中草藥二萜類與多醣體類化合物對小鼠降血糖作用之研究

Hypoglycemic action of diterpenoids and polysaccharides from herbal medicine in mice.

阮琪惇(Chi-Tun Ruan)

指導教授 ： 蘇銘嘉

國立臺灣大學/醫學院/藥理學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.03403

摘要

名列十大死因的新陳代謝性疾病──糖尿病，常會導致神經與血管併發症，嚴重的話還可能導致截肢。由於人口增長、邁向老年化社會、城市化、不健康的飲食習慣、肥胖人數增加與久坐的生活習慣，讓糖尿病的人口數預計在2000年到2030年間呈倍數增加。根據先前文獻研究指出，糖尿病與胰島素的異常釋放及胰島素敏感度降低有關，從而提高肝臟葡萄糖產生並減少肌肉對於葡萄糖的攝取。如今，已有多種口服降血糖藥物問世，提供糖尿病的相關治療，如：磺胺尿素類、雙胍類、meglitinide衍生物、thiazolidinediones (TZD) 類衍生物、甲型葡萄糖苷酶抑制劑、腸泌素調節劑等。此外，自古以來，中草藥廣泛應用於治療糖尿病。例如，於亞洲及非洲，青牛膽屬的寬筋藤已被廣泛運用於治療糖尿病患。本篇論文則探討分離自寬筋藤的borapetoside C與borapetoside A及由中草藥之萃取物GYT-067之降血糖功效。
首先，borapetoside C是一種具有clerodane結構的類萜類衍生物，分離自寬筋藤中的一個二萜類降血糖化合物。於小鼠實驗中發現borapetoside C在腹腔注射投與0.5 mg/kg六十分鐘後即能有效改善血糖值。而此降血糖作用於正常與第二型糖尿病小鼠模式中，可能和藥物投與後增加血液中胰島素含量有關，然而在胰島素分泌缺乏的第一型糖尿病小鼠模式中，borapetoside C並不會提高血中胰島素含量。在正常及第二型糖尿病小鼠，borapetoside C不但可以降低腹腔注射葡萄糖耐受性試驗之血糖值，還可促進骨骼肌肝醣合成量。在七天治療後，borapetoside C還能降低第一型糖尿病小鼠因病態提升的肝臟中磷酸烯醇式丙酮酸羧激酶 (PEPCK) 之蛋白表現量。
其次，腹腔注射 5 mg/kg borapetoside C 單次投與正常與第二型糖尿病小鼠後，亦能降低口服葡萄糖耐受性試驗血糖值。若與正對照組──胰島素 (1 IU/kg, i.p.) 相比，borapetoside C更能增加第二型糖尿病小鼠骨骼肌肝醣合成量。七天連續治療後，borapetoside C能增加胰島素受體 (IR)、蛋白質激酶B (Akt) 磷酸化與第二亞型葡萄糖轉運蛋白的表現量。另一方面，低劑量 (0.1 mg/kg) borapetoside C 與胰島素併用之下，能增加胰島素誘發之降血糖能力與胰島素促使肌肉肝醣合成增加之現象。而在七天治療後發現，borapetoside C與胰島素併用組別，更能增加胰島素受體、蛋白質激酶磷酸化與第二亞型葡萄糖轉運蛋白表現量。因此，此部分實驗證明borapetoside C能增加葡萄糖利用率、延緩胰島素阻抗的進程並增加胰島素敏感性。當糖尿病小鼠體內之胰島素受體─蛋白質激酶B─葡萄糖轉運蛋白的訊息傳遞路徑增強，進一步增加胰島素敏感性可能是borapetoside C達到降血糖功效之機轉。
另外，從寬筋藤中得到的較微量成分borapetoside A在10-8至10-7 M濃度下即能有效增加培養細胞 (C2C12及Hep3B) 之肝醣合成。在動物體之降血糖作用評估，亦發現在正常與第二型糖尿病小鼠模式中，borapetoside A透過增加胰島素分泌而達到降血糖功效；然而，在第一型糖尿病小鼠模式中，borapetoside A不會增加體內胰島素含量，但在腹腔注射投與10 mg/kg之降血糖功效與正對照組 metformin 300 mg/kg 相當。另外，borapetoside A不但具有改善腹腔注射葡萄糖試驗之血糖值，亦可增加肌肉肝醣含量。此外，在每天兩次、共計七日療程後，borapetoside A可以改善肝臟胰島素訊息傳遞途徑與病態增加的磷酸烯醇式丙酮酸羧激酶之蛋白表現量。
另，從植物中萃取出的GYT-067能明顯降低實驗動物體重、攝食與飲水量。GYT-067亦能改善腹腔注射葡萄糖耐受性試驗中血糖提升之現象。另外，二十八天治療後，GYT-067能顯著降低肝臟磷酸烯醇式丙酮酸羧激酶 (PEPCK) 的蛋白表現量；在脂肪組織處，GYT-067組之脂肪細胞尺寸明顯小於負對照組。因此，我們進行一連串GYT-067分離流程中各萃取物質之藥理活性測試，以期找出有效部分。
基於上述各部分實驗，我們可以知道borapetoside C和borapetoside A不但具有胰島素依存性，亦具有非胰島素依存性的作用。此外，borapetoside C還具有延緩胰島素阻抗、增加胰島素敏感性之功效。另一方面，於第二型糖尿病小鼠模式中，GYT-067萃取物與metformin有相似之降血糖作用。雖然針對borapetoside C、borapetoside A與GYT-067 等藥物之詳細的降血糖機制尚未完全明瞭，但由目前研究結果顯示這些藥物具優秀潛力開發成為新降低血糖藥物。

關鍵字

Tinospora crispa ； borapetoside C ； borapetoside A ； 降血糖作用 ； 糖尿病小鼠

並列摘要

Diabetes mellitus (DM), one of the metabolic disorders ranked among the top ten causes of mortality, often leads to neurological and vascular complications, and in severe conditions could result in limb amputation. The number of DM patient is expected to double at 2030 comparing to that in 2000 due to population growth, increase in aging population, urbanization, unhealthy diet, and soaring incidences of obesities and sedentary lifestyles. Numerous prior studies have proven that diabetes is associated with abnormal insulin release and sensitivity which result in increased hepatic glucose production and reduced glucose uptake in muscles. Nowadays, several classes of oral antidiabetic agents are available. However, the use of herbal remedies as DM treatments has been practiced since ancient times. For example, Tinospora crispa has been widely used in Asia and Africa as a remedy for diabetes and other diseases. In this study, the hypoglycemic effects of borapetoside C and borapetoside A, the pure active principles from Tinospora crispa, and GYT-067, a crude extract from a herbal plant, are explored.
Borapetoside C is a clerodane type terpene derivative isolated from Tinospora crispa. When injected intraperitoneally at doses ranging from 0.5 to 5 mg/kg in mice, borapetoside C decreased plasma glucose concentration within one hour. Such hypoglycemic effect seemed to be associated with an increase in the plasma insulin in normal and type 2 DM (T2DM) mice, but not in type 1 DM (T1DM) mice, where the insulin level remained constant. Borapetoside C treatment not only attenuated the elevation of plasma glucose level induced by an intraperitoneal glucose tolerance test (IPGTT), but also stimulated glycogen synthesis. Moreover, the elevated protein level of phosphoenolpyruvate carboxykinase (PEPCK) in the liver of T1DM mice was reversed after borapetoside C treatment.
Acute treatment with borapetoside C (5 mg/kg) attenuated the elevated plasma glucose induced by oral glucose challenge in normal and T2DM mice. Compared to the effect of insulin, borapetoside C caused a more prominent increase of glycogen content in skeletal muscle of T2DM mice. Continuous treatment with 5 mg/kg borapetoside C for 7 days increased phosphorylation of insulin receptor (IR) and protein kinase B (Akt) as well as the expression of glucose transporter-2 (GLUT2) in T1DM mice. Combined treatment of a low dose borapetoside C (0.1 mg/kg) with insulin enhanced insulin-induced lowering of the plasma glucose level and insulin-induced increase of muscle glycogen content. Such treatment for 7 days enhanced insulin-induced IR and Akt phosphorylation and GLUT2 expression in the liver of T1DM mice.
Thirdly, borapetoside A, a minor diterpenoid glycoside from T. crispa, was shown to increase the glycogen content and decrease the plasma glucose concentration in a dose-dependent manner both in vitro and in vivo. The hypoglycemic effects of borapetoside A in the normal healthy mice and T2DM mice were associated with the increases of the plasma insulin levels; whereas, the insulin levels remained unchanged in T1DM mice. Borapetoside A not only attenuated the elevation of plasma glucose induced by an IPGTT, but also increased the glycogen synthesis. Moreover, the elevated protein expression level of PEPCK, which is caused by the dysfunction of insulin signal pathway in the liver of T1DM mice, was reversed after borapetoside A treatment.
Treatment with GYT-067, a polysaccharide containing extract from a medical herb, produced a significant reduction in body weight, food and water intake. GYT-067 attenuated the elevation of plasma glucose induced by an IPGTT and increased glycogen synthesis. Also, GYT-067 significantly reversed the elevated hepatic protein levels of PEPCK after treatment with GYT-067 daily for 28 days. Moreover, after 4-week treatment of GYT-067, the adipocyte size was smaller than vehicle-treatment group. Therefore, we checked the hypoglycemic activity of sequential purification GYT-067 in the process to find the active components.
Based on the studies of hypoglycemic activity of chemical principles from T. crispa, we conclude that the mechanisms involved in the hypoglycemic effect of borapetodie C and borapetoside A include insulin-dependent and -independent pathways. Moreover, borapetoside C delayed the development of insulin resistance in association with increased insulin sensitivity. On the other hand, GYT-067, a crude extract from other plant, exerts similar mode of hypoglycemic actin as metformin in T2DM mice. Although the underlying mechanisms for the hypoglycemic activities of borapetoside C, borapetoside A, and GYT-067 remain to be determined, their well-characterized antidiabetic activities suggest that they highly potential be used as alternative agent for the treatment of diabetes in the future.

並列關鍵字

Tinospora crispa ； borapetoside C ； borapetoside A ； hypoglycemia effect ； diabetic mice

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