CN1045208C - 异戊他定化合物、其制备方法及生产该化合物的微生物 - Google Patents
异戊他定化合物、其制备方法及生产该化合物的微生物 Download PDFInfo
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Abstract
本发明涉及的是具有通式(Ⅰ)或(Ⅱ)的新的α-淀粉酶抑制剂异戊他定系列化合物,其制备方法及其在减肥、治疗糖尿病等方面的应用。本发明还涉及能够产生该系列化合物的新的微生物菌株。
Description
本发明涉及的是一类新的α-淀粉酶抑制剂即异戊他定系列化合物,该系列化合物的制备方法以及该系列化合物在治疗糖尿病等疾病方面的应用。本发明还涉及产生异戊他定系列化合物的新的微生物菌株。
由于营养的过剩及体力劳动的减轻,近年来发胖及患糖尿病的人数呈增加的趋势。这是由于食物中的淀粉等糖类在胃肠道中经α-淀粉酶及其他α-葡萄糖苷酶(如蔗糖酶和麦芽糖酶)作用而消化,变成葡萄糖后被体内吸收,使血糖值上升并刺激胰脏分泌胰岛素。在正常情况下,血中的葡萄糖很快被代谢变成热量释放,但当营养过剩(摄取的能量过多)和活动量少(消耗的能量过少)的时侯,在胰岛素的作用下,促使脂肪细胞摄取血中″入超″的葡萄糖,并将其转化为脂肪,引起脂肪细胞的肥大及全身脂肪的异常蓄积,从而发生肥胖。如果体内胰岛素分泌不足,或胰岛素的作用被钝化,则出现持续高血糖,在饮食不加控制的情况下,食后血糖更加异常升高,超过一定的阀值后便从尿中排泄出糖,形成所谓糖尿病。糖尿病的高血糖还将引起糖尿病性动脉硬化、肾病变、微血管病变、视网膜病变等,诱发一系列并发症。因此,控制血糖对于预防和治疗肥胖病、糖尿病都是一项首要任务。
目前,控制血糖的首选方法是节制饮食,特别是糖类食物的摄入,但这一方法需抑制食欲及忍受饥饿,难于长期坚持下去,甚至由于过分节食造成营养不良,诱发其他疾病。α-淀粉酶抑制剂的发明和应用提供了一种理想的控制血糖的方法,它只阻滞淀粉类食物在胃肠道中的消化、吸收,而不影响其他营养物质的吸收和代谢,故能选择性地减少能量的摄取,降低食后血糖,避免或减少糖类向脂肪的转化,起到减肥和预防肥胖的作用,并在改善糖尿病患者的饮食生活的同时使高血糖得以减轻,防止并发症的发生。α-淀粉酶抑制剂还能阻止口腔中淀粉类食物的糖化,减少由糖类形成的齿垢从而预防龋齿。此外,最近的报道说明,α-淀粉酶抑制剂的应用使肠道内残留的糖类增加,有利于肠道有益菌群的生存,并将残留的糖发酵产生乳酸,使肠道趋于酸性环境,从而促使食物中钙的溶解和吸收,产生预防和治疗骨质疏松症的效果(欧洲专利1990,EP36496)
迄今已发现的天然α-淀粉酶抑制剂大体上可分成多肽和拟低聚糖两大类。前者广泛存在于菜豆、小麦、高粱等豆、粮作物种子中,也有许多来源于微生物代谢产物(上田等;Agric.Biol.Chem.1973,37:2025;Oeding,V.etal,:Ger.offen.1987,2701890;村尾等:Agric.Biol.Chem.1980,44:1679;村尾等:Agric.Biol.Chem.1983,47:453;俊藤等:日本公开特许昭1979,54-11395;原田等:日本公开特许昭1984,59-10193;宫川等:日本公开特许昭1986,61-74587;村尾等:日本公开特许平1990,2-67299)。后者有微生物产生的阿卡糖(acarbose)及其同系物(Naturwissenschaften,1977,64:535);trestatins(J.Antibiot.1983,36:1157),adiposins(J.Antibiot.1982,35:1234),amylostatins(Agric.Biol.Chem.1982,46:1941),oligostatins(J.Antibiotics,1981,34:1424),AI-5662(欧洲专利1986,EP173950)等。多肽类的α-淀粉酶抑制剂由于在胃肠道容易被蛋白酶水解失活而难于获得药用价值,拟低聚糖类化合物在体内的稳定性好,已有用于临床的报告(Eichler,H.G.et al.:Di-abetologia 26:278(1984);Tappy,L.et al.:Int.J.Obes.10:185(1986))。因此,今天仍有必要寻找更多更好的拟低聚糖类α-淀粉酶抑制剂,并开发低成本的制造方法及多方面的药用价值。
本发明的目的就是要提供一族新的拟低聚糖类α-淀粉酶抑制剂异戊他定系列化合物及其制造方法,并将其应用于治疗肥胖、糖尿病等疾病。
一方面,本发明提供了具有如下通式(Ⅰ)的异戊他定系列化合物:
其中m为1~5的整数。
在上述异戊他定系列化合物中,优选的是其中m为4、3、2的化合物,它们分别被称作异戊他定C、D、F。
在式(Ⅱ)所示的异戊他定化合物中,优选其中n为6、4的化合物,它们分别被称作异戊他定B、E。
上述化合物分子中都含有异戊酸葡萄糖酯,区别于迄今已发现的拟低聚糖类α-淀粉酶抑制剂,因而是一类新的具有α-淀粉酶抑制活性的拟低聚糖类化合物。
上述式(Ⅰ)和式(Ⅱ)所示的异戊他定系列化合物的混合物为白色无定形粉末,易溶于水和二甲基亚砜,稍溶于甲醇、乙醇和丙酮,不溶于其他有机溶剂。
异戊他定系列化合物的生物学性质
异戊他定系列化合物以B、C、D、E、F为主要组成分的混合物(以下简称异戊他定混合物)强烈抑制猪胰、牛胰、兔胰、人唾液、人血(来源于胰脏)、酵母和霉菌的α-淀粉酶,而对细菌来源的α-淀粉酶(如枯草杆菌α-淀粉酶)无作用。异戊他定对猪胰α-淀粉酶(Sigma公司产品)的抑制作用类型为可逆紧密结合反竞争性抑制,抑制活性为30000~50000IU(淀粉酶活性抑制单位)/mg(依组成分比例不同而异)。
异戊他定混合物对枯草杆菌(Bacillus subtilis)、大肠杆菌(Es-cherichia coli)、绿脓杆菌(Psedomonas aeruginosa)、草分枝杆菌(Mycobacterium phlei)、白色假丝酵母(Candida albiccans)、鲁氏毛菌(Mucor rouxianus)等微生物均无抗菌活性(MIC>1000μg/ml)异戊他定混合物对体重18~22g的昆明种小鼠10只(♀♂各半)以40g/kg体重的剂量灌胃给药,未观察到动物死亡及任何异常表现,提示LD50(P.O.)>40g/kg。另取同样小鼠10只(♀♂各半),以20g/kg体重的剂量自尾静脉给药,也未观察到动物死亡及任何异常表现,说明LD50(I.V.)>20g/kg。
异戊他定混合物对鼠伤寒沙门氏菌(Salmonella typhimurium)无致突变作用。
异戊他定混合物以0.01~1mg/kg体重的剂量对禁食12小时的Laca小鼠在淀粉负荷后灌胃给药,30分种后断头取血测血糖,实验结果表明,0.5mg/kg的剂量就有显著的抑制食后血糖上升的作用(表1)。
表1.异戊他定混合物对小鼠食后血糖的抑制作用
异戊他定剂量(mg/kg) 淀粉负荷(g/kg) N 血糖(mg/dl)
- - 10 92.7±16.2
- 10 10 128.3±13.4
0.01 10 10 119.54±26.7
0.05 10 10 124.7±16.3
0.5 10 10 80.03±18.5
1 10 10 99.2±17.0
用异戊他定C、D、E和异戊他定混合物各按1mg/kg体重的剂量同上法进行实验,结果表明以上三个单组分与混合物之间的抑血糖作用没有显著的差异(表2)。
表2.异戊他定C、D、E及其混合物对小鼠食后血糖的抑制作用异戊他定组分
淀粉负荷 给药剂量 血糖浓度
(g/kg) (mg/kg) (mg/dl) N
空白 - - 91.1±22.4 10
对照 10 - 132.7±14.9 10
混合物 10 1 94.7±22.8 10
C 10 1 88.0±19.8 10
D 10 1 87.3±22.8 10
E 10 1 82.4±22.0 10
异戊他定混合物以100mg/kg体重的剂量给小鼠灌胃给药,测量给药后0.5,1,2和4小时的血中药物浓度均为零,说明异戊他定不经胃肠道吸收。
异戊他定混合物以1~50mg/kg体重的剂量在淀粉负荷后立即灌胃给药,可
使因静注四氧嘧啶(alloxan)所致高血糖小鼠的食后血糖明显降低,作用随剂量增加而增强(表3、表4)。
表3.异戊他定混合物对四氧嘧啶高血糖小鼠的降血糖作用(一)四氧嘧啶 淀粉 异戊他定 血糖(mg/kg) (g/kg) (mg/kg) N (mg/dl)
- - - 10 45.8±7.0
100 - - 10 407.2±56.1
100 10 - 10 592.2±133.7
100 10 1 10 445.9±57.8
100 10 5 10 352.6±93.0
100 10 25 10 174.5±48.0
100 10 50 10 134.4±57.8
表4.异戊他定混合物对四氧嘧啶高血糖小鼠的降血糖作用(二)四氧嘧啶 淀粉 异戊他定 血糖(mg/kg) (g/kg) (mg/kg) N (mg/dl)
- - - 10 157.9±32.3
100 - - 10 603.3±63.3
100 10 - 10 664.9±80.5
100 10 10 10 452.5±45.4
100 10 20 10 444.4±56.7
100 10 40 10 267.4±119.3
异戊他定产生菌
许多链霉菌属的放线菌产生异戊他定,下面仅以蓝色链霉菌井陉亚种DM572(Streptomgces cyaneus nov.subsp.jingxingensis DM572)为一例进行说明。
(1)产生菌来源这一产生菌于1987年从采自河北省井陉县境内山区的土样中分离得到,菌株号为DM572。
(2)形态特性基生菌丝和气生菌丝均呈单纯分枝,培养中未见断裂。气生菌丝上生成短小的孢子柄,顶端着生1~3根孢子丝。孢子丝呈螺旋状,由十几至二十几个孢子组成。孢子为圆柱状,个别弯曲,表面光滑,大小为(0.4~0.6)×(1.0~1.5)μm。未见孢子囊生成。
(3)培养特性DM572菌株在各种琼脂培养基上于28℃培养14~28天的生长特征如表5所列。
表5.DM572菌株在各种琼脂培养基上的生长特征
培养基 基生菌丝 孢子层 可溶色素酵母膏-麦芽汁 酱紫 浅乌贼黑 无
燕麦片 淡桔橙 浅鹤灰 无高氏合成1号 深灰蓝 浅象灰 无无机盐-淀粉 酱紫 中灰驼 无
酪氨酸 燕颌蓝 浅松烟 无
(4)生理生化反应DM572菌株的生理生化反应如表6所列。
表6.DM572菌株的生理生化反应
黑色素生成 牛奶凝固 牛奶胨化 纤维素水解 明胶液化 淀粉水解
- - + + - +
+.阳性反应,-.阴性反应。
(5)碳源利用DM572菌株对葡萄糖、果糖、阿拉伯糖、棉子糖、鼠李糖、肌醇、未糖和水杨苷利用良好,对蔗糖利用较差,而不利用松三糖、木糖醇和阿东糖醇。
(6)降解活性DM572菌株对木聚糖、酪氨酸和次黄嘌呤,有很强的降解活性,对睾酮稍能降解,而不降解弹性蛋白、黄嘌呤和鸟嘌呤。
(7)细胞成分分析DM572菌株的细胞壁中含有LL-二氨基庚二酸,为胞壁Ⅰ型。甲基萘醌的组成以MK-9(H8)、MK-9(H2)、MK-9(H6)和MK-9(H10)为主。磷酸成分中含磷脂酰乙醇氨,为磷脂Ⅱ型。
根据以上形态、生理生化和细胞成分的特征,可将DM572菌株归入链霉菌属中的蓝色类群,并与其中的蓝色链霉菌最为接近。但因DM572菌株对细菌无拮抗性,并有很强的淀粉水解能力,这两方面与中国科学院微生物研究所放线菌分类组编著的《链霉菌鉴定手册》(北京,科学出版社,1975)所描述的蓝色链霉菌不同,故定名为蓝色链霉菌井陉亚种(Streptomyces cyaneus nor.subsp.jingxingensis DM572)。
DM572菌株已于1992年12月26日保藏于中国典型培养物保藏中心,其保藏登记号为CCTCC M920042。
异戊他定的制备方法
异戊他定可以产生由它的链霉菌属放线菌特别是蓝色链霉菌井陉亚种DM572发酵,再由发酵液中提取、精制得到。
(1)发酵DM572菌株可以利用淀粉、糊精、麦芽糖、葡萄糖或植物油作为碳源,麸质粉、花生饼粉、黄豆饼粉、棉籽饼粉、菜籽饼粉或玉米浆作为氮源,同时加入磷酸盐、碳酸钙、硫酸镁、氯化钠等无机盐,制成液体培养基,于120℃下灭菌30分钟,冷却后接种预先培养好的DM572菌株孢子或菌丝,于25~32℃下(最好是28~30℃)通气、搅拌发酵120~144小时,在发酵液中可积累一定量的异戊他定系列化合物。
(2)提取将以上所得发酵液用无机酸或有机酸(最好是有机酸)酸化至PH2~4(最好是3.0~3.5),充分搅匀后过滤。滤液调节PH7~9(最好是7.5~8.5),以一定流速通入大孔吸附树脂柱,使树脂饱和后,充分水洗至电导率不继续下降,再用1~5%的丙酮水溶液洗涤。直至洗出液的颜色不再变浅,然后用10~30%丙酮水溶液解吸,直至流出液不再有蒽酮反应。解吸液用活性碳或脱色树脂脱色,脱色后的解吸液减压浓缩,在浓缩液中加入8~12倍的丙酮-甲醇(2∶1V/V)混合溶媒,搅均后于4℃左右温度下静置2~6小时,过滤得析出的沉淀,减压干燥,研碎,即为异戊他定混合物粗品。
(3)精制用天津第二试剂厂出品的颗粒直径为15μ的C18层析介质填装成高径比约20∶1的反相层析柱,通入适量用蒸馏水溶解并经0.45μm超滤膜过滤的上述异戊他定混合物粗品,随后用10%甲醇-水溶液借助蠕动泵进行展层,收集抑酶反应阳性部分,减压浓缩至干,即得精制的异戊他定混合物。
(4)单组分分离用Waters公司的高效液相层析仪和μBondapakC18柱,上述精制样品用蒸馏水溶解并经0.45μm膜超滤后注入该系统,在室温下以8~15%的乙腈-水溶液展层,紫外210nm检测,收集单峰,减压浓缩至干,可得异戊他定B、C、D、E、F等单组分。
异戊他定对α-淀粉酶抑制活性的检测
在含0.5ml蒸馏水的空白管和对照管及含0.5ml适量浓度的样品液的样品管中,各加入2~2.5AU/ml的猪胰α-淀粉酶(Sig-ma公司)溶液0.5ml,于37℃保温20分钟,除空白管外,各管再加入2%可溶性淀粉溶液1ml,于37℃准确保温5分钟,然后按Bern-feld的经典方法(Bernfeld,P.:Methods Enzymol.1955,1:149),迅速在各管中加入2ml1%3,5-二硝基水杨酸溶液,空白管再补加1ml淀粉溶液,置100℃水浴中加热5分钟,取出放冰浴中冷却至室温后,各加蒸馏水6ml,摇匀,用752C分光光度计(上海分析仪器三厂),于540nm下测定对照管和样品管相对于空白管的消光值。
式中Ec=对照管相对于空白管的消光值
Ei=样品管相对于空白管的消光值
抑制剂活性的计算式为:
式中IU=样品中的抑制剂活性,1IU为抑制2AU淀粉酶活力
50%的抑制剂量。
I50=在测定系统中抑制率为50%时的抑制剂量。AU=在测定系统中的淀粉酶活力,1AU为在上述测定条件下每分
钟催化淀粉水解产生相当于1μmol麦芽糖的淀粉酶量。
异戊他定的制造实施例
实例一:在100L容积的不锈钢通用型机械搅拌发酵罐中,投入60L如下组成的培养基(g/L):淀粉40,玉米麸质粉35,磷酸二氢钾0.3,碳酸钙4,玉米油4,泡敌0.1,pH自然,于120℃灭菌30分钟,冷却至30℃,接种预先在750mL摇瓶中(内装120mL如下组成的培养基(g/L):淀粉15,葡萄糖5,玉米麸质粉25,玉米浆5,碳酸钙4,pH用NaOH溶液调节到6.8~7.0,于120℃灭菌30分钟,冷却后接种DM572菌株斜面孢子,置于200rpm、25mm偏心矩的旋转式摇瓶机上)28℃培养48小时的摇瓶种子5瓶,在28~30℃、1V/V/M通气量和270rpm搅拌转速下发酵144小时,发酵液加草酸酸化至pH3.0,过滤得滤液50L,调节pH为8.0左右,α-淀粉酶抑制剂活性为54247IU/mL,总活性为27.12亿IU。
实例二:在Φ50×1200mm的玻璃层析柱,填装2L312号大孔吸附树脂(山东淄博化工厂产),先后用丙酮和蒸馏水充分洗涤后,以40mL/min的流速通入上述发酵滤液,流出液弃去,用20L蒸馏水以60mL/min的流速洗柱,再用15L10%丙酮-水溶液以同样流速洗,然后用12L20%丙酮-水溶液以40mL/min流速解吸,收集到10L解吸液,活性为63527IU/mL,总亿6.35亿IU,收率23.4%,将此解吸液通过间苯二胺树脂柱脱色,脱色液减压浓缩至1L,加入8L丙酮和2L甲醇,充分搅拌后于4℃下静置四小时,过率得白色沉淀物,减压干燥,研碎,得13.5g白色无定形粉末,活性41842IU/mg,总亿5.65亿IU,收得率89.0%。相对于发酵滤液的总收率为20.8%。异戊他定系列化合物成分组成情况,由HPLC检测得如图1所示结果。
实例三:用颗粒直径为15μm的C18反相HPLC用层析介质(天津第二试剂厂)60mL,装入Φ13×380mm玻璃层析柱中,用甲醇和重蒸馏水充分洗涤。取实例二得到的白色无定形粉末2.00g,溶于20mL重蒸馏水中,以孔径为0.45μm滤膜过滤,注入层析柱的顶端,用100mL重蒸馏水以2mL/min流速洗涤后,再依次用10%甲醇-水溶液和20%甲醇-水溶液各50mL借助蠕动泵以1ml/min流速展层,收集20%甲醇-水溶液展层的流出液,减压浓缩至干,得0.91g白色无定形粉末,活性44900IU/mg,收得率48.8%。异戊他定系列化合物成分组成情况,由HPLC检测得如图2所示结果。
实例四:取实例三所得样品50.0mg,溶于2.5mL超纯水中,经0.45μm滤膜过滤,分5次注入HPLC系统中,按以下条件进行单组分分离:
仪器:Waters公司M481系列高效液相层析仪
柱:μBondapakC18,Φ7.8×300mm
流动相:乙腈-水(13∶87)
流速:3mL/min
检测:UV210nm
经过反复5次操作,收集得五个主要组成分B5.2mg,C6.7mg,D3.5mg,E5.3mg和F3.2mg,活性分别为B32365IU/mg,C23654IU/mg,D30484IU/mg,E31656IU/mg和F25586IU/mg。重量总收率47.6%,活性总收率30.4%。
FAB质谱分析证明,上述五个主要组成分的分子量分别为:B=1844(Ⅱ式中n=6),C=1378(Ⅰ式中m=4),D=1216(Ⅰ式中m=3),E=1519(Ⅱ式中n=4)和F=1053(Ⅰ式中m=2)。
其中对异戊他定C进行了详细的理化性质研究,结果如下:
性状:白色无定形粉末,略带甜味。
熔点:203~208℃
分子式与分子量:C54H91O39N=1378
比旋光度(C1.0,H2O)∶〔α〕24D=+156.8
紫外及可见光谱:末端吸收(图3)
红外光谱(KBr压片,cm-1):3390,2900,1720,1640,1450,1280,1250,1180,1150,1080,1030,900,830。(图4)
质谱(FAB-MS):M/Z1379(M+H+)(图5)
1H-核磁共振谱(500MHz,D2O):δ(ppm)0.96355,0.97691,1.31307,1.32568,2.05222,2.06592,2.07970,2.09332,2.10704,2.34574,2.35768,3.2~4.2,5.23302,5.24056,5.26208,5.27997,5.5.28661,5.37172,5.38785,5.39587,5.40232,5.41020,5.98272,5.99166(图6)
13C-核磁共振谱(500MHz,D2O):δ(ppm)20.142,24.372,28.097,45.566,57.726,58.558,63.524,64.703,66.139,67.067,71.681,72.186,72.275,72.426,72.781,73.905,74.024,74.180,74.254,74.344,74.556,75.482,75.694,75.885,76.007,76.169,76.778,77.354,78.872,79.686,80.018,80.083,80.301,80.867,94.656,98.557,100.485,102.279,102.488,102.571,103.278,129.026,139.282,178.620(图7)
溶解性:易溶于水和二甲基亚砜,稍溶于甲醇、乙醇和丙酮,不溶于其他有机溶剂。
颜色反应:AgNO3+NH3(-),H2SO4(+),草酸苯胺(-),蒽酮(+)。
元素分析〔实验值/理论值(%)〕:C44.21/44.18,H6.35/6.94,N1.29/0.95
生化活性:异戊他定C对猪胰α-淀粉酶(Sigma公司)的抑制常数Ki=5.74nmol/L。
Claims (6)
2.根据权利要求1所述的异戊他定化合物,其中m为4、3或2。
3.根据权利要求1所述的异戊他定化合物,其中n为6或4。
4.根据权利要求1~3中任一项所述的异戊他定化合物,其特征在于该化合物用于制备治疗糖尿病和肥胖病的药物。
该方法包括以下步骤:
-培养能够产生异戊他定的微生物,该微生物为蓝色链霉菌井陉亚种DM572菌株CCTCC M920042;
-用大孔吸附树脂自上述发酵液的滤液中吸附异戊他定化合物,再经有机溶剂解吸、树脂或活性炭脱色、减压干燥处理后制得异戊他定混合物粗品;
-对上述异戊他定混合物粗品进行反相柱层析,收集抑酶反应阳性部分,经减压浓缩后制得异戊他定混合物精品;并根据需要
-对异戊他定混合物精品进行高效液相层析,收集单峰,减压浓缩至于,分别得到m=4、3、2的式(Ⅰ)化合物及n=6和4的式(Ⅱ)化合物。
6.一种微生物,该微生物为蓝色链霉菌井陉亚种DM572菌株CCTCC M920042。
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US4316894A (en) * | 1979-09-19 | 1982-02-23 | Meiji Seika Kaisha, Ltd. | Antibiotic SF-1130-x3 3 substance and production and use thereof |
EP0173950A2 (de) * | 1984-09-04 | 1986-03-12 | Hoechst Aktiengesellschaft | Neuer alpha-Glukosidase Inhibitor, Verfahren zur seiner Herstellung, seine Verwendung und pharmazeutische Präparate |
EP0543076A2 (en) * | 1991-11-18 | 1993-05-26 | Yakurigaku Chuo Kenkyusho | Wheat alpha-amylase inhibitor for the treatment of diabetes |
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US4316894A (en) * | 1979-09-19 | 1982-02-23 | Meiji Seika Kaisha, Ltd. | Antibiotic SF-1130-x3 3 substance and production and use thereof |
EP0173950A2 (de) * | 1984-09-04 | 1986-03-12 | Hoechst Aktiengesellschaft | Neuer alpha-Glukosidase Inhibitor, Verfahren zur seiner Herstellung, seine Verwendung und pharmazeutische Präparate |
EP0543076A2 (en) * | 1991-11-18 | 1993-05-26 | Yakurigaku Chuo Kenkyusho | Wheat alpha-amylase inhibitor for the treatment of diabetes |
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