柑橘屬植物檸檬苦素分子與機能之研究

Abstract

台灣栽培多種遺傳多樣性豐富的柑橘類果樹，但近年來，因生產過剩導致農產廢棄物的增加，間接帶來許多不必要的農業成本。為了解決柑橘類農產廢棄物問題，本研究第一部份利用分子標誌分析12種柑橘樣品之葉綠體trnL intron及trnL-trnF IGS區域與去苦味酵素基因limonoid glucosyltransferase (LGTase)親緣性之研究，結果發現LGTase基因可用以區分葡萄柚和檸檬。同時，也可區分柚類和其他柑橘屬植物。另外，將LGTase基因1620 bp片段進行其基因特性之研究，此片段為完整開放讀架 (open reading frame)。並將轉換成amino acid序列，以motifscan, prosite軟體對其活性位置作預測，得知，此段序列含有一段40胺基酸的UDP- Glycosyltransferase signature活性區域。第二部份對柑橘屬植物中所含的兩種型態Citlgt Type I和Citlgt Type II進行研究。其中Citlgt Type I只在果實生長後期表現，而Citlgt TypeII則是在果實的生長過程持續表現。本研究發現在cDNA測定下，分別對開花至成熟果LGTase基因之表現情形作探討，利用所設計的LGTF/LGTR引子進行PCR擴增，得到此段基因於果實生長任何階段皆會表現。進一步利用變性梯度凝膠電泳技術(denatured gradient gel electrophoresis, DGGE)探討兩型基因，DGGE技術可將柑橘LGTase基因分為兩條帶，再利用染劑SYBR Green®與Universal ProbeLibrary™兩方法進行定量分析。其中，Universal ProbeLibrary™在使用LGTF1/LGTA1與LGTF2/ LGTA2引子，能將此二型基因分別擴增，並證實可利用此方法測其生長表現量。本研究第三部份針對台灣柚類(pummelo)果實的機能抗氧化性和美白功效性進行評估。結果發現，蜜柚、台北柚和斗柚之中果皮甲醇萃取物，酚類化合物為8.02-9.99 mg/g。總抗氧化TEAC測定以石頭柚9.3 mg TEAC/g最高; DPPH清除自由基能力方面，EC50值分別為斗柚0.39 mg/mL和台北柚0.95 mg/mLy最好; 螯合亞鐵離子能力之EC50以斗柚(0.78 mg/mL)和文旦柚(0.92 mg/mL)最高。酪胺酸酶活性測定，斗柚抑制酪胺酸酶活性可達90.8% (10 mg/mL)，與麴酸有相近之抑制效果。顯示柑橘廢棄物具有開發作為天然抗氧化劑和防止皮膚色素沉澱原料的潛力。第四部份探討LGTase酵素之反應物(limonin)與產物(limonoid glucoside)對癌細胞之影響。在細胞毒性方面，以乳癌MCF-7細胞株，採用MTS法測試化合物之細胞毒性，可得知兩化合物在50 μmol/L濃度下對MCF-7細胞的存活率有明顯下降。由MTS測定可得知limonin與limonin glucoside對MCF-7細胞具有抑制能力，更進一步看其細胞凋亡之型態，Limonin 與 limonoid glucoside作用使細胞走向早期細胞凋亡的比例分別為5.78%和5.34%，走向晚期細胞凋亡的比例分別為5.85%和7.18%，使細胞走向壞死階段分別佔23.4%和23%。兩化合物對MCF-7細胞確實具有殺死癌細胞能力與誘導細胞走向凋亡路徑，具作為天然藥物或預防劑的開發潛力。

In recent years, the overproduction of citrus fruits has caused an increase of agricultural wastes in Taiwan. In an attempt to handle these potentially valuable wastes. In the first part, we have analyzed 12 Citrus sample using their chloroplast trnL intron trnL-trnF IGS region and limonoid glucosyltransferase gene (LGTase). LGTase gene was found suitable to differentiate grapefruit, lemon and other Citrus plants. LGT-GF / LGT-GR primers amplifies a 1620 bp open reading frame containing a sequence of 40 amino acid UDP-Glycosyltransferase signature. In the second part, we have analyzed Citrus plants contain two types LGTase gene which expressed at different growing stages. We have also identified these two genes using denatured gradient gel electrophoresis (DGGE) using SYBR Green® and Universal ProbeLibrary™ for their quantitative analysis. In the third part, we have evaluated the antioxidant and skin whitening properties of Taiwanese pummelo. The methanol extract of citrus fruits were performed as an ideal antioxidant substances. Citrus grandis Osbeck Miyu (Miyu) showed highest phenolic content (9.99 mg /g), C. grandis Osbeck Shihtouyu (Shihtouyu) gave highest of 2, 2-azino-bis-(3- ethylbenzthiazoline -6-sulfonic acid) content (9.3 mg trolox /g) while C. grandis Osbeck Taipeiyu (Taipeiyu) showed the highest 1,1-diphenyl-2-picrylhydrazyl content. The ferrous-ion chelating effect of C. grandis Osbeck Touyu (Touyu) and C. grandis Osbeck Wentan (Wentan) were found to be 0.78 and 0.92 mg/mL, respectively. Touyu inhibited tyrosinase up to 90.8% (10 mg/mL), which was almost similar to the 95% inhibition effect given by kojic acid (10 mg/mL). Thus, the components of pummelo have high potential to be applied in skin product. Inhibitory effects of limonin and limonoid glucoside on breast cancer MCF-7 cell were evaluated using MTS method. MCF-7 cell viability decreased significantly in concentration of 50 μmol/L. Limonin and limonoid glucoside induced 5.78% and 5.34% of cell early apoptosis, respectively, while 5.85% and 7.18% in late apoptosis. These two extracts induced 23.4% and 23% of cell death. These two compounds were found to inhibit MCF-7 cell and potential to be further applied in pharmaceutical area.