慢性肾病及其并发症的危害,本发明所涉及的化合物预计将在慢性肾病干预过程中扮演重要角色。
Claims (5)
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99:1,98:2,97:3,96:4,95:5,94:6,93:7,92:8,90:10,85:15,80:20,50:50梯度洗脱,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,组份3经MCI gel CHP20P柱层析,甲醇-水(20–100%)洗脱得11个亚组份,其中组份3.10经SephadexLH-20(MeOH)柱层析,TLC检测,合并相同斑点组份后经反相RP-18柱层析,甲醇-水(40-60%)洗脱,三氯化铁试剂阳性斑点合并,然后经制备薄层层析(CHCl3/Me2CO,12:1),得化合物3及组份3.10.1,该组份经半制备反相RP-18HPLC(MeCN/H2O,35:65)纯化获得化合物1,组份3.8经MCI gel CHP20P柱层析(MeOH/H2O,40-100%)得五个亚组份,组份3.8.1经Sephadex LH-20(MeOH)纯化,三氯化铁试剂显色跟踪,合并组份再经真空柱层析(petroleum ether/Me2CO,10:1-1:1)纯化得化合物2,化合物1-3均为消旋体,化合物1经手性材料半制备HPLC(n-hexane/dioxane,70:30,流速:1mL/min)纯化得化合物(+)-1和(-)-1;化合物2同样经手性材料半制备HPLC(n-hexane/dioxane,60:40,流速:1mL/min)获得(+)-2和(-)-2;化合物3经手性材料半制备HPLC(n-hexane/dioxane,88:12,流速:1mL/min)获得(+)-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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