- 學位論文
土肉桂細枝70%丙酮抽出物之降血糖及抗糖尿病活性
Antihyperglycemic and Antidiabetic Activities of 70% Acetone Extracts from Cinnamomum osmophloeum Twigs
摘要
近年來肉桂 (Cinnamon) 被視為具有治療第二型糖尿病 (Type 2 diabetes) 之功效，其中，菌桂 (Cinnamomum cassia) 及錫蘭肉桂 (C. zeylanicum) 為最常見市售肉桂商品，然而，除了臺灣本土樟屬樹種－土肉桂 (C. osmophloeum) 葉子外，其餘部位之降血糖活性仍未被研究。 本研究先利用抑制糖質代謝酶活性試驗，評估不同部位之土肉桂70%丙酮抽出物，得知細枝70%丙酮抽出物 (CoTE) 抑制葡萄糖苷酶 (IC50 = 3.8 μg/mL) 及澱粉酶 (IC50 = 84.5 μg/mL) 之活性較葉子、2 cm及5 cm枝條的為佳。此外，不同葉子精油品系 (肉桂醛型、混合型及伽羅木醇型) 之土肉桂細枝的降血糖活性無差異。利用液相液相分離CoTE得到4個可溶部，先以抑制糖質代謝酶及蛋白酪胺酸去磷酸酶 (Protein tyrosine phosphatase 1B, PTP1B) 活性試驗，篩選可溶部之降血糖活性成分，再以DPPH (2,2-Diphenyl-1-picrylhydrazyl) 自由基捕捉能力及亞鐵離子螯合能力，篩選可溶部之抗氧化活性成分。得知以正丁醇可溶部 (n-Butanol soluble fraction, BSF) 之原花青素 (561.1 mg/g) 及縮合單寧 (331.5 mg/g) 含量最豐富，具有最優良之抗氧化及降血糖活性，且當使用濃度為200 μg/mL時，對前脂肪細胞及成熟脂肪細胞皆無毒性。 進一步以管柱層析將BSF分離得到10個次分離部，再進行上述活性試驗，得知原花青素含量豐富之F7 (643.3 mg/g) 及F8 (604.7 mg/g) 的抗氧化及降血糖活性最優良。接著利用介質輔助雷射脫附游離/飛行時間質譜 (Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry, MALDI-TOF/MS) 分析F6至F10所含原花青素之結構，得知F6至F10之縮合單寧平均聚合度為3.5至5.1，且聚合度最高為8；F6至F10之A-type鍵結比例為18.9至26.3%，特徵為每個縮合單寧皆含有一個A-type鍵結，其餘則為B-type鍵結。 本研究並以高血糖模式大鼠探討CoTE 之降血糖活性，其中，餵食大鼠150 mg/kg CoTE之組別 (HCO)，能夠改善高血糖大鼠之葡萄糖耐受性曲線下面積 (Areas under the curve of oral glucose tolerance test, AUC of OGTT)、減重、抑制腸道中葡萄糖苷酶、澱粉酶、脂解酶活性及提升血液中高密度脂蛋白含量 (High-density lipoprotein, HDL)，且餵食高劑量 (150 mg/kg) 之CoTE對糖尿病大鼠無毒性。 綜合上述試驗結果顯示，CoTE因富含有A-type鍵結之縮合單寧，能夠減緩腸道中澱粉及油脂分解，進而具有降低餐後血糖上升及減重之功效；此外，增加血液中HDL含量能夠改善血糖、血脂及降低心血管疾病風險，因此，土肉桂細枝70%丙酮抽出物具有開發為降血糖之天然保健食品潛力。
並列摘要
Cinnamon is currently being investigated as a potential preventative supplement to treat type 2 diabetes mellitus. Cinnamomum cassia and C. zeylanicum are the most widely available merchandise of cinnamon. However, C. osmophloeum is an endemic tree in Taiwan, which has not been investigated its antihyperglycemic activity except for leaf. Antihyperglycemic activities of 70% acetone extracts from different plant parts of C. osmophloeum were examined by enzymes of glucose metabolism of inhibitory assays. The C. osmophloeum twig extract (CoTE) showed the best inhibitory activities against α-glucosidase (IC50 = 3.8 μg/mL) and α-amylase (IC50 = 84.5 μg/mL), which are better than those of leaf, 2-cm branch and 5-cm branch extracts. CoTE of 3 different chemotypes, including cinnamaldehyde, linalool and mixed type, were proven to have no significant difference in antihyperglycemic activities. Among all the 4 soluble fractions from CoTE, the proanthocyanidins-rich n-butanol soluble fraction (BSF) exhibited the best inhibitory activities against α-glucosidase (IC50 = 3.6 μg/mL), α-amylase (IC50 = 57.4 μg/mL) and protein tyrosine phosphatase 1B (PTP1B) (IC50 = 1.7 μg/mL). Simultaneously, the BSF showed the best DPPH free radical scavenging activity (IC50 = 11.3 μg/mL) and ferrous ions chelating activity (IC50 = 17.7 μg/mL). Meanwhile, CoTE had no toxicity on both 3T3-L1 preadiocytes and mature adipocytes at the dosage of 200 μg/mL. BSF was separated into 10 subfractions (F1-F10) by Sephadex LH-20 column chromatography, followed by testing using the above-mentioned assays. Subfractions F7 and F8 showed the best antioxidant and antihyperglycemic activities. In addition, proanthocyanidins-rich subfractions F6-F10 were analyzed by matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) for determining the structure of proanthocyanidins. It showed that the mean degree of polymerization (mDP) of condensed tannins from subfractions F6-F10 ranged from 3.5-5.1, and the highest DP of condensed tannins was up to 8. These condensed tannins contained major B-type and at least 1 A-type linkages. Antihyperglycemic activity of CoTE was examined further by type 2 diabetes model of Wistar rats. The results obtained showed that rats fed 150 mg/kg/day CoTE during 3 months decreased both the areas under curve of oral glucose tolerance test (AUC of OGTT) and the body weight. Simultaneously, CoTE inhibited the activities of intestinal maltase, amylase and lipase and increased the contents of high-density lipoprotein (HDL) of diabetic rats. The high dosage (150 mg/kg) of CoTE had no toxicity on diabetic rats. According to the above-mentioned results, it demonstrated that CoTE had potential for reducing blood glucose levels and preventing dietary fat absorption due mainly to inhibiting the acitivities of intestinal enzymes. CoTE can increase the serum HDL content, suggesting that it has potential for improvement of glucose and lipid metabolism, and reducing the risk of atherosclerosis. Therefore, CoTE shows great potential as a natural health product for type 2 diabetes therapy.