CN114560842B - 砂仁素a~d及其药物组合物与其制备方法和应用 - Google Patents
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Abstract
本发明提供结构式(I)所示的4个新的化合物砂仁素A~D(amovillosumins A‒D,1–4),以其为活性成份的药物组合物,及其制备方法和应用,属于药物技术领域。本发明的化合物可显著促进GLP‑1分泌,能够与可药用载体组成药物组合物,能够用于制备降血糖药物或保健食品。
Description
技术领域:
本发明属于药物技术领域。具体地,涉及4个新的降木脂素化合物,砂仁素A~D(amovillosumins A-D,1–4),以其为有效成分的药物组合物,它们的制备方法,及其在制备GLP-1促泌剂药物中的应用,和在制备降血糖药物中的应用。
背景技术:
糖尿病是一种由于胰岛素分泌不足或反应性减弱引起的慢性疾病,其主要特征是血糖升高、碳水化合物以及脂肪、蛋白质代谢紊乱。糖尿病分为1型和2型糖尿病,妊娠糖尿病以及其他类型糖尿病,其中90%的病例属于2型糖尿病。2型糖尿病是由于胰岛素抵抗、胰岛素作用受损或者两种病因共同作用引起的,易引发心血管疾病、肾功能衰竭和肌肉骨骼疾病等并发症。临床上,除了外源性胰岛素之外,口服降血糖药物是治疗2型糖尿病的主要方法,其类型主要有双胍类、α-葡萄糖苷酶抑制剂、胰岛素促泌素、胰岛素增敏剂、胰高血糖素样肽-1(GLP-1)受体激动剂、二肽基肽酶(DPP-4)抑制剂、钠-葡萄糖协同转运蛋白-2(SGLT-2)抑制剂和胰淀素类似物等。
胰高血糖素样肽-1(GLP-1)是小肠上皮细胞分泌的肠促胰岛素激素,具有促进胰岛素分泌、抑制胰高血糖素分泌和胃排空、产生饱腹感、刺激β细胞新生等多重作用。但是内源性GLP-1在体内会被DPP4酶迅速降解,因此其血浆半衰期很短,约为2-3分钟。目前在临床上使用的GLP-1相关药物主要有两种,即GLP-1类似物和DPP-4抑制剂,但是由于具有注射给药的缺点、以及胃肠道反应和损伤胰腺的不良反应,从而阻碍了它们的使用,因此发现新颖的小分子GLP-1促泌剂可能为抗糖尿病提供一种全新的治疗策略。
砂仁为姜科(Zingiberaceae)豆蔻属(Amomum)多年生草本植物阳春砂、绿壳砂或海南砂的干燥成熟果实,主要分布于我国的海南、广东、广西、云南等地。其主要化学成分可分为挥发性和非挥发性成分两类,挥发性成分主要为挥发油;非挥发性成分主要有萜类、二苯基庚烷类、黄酮类、有机酸、多糖、酚类物质以及无机物等。近现代药理学研究表明,砂仁具有胃肠保护、降血糖、抗炎镇痛、抗菌以及抗氧化等多种药理活性。此前,Park和Kwon研究发现,砂仁水提取物可以预防四氧嘧啶诱导的糖尿病小鼠的高血糖和低胰岛素血症,并通过抑制NF-κB活化降低IL-1β和IFN-γ介导的大鼠胰岛素瘤细胞系(RINm5F)的细胞毒性,但其降血糖活性成分尚不明确。本发明人研究发现,砂仁提取物对db/db小鼠具有降血糖作用,以活性为导向,从中分离得到了4个结构新颖且可以促进GLP-1分泌活性的降木脂素,砂仁素A~D(amovillosumins A-D,1–4)。
迄今为止,现有技术无化合物砂仁素A~D(amovillosumins A-D,1–4)的报道,也没有其药用活性的报道,更没有化合物1-4及其药物组合物作为GLP-1促泌剂的报道,及在制备降血糖药物中的应用的报道。
发明内容:
本发明的目的在于提供一类新的具有药用价值如式(I)所示的新化合物砂仁素A~D(amovillosumins A-D,1–4),它们的制备方法,化合物1–4及其药物组合物作为GLP-1促泌剂,及其在制备降血糖药物中的应用。
为了实现本发明的上述目的,本发明提供了如下的技术方案:
结构式(I)所示的化合物,砂仁素A~D(amovillosumins A-D,1–4),
本发明另外还提供了制备所述的式(I)化合物1–4的方法,将干燥砂仁粉碎成粗粉10kg,用90%乙醇冷浸提取三次,每次50升,合并提取液减压浓缩后得到粗浸膏,将浸膏均匀分散于水中,用乙酸乙酯进行萃取,减压浓缩得到乙酸乙酯部分,将乙酸乙酯部分用甲醇溶解,吸附于200–300目硅胶,室温放置,挥干溶剂;用200–300目硅胶进行柱层析,以体积比为0:100、5:95、10:90、20:80的MeOH-CHCl3以及MeOH进行梯度洗脱,所得流分经TLC检测,合并得到8个流分,分别为Fr.A-1~Fr.A-8;Fr.A-5用甲醇溶解后均匀吸附于聚酰胺,室温放置,挥干溶剂,以MCI gel CHP 20P为吸附剂进行柱层析,以体积比为60:40、80:20和100:0的MeOH-H2O为洗脱剂进行梯度洗脱,TLC检测,合并得到3个流分:Fr.A-5-1~Fr.A-5-3;Fr.A-5-1经硅胶柱层析,以体积比为20:80、30:70和60:40的丙酮-石油醚以及丙酮为洗脱剂进行梯度洗脱,得到7个亚流分A-5-1-1~A-5-1-7;Fr.A-5-1-3经Sephadex LH-20柱层析,用MeOH-CHCl3,50:50洗脱,经TLC检查合并相同流分,经半制备HPLC纯化,色谱柱为Agilent XDB-C18柱,流动相为体积比为48:52的MeCN-H2O,得到化合物1和4;Fr.A-5-1-4经Sephadex LH-20柱层析,用MeOH-CHCl3,50:50洗脱,经TLC检查合并相同流分,经半制备HPLC纯化,色谱柱为Agilent XDB-C18柱,流动相为体积比为43:57的MeCN-H2O,得到化合物2和3。
本发明提供了上述技术方案所述化合物1–4在制备GLP-1促泌剂药物中的应用。
本发明提供了上述技术方案所述化合物1–4在制备降血糖药物中的应用。
本发明提供了一种药物组合物,所述药物组合物包括上述技术方案所述化合物1–4中的至少一种和药学上可接受的载体或赋型剂。
本发明提供了上述技术方案所述药物组合物在制备GLP-1促泌剂药物中的应用。
本发明提供了上述技术方案所述药物组合物在制备降血糖药物中的应用。
制备含化合物1–4的药物组合物的方法是,以化合物1–4中的至少一种为原料,加入可药用载体。所述的药用载体是一种或多种固体、半固体和液体稀释剂、填料以及药物制品辅剂。
本发明化合物1–4用作GLP-1促泌剂或药物时,可以直接使用,或者以药物组合物的形式使用。该药物组合物含有0.1~99%,优选为0.5~90%的化合物1–4,其余为药物学上可接受的,对人和动物无毒和惰性的可药用载体。将本发明的药物组合物以单位体重服用量的形式使用。本发明的药物可经注射(静注、肌注)和口服两种形式给药。
与现有技术相比,本发明具备如下的优益性:
1.本发明提供了一类新的降木脂素化合物,即化合物砂仁素A~D(amovillosumins A-D,1–4),为首次报道的新化合物。
2.本发明从砂仁中分离得到4个新的化合物1-4,在浓度为25.0和12.5μmol/L时,化合物1和2明显地刺激STC-1细胞中GLP-1的分泌。本发明表明砂仁中化合物1-4具有降血糖作用。
3.本发明的制备方法简单易行,操作方便,得率较高,环保安全,具有较高的可行性。
附图说明:
图1为本发明化合物1–4的结构式示意图;
图2.为本发明化合物1-4对STC-1细胞中GLP-1分泌的促进作用,油酰乙醇胺(Oleoyl ethanolamine,OEA)用作阳性对照。数值用平均值±标准偏差表示(n=3)。
具体实施方式:
为了更好地理解本发明的实质,下面结合附图,用本发明的试验例和实施例来进一步说明本发明砂仁素A~D(amovillosumins A-D,1–4)的制备方法、结构鉴定、药理作用,以及本发明的制备方法及药物组成,但不以此试验例和实施例来限定本发明。
实施例1:
化合物砂仁素A~D(amovillosumins A-D)(化合物1–4)的制备:
将干燥砂仁粉碎成粗粉10kg,用90%乙醇冷浸提取三次,每次50升,合并提取液减压浓缩后得到粗浸膏,将浸膏均匀分散于水中,用乙酸乙酯进行萃取,减压浓缩得到乙酸乙酯部分,将乙酸乙酯部分用甲醇溶解,吸附于200–300目硅胶,室温放置,挥干溶剂;用200–300目硅胶进行柱层析,以体积比为0:100、5:95、10:90、20:80的MeOH-CHCl3以及MeOH进行梯度洗脱,所得流分经TLC检测,合并得到8个流分,分别为Fr.A-1~Fr.A-8;Fr.A-5用甲醇溶解后均匀吸附于聚酰胺,室温放置,挥干溶剂,以MCI gel CHP 20P为吸附剂进行柱层析,以体积比为60:40、80:20和100:0的MeOH-H2O为洗脱剂进行梯度洗脱,TLC检测,合并得到3个流分:Fr.A-5-1~Fr.A-5-3;Fr.A-5-1经硅胶柱层析,以体积比为20:80、30:70和60:40的丙酮-石油醚以及丙酮为洗脱剂进行梯度洗脱,得到7个亚流分A-5-1-1~A-5-1-7;Fr.A-5-1-3经Sephadex LH-20柱层析,用MeOH-CHCl3,50:50洗脱,经TLC检查合并相同流分,经半制备HPLC纯化,色谱柱为Agilent XDB-C18柱,流动相为体积比为48:52的MeCN-H2O,得到化合物1和4;Fr.A-5-1-4经Sephadex LH-20柱层析,用MeOH-CHCl3,50:50洗脱,经TLC检查合并相同流分,经半制备HPLC纯化,色谱柱为Agilent XDB-C18柱,流动相为体积比为43:57的MeCN-H2O,得到化合物2和3。
化合物1–4结构数据:
旋光由Jasco model 1020旋光仪(Horiba,Tokyo,Japan)测定;红外光谱(IR)采用KBr压片法,由Bio-Rad FTS-135型红外光谱仪(Hercules,California,USA)测定;紫外光谱由UV-2401PC型紫外光谱仪(Shimadzu,Kyoto,Japan)测定。ECD谱由Applied Photophysics圆二色谱仪(Agilent,Santa Clara,United States)测定;核磁共振谱(1D和2D NMR)用Avance III-600型超导核磁共振仪(Bruker,Bremerhaven,Germany)测定,以氘代甲醇和氘代氯仿作为溶剂;高分辨质谱(HRMS)用LCMS-IT-TOF型质谱仪(Shimadzu,Kyoto,Japan)测定;薄层色谱硅胶、柱层析硅胶(200-300目)购自青岛美高及青岛海洋化工集团有限公司,葡聚糖凝胶LH-20(Sephadex LH-20)购自Amersham Bioscience(瑞典)。CHP20P MCI凝胶购自Mitsubishi Chemical Corporation(Tokyo,Japan)。
砂仁素A(1)
分子式:C19H22O6
分子量:346
性状:白色粉末
HRESIMS m/z:347.1483[M+H]+(calcd.for C19H22O6,347.1477);
UV(MeOH)λmax(logε)209(4.95)nm;
IR(KBr)νmax 3428,2937,1615,1601,1510,1463,1338,1226,1149,1106,1048,818cm-1;1H-NMR和13C-NMR(DEPT)数据见表1和2。
砂仁素B(2)
分子式:C18H20O6
分子量:332
性状:白色粉末
HRESIMS m/z 333.1332[M+H]+(calcd.for C18H20O6,333.1331);
UV(MeOH)λmax(logε)209(5.95)nm;
IR(KBr)νmax 3429,2938,1615,1600,1510,1463,1341,1216,1118,1082cm-1;1H-NMR和13C-NMR(DEPT)数据见表1和2。
砂仁素C(3)
分子式:C19H22O5
分子量:330
性状:白色粉末
HRESIMS m/z 331.1532[M+H]+(calcd.for C19H22O5,331.1524);
UV(MeOH)λmax(logε)198(5.44)nm;
IR(KBr)νmax 3453,2938,1608,1590,1504,1466,1332,1272,1239,1126,1036,817cm-1;1H-NMR和13C-NMR(DEPT)数据见表1和2。
砂仁素D(4)
分子式:C18H20O6,
分子量:332
性状:白色粉末
HRESIMS m/z 333.1334[M+H]+(calcd.for C18H20O6,333.1335);
UV(MeOH)λmax(logε)209(4.86)nm;
IR(KBr)νmax 3428,2937,1614,1521,1476,1383,1252,1118,1098cm-1;
ECD(MeOH)λmax(Δε)200(+2.56)nm;
(c0.1,MeOH);
1H-NMR和13C-NMR(DEPT)数据见表1和2。
表1化合物1-4的1H NMR(600MHz,CD3OD,δin ppm,J in Hz)数据.
a测试溶剂为CD3OD,b测试溶剂为CDCl3.
表2.化合物1–4的13C NMR(150MHz,CD3OD)数据.
a测试溶剂为CD3OD,b测试溶剂为CDCl3.
通过对化合物1~4的手性分析,发现化合物1~3以对映异构体的形式存在,因此本发明对化合物1~3进行手性拆分。实验步骤如下:化合物1经半制备HPLC(C1-5柱,MeCN-H2O,48:52)拆分得到1a和1b;化合物2经半制备HPLC(/>C1-5柱,MeCN-H2O,65:35)拆分得到2a和2b;化合物3经半制备HPLC(/>C1-5柱,MeOH-H2O,30:70)拆分得到3a和3b。化合物数据如下:
ECD(MeOH)λmax(Δε)207(-11.75),217(+5.80),238(-1.05)nm
(c0.2,MeOH);
ECD(MeOH)λmax(Δε)207(+21.73),217(-2.99),238(+4.65)nm;
(c0.1,MeOH);
ECD(MeOH)λmax(Δε)197(+4.89),210(-27.67),220(-0.18),237(-6.26)nm;
(c0.5,MeOH);
ECD(MeOH)λmax(Δε)209(+20.16),219(+3.28),235(+5.66)nm;
(c0.8,MeOH);
ECD(MeOH)λmax(Δε)198(+8.33),208(-10.88),220(-0.28)nm;
(c0.3,MeOH);
ECD(MeOH)λmax(Δε)198(-10.00),208(+20.27),220(+3.85)nm;
(c0.8,MeOH);
实施例2:
化合物1~4促GLP-1分泌活性。
1材料和方法
1.1材料
STC-1细胞购自美国ATCC;糖原购自美伦生物科技有限公司(大连);α-D-葡萄糖1-磷酸二钠盐(St.Louis,MO,U.S.A.);钼酸铵购自上海九鼎化学有限公司(上海);孔雀石绿购自中国北京八菱威科技有限公司(北京);牛血清购自上海龙田生物技术有限公司(上海);小鼠GLP-1酶联免疫吸附测定试剂盒购自北京太阳生物科技有限公司科技有限公司(北京)。
1.2仪器
Flex Station 3台式多功能酶标仪(Bio-RAD 680,美国);分析天平(AG135,Metler Toledo,中国);恒温箱(DHP-9082,上海)。
1.3实验过程
GLP-1分泌实验:将用STC-1细胞用含有10%胎牛血清和1%青霉素/链霉素的培养基在37℃和5%CO2饱和湿度的培养箱中培养。将细胞浓度为1×106个/每孔的细胞接种于24孔板中继续培养24h。细胞融合度达到80%-90%后,用含0.1%(w/v)无脂肪酸BSA的Krebs-Ringer碳酸氢盐缓冲液(KRB:134.5mM NaCl,10mM Hepes,3.6mM KCl,5mM NaHCO3,1.5mMCaCl2,0.5mM NaH2PO4,0.5mM MgCl2)饥饿1h,然后再用不同浓度待测化合物处理的含有0.1%(w/v)无脂肪酸BSA和55.5mM葡萄糖的Krebs-Ringer碳酸氢盐缓冲液孵育1h。处理结束后,在4℃800g下离心5min,收集上清液,然后用小鼠GLP-1ELISA试剂盒检测上清液中的GLP-1含量。
2.结果:
在浓度为25.0和12.5μmol/L时,评价了化合物1-4刺激STC-1细胞中GLP-1分泌情况。在用不同浓度的化合物处理后,与对照组相比,化合物明显的增加了GLP-1分泌(图2和表3)。化合物1和2明显的刺激GLP-1分泌,当浓度为25.0μmol/L时,促泌率分别为375.1%和222.7%,当浓度为12.5μmol/L时,促泌率分别为166.9%和62.7%,强于阳性油酰乙醇胺(163.1%,25.0μmol/L)。表明该类化合物具有促GLP-1分泌活性,具有明显的降血糖作用。
表3.化合物对STC-1细胞GLP-1分泌的促进作用
a促泌率表示为GLP-1含量较对照组增加的百分比.
3、结论:
本发明从砂仁中分离得到4个新的降木脂素,砂仁素A~D(1–4),在浓度为25.0和12.5μmol/L时,化合物1和2明显地刺激STC-1细胞中GLP-1的分泌。本发明表明砂仁中降木脂素具有降血糖作用。
实施例3:
制剂实施例:
1.取化合物1–4任其一或其任意组合,用少量的DMSO溶解后,按常规加注射用水,精滤,灌封灭菌制成注射液。
2.取化合物1–4任其一或其任意组合,用少量的DMSO溶解后,将其溶于无菌注射用水中,搅拌使溶解,用无菌抽滤漏斗过滤,再无菌精滤,分装于安瓿中,低温冷冻干燥后无菌熔封得粉针剂。
3.取化合物1–4任其一或其任意组合,按其与赋形剂重量比为9:1的比例加入赋形剂,制成粉剂。
4.取化合物1–4任其一或其任意组合,按其与赋形剂重量比为5:1的比例加入赋形剂,制粒压片。
5.取化合物1–4任其一或其任意组合,按常规口服液制法制成口服液。
6.取化合物1–4任其一或其任意组合,按其与赋形剂重量比为5:1的比例加入赋形剂,制成胶囊。
7.取化合物1–4任其一或其任意组合,按其与赋形剂重量比为3:1的比例加入赋形剂,制成胶囊。
8.取化合物1–4任其一或其任意组合,按其与赋形剂重量比为5:1的比例加入赋形剂,制成颗粒剂。
Claims (8)
1.如下结构式所示的化合物砂仁素1–4,
2.权利要求1所述的结构式所示的化合物砂仁素1–4的制备方法,其特征在于,该方法包括以下步骤:干燥砂仁粉碎成粗粉10kg,用90%乙醇冷浸提取三次,每次50升,合并提取液减压浓缩后得到粗浸膏,将浸膏均匀分散于水中,用乙酸乙酯进行萃取,减压浓缩得到乙酸乙酯部分,将乙酸乙酯部分用甲醇溶解,吸附于200–300目硅胶,室温放置,挥干溶剂;用200–300目硅胶进行柱层析,以体积比为0:100、5:95、10:90、20:80的MeOH-CHCl3以及MeOH进行梯度洗脱,所得流分经TLC检测,合并得到8个流分,分别为Fr.A-1~Fr.A-8;Fr.A-5用甲醇溶解后均匀吸附于聚酰胺,室温放置,挥干溶剂,以MCI gel CHP 20P为吸附剂进行柱层析,以体积比为60:40、80:20和100:0的MeOH-H2O为洗脱剂进行梯度洗脱,TLC检测,合并得到3个流分:Fr.A-5-1~Fr.A-5-3;Fr.A-5-1经硅胶柱层析,以体积比为20:80、30:70和60:40的丙酮-石油醚以及丙酮为洗脱剂进行梯度洗脱,得到7个亚流分A-5-1-1~A-5-1-7;Fr.A-5-1-3经Sephadex LH-20柱层析,用MeOH-CHCl3,50:50洗脱,经TLC检查合并相同流分,经半制备HPLC纯化,色谱柱为Agilent XDB-C18柱,流动相为体积比为48:52的MeCN-H2O,得到化合物1和4;Fr.A-5-1-4经Sephadex LH-20柱层析,用MeOH-CHCl3,50:50洗脱,经TLC检查合并相同流分,经半制备HPLC纯化,色谱柱为Agilent XDB-C18柱,流动相为体积比为43:57的MeCN-H2O,得到化合物2和3。
3.权利要求1所述的结构式所示的化合物砂仁素1–4在制备GLP-1促泌剂药物中的应用。
4.权利要求1所述的结构式所示的化合物砂仁素1–4在制备降血糖药物中的应用。
5.一种药物组合物,其特征在于,所述药物组合物包括权利要求1所述的结构式所示的化合物砂仁素1–4中的至少一种和药学上可接受的载体。
6.制备权利要求5所述的药物组合物的方法,其特征在于,该方法包括以下步骤:干燥砂仁粉碎成粗粉10kg,用90%乙醇冷浸提取三次,每次50升,合并提取液减压浓缩后得到粗浸膏,将浸膏均匀分散于水中,用乙酸乙酯进行萃取,减压浓缩得到乙酸乙酯部分,将乙酸乙酯部分用甲醇溶解,吸附于200–300目硅胶,室温放置,挥干溶剂;用200–300目硅胶进行柱层析,以体积比为0:100、5:95、10:90、20:80的MeOH-CHCl3以及MeOH进行梯度洗脱,所得流分经TLC检测,合并得到8个流分,分别为Fr.A-1~Fr.A-8;Fr.A-5用甲醇溶解后均匀吸附于聚酰胺,室温放置,挥干溶剂,以MCI gel CHP 20P为吸附剂进行柱层析,以体积比为60:40、80:20和100:0的MeOH-H2O为洗脱剂进行梯度洗脱,TLC检测,合并得到3个流分:Fr.A-5-1~Fr.A-5-3;Fr.A-5-1经硅胶柱层析,以体积比为20:80、30:70和60:40的丙酮-石油醚以及丙酮为洗脱剂进行梯度洗脱,得到7个亚流分A-5-1-1~A-5-1-7;Fr.A-5-1-3经SephadexLH-20柱层析,用MeOH-CHCl3,50:50洗脱,经TLC检查合并相同流分,经半制备HPLC纯化,色谱柱为Agilent XDB-C18柱,流动相为体积比为48:52的MeCN-H2O,得到化合物1和4;Fr.A-5-1-4经Sephadex LH-20柱层析,用MeOH-CHCl3,50:50洗脱,经TLC检查合并相同流分,经半制备HPLC纯化,色谱柱为Agilent XDB-C18柱,流动相为体积比为43:57的MeCN-H2O,得到化合物2和3;再以化合物1–4的至少一种或其任意组合为原料加入一定比例的可药用载体。
7.权利要求5所述的药物组合物在制备GLP-1促泌剂药物中的应用。
8.权利要求5所述的药物组合物在制备降血糖药物中的应用。
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