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標題: | 芽孢桿菌發酵梔子對D-galactose誘導氧化傷害小鼠之評估 Study of Bacillus fermented Gardenia jasminoides on D-galactose induced oxidative damage in mice |
作者: | You-Jia Chen 陳又嘉 |
指導教授: | 劉?睿(Je-Ruei Liu) |
關鍵字: | 梔子,梔子?,貝萊斯芽孢桿菌,氧化壓力,D-半乳糖誘導氧化傷害, D-galactose,oxidative stress,Gardenia jasminoides,Bacillus velenzenssis, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 梔子(Gardenia jasminoides Ellis, GJE)為一種清熱解毒的中藥材,具有抗氧化、抗發炎、護肝、保護神經等多種功效;根據先前實驗室對梔子的研究結果發現,將梔子以實驗室分離出來的菌Bacillus sp. AC發酵後,其對於1,1-二苯基-2-三硝基苯肼自由基與氫氧自由基的清除能力,皆較未發酵的梔子高;將發酵後梔子共處理於老化之人類胚肺細胞(MRC-5)後,也提高了細胞抗氧化酵素的活性。本論文的目的為接續先前細胞實驗結果,將發酵後梔子實驗進行D-半乳糖誘導氧化傷害之動物模式評估。在AC菌種的鑑定實驗中,以持家基因DNA gyrase subunit B(gyrB)畫出的親緣關係樹得知,AC與Bacillus velezensis的親緣關係較為相近,而經梔子經AC發酵後,梔子中指標成分梔子苷的含量與發酵前相比顯著下降。動物實驗則採用過量注射D-半乳糖的方式誘導小鼠產生氧化傷害;第一批實驗使用BALB/c小鼠,以每kg體重每天注射300 mg的D-半乳糖誘導小鼠產生氧化傷害,並分別同時管餵水、LB Broth、AC菌液、梔子液(發酵前每kg體重小鼠每天可攝取到40 mg之梔子苷含量)或梔子發酵液,探討發酵梔子對於延緩D-半乳糖誘導氧化傷的效果。在氧化指標測定項目中,血清丙二醛濃度於10周處理後,負控制組與誘導氧化控制組間有顯著差異,但管餵發酵梔子的組別與誘導氧化控制組間沒有統計上的差異,而其他氧化傷害指標,如水迷宮試驗、肝臟中過氧化氫酶及穀胱甘肽酶活性、肝臟中丙二醛含量,以及腦部丙二醛含量的數值,於負控制組、誘導氧化控制組,以及各個處理組間皆沒有顯著差異。基於推測此批小鼠誘導氧化傷害的狀況並不顯著,之後實驗的試驗設計參考先前文獻,改以C57BL/6小鼠進行第二批動物實驗。第二批以C57BL/6進行的試驗中,每kg小鼠每天注射200 mg之D-半乳糖溶液誘導小鼠產生氧化傷害;經過10周的誘導後,肝臟中超氧化物歧化酶活性於負控制組與誘導氧化控制組間有顯著差異,但管餵發酵梔子的組別與誘導氧化控制組間沒有統計上的差異;而其他氧化傷害指標,如水迷宮試驗、血清及腦中丙二醛含量、肝臟及腦中過氧化氫酶及穀胱甘肽酶活性,以及腦中超氧化物歧化酶活性於各個處理組間,皆沒有顯著差異。綜合以上兩批小鼠的實驗結果,D-半乳糖誘導氧化傷害的過程於兩批動物實驗效果皆不顯著,而推測造成此狀況的原因為D-半乳糖誘導劑量不足所致;因此,之後若要以此誘導模式再次進行試驗,需先做多個不同的誘導條件,以測試出D-半乳糖於實驗環境中的有效劑量,亦或是換一種老化老鼠模型,待建立出條件可行的模式後,再進行發酵梔子的抗氧化或是抗老化實驗。 Gardenia jasminoides (GJE) is a kind of traditional Chinese herbal medicine (CHM) which is known for its anti-oxidative, anti-inflammatory, and neuroprotective characteristics. In a previous in vitro study, fermented GJE showed greater anti-oxidant ability than unfermented GJE through 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and deoxyribose radical assays. Treated premature-senescence of MRC-5 cell with fermented GJE also increased the antioxidant enzymes activity of cell. The objective of this study is to investigate the in vivo anti-aging property of GJE fermented with Bacillus sp. AC. The species of AC closed related to Bacillus velezensis through phylogenic tree built up by DNA gyrase subunit B (gyrB). The geniposide content of Gardenia jasminoides decreased significantly after fermented with AC. In the first trial of in vivo test, male BALB/c mice were subcutaneously injected daily with D-galactose (300 mg/kg of body weight) to mimic the oxidative damage of mice. Mice were orally administrated with either water, LB broth, AC, GJE (containing 40 mg/kg body weight of geniposide before fermentation) or fermented GJE at the same time. After 10 weeks of treatments, the level of serum malondialdehyde (MDA) in control group showed significantly different compared with D-galactose induced oxidative control group. However, orally administrated with fermented GJE showed no significantly different compared with D-galactose induced oxidative control group. In addition, several aging markers had no significant difference among all the treatment groups. Since the failure of the oxidative damage induced process had been assumed, I altered some experimental processes according to previous studies. In the second trial of in vivo test, male C57BL/6 mice were adopted in this experiment. Mice were subcutaneously injected daily with D-galactose (200 mg/kg of body weight) for 10 weeks. Although the SOD activity of liver in control group increased significantly compared with D-galactose induced oxidative damage group, there was no significant difference among all the groups in other aging biomarkers. In conclusion, I assumed that the D-galactose induced oxidative damage processes fail in both trails. In order to establish the effective model of mice, further experiments will be needed to investigate the adequate volume of D-galactose to induce the oxidative damage process, or change another effective aging model, to investigate the alleviation effective of fermented GJE. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21151 |
DOI: | 10.6342/NTU201803854 |
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顯示於系所單位: | 生物科技研究所 |
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