Analysis of Physiologically Activated Compounds and Cloning of Related Genes Involving Developmental Stages of Angelica dahurica

白芷具有美白潤膚之功效。已知其中的一個美白成分為氫氧基化佛手柑內酯 (8-hydroxybergapten)，由苯基丙烷類代謝路徑 (phenylpropanoids) 合成。為選殖及探討參與合成8-hydroxybergapten之酵素基因特性，利用5´-與3´-RACE-PCR對白芷根Cinnamate 4-hydroxylase (AdC4H) 及bergaptol -O-methyltransferase (AdBMT) cDNA片段進行聚合酶連鎖增幅反應取得序列全長。含有AdBMT基因之大腸桿菌可簡便有效率的轉化佛手柑內醇 (bergaptol) 成大量的佛手柑內酯 (bergapten)，可成為有潛力的生物反應器。將AdC4H可編譯區cDNA建構在pYES2/NT，另外選殖並建構阿拉伯芥cytochrome P450 reductase ATR1全長於pYES3/CT，以缺乏色胺酸及尿嘧啶之培養基篩選，取得併存二載體 (pYES2/NT-C4H及pYES3/CT-ATR1) 之轉形酵母菌 (Saccharomyces cerevisiae)，可提高AdC4H催化效率。於200 μM trans-cinnamic acid培養基液中培養含AdC4H基因之酵母菌，以30℃培養24小時，即可得到122 μM ρ-coumaric acid。使用UPLC管柱為ethylene bridged hybrid (BEH) C18，在酸化水 (pH 3.0) 沖提下變化氰甲烷 (acetoniltrile) 濃度由15%至95%使成梯度流洗，可在12分鐘之內同時有效率的分離並定量七種香豆素。呋喃香豆素psoralen、xanthotoxin、bergapten及8-hydroxybergapten皆可在種植9個月的白芷根部偵測到，其中8-hydroxybergapten含量皆高過其它呋喃香豆素10餘倍。種植3個月及6個月之白芷莖抑制酪胺酸酶能力為最佳。白芷AdC4H mRNA主要表現於種植9個月之根、莖及葉。AdBMT mRNA表現於各器官，以種植6個月之莖及9個月之葉比較多。白芷莖部之高抑制酪胺酸酶能力，未來可萃取白芷莖香豆素應用於化妝品工業。Bai Zhi (Angelica dahurica) has long been used as a face cream for skin-whitening purposes. One of the known skin-whitening components, 8-hydroxybergapten is synthesized from phenylpropanoids pathway. To clone and characterize of related genes involved in 8-hydroxybergapten synthesis, the tandem 5´- and 3´-rapid amplification of cDNA ends via polymerase chain reaction was used to obtain the full-length cinnamate 4-hydroxylase (AdC4H) and bergaptol-O-methyltransferase (AdBMT) cDNA sequences from Bai Zhi root. A simple and efficient production of bergapten in the E. coli culture overexpressing AdBMT was performed. With the supply of bergaptol in the medium, E. coli cells can be used as a potential bioreactor to produce bergapten. The full length of AdC4H coding region was introduced into pYES2/NT vector. Besides, the full length of cytochrome P450 reductase ATR1 cloned from Arabidopsis thaliana was introduced into pYES3/CT vector. The transformed yeast cell (Saccharomyces cerevisiae) harboring two different plasmids (pYES2/NT-C4H and pYES3/CT-ATR1) were obtained by growing in the medium without containing tryptophan and uracil. The AdC4H together with ATR1 overexpression showed highly efficient catalysis of 4-hydroxylation of cinnamate. The amount of 122 μM ρ-coumaric acid could be obtained from yeast harboring two different plasmids that cultured in medium containing 200 μΜ trans-cinnamic acid at 30℃ for 24 hours. An improved ultra performance liquid chromatography method was developed to simultaneously quantify seven coumarins isolated from various Bai Zhi organs. It can be achieved within 12 min using ethylene bridged hybrid (BEH) C18 column and a gradient elution system at acetonitrile concentrations varying from 15 to 95% (v/v) in acidic water (pH 3.0) eluent. The furanocoumarins such as psoralen、xanthotoxin、bergapten and 8-hydroxybergapten were detected in 9 month-old Bai Zhi roots, in which the content of 8-hydroxybergapten was 10-fold higher than other coumarins. Nevertheless, stems from 3 and 6 month-old Bai Zhi exhibited the highest inhibitory ability on tyrosinase. RNA blot analysis indicated that AdC4H mRNAs was predominantly expressed in 9 month-old Bai Zhi roots, stems, and leaf organs, whereas AdBMT mRNAs was expressed in every organ of the three different organs with higher expression in 6 month-old Bai Zhi stem and 9 month-old Bai Zhi leaf. In the future, the Bai Zhi stem coumarins with high inhibitory effect on tyrosinase could be further extracted and used in cosmetic industry.目 錄 前言 1 第一章 白芷佛手柑內醇甲基轉移酶之分子選殖、功能性分析及利用大腸桿菌有效率的量產佛手柑內酯 摘要 2 Abstract 3 一、前人研究 5 白芷美白功效驗證 5 O-甲基轉移酶 (O-methyltransferase, OMTs) 5 二、材料與方法 9 實驗藥材 9 專一性片段及全長cDNA之選殖 9 重組蛋白表現與純化 15 AdBMT重組酵素活性檢測 18 西方墨跡法 (Western hybridization) 20 In vivo 產物動力學測定 22 Ultra Performance Liquid Chromatography (UPLC) 23 三、結果 24 白芷BMT全長基因序列之選殖和序列分析 24 重組蛋白之表達與純化 25 重組蛋白之特性分析 26 Bergapten量產研究 28 四、討論 29 五、參考文獻 33 六、圖表 36 第二章 白芷cinnamate 4-hydroxylase基因之選殖與功能性分析 摘要 55 Abstract 56 一、前人研究 58 Cytochrome P450之分類 58 Cyt P450的基因與胺基酸保守區域 59 Cyt P450催化與功能反應 60 二、材料與方法 63 實驗藥材 63 專一性片段及全長cDNA之選殖 63 重組蛋白表現與純化 64 西方墨跡法 67 Ultra Performance Liquid Chromatography (UPLC) 68 本研究所使用的分析軟體 68 三、結果 69 白芷AdC4H全長基因序列之選殖 69 AdC4H重組酵素之功能性表達 70 四、討論 72 五、參考文獻 76 六、圖表 79 第三章 白芷香豆素美白效能之分析 摘要 95 Abstract 96 一、前人研究 97 香豆素萃取物與療效 97 香豆素之合成和美白能力 99 酪胺酸酶 101 香豆素在醫美化工產業之研究與應用 102 二、材料與方法 105 實驗藥材 105 實驗儀器及藥品 105 白芷活性成分之萃取與檢測 105 北方墨跡法 107 三、結果 110 UPLC分析條件之確立 110 白芷不同成熟時期各器官之香豆素含量 110 白芷不同成熟時期各器官抑制酪胺酸酶能力 111 白芷不同器官和時期之AdC4H及AdBMT基因表現 111 四、討論 112 五、參考文獻 117 六、圖表 121 發表文章 12

(62(4):382-388) Changes in Firmness and Pectin Properties of Different Peach Types during Ripening

果膠分子間之鍵結被認為是影響蔬果質地變化之重要因素。為探討桃不同果肉類型採收後，果實軟化與果膠層化學鍵結之間的相關變化，選擇果肉溶質型之「春蜜」及「台農甜蜜」和硬肉類型之「鶯歌桃」，測定採後12 d內之果實硬度、果膠酯化度（degree of esterification；DE）、結合態鈣（bound calcium）及非甲氧基酯鍵（non-methoxy ester linkage；NME）等含量變化。結果顯示，採後第3 d，「春蜜」、「台農甜蜜」硬度急速下降，低於「鶯歌桃」3倍，由硬度差異變化顯示其果肉特性。三品種桃子隨著貯藏時間增長，總果膠含量及DE值隨之下降，果膠含量由原每克酒精不溶物（alcohol insoluble solids；AIS）含513 mg AGA （anhydrogalacturonic acid）降至350 mg AGA，而DE值由45%下降至29%，以「春蜜」降幅最大。對硬度有正面貢獻之結合態鈣含量，則隨貯藏時間增長而呈現先增後降之變化，以貯藏第3 d之「鶯歌桃」為最高，達6.7 mg g^(-1)AIS^(-1)，隨貯藏時間增長，三品種桃皆降至2 mg g^(-1)AIS^(-1)左右。另一對硬度有正面貢獻之NME，其含量及變化則依各果肉類型而異，在相同類型之桃子中，總酯鍵、NME之變化趨勢相同，「春蜜」和「台農甜蜜」桃隨貯藏時間之增長有先增後降之趨勢，而「鶯歌桃」則隨貯藏時間延長而增加。若能進一步建立桃不同類型採後初期果膠分子與果膠酵素活性變化之相關性，可作為未來選用貯運、加工及保鮮方式之重要參考依據。The interaction between pectin molecules affects the changes of texture in both fruit and vegetables. To investigate the linkage of firmness and pectin to texture changes in different flesh types of peach, traits of firmness, degree of esterification (DE), bound calcium content, and non-methoxy ester linkage (NME) content of melting flesh peach ('Spring Honey' and 'Premier') and stony hard peach ('Yinggetao') were analyzed during twelve-day ripening period after harvest. Results indicated that firmness of 'Spring Honey' and 'Premier' decreased rapidly and their values were three-fold lower than 'Yinggetao' in three days after harvest, suggesting a different flesh characteristic between these two types of peach. The total pectin content and DE value of all peach cultivars were decreased after harvest, from 513 to 350 mg anhydrogalacturonic acid g^(-1) alcohol insoluble solids^(-1) in total pectin and from 45 to 29% in DE value, respectively. 'Spring Honey' showed the largest decreased. The bound calcium had a positive effect on firmness and was decreased with the increasing of ripening after rising in the early stage. 'Yingtao' contained highest bound calcium, 6.7 mg g^(-1) AIS^(-1), at three days after harvest, but its content decreased during ripening, reached to about 2 mg g^(-1) AIS^(-1) at the end of experiment as other peach cultivars. NME also had a positive effect on firmness, whereas, different types of peach had vary contents and changing patterns during ripening period. The total ester linkages and NME followed the same changing pattern in the same peach type; 'Spring Honey' and 'Premier' were first increased and then decreased, while 'Yingtao' was increased throughout the storage. As a result, for establishing a better relationship between pectin molecules and pectinase activity for different types of peach during initial phase of storage after harvest, storing conditions and methods of processing and preservation should be taken into account altogether

Functionalities of Coumarins

香豆素（coumarins）為植物重要的二級代謝產物，具有生理活性對人體健康有助益。香豆素為植物特有的多酚類化合物，主要存在於繖形花科（Apiaceae）及芸香科（Rutaceae）植物中，其中部分植物已成為人類日常生活食用之香料及蔬菜。香豆素由類苯基丙烷代謝路徑生合成，主體結構為一苯環加上一吡喃酮，藉此衍生出各種香豆素。香豆素衍生物具有抗發炎、抗菌、抗癌及美白等多種功效。在醫學上，利用香豆素之光敏性治療皮膚性疾病，或利用化學合成方式將香豆素衍生為抗凝血用藥。除此之外，香豆素亦廣泛應用於多種商業用途。由於生物食入會造成體內解毒酵素cytochorme P450活性增加，因此被認為具毒害性質。未來香豆素之保健功能與毒害作用如能再進一步研究釐清，並將其相關植物進行加工多元化利用，將可提高植物之利用價值。 Coumarins are one of the plant secondary metabolites demonstrated to have physiological activities on human health. They belong to one of the plant specific polyphenolic compounds found primarily in Apiaceae and Rutaceae. Several of these plants are used as spices and vegetables in the human diet. Coumarins are synthesized from the phenylpropanoids pathway, all of which consist of a benzene ring joined to a pyrone and various complex coumarins are derived. Coumarin derivatives have many bioactive effects, such as anti-inflammatory, anti-bacterial, anti-cancer, whitening and other effects. In the pharmaceutical industry, coumarin therapy is used for skin diseases due to its photosensitivity, while chemically synthesized coumarin derivatives are utilized in anti-clotting medication. Coumarins also have many commercial applications. Since coumarin causes cytochrome P450 activity to increase when insects ingest it, it is considered toxic to organisms. The health benefits and toxic effects of coumarins to humans should be clarified and the development of diversified processing products in the future will enhance the value of the plants

(63(2):159-166) An Improved Ultra Performance Liquid Chromatography Method for Analyzing Lutein Content of Vegetables in Taiwan

葉黃素可延緩眼睛老化及黃斑部病變，但人體無法自行合成，需由飲食中補充。為建立快速分析葉黃素之方法與台灣常見市售蔬菜葉黃素含量之資料庫，本研究利用超高壓液相層析儀，使用管柱ethylene bridged hybrid (BEH) C18，以氰甲烷和甲醇 (70 : 30) 之混合液為移動相，等位沖提流洗下，可在5 min 之內定量標的物葉黃素。此外，α- 及β-胡蘿蔔素也可利用此方法一併分析定量。本研究分析結果顯示，50 件蔬菜樣本中葉黃素含量介於0.16–1.77 mg g-1 乾重，其中葉黃素含量大過1 mg g-1 乾重者有8 件樣本，多屬深綠色之綠葉蔬菜，包括香菜、空心菜、油菜及芥藍等，其中以香菜葉含量最高，達1.77 mg g-1 乾重。綠葉蔬菜之葉部含量高於莖部，且皆高於黃色蔬菜及淡色蔬菜，惟淡色蔬菜不被食用之深綠色外葉葉黃素含量大過食用部位。瓜果類蔬菜如大黃瓜，其綠色外皮含量亦高過於白色果肉組織，相差達2.6 倍。藉由蔬菜外觀色澤和葉黃素含量差異，消費者可選用深綠色之綠葉蔬菜，以增加日常葉黃素攝取量。許多未食用之農業廢棄蔬菜，葉黃素含量高於食用部位，應可進一步作加值利用。 Lutein can mitigate eyes’ aging and macular degeneration, but cannot be synthesized in the human body. It must be supplemented from diet. An efficient method for separation and quantification of lutein was developed to establish the database of lutein contents of various commercial vegetables in Taiwan. By ultra performance liquid chromatography, lutein can be analyzed within 5 min using ethylene bridged hybrid (BEH) C18 as a column and an isocratic elution solution of acetonitrile/ methanol (70 : 30, v/v) at a flow rate 0.25 mL min-1. The α-carotene and β-carotene could also be determined simultaneously. The results showed that lutein contents ranged between 0.16 to 1.77 mg g-1 dry weights among 50 vegetable samples. The lutein contents of eight samples were higher than 1 mg g-1 dry weight, and most of them were dark green leafy vegetables, including coriander, water spinach, rapeseed and Chinese kale. The highest was coriander’s leaves, up to 1.77 mg g-1 dry weight. The contents of lutein in the dark green leafy parts were higher than the stems of the yellow and white vegetables. However, the contents of lutein in leaves of the inedible parts of white vegetables were higher than the edible parts. The lutein content of cucumber peel was higher than the pulp up to 2.6 times. According to the differences between vegetable colors and lutein contents, consumers can choose dark green leafy vegetables to increase daily intake of lutein. The content of lutein in the inedible parts of vegetables, usually regarded as agricultural wastes, is higher than in the edible parts. Therefore, these wastes are valuable for value-adding applications in the future

(66(2):105-112)Simultaneous Quantification of Seven Coumarins in Angelica dahurica (Bai Zhi) Using Ultra Performance Liquid Chromatography and Its Whitening Efficiency Assessment

本研究建立同時分析7 種香豆素的快速方法，並探討白芷栽種時期各部位之香豆素含量變化和抑制酪胺酸酶活性。超高效能液相層析儀 (UPLC) 使用管柱為ethylene bridged hybrid (BEH) C18，在酸化水 (pH 3.0) 沖提下，改變氰甲烷 (acetoniltrile) 濃度由15% 至95% 使成梯度沖提，可在12 min 之內，同時有效率的分離並定量香豆素的含量。分析結果顯示，呋喃香豆素，包括psoralen、xanthotoxin、bergapten 及8-hydroxybergapte 等成分皆可在種植9 mo 的白芷根部檢測其含量，其中8-hydroxybergapten 含量皆高過其他呋喃香豆素10 餘倍。種植3 mo 及6 mo 之白芷莖部抑制酪胺酸酶能力為最佳。高抑制酪胺酸酶之白芷莖部香豆素，未來可進行萃取並應用於化妝品工業。 An improved ultra performance liquid chromatography method was developed to simultaneously quantify seven coumarins isolated from various organs of Bai Zhi [Angelica dahurica (Fish.) BENTH. et HOOK]. An efficient separation and quantification of coumarin can be established within 12 min using ethylene bridged hybrid (BEH) C18 column and a gradient elution system with a acetonitrile concentrations varying from 15 to 95% (v/v) in acidic water (pH 3.0) elution. The furanocoumarins such as psoralen, xanthotoxin, bergapten, and 8-hydroxybergapten were detected in 9 month-old Bai Zhi roots, in which the content of 8-hydroxybergapten was 10-fold higher than other coumarins. Nevertheless, stems from 3 and 6 month-old Bai Zhi exhibited the highest inhibitory ability on tyrosinase activity. In the future, coumarins extracted from the stems of Bai Zhi with high inhibitory effect on tyrosinase could be further used in the cosmetic industry

(52(3):218-227)A Study on Capsaicin Contents of Different Pepper Lines and the Manufacturing for Pepper Pastes

本試驗利用本所栽種之多種辣椒品系，進行辣椒素含量之分析及辣味的感官品評以探討其相關性，結果顯示辣椒素含量介於0～1％間，和感官品評出辣味數據二者之間有高度正相關(r=0.93)。辣椒醬之醃漬條件及調味配方方面：所需鹽度14 ％以上，醃漬時間需30天，配方以蒜頭10％、米酒10％、味精3％、糖3％、沙拉油20％為最佳，添加入茴香、花椒者會使評分降低，添加豆豉5％者則會使顏色變暗，口味上較偏向豆瓣醬。The correlations between capsaicinoid content and sensory evaluation, and the effect of different sodium chloride concentration and additive agents on the quality of pepper paste have been studied for providing more choice to the manufacturers and consumers. It was found that the capsaicinoid content and sensory evaluation was highly positively correlated (r=0.93). This indicated that the degree of pungency in pepper could be determined by instrumental analysis. Rancidity didn’t appear on pepper pastes under the condition of 14% sodium chloride or above for 30 days. The best condition for making pepper paste was found to be 10% garlic, 10% rice wine, 3% monosodium glutamate, 3% sugar, and 20% soybean oil. However, anise, chinese prickly ash would lower the sensory rating. Addition of 5% pickled black bean would make the pepper paste darker in color and tasted somewhat like bean paste, but still favorably accepted by consumer panel