- 學位論文
台灣藜飲品之開發與其胃黏膜保護功效之評估
Development of djulis beverage and evaluation of its protective effects on gastric mucosa
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摘要
胃潰瘍是一種常見的疾病，影響全世界大量的人口，而造成胃潰瘍的原因有服用過多的非類固醇抗發炎藥 (Nonsteroid anti-inflammatory drugs, NSAIDs)、長期酗酒、壓力以及胃幽門螺旋桿菌 (Helicobacter pylori) 感染等。Indomethacin (INDO) 為一種NSAIDs，可用於減緩疼痛及發炎，然而NSAIDs容易引起消化道的潰瘍。台灣藜 (Chenopodium formosanum, djulis) 為一種假穀物，原生於台灣，且富含膳食纖維、蛋白質、酚類物質和黃酮類化合物等成分，故本研究之目的為開發台灣藜飲品 (Djulis beverage, DB) 並探討其胃黏膜保護之功效。實驗採用田口法，利用多種酵素水解台灣藜，以得到DB之最佳蛋白質和胜肽含量之最適製備條件，並利用相同之最適條件水解燕麥得到燕麥飲品 (Oat beverage, OB)。後續將DB與OB進行總酚含量 (Total phenolic content, TPC)、總類黃酮含量 (Total flavonoid content, TFC) 以及營養成分測定，並以高效能液相層析法 (High performance liquid chromatography, HPLC) 分析相關酚酸與類黃酮之含量，最後將DB進行感官品評。在動物實驗方面，將雄性Wistar大鼠分為六組，分別為Control組、INDO組 (100 mg/kg INDO)、DBL組 (100 mg/kg DB + 100 mg/kg INDO)、DBH組 (200 mg/kg DB + 100 mg/kg INDO)、OBH組 (200 mg/kg OB + 100 mg/kg INDO) 以及OM組 (30 mg/kg Omeprazole + 100 mg/kg INDO)，連續管餵七天，並於第七天將動物犧牲後量化大鼠胃黏膜損傷面積及計算保護指數，再利用蘇木精-伊紅 (H&E) 染色觀察胃部組織病變，後續分析胃黏膜組織中抗氧化成分及脂質過氧化程度，最後利用西方墨點法 (western blot) 分析促發炎因子與環氧化酶相關之蛋白表現量，並以分析套組測定前列腺素E2 (Prostaglandin-E2, PGE2) 之含量。田口法的結果顯示，水解台灣藜之最適條件為70℃、pH 8及90分鐘。在TPC、TFC以及營養成分分析結果中，DB的含量皆高於OB。此外，於HPLC分析結果中可以發現DB之酚酸與類黃酮之總含量顯著高於OB (p<0.05)。動物實驗的結果顯示，經過100 mg/kg及200 mg/kg 的DB預處理可以顯著改善INDO所引起的大鼠胃黏膜損傷，其潰瘍面積由18.81 ± 2.35 mm2 (INDO組) 分別降低至8.95 ± 0.85 mm2 (DBL組) (p<0.05) 及3.30 ± 0.38 mm2 (DBH組) (p<0.05)，且DBH組與潰瘍面積為7.82 ± 0.67 mm2的OBH組相比，DBH組改善潰瘍面積的作用較為顯著 (p<0.05)。組織病理學結果也指出，DB的預處理能夠減少胃黏膜組織上皮細胞流失、炎性細胞浸潤與黏膜下層水腫的現象。在抗氧化分析中，發現與INDO組相比，給予DB能夠顯著提升穀胱甘肽 (Glutathione, GSH) 之含量與穀胱甘肽過氧化物酶 (Glutathione peroxidase, GPx)、穀胱甘肽還原酶 (Glutathione reductase, GRd) 和超氧化物歧化酶 (Superoxidase dismutase, SOD) 之活性 (p<0.05)。此外，DBH組之GSH含量、GPx活性以及SOD活性也皆顯著高於OBH組 (p<0.05)，且與OM組結果相似。另外，經過DB預處理後，可以降低大鼠胃黏膜中丙二醛 (Malondialdehyde, MDA) 之含量，結果發現DBH組MDA含量顯著低於OBH組 (p<0.05)，且改善脂質過氧化的程度與OM組有相似的結果。在western blot的結果顯示，給予DB能夠分別降低核轉錄因子-κB (Factor nuclear factor-kappa B, NF-κB)、腫瘤壞死因子-α (Tumor necrosis factor-α, TNF-α)、介白素-6 (Interleukin-6, IL-6) 及一氧化氮合成酶 (Inducible nitric oxide synthase, iNOS) 之蛋白表現量，且隨著DB劑量增加，促發炎因子之表現量隨之下降，具劑量依賴性 (p<0.05)。此外，DB可以提升環氧化酶-1 (Cyclooxygenase-1, COX-1) 表現量，並降低環氧化酶-2 (Cyclooxygenase-2, COX-2) 表現量，藉此提升PGE2之含量，DBH組之PGE2含量顯著性高於OBH組與INDO組 (p<0.05)，且與OM組含量結果相似。最後，在感官品評的結果可以發現DB與市售相關產品相比，於視覺、嗅覺與質地上的差異性較小，而味覺及整體接受度得分則略低。綜合上述可知，給予DB能夠減輕INDO所引起的胃黏膜損傷，也能降低促發炎因子與調節環氧化酶相關蛋白之表現，並提升抗氧化物質，以達到保護胃黏膜的效果。
並列摘要
Gastric ulcer is a common disease that affects a large number of people worldwide and is caused by an overdose of nonsteroid anti-inflammatory drugs (NSAIDs), chronic alcoholism, stress, and gastric Helicobacter pylori infection. Indomethacin (INDO) is a kind of NSAIDs that is used to relieve pain and inflammation, however, NSAIDs may easily cause ulcers in the digestive tract. Djulis (Chenopodium formosanum) is a pseudocereal native to Taiwan and rich in dietary fiber, proteins, phenolics, and flavonoids. Therefore, the purpose of this study was to develop djulis beverage (DB) and explore its gastric mucosal protective efficacy. The experiment adopted the Taguchi method and used a variety of enzymes to hydrolyze djulis to obtain the optimal preparation conditions for DB, and using the same optimal conditions to hydrolyze oats to obtain an oat beverage (OB). Subsequently, total phenolic content (TPC), total flavonoid content (TFC), and nutritional components of DB and OB were measured, and the content of phenolic acids and flavonoids was analyzed by high-performance liquid chromatography (HPLC). Finally, DB was subjected to sensory evaluation. In animal experiments, male Wistar rats were divided into six groups, namely Control group, INDO group (100 mg/kg INDO), DBL group (100 mg/kg DB + 100 mg/kg INDO), DBH group (200 mg/kg DB + 100 mg/kg INDO), OBH group (200 mg/kg OB + 100 mg/kg INDO) and OM group (30 mg/kg Omeprazole + 100 mg/kg INDO), administration of the samples for seven days, and after the animals were sacrificed on the seventh day, the damage area of the gastric mucosa of the rats was quantified and the protection index was calculated. Hematoxylin-eosin (H&E) staining was then used to observe the gastric tissue lesions. The antioxidant components and lipids peroxidation in the gastric mucosal tissue were subsequently analyzed. Finally, western blot was used to analyze the expression of proteins related to pro-inflammatory factors and cyclooxygenase, and an analysis kit was used to determine the content of prostaglandin E2 (PGE2). The results of the Taguchi method showed that the optimal conditions for hydrolysis of djulis were 70°C, pH 8, and 90 min. In the analysis results of TPC, TFC, and nutritional components, DB has a higher content than OB. In addition, the HPLC analysis results showed that the total contents of phenolic acids and flavonoids in DB were significantly higher than that in OB (p<0.05). The results of animal experiments showed that 100 mg/kg and 200 mg/kg DB pretreatment significantly improved the gastric mucosal damage caused by INDO in rats, and the ulcer area was reduced from 18.81 ± 2.35 mm2 (INDO group) to 8.95 ± 0.85 mm2 (DBL group) (p<0.05) and 3.30 ± 0.38 mm2 (200 mg/kg DBH group) (p<0.05), respectively. Compared with the OBH group with an ulcer area of 7.82 ± 0.67 mm2, the DBH group showed a more significant effect on improving the ulcer area (p<0.05). The histopathological results also pointed out that the pretreatment with DB reduced the loss of epithelial cells, inflammatory cell infiltration, and submucosal edema in gastric mucosa tissue. In the analysis of antioxidants, it was found that administration of DB significantly increased the content of glutathione (GSH) and the activity of glutathione peroxidase (GPx), glutathione reductase (GRd) and superoxidase dismutase (SOD) compared with the INDO group (p<0.05). Moreover, the GSH content, GPx activity and SOD activity of the DBH group were also significantly higher than those of the OBH group (p<0.05), and the results were similar to those of the OM group. In addition, DB pretreatment reduced the content of malondialdehyde (MDA) in the gastric mucosa of rats. It was found that the MDA content in the DBH group was significantly lower than that in the OBH group (p<0.05), and the improvement of lipid peroxidation status was similar to that in the OM group results. The results of western blot showed that administration of DB reduced the expression levels of nuclear transcription factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nitric oxide synthase (Inducible nitric oxide synthase (iNOS), and as the dose of DB increased, the expression of pro-inflammatory factors dose-dependently decreased (p<0.05). Furthermore, DB increased the expression of cyclooxygenase-1 (COX-1) and reduced the expression of cyclooxygenase-2 (COX-2), in this way, the PGE2 content was increased. The PGE2 content of the DBH group was significantly greater than that of the OBH group and INDO group (p<0.05), and the values were similar to those of the OM group. Finally, from the sensory evaluation results, it can be found that compared with related products on the market, the differences in vision, smell, and texture of DB are smaller, while the taste and overall acceptance scores are slightly lower. Based on the above, it can be seen that administration of DB reduced gastric mucosal damage caused by INDO, decreased pro-inflammatory-related factors, regulated the expression of cyclooxygenase proteins, and increased antioxidants to achieve the protective effects on gastric mucosa.
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