臺灣博碩士論文加值系統

馬藍為中國傳統的藥用作物，也常被用於染料上的利用，其中的主要染料成分為indigo與indirubin。本研究將探討在不同栽培條件下可提升馬藍中indigo的生合成，以及不同製藍條件下藍靛的生成效率。試驗結果發現植株內indigo的含量在光強度60 μmol·m-2·s-1白光下較高，約為211 μg·g-1 FW，indirubin的含量則隨光強度越強時其含量越高。而馬藍植株內indigo的含量在藍光照射下有著最高的量，約為84.37 μg·g-1 FW，indirubin則是在白光照射下有著最高的量，約為1.60 μg·g-1 FW。光週期的結果顯示，植株在光照：黑暗＝6 h：18 h的條件下其indigo含量最高，indirubin則是在光照：黑暗＝12 h：12 h下的含量最高。在施肥的試驗中呈現植株在沒有施肥與每週施用濃度1 g / L 葉綠精的處理下，其indigo的含量較高約為310 μg·g-1 FW，而施肥量的不同會造成indigo/indirubin比例上的改變，進而導致製出藍靛的顏色上產生差異。將植株在不同溫度下栽培，可以發現在23℃的條件下生長條件下植株ingido的含量最高。另外，在製藍的試驗中發現打藍處理對於indigo形成有極顯著的影響，在45℃以上、pH 8-10、打藍10-15分鐘產生的藍靛的indigo與indirubin含量較多。而浸泡相關試驗中發現於pH值3與11浸泡時indigo與indirubin有著最高的生成量，以20℃下浸泡兩天所製作出的藍靛中indigo的量與藍靛純度百分比（indigo重/藍靛總重）最高。而indirubin的量在20℃或50℃下浸泡兩天時較高，但隨著浸泡時間越長其量也逐漸越低。

Strobilanthes cusia, a traditional Chinese medicinal crop, was widely used as dyeing material owing to containing the main dye compounds - indigo and indirubin. In this study, the effects of different cultivation and manufacturing conditions on elevation of indigo biosynthesis in S. cusia and production rate of indigoids will be investigated. Experimental results show that the indigo content in S. cusia is higher at light intensity (60 μmol·m-2·s-1), circa 211 μg·g-1 FW. The content of indirubin in S. cusia increases following with the treatment under higher light intensity. The content of indigo in S. cusia is highest (~84.37 μg·g-1 FW) treated in blue light, whereas the highest indirubin content (~1.60 μg·g-1 FW) obtained in white light condition. It reveals that indigo content in S. cusia is highest under the photoperiod of light : dark = 6 h：18 h, whereas the highest indirubin content obtained under light : dark = 12 h：12 h. Fertilizer experiments have shown that the higher indigo content (~310 μg·g-1 FW) of S. cusia was obtained without additional fertilizer application or treated with Hakaphos® blue 1 g / L once per week. The levels of fertilizer will change indigo/indirubin ratio in S. cusia and lead to color variation of indigoids. Experimental materials have highest indigo content cultivated at 23℃ among different temperature treatments. In addition, it exhibits that the effect of aero-oxidation during indigoids manufacture on indigo production is significant. Under the condition at > 45 ℃, pH 8-10 and aero-oxidation for 10-15 mins the content of indigo and indirubin of indigoids will be produced more. Soaking experiments have shown that the highest amount of indigo and indirubin will be synthesized at pH 3.0 and pH 11.0. Indigo weight and purity percentage of indigoids (indigo weight / total indigoids) was obtained maximally at 20℃ and soaking for 2 days. The amount of indirubin was higher at 20℃ or 50℃ and soaking for 2 days. But it will reduce gradually in propotion to the soaking time.

致謝 i
中文摘要 ii
Abstract iv
目次 vi
中文表目次 x
英文表目次 xi
中文圖目次 xii
英文圖目次 xv
壹、前言 1
貳、前人研究 3
一、藍染作物馬藍之介紹 3
二、馬藍活性成分特性 4
三、藍染作物之代謝途徑 7
四、影響indigo生合成的非生物性因子 9
五、傳統藍靛製作方法 10
六、藍靛發酵方法 12
七、影響製藍的因子 14
參、材料與方法 15
試驗植物材料 15
一、週年性調查試驗 16
二、施肥量處理試驗 16
三、光強度處理試驗 17
四、光週期處理試驗 17
五、光質處理試驗 17
六、溫度處理試驗 17
七、栽培條件最佳化試驗 18
建藍條件試驗 19
一、製藍步驟影響性試驗 19
二、浸泡時間與溫度對藍靛純度複因子試驗 20
三、浸泡pH值對藍靛純度之影響 20
四、打藍pH值對藍靛純度試驗 21
五、浸泡複因子試驗 21
六、打藍複因子試驗 21
馬藍樣品中靛藍（indigo）與靛玉紅（indirubin）萃取: 22
高效液相層析儀（HPLC）分析條件: 22
標準品檢量線製作 23
統計分析方法 23
肆、結果 24
栽培條件試驗 24
一、週年性調查試驗結果 24
二、施肥量對馬藍活性成分含量之影響 30
三、光強度對馬藍活性成分含量之影響 33
四、光週期對馬藍活性成分含量之影響 35
五、光質對馬藍活性成分含量之影響 37
六、溫度對馬藍活性成分含量之影響 39
七、 栽培條件最佳化試驗 41
建藍條件試驗 43
一、製藍步驟對藍靛製作之重要性 43
二、浸泡時間與溫度對藍靛純度之影響 44
三、浸泡pH值對藍靛純度之影響 48
四、打藍pH值對藍靛純度之影響 50
五、浸泡複因子試驗 52
六、打藍複因子試驗 56
伍、討論 60
陸、參考文獻 64
柒、附錄 67


中文表目次
表1、 打藍前與打藍後在馬藍葉片浸泡液中藍靛的indigo與indirubin之含量與純度 43
表2、 馬藍（Strobilanthes cusia）葉片於不同溫度與浸泡時間下，所獲得藍靛indigo之變方分析 45
表3、 馬藍（Strobilanthes cusia）葉片於不同溫度與浸泡時間下，所獲得藍靛indirubin之變方分析 45
表4、 馬藍（Strobilanthes cusia）葉片於不同pH值、溫度與浸泡時間的參數處理下，所獲得藍靛中indigo之變方分析 54
表5、 馬藍（Strobilanthes cusia）葉片於不同pH值、溫度與浸泡時間的參數處理下，所獲得藍靛中indirubin之變方分析 54
表6、 馬藍（Strobilanthes cusia）葉片於不同pH值、溫度與打藍時間的參數處理下，所獲得藍靛中indigo之變方分析 58
表7、 馬藍（Strobilanthes cusia）葉片於不同pH值、溫度與打藍時間的參數處理下，所獲得藍靛中indirubin之變方分析 58

英文表目次
Table 1. The content and purity of indigo and indirubin in indigoids of leaves in S. cusia before and after aero-oxidation 43
Table 2. Analysis of Variance (ANOVA) for indigo contents of indigoids manufactured from leaves of S. cusia affected by different treatments of soaking temperature and reactoin time parameters. 45
Table 3. Analysis of Variance (ANOVA) for indirubin contents of indigoids manufactured from leaves of S. cusia affected by different treatments of soaking temperature and reactoin time parameters. 45
Table 4. Analysis of Variance (ANOVA) for indigo contents of indigoids manufactured from leaves of S. cusia affected by different treatments of pH value, temperature and soaking time parameters. 54
Table 5. Analysis of Variance (ANOVA) for indirubin contents of indigoids manufactured from leaves of S. cusia affected by different treatments of pH value, temperature and soaking time parameters. 54
Table 6. Analysis of Variance (ANOVA) for indigo contents of indigoids manufactured from leaves of S. cusia affected by different treatments of pH value, temperature and aero-oxidation time parameters. 58
Table 7. Analysis of Variance (ANOVA) for indirubin contents of indigoids manufactured from leaves of S. cusia affected by different treatments of pH value, temperature and aero-oxidation time parameters. 58

中文圖目次
圖1、 靛藍化學結構圖 5
圖2、 Pfleger的靛藍化學合成步驟 5
圖3、 靛玉紅化學結構圖 6
圖4、 菘藍中靛藍及其同分異構物生合成代謝途徑 8
圖5、 馬藍於溫室中栽培60天之幼苗的生長情形種子苗（A）及扦插苗（B） 15
圖6、 中寮鄉2015/5–2015/10之降雨量（mm）與氣溫（℃）資料 26
圖7、 馬藍（Strobilanthes cusia）在田間（2015/5–2015/10）週年性株高變化情形。 26
圖8、 馬藍（Strobilanthes cusia）在田間（2015/5–2015/10）週年性鮮重調查。 27
圖9、 馬藍（Strobilanthes cusia）在田間（2015/5–2015/10）週年性indigo含量調查。 24
圖10、 馬藍（Strobilanthes cusia）在田間（2015/5–2015/10）週年性indirubin含量調查。 28
圖11、 馬藍（Strobilanthes cusia）在溫室（2014/06/19～2015/05/19）週年性株高調查。 28
圖12、 馬藍（Strobilanthes cusia）在溫室（2014/06/19～2015/05/19）週年性鮮重與地上部鮮重調查。 29
圖13、 馬藍（Strobilanthes cusia）在溫室（2014/06/19～2015/05/19）週年性葉部indigoids含量調查。 29
圖14、 田間不同施肥處理對馬藍（Strobilanthes cusia）株高（A）、葉內indigo含量（B）及葉內indirubin含量（C）的影響。 31
圖15、 溫室栽培下不同施肥處理對馬藍（Strobilanthes cusia）株高（A）、鮮重（B）、葉內indigo含量（C）及indirubin含量（D）的影響。 32
圖16、 在生長箱中培養2個月以不同的光強度處理對馬藍（Strobilanthes cusia）株高（A）、鮮重（B）、葉內indigo含量（C）及indirubin含量（D）的影響。 34
圖17、 在生長箱中培養2個月以不同的光週期處理對馬藍（Strobilanthes cusia）株高（A）、鮮重（B）、葉內indigo含量（C）及indirubin含量（D）的影響。 36
圖18、 在生長箱中培養2個月以不同的光質處理對馬藍（Strobilanthes cusia）株高（A）、鮮重（B）、葉內indigo含量（C）及indirubin含量（D）的影響。 38
圖19、 在生長箱中培養2個月以不同的溫度處理對馬藍（Strobilanthes cusia）株高（A）、鮮重（B）、葉內indigo含量（C）及indirubin含量（D）的影響。 40
圖20、 馬藍（Strobilanthes cusia）光強度（a）、施肥量（b）與溫度（c）處理對indigo含量之反應曲線。 41
圖21、 在生長箱中培養2個月以不同栽培條件處理對馬藍（Strobilanthes cusia）株高（A）鮮重（B）葉內indigo含量（C）indirubin含量（D）的影響。 42
圖22、 馬藍（Strobilanthes cusia）葉片在不同溫度及浸泡時間處理下對其藍靛中indigo含量（A）、indirubin含量（B）及純度（C）之影響。 46
圖23、 馬藍（Strobilanthes cusia）葉片在不同溫度及浸泡時間下製作所得藍靛之顏色。不同的處理條件（由左至右）分別為：20℃48hrs、20℃72hrs、20℃96hrs，35℃48hrs、35℃72hrs、35℃96hrs，50℃48hrs、50℃72hrs及50℃96hrs。 47
圖24、 馬藍（Strobilanthes cusia）葉片在不同pH值下浸泡48 hrs的處理，對製作所得藍靛中indigo含量（A）、indirubin含量（B）及純度（C）之影響。 49
圖25、 馬藍（Strobilanthes cusia）葉片浸泡液在不同pH值下打藍處理下，對製作所得藍靛中indigo含量（A）、indirubin含量（B）及純度（C）之影響。 51
圖26、 馬藍（Strobilanthes cusia）葉片浸泡液在不同pH值、溫度與浸泡時間處理下，對製作所得藍靛中indigo含量（A）及indirubin含量（B）之影響。 53
圖27、 馬藍（Strobilanthes cusia）葉片於不同pH值、溫度與浸泡時間參數處理下，所獲得藍靛中indigo（a）及indirubin（b）含量（μg•g-1 FW）之反應曲面等高線圖。 55
圖28、 馬藍（Strobilanthes cusia）葉片浸泡液在不同pH值、溫度與打藍時間處理下，對製作所得藍靛中indigo含量（A）及indirubin含量（B）之影響。 57
圖29、 馬藍（Strobilanthes cusia）葉片於不同pH值、溫度與打藍時間參數處理下，所獲得藍靛中indigo（a）及indirubin（b）含量（μg•g-1 FW）之反應曲面等高線圖。 59
附圖1、 以HPLC分析兩種標準品indigo（a）與indirubin（b）所建立之檢量線 67
附圖2、 HPLC分析之indigo及 indirubin標準品（a）與馬藍樣品（b）的指紋圖譜 68

英文圖目次
Fig. 1 Chemical structure of indigo. 5
Fig. 2 Pfleger''s synthesis of indigo. 5
Fig. 3 Chemical structure of indirubin. 6
Fig. 4 Pigment formation and biosynthetic pathway of its isomers in Isatis tinctoria. 8
Fig. 5 The growth condition of Strobilanthes cusia cultivated in greenhouse. Seed germinating seedling (A) and Cutting seedling (B). 15
Fig. 6 Precipitation（mm）and mean temperatures（℃） in Zhongliao during 2014/5–2015/10. 26
Fig. 7 Annual investigation of plant height of S. cusia in field during 2014/5–2015/10. 26
Fig. 8 Annual investigation of fresh weight of S. cusia in field during 2014/5–2015/10. 27
Fig. 9 Annual investigation of indigo content of S. cusia in field during 2014/5–2015/10. 27
Fig. 10 Annual investigation of indirubin content of S. cusia in field during 2014/5–2015/10. 28
Fig. 11 Annual investigation of plant height of S. cusia in green house during 2014/06/19－2015/05/19. 28
Fig. 12 Annual investigation of fresh weight and shoot fresh weight of S. cusia in green house during 2014/06/19－2015/05/19. 29
Fig. 13 Annual investigation of indigoids content of S. cusia in green house during 2014/06/19－2015/05/19. 29
Fig. 14 Effects of different treatments of fertilizers on plant height (A), the indigo content (B) and the indirubin content (C) of leaves in S. cusia in field.. 31
Fig. 15 Effects of different treatments of fertilizers on plant height (A), fresh weight (B)and the indigo content (C) the indirubin content (D) of leaves in S. cusia cultivated in greenhouse. 32
Fig. 16 Effects of different treatments of light intensities on plant height (A), fresh weight (B)and the indigo content (C) the indirubin content (D) of leaves in S. cusia cultivated in growth chamber for 2 months. 34
Fig. 17 Effects of different treatments of photoperiod on plant height (A), fresh weight (B)and the indigo content (C) the indirubin content (D) of leaves in S. cusia cultivated in growth chamber for 2 months. 36
Fig. 18 Effects of different treatments of light quality on plant height (A), fresh weight (B)and the indigo content (C) the indirubin content (D) of leaves in S. cusia cultivated in growth chamber for 2 months. 38
Fig. 19 Effects of different treatments of temperature on plant height (A), fresh weight (B)and the indigo content (C) he indirubin content (D) of leaves in S. cusia cultivated in growth chamber for 2 months. 40
Fig. 20 Quadratic response of light intensity (a) fertilization (b) temperature (c) on indigo content. 41
Fig. 21 Effects of different cultivation conditions on plant height (A), fresh weight (B), the indigo content (C) and the indirubin content (D) of leaves in S. cusia cultivated in growth chamber for 2 months. 42
Fig. 22 Effects of different treatments of temperature and soaking time on the indigo content (A), the indirubin content (B) and purity (C) of indigoids manufactured from leaves of S. cusia.. 46
Fig. 23 Indigoids color manufactured from leaves of S. cusia under different treatments of temperature and soaking time.Conditions of treatments(from left to right): 20℃48hrs、20℃72hrs、20℃96hrs，35℃48hrs、35℃72hrs、35℃96hrs，50℃48hrs、50℃72hrs and 50℃96hrs, respectively. 47
Fig. 24 Effects of different treatments of pH value and soaking for 48 hrs on the indigo content (A), the indirubin content (B) and purity (C) of indigoids manufactured from leaves of S. cusia. 49
Fig. 25 Effects of different treatments of soaking pH values and aero-oxidation on the indigo content (A), the indirubin content (B) and purity (C) of indigoids manufactured from leaves of S. cusia. 51
Fig. 26 Effects of different treatments of pH value、temperature and soaking time on the indigo content (A) and the indirubin content (B) of indigoids manufactured from leaves of S. cusia. 53
Fig. 27 The contour diagrams of response surface method for indigo (a) and indirubin(b) content of indigoids manufactured from leaves of S. cusia under different parameters of pH value、temperature and soaking time. 55
Fig. 28 Effects of different treatments of soaking pH value、temperature and aero-oxidation n the indigo content (A) and the indirubin content (B) of indigoids manufactured from leaves of S. cusia. 57
Fig. 29 The contour diagrams of response surface method for indigo (a) and indirubin (b) content of indigoids manufactured from leaves of S. cusia under different parameters of pH value、temperature and aero-oxidation time. 59
Supplement Fig. 1 Calibration curves established from 2 standard compounds analyzed by HPLC. indigo（a）and indirubin（b） 67
Supplement Fig. 2. HPLC fingerprints of indigo and indirubin standard（a）and sample of Strobilanthes cusia（b）. 68

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