九翅豆蔻根茎中抑制α-葡萄糖苷酶的有效成分及其降血糖作用机制研究

Diabetes has become one of the major chronic diseases, which undermines patient’s health, as well as poses a heavy burden on the State, society and the family. It is still imminent for development of new antidiabetic drugs, as the present antidiabetic drugs are very not enough to meet the clinical needs. Intestinal α-glucosidase inhibitors (GI) can effectively regulate the postprandial blood glucose, which plays a key role in management of type 2 diabetes, and three GI have been used in clinical practice. The results of our studies indicate that ethanol extract from rhizome of Amomum maximum (ERAM) possesses a potential hypoglycemic effect, as it could significantly inhibit α-glucosidase activity in vitro, as well as reduce the postprandial blood glucose level in diabetes rats. Furthermore, the lipid soluble fractions of ERAM, but not water soluble fraction, were conformed as the antihypercemic active fraction. However, our recognition on chemical compositions of A. maximum is very limited, and its hypoglycemic effect has not yet been reported. Thus, in present project, the chemical components of lipid fraction of ERAM will be systematically separated, purified, and identified, then the inhibitory effects of these compounds on α-glucosidase activity will be in vitro/in vivo evaluated, furthermore, the anti-hyperglycemic effects of these compounds will be studied in diabetes mice and Caco-2 cell line. Eventually, present study will reveal the hypoglycemic ingredients in rhizome of A. maximum, as well as their underlying mechanism, which will provide a solid scientific foundation for development of novel GI.

糖尿病已成为最主要的慢性病之一，给国家、社会和家庭造成沉重负担。然而现有治疗药物远远不能满足临床需要，开发新型糖尿病治疗药物仍迫在眉睫。肠道α-葡萄糖苷酶（GA）抑制剂可以有效调节餐后血糖波动，对管理2型糖尿病具有十分重要的意义，并已有三个该类药物运用于临床。我们的研究发现，九翅豆蔻根茎乙醇提取物体外能够显著抑制GA的活性，并能有效降糖尿病大鼠餐后血糖的波动水平，表明其具有潜在治疗糖尿病的效果。进一步的研究表明其抑制GA的活性部位为脂溶性馏分，而不是水溶性馏分。然而，目前对于九翅豆蔻化学成分的认识非常有限，其降血糖效果也尚未有报道。因此，本项目拟对九翅豆蔻根茎脂溶性部位的化学成分展开系统的分离纯化和结构鉴定，并评价各成分体内外对GA的抑制效果、以及改善糖尿病小鼠高血糖状态的效果，明确九翅豆蔻根茎降血糖的有效成分和作用机制，为开发新型GA抑制剂类糖尿病治疗药物提供坚实的科学基础。

本项目对九翅豆蔻根茎中抑制α-葡萄糖苷酶（AG）的有效成分展开了研究，从中共计分离得到17个化合物。并发现其中五个化合物5,6-dehydrokawain、ottensinin、zerumin、hannokinol和amomax B在100 µg/ml浓度下对AG的抑制率高于50%，具有明显的抑制作用；而其它12个化合物在该浓度下对AG的抑制率远远低于50%，表明对AG酶的抑制作用较弱。进一步的研究发现两个半日花烷型二萜zerumin和ottensinin对AG的半数抑制浓度（IC50）与阳性对照药物阿卡波糖的相比无统计学差异（P>0.05）；而5,6-dehydrokawain、hannokinol和amomax B的IC50值都显著高于阳性对照药物（P<0.05）。对AG和α-淀粉酶的抑制缺乏选择性，是阿卡波糖临床应用产生腹胀腹泻等不良反应的主要原因。与阿卡波糖不同，zerumin和ottensinin对α-淀粉酶的抑制作用明显低于对AG抑制作用（P<0.05），表明这两个化合物产生腹胀腹泻等不良反应的机率大大降低。同时，体内活性测试结果也表明服用ottensinin和zerumin两组小鼠餐后各时间点（30、60、120和180 min）的血糖水平明显低于模型组小鼠的血糖值（p<0.05），而与阿卡波糖（20 mg/kg）组小鼠的血糖水平间没有统计数差异（p>0.05）。化合物amomax B、 hannokinol 和5,6-dehydrokawain虽然能够降低小鼠餐后的血糖值，但是与模型组比较没有统计学意义（5,6-dehydrokawain干预组小鼠餐后30 min的血糖值除外）。进一步的Caco-2细胞实验证实化合物ottensinin和zerumin能够浓度依赖性地抑制Caco-2细胞中双糖酶的活性，并显著抑制葡萄糖的摄入。综上所述，ottensinin和zerumin两个化合物是九翅豆蔻根茎中抑制AG的主要活性成分，能够有效抑制糖尿病小鼠的餐后血糖波动。同时，对AG抑制具有较好的选择性，有望克服阿卡波糖临床使用中出现的不良反应，显示出较好的开发潜力，可作为先导化合物为AG抑制剂类降血糖药物的开发提供化学实体。