絲瓜乾瓜體纖維的幾丁質來源並應用於生物醫學材料
New chitinous source from dried fruit fiber of Luffa aegyptiaca for biomaterials
中文摘要
絲瓜 (Luffa aegyptiaca)是台灣相當常見的蔬菜，其海綿狀之乾瓜體纖維可作為刷洗碗盤或 身體之用具。本實驗之目的是利用絲瓜乾瓜體纖維(菜瓜布)做為一項新的幾丁質來源，並製成生 醫材料之傷口敷料 (wound dressing)。
將購自傳統市場之菜瓜布，裁剪出大約1 公分長之纖維，再於濃度 1 N 的氫氧化鈉 (NaOH)之 溶液加熱至85 ℃處理 4 小時，以 25 %之漂白水 (sodium hypochloride)將此紙漿狀之懸浮 液漂白，使其纖維褪色後再以蒸餾水清洗以去除次氯酸鹽(hypochloride)，最後將此白色懸浮 液過濾及冷凍乾燥，製成紙狀的薄膜。將此薄膜取5 mg 分別加入 2 M 鹽酸 (HCl)或三氟醋酸 (trifluoroacetic)置入安培瓶 (ampule)並熔封，於 100 ℃下作用 5、10、15 小時將醣類水解，
再將水解之產物以薄層液相色層分析法 (TLC)與高效能液相色層分析儀 (HPLC)分析。利用 Elson-Morgen reagent 呈色後，結果顯示絲瓜薄膜含有幾丁質之單醣：N-acetyl-D- glucosamine，其水解之產物也含有六碳醣，是以葡萄糖為主。
紅外光譜 (IR)分析之絲瓜薄膜，顯示其具有一級胺之乙醯基，此為幾丁質結構之特色。絲瓜薄 膜再經由甲基化、水解、還原及乙醯化作用之流程，此設計之目的為確定其醣類之鍵結方式，利 用氣相色層分析及質譜儀 (GC/MS)分析此甲基化及乙醯化作用之產物，顯示出有一波峰是以 [β-1,4-N-acetyl-D- glucosamine]之鍵結方式。此結果指出絲瓜乾瓜體纖維約含有 39.7 %之 幾丁質，而由絲瓜製成薄膜之產率約70 %。
利用8 週齡、體重至少 300g 之大白鼠 (Wistar rats)做動物實驗，觀察促進傷口癒合之效果。
先用巴比妥酸鹽 (barbiturate)將動物麻醉，再分別於脊髓兩側以施以全皮膚切除(full thickness wound)切兩個 2 cm x 2 cm 鏡相之傷口，再將絲瓜薄膜與 SACCHACHITIN 或紗
布分別貼覆於傷口上，並於切除後每3~4 天觀察癒合情形及傷口大小。結果指出菜瓜薄膜能促
進傷口癒合(25 天癒合完全)，效果比紗布(大於 30 天)快速，但其癒合時間較靈芝薄膜(21 天) 長。然而絲瓜薄膜仍然具有高產率、方便製作以及較精美外觀等優點。
英文摘要
Luffa aegyptiaca is a common vegetable in Taiwan and it produced sponge-like dry fruit for cleaning dished or body. The purpose of the present study was to use the dried fruit of L. aegyptiaca (Loofa) as a new source of chitin for a potential wound dressing biomedical material. Loofa obtained from traditional market was cut into approximate 1 cm in length and then treated with 1 N sodium hydroxide (NaOH) at 85 oC for 4 hours. The pulp like suspension was bleached by 25 % sodium hypochloride until the fiber turned colorless and the pulp was washed by distilled water to remove excess hypochlorite. The white pulp was again filtered and lyophilized to make into a paper-like membrane. The sugar composition of the
membrane was carried out by hydrolyzing 5 mg of the membrane in sealed ampules by either 2 M HCl or trifluoroacetic acid at 100 °C for 5, 10, and 15 hr and then the dried hydrolysate was analyzed by thin layer chromatography (TLC) as well as high performance liquid chromatography (HPLC). The results indicated that L. aegyptiaca membrane contained N-acetyl-D-glucosamine, the monomer of chitin, visualized by Elson-Morgen reagent. The hydrolysate also contained
hexoses mainly glucose. Infrared spectrometry (IR) of the membrane showed signal of acetyl group of primary amine that characterized the structure of chitin.
Methylation of the white pulp with followed hydrolysis, reduction and acetylation procedures was designed to determine the linkage type of sugar residues and GC/MS analysis revealed a linkage type of [β-1,4-N-acetyl-D -glucsamine] in one of the peak of the methylated and acetylated product. The results indicated that loofa contained 39.7% of chitin in total fiber of the dried
fruit with a yield of 70% from the vegetable sponge to membrane. Animal model was carried out by using Wistar rats of 8 week in age, with at least 300 g in weight. For the test of wound healing enhancement, the animals were
anesthetized by barbiturate and then two 2 cm x 2 cm full thickness mirror-image wounds was cut along the both side of spinal cord. Then membranes of loofa and SACCHACHITIN as well as cotton gauge were applied onto the wound area of Wistar rats. The wound size and the healing progression were observed for each 3~4 days after treatment. The results indicated that L. aegyptiaca membrane showed significant healing enhancement (25 days to complete heal) when compared to cotton gauge (>30 days), but the healing period was longer than that of SACCHACHITIN (21 days). However, the membrane of loofa was still advantageous in high yield, better physical properties for fabrication and with a more attractive appearance.
