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研究生:凃玲玲
研究生(外文):Lin-Lin Tu
論文名稱:混合膠體系統於流體式口服胰島素遞送載體之應用
論文名稱(外文):Applications of hydrogel systems as a fluid-type carrier for oral insulin delivery
指導教授:梁弘人梁弘人引用關係
指導教授(外文):Hong-Jen Liang
學位類別:碩士
校院名稱:元培科技大學
系所名稱:食品科學研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
畢業學年度:99
語文別:中文
論文頁數:56
中文關鍵詞:胰島素褐藻膠關華豆膠刺槐豆膠三仙膠
外文關鍵詞:InsulinAlginateGuar gumLocust bean gumXanthan gum
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糖尿病為慢性新陳代謝疾病,統計顯示國內四十歲以上人口的糖尿病盛行率約為十分之一,且近年來高居國人十大死因前五名。臨床上治療糖尿病,患者必須服用降血糖藥物或以靜脈或皮下注射方式施打胰島素以確保血糖正常,不但易引起副作用如注射部位局部紅腫、搔癢等過敏症狀,以及局部皮下脂質增生等副作用,亦造成病患疼痛與不便感,且注射部位若消毒不全,也可能造成金黃色葡萄球菌(Staphylococcus aureus)、龜分枝桿菌(Mycobacterium chelonae) 以及堪薩斯分枝桿菌(M. kansasii)等細菌感染,是臨床治療上很大的阻礙。
口服胰島素是一理想的給藥方式,其優點為方便服用以及能免去打針及感染之過程,因此病人的接受度高。但口服胰島素與大多數口服藥物因需經由消化道途徑進入人體,均有生物利用率不佳等缺點。多數研究以多醣或聚合物微粒製備方式包埋胰島素,雖可抵抗腸胃道酸液及消化酶的破壞,但製備成本過高,且製備過程需用到有機溶劑,易造成生物毒性,因此本研究嘗試以酸誘導多醣凝膠特性研發流體式胰島素口服混合膠體遞送系統,無需微粒製備成本,提高口服胰島素應用的可能性。
本研究嘗試開發兩種流體式混合膠體以保護胰島素抵抗酸液及消化酵素破壞其活性之可能性:酸誘導凝膠N3系統及非酸誘導凝膠nN3系統。酸誘導凝膠N3系統是以褐藻膠為主的混合膠體,而非酸誘導凝膠nN3系統是以關華豆膠、刺槐豆膠、三仙膠等非凝膠性質混合膠體。首先以BSA為目標測試蛋白質,以此兩種混合膠體包覆BSA(Bovine Serum Albumin,BSA)進行酸液中蛋白質的釋放試驗。並以兩種膠體系統包覆澱粉酶,在酸液及消化酵素中,進行不同時段區間澱粉酶活性試驗,以評估兩種膠體系統對蛋白質在模擬消化道環境中的釋放及保護效果。最後我們以(Streptozotocin,STZ)破壞SD (Sprague Dawley)大鼠胰臟的β細胞,使其患得糖尿病後,再以混合膠體包覆胰島素製成口服胰島素後,餵予患有糖尿病的SD大鼠,以評估活體內兩種膠體系統傳遞胰島素及降血糖之效果。
實驗結果顯示N3膠對BSA的包覆效果達到96%,且對酸鹼度具有敏感性,能在中性環境下緩慢地將蛋白質釋放出來。nN3膠能有效保護澱粉酶活性免於被酵素與胃酸破壞,且隨著nN3-G的濃度增加效果越顯著。此外蛋白質電泳分析發現,nN3-G20包能夠保護BSA免於被胃蛋白酵素與酸破壞。最後活體實驗證實,以N3混合膠體系統製成之口服胰島素,口服給藥8小時內糖尿病大鼠的平均血糖只下降19.91±11.93%。然而,以nN3混合膠體系統製成之口服胰島素,口服給藥8小時內糖尿病大鼠的平均血糖大幅下降63.38±16.34%,其效果與皮下注射胰島素相當。單獨以nN3-G10製成之口服胰島素,給藥後4小時即見降血糖之功效,8小時內糖尿病大鼠的平均血糖也顯著下降64.27±11.39%。因此我們認為兩種膠體系統應用在口服載體上具有截然不同的功能,以N3酸誘導系統可因酸鹼度變化凝膠或解膠;nN3-G系列非酸誘導系統對於蛋白質有直接的保護作用,更具有開發成為液體式口服胰島素遞送載體的潛力。

Nowadays the estimated prevalence of diabetes mellitus, a worldwide chronic metabolic disease, is nearly one tenth among the population over 40s in Taiwan. For the past few years, diabetes mellitus has been the fifth or fourth major leading cause of death for people in Taiwan. Clinically, for normalizing the excessively high blood sugar, anti-hyperglycaemic medicines are often orally given, or recombinant insulin intravenously or subcutaneously in diabetes patients. However, in addition to the fear of pain and inconvenience, it has been reported that some skin allergic reaction such as local erythema and swelling could be elicited at the injection site. Furthermore, continuous injection of insulin at the same site causes lipohypertrophy, a lump under the skin caused by accumulation of extra fat. Without the proper sterilization of needle before injection, patient would be at the risk of bacterial infection by Staphylococcus aureus, Mycobacterium chelonae or M. kansasii.
Orally administration, which is pain-free and low risk of infection, is one of the best routes for insulin delivery. However, like most of oral medicines, the bioavailability of orally delivered insulin is low due to acidic environment and several gastric enzymes which digest peptide-like materials such as insulin. Numerous studies have focus on the encapsulation of insulin by either non-digestible polysaccharides or biopolymers that encapsulate the insulin against acidic environment and digestive enzymes. However expensive cost and organic solvents required during encapsulation preparation are the main obstacles for the development of oral insulin delivery. The aim of the study is attempt to develop a novel fluidic type carrier for oral insulin delivery using non-digestible polysaccharide which forms gelation in response to acidic pH.
Two types of fluidic-like carriers, acid-induced gelation N3 hydrogel and non acid-induced gelation nN3 hydrogel that formulated various polysaccharides were designed for protection of insulin from attack of acidic and enzymatic digestion. Bovine serum albumin (BSA) was used as the testing protein and encapsulated by the designed carriers. In addition, activity of α-amylase encapsulated with the designed hydrogels was determined after treatment of stimulated gastric fluid (SGF) for different periods of time, evaluating the protective efficacy of the gels under gastric environment. Finally, streptozotocin-induced diabetic Sprague Dawley rats were fed with insulin-loaded hydrogels and the plasma glucose was monitored for evaluating the anti-hyperglycaemic efficiency in vivo.
Our results indicated the loading efficiency of N3 hydrogel for BSA is about 96 %. Furthermore, BSA was slowly released from the hydrogels under neutralized solution. nN3 hydrogel, on the other hand, has ability to protect BSA from acid and enzymatic digestion, which is proportional to the concentration of G in the nN3 gel.Moreover, nN3-G20 prevented BSA from the destructive hydrolysis by pepsin and acid, as shown in a clear protein band of SDS-PAGE following coomassie blue staining. In vivo experimental results showed that insulin-loaded N3 hydrogel only reduced 19.91±11.93% plasma glucose level within 8 hours after orally administration. Nevertheless, insulin-loaded nN3 hydrogel could significantly reduce plasma glucose level to the extent of 63.38±16.34% after orally administration. A significant decrease of plasma glucose in rats was observed 4 hours after feeding diabetic rats with insulin-loaded nN3-G10, and it reduced to the extent of 64.27±11.39% within 8 hours. In conclusion, our results indicated that N3 and nN3-G carriers function differently. The former is pH-responsive and able to encapsulate protein effectively in acidic environment. The latter, however, can directly protect insulin from acidic and enzymatic attack. It is highly potential for nN3-G developed as an effective carrier for oral insulin delivery.
致謝.............................................I
中文摘要 ........................................II
英文摘要 ........................................IV
目錄 ........................................VI
圖目錄 ......................................VIII
表目錄 .........................................X
第一章 緒論.......................................1
1.1 糖尿病的現況..................................1
1.1.1 糖尿病定義 .................................1
1.1.2 糖尿病診斷標準...............................1
1.1.3 糖尿病分類..................................2
1.1.4 糖尿病治療..................................3
1.2 胰島素簡介....................................4
1.2.1 胰島素組成與作用.............................4
1.2.2 胰島素傳送途徑...............................5
1.2.3 口服胰島素...................................5
1.3 多醣特性.......................................7
1.3.1 阿拉伯膠(Gum arabic)........................8
1.3.2 褐藻酸(Alginic acid)........................9
1.3.3 關華豆膠(Guar gum)..........................12
1.3.4 刺槐豆膠(Locust bean gum)...................14
1.3.5三仙膠(Xanthan gum)..........................14
第二章 實驗目的....................................16
第三章 材料與方法...................................17
3.1 藥品與試劑.....................................17
3.1.1 模擬胃酸溶液.................................18
3.1.2 0.2M 碳酸鈉(Na2CO3)中和溶液..................19
3.1.3 0.05M 檸檬酸溶液(Citrate buffer).............19
3.2 多醣混合膠體之配製..............................19
3.3 BSA體外釋放試驗................................20
3.4 澱粉酶耐受性試驗................................20
3.5 SDS-PAGE 蛋白質電泳實驗.........................20
3.6 胰島素體內釋放實驗...............................22
3.7 動物...........................................22
3.8 統計方法........................................23
第四章 實驗結果......................................24
4.1 BSA體外釋放試驗..................................24
4.2胃蛋白酶酵素耐受試驗...............................25
4.3 N3與nN3膠體澱粉酶酵素活性保護試驗..................26
4.4 N3與nN3膠體混合胰島素之口服試驗 ....................27
4.5 nN3-G之澱粉酶酵素活性保護試驗......................28
4.6蛋白質電泳分析.....................................29
4.7 nN3-G之混合胰島素口服試驗..........................30
4.8 nN3-G與BSA形成沉澱錯合物..........................30
第五章 結論..........................................52
第六章 參考文獻.......................................54

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