臺灣博碩士論文加值系統

地球之臭氧層日漸稀薄，使得陽光中之UV-B透過大氣層的部分逐漸提高，而植物為了減少胞器受到UV-B之傷害，會促使細胞合成更多的活性化合物累積在葉子中。這些化合物中，有些黃酮類化合物除了可吸收UV-B能量外，更可進一步清除UV-B照射所形成之自由基。此機制可使葉子產生更多活性化合物，在植物利用上具有開發之潛力。臺灣重要經濟樹種之一土肉桂（Cinnamomum osmophloeum Kanehira）葉子含有多種Kaempferol類黃酮醣苷，具有抗氧化及降低細胞血糖等作用等多種生物活性，本研究以土肉桂作為試材，測定UV-B照射後葉子化合物含量之變化。
本研究首先將土肉桂照射4 h UV-B後，採收距燈源不同距離之葉子進行萃取。試驗結果顯示，光照距離為30 cm（UV-B能量為4.20 W m-2）之組別，在照射後其總黃酮類化合物有最大增幅（1.21倍），故以30 cm為試驗光照距離。除了照射4 h UV-B外亦測試不同光源之光照順序，其中僅有照射2 h藍光接續2 h UV-B組別之總黃酮類化合物含量有顯著的增加，為照射前的1.18倍，與照射4 h UV-B組別之總黃酮類化合物含量增加結果相近。
為測試單日光照中土肉桂葉子之活性化合物最大改變倍率，因此改變UV-B照射時數為2、4、6及8 h，測得照射4 h及8 h UV-B時會有較高的單日總黃酮類化合物增加倍率，其中照射4 h UV-B結果較佳。此外，也對土肉桂葉子水可溶部中主要的5種黃酮類化合物之改變倍率進行測試，包含Kaempferol-3-O-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranoside（F1）、Kaempferol-3-O-β-D-apiofuranosyl-(1→2)-α-L -arabinofuranosyl-7-O-α-L-rhamnopyranoside（F2）、Kaempferitrin（F3）、Kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinofuranosyl-7-O-α-L -rhamnopyranoside（F4）及Kaempferol-3-O-α-L-rhamnopyranoside（F5）。試驗結果顯示，照射UV-B時數對F1-F5化合物改變倍率並無顯著影響。因此，後續試驗條件仍為照射4 h UV-B。
更進一步將單日照射4 h UV-B之過程重複2、4及8 d，結果顯示，照射4 d UV-B時總黃酮類化合物改變倍率會更進一步增加為1.39倍，但與照射8 d UV-B組別之增加倍率沒有顯著差異。至於總酚類化合物，則是於照射4 d UV-B之組別增加為1.20倍，延長照射時間為8 d UV-B後，總酚類化合物顯著增為1.37倍。水可溶部5種化合物F1-F5照射後之改變倍率也與總黃酮類化合物試驗結果相似。照射4 d UV-B後F1、F2及F3化合物皆顯著增為約1.42倍，但照射4 d UV-B卻與照射8 d組別之增加倍率沒有顯著差異。
綜合所有結果顯示，無論是總黃酮類化合物、總酚類化合物及水可溶部黃酮醣苷之含量，皆顯示距離燈源30 cm時，照射4 h UV-B連續4 d是最有效率的增加化合物含量之照射條件。此條件下，照射後總黃酮類化合物增為1.39倍、總酚類化合物增為1.20倍、化合物F1、F2及F3可增為照射前之1.42倍。

The depletion of ozone in the atmosphere has resulted in an increase in UV-B irradiation. A high UV-B dosage provokes damage to organism. Plants may attenuate the impact of UV-B irradiation through the accumulation of different types of phenolics produced. Most of these compounds are flavonoid, which can act as an effective UV-B screen. Moreover, some flavonoids may also eliminate free radicals which caused by UV-B radiation. This mechanism will make leaves to synthesis more active compounds, which can be used potentially. Cinnamomum osmophloeum is one of the indigenous trees in Taiwan. This tree contains aboundant kaempferol glycoside compounds in its leaves. These compounds were proved to have antioxidant activity and hypoglycemic effect. This study would irradiate C. osmophloeum leaves with UV-B in various conditions to test the variations of activity compound contents in the leaves.
First, we irradiate leaves with 4 h UV-B by different irradiation distances, and the changing ratio of the total flavonoid contents increase to the maximum 1.21 fold when irradiated at 30 cm (UV-B energy: 4.20 W m-2). We decided to use 30 cm as the irradiation distance for all other tests. Besides, we change 4 h UV-B irradiation to different order of different light irradiation. Only the test group of 2 h blue light + 2 h UV-B showed increased total flavonoid content ratio to 1.18 fold which is similar to the result when trees irradiated with UV-B for 4 h.
Furthermore, in order to know the maximum changing ratios of the active compounds per day, we treat C. osmophloeum leaves with 2, 4, 6 and 8 h irradiation. The changing ratios of the total flavonoid contents were better when irradiating with 4 or 8 h UV-B per day, with the best result when irradiating UV-B for 4 h. Furthermore, in investigating the changing ratio from the UV-B irradiation of the 5 major flavonoid compound, kaempferol-3-O-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl -7-O-α-L-rhamnopyranoside (F1), kaempferol-3-O-β-D-apiofuranosyl-(1→2)-α-L -arabinofuranosyl-7-O-α-L-rhamnopyranoside (F2), kaempferitrin (F3), kaempferol-3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinofuranosyl-7-O-α-L -rhamnopyranoside (F4) and kaempferol-3-O-α-L-rhamnopyranoside (F5) in the water soluble fraction of C. osmophloeum leaves, the results showed that there were no significant differences between the changing ratios of compound F1-F5 from the irradiation with different hours of UV-B. As the result, we decided to treat leaves with 4 h UV-B irradiation as the continuous irradiation condition.
When the trees continuously irradiated UV-B for 4 h per day and lasted for 2, 4 and 8 d, the changing ratios of total flavonoid contents would significantly increase to 1.39 fold after irradiated UV-B for 4 d. But the result for irradiation for 4 d has no difference to that of irradiation UV-B for 8 d. The changing ratio of the total phenolic contents is significantly increased to 1.20 fold when irradiating UV-B for 4 d. Besides, when the tree irradiatind UV-B for 8 d, the changing ratio of total phenolic contents has significant increased to 1.37 fold. The 5 compounds, F1-F5, in the water fraction showed similar changing ratios to that of the total flavonoid contents. The changing ratios of compound F1, F2 and F3 would increase to 1.42 fold after irradiated UV-B for 4 d, but the result for irradiation for 4 d has no difference to that of 8 d.
As the result, we have concluded that to increase total flavonoid contents, total phenolic contents and the flavonol glycoside form water fraction, the most effective condition is to irradiate trees with UV-B at a 30 cm diatance for 4 h per day and last for 4 d. The total flavonoid contents would increase to 1.39 fold, the total phenolic contents would increase to 1.20 fold, the compound F1, F2 and F3 would increase to 1.42 fold when irradiating with this condition.

謝誌...............................................................................................................................I
摘要.............................................................................................................................III
Abstract.........................................................................................................................V
目錄............................................................................................................................VII
圖目錄.........................................................................................................................IX
表目錄........................................................................................................................XII
I. 前言......................................................................................................................1
II. 文獻回顧..............................................................................................................2
2.1土肉桂..............................................................................................................2
2.2 UV-B照射對植物體之影響........................................................................... 5
2.2.1 UV-B照射對植物細胞之生理影響........................................................5
2.2.2UV-B照射下植物體內活性化合物之作用.............................................6
2.3 UV-B照射對植物體內活性化合物含量之影響..........................................10
2.3.1單位時間內接收不同能量之UV-B照射之影響................................. 10
2.3.2不同光照順序對植株活性化合物含量之影響.................................... 12
2.3.3不同照射時間之UV-B對植株活性化合物含量之影響.....................14
2.3.4重覆每日照射UV-B對植株之影響.....................................................16
III. 研究目的............................................................................................................21
IV. 材料與方法........................................................................................................22
4.1試驗材料........................................................................................................ 22
4.2 光照環境…………........................................................................................22
4.2.1光照距離測定試驗.................................................................................22
4.2.2光照順序試驗.........................................................................................22
4.2.3不同時數光照試驗.................................................................................23
4.2.4不同天數連續光照試驗.......................................................................23
4.3土肉桂葉子抽出物之萃取........................................................................... 23
4.4水可溶部化合物提取................................................................................... 23
4.5化合物含量分析........................................................................................... 24
4.5.1總酚類化合物含量............................................................................... 24
4.5.2總黃酮類化合物含量........................................................................... 24
4.5.3水可溶部化合物絕對定量……........................................................... 25
4.6統計分析...................................................................................................... 26
V. 結果與討論.......................................................................................................27
5.1單日照射條件對土肉桂葉子活性化合物含量之影響............................... 27
5.1.1光照距離測定....................................................................................... 27
5.1.2光照順序對活性化合物含量之影響................................................... 30
5.2照射時數對活性化合物增加倍率之影響................................................... 33
5.3光照後水可溶部黃酮醣苷化合物含量變化..................................................36
5.3.1水可溶部化合物................................................................................... 36
5.3.2單日光照試驗水可溶部化合物含量變化............................................39
5.4連續光照後化合物含量變化..........................................................................40
5.4.1連續光照對活性化合物改變倍率之影響.............................................40
5.4.2連續光照對水可溶部化合物增加倍率影響及推論.............................43
VI. 結論與建議........................................................................................................46
VII. 參考文獻............................................................................................................48

 
圖目錄
圖1、土肉桂葉子之黃酮類化合物
Figure 1. The flavonoids in leaves of C. osmophloeum................................................4
圖2、UVR8調節UV-B訊號之生理作用
Figure 2. Physiological roles of UVR8-mediated UV-B signaling...............................6
圖3、 野生型阿拉伯芥及對UV敏感之突變株對UV-B照射之感受性
Figure 3. Sensitivity of wild-type and UV-sensitive mutant Arabidopisis to UV-B radiation exposure..............................................................................................7
圖4、（A）野生型阿拉伯芥及（B）對UV敏感突變株分別加入UV（+UV）照射與不加入UV（-UV）照射處理後抽出物之吸收光譜
Figure 4. Absorption spectra of extracts from UV-irradiated (+UV) and non-UV-irradiated (-UV) Arabidopsis from either the (A)wild type and (B) UV-sensitive mutant............................................................................................. 8
圖5、黃酮類化合物在生長及光照壓力下保護作用之示意圖。（A）黃酮類化合物清除H2O2機制之示意圖。（B）液泡之過氧化酶可清除自然擴散之H2O2 （C）含有多量黃酮類合物的表皮細胞可清除葉肉細胞中由光照引起之H2O2
Figure 5. A proposed diagram for protective function of flavonoids during growth and light stress. (A) Scheme of the H2O2-scavenging mechanism by flavonoids. (B) The diffusive nature of H2O2 enables vPX to scavenge it in vacuoles. (C) The photoproduced H2O2 may leak out from mesophyll cells and be scavenged in epidermal cells that have a high flavonoid content............................................................................................................9
圖6、增加UV-B照射能量後Vigna mungo及V. acontifolia之幼苗葉片（a）總黃酮類化合物含量（Total flavonoid contents, TFC）及（b）總酚類化合物含量（Total phenolic contents, TPC）變化
Figure 6. (a) Total flavonoid contents (TFC) (b) Total phenolic contents (TPC) in the leaves of Vigna mungo and Vigna acontifolia seedlings exposed to enhanced UV-B radiation................................................................................................. 11
圖7、兩種基因型之藍莓葉子於不同時間及能量（UV-B 0.07 Wm-2; 0.12 Wm-2 and 0.19 Wm-2）之UV-B照射後總酚類化合物含量（Total phenolic contents, TPC）
Figure 7. Total phenolic contents (TPC) of leaves of two highbush blueberry genotypes exposed for 72 h to different UV-B biological effective doses (UV-B 0.07 Wm-2; 0.12 Wm-2 and 0.19 Wm-2).........................................15
圖8、Bell pepper葉子之於低溫處理（LT）及照射UV-B後總黃酮類化合物含量（mg of quercetin per 100g of fresh weight）
Figure 8. Total flavonoid content in bell pepper leaves exposed to low temperature and UV-B radiation expressed as mg of quercetin per 100g of fresh weight...........................................................................................................18
圖9、Bell pepper葉子之Luteolin-7-O-glucoside及Apigenin-7-O-glucoside含量於低溫處理及照射UV-B後之含量
Figure 9. Concentrations of luteolin-7-O-glucoside (L-7-G) and apigenin-7-O -glucoside (A-7-G) in bell pepper leaves exposed to low temperature and UV-B radiation..........................................................................................19
圖10、阿拉伯芥於未照射UV（空心圓點）及照射UV（實心圓點）之黃酮醣苷含量變化
Figure 10. Temporal flavonol glycoside changes in UV (open circles) and non-UV treated (closed circles) Arabidopsis thaliana............................................20
圖11、UV-B不同光照時數對土肉桂葉子總黃酮類化合物含量（Total flavonoid contents, TFC）之改變倍率
Figure 11. Changing ratios of total flavonoid contents (TFC) of C. osmophloeum leaves from irradiation with different hours of UV-B...........................34
圖12、UV-B不同光照時數對土肉桂葉子總酚類化合物含量（Total phenolic contents, TPC）之改變倍率
Figure12. Changing ratios of total phenolic contents (TPC) of C. osmophloeum leaves from irradiation with different hours of UV-B............................................35
圖13、土肉桂葉子抽出物之水可溶部之HPLC圖譜
Figure 13. HPLC chromatogram of water fraction of C. osmophloeum leaves extraction...................................................................................................37
圖14、土肉桂葉子抽出物之水可溶部化合物F1-F5之結構
Figure 14. Structure of compound F1-F5 from the water fraction of C. osmophloeum leaves extracts............................................................................................38
圖15、UV-B光照不同時數對水可溶部化合物之改變倍率
Figure 15. Changing ratios of the water fraction compounds from irradiation with different hours of UV-B.............................................................................39
圖16、UV-B光照不同天數對土肉桂葉子總黃酮類化合物含量（Total flavonoid contents, TFC）之改變倍率
Figure 16. Changing ratios of total flavonoid contents (TFC) of C. osmophloeum leaves from irradiattion with different days of UV-B................................41
圖17、UV-B光照不同天數對土肉桂葉子總酚類化合物含量（Total phenolic contents, TPC）之改變倍率
Figure 17. Changing ratios of total phenolic contents (TPC) of C. osmophloeum leaves from irradiation with different days of UV-B.................................42
圖18、UV-B光照不同天數對水可溶部化合物之改變倍率
Figure 18. Changing ratios of the water fraction compounds from irradiation with different days of UV-B.............................................................................. 44
表目錄
表1、兩種基因型之藍莓葉子於照射UV-B後其酚類化合物與黃酮類化合物之含量
Table 1. Concentrations of phenolic acids and flavonoids in leaves of two genotypes of highbush blueberry exposed to UV-B........................................................12
表2、連續照射紅光、遠紅光及藍光於照射UV-B前後之比較結果
Table 2. Comparison between the effects of continuous red (R), far-red (FR) or blue (B) given before or after the UV irradiation................................................13
表3、UV-B照射對綠豆其總黃酮類化合物含量（Total flavonoid contents, TFC）及總酚類化合物含量（Total phenolic contents, TPC）變化
Table 3. The effect of UV-B radiation on total phenolic contents (TPC) and total flavonoids contents (TFC) in mung bean sprouts........................................14
表4、不同採收前處理白菜葉片黃酮類化合物含量（mg g-1 dry matter）
Table 4. Contents of flavonoids in leaf blades of different pre-harvest-treated pak choi plants..............................................................................................................17
表5、HPLC溶液
Table 5. HPLC solvent.................................................................................................26
表6、不同距離下UV-B照射對土肉桂葉子中總黃酮類化合物含量影響
Table 6. Change of total flavonoid contents (TFC) of C. osmophloeum leaves from UV-B irradiation with different distances..................................................... 28
表7、不同距離下UV-B照射對土肉桂葉子中總酚類化合物含量影響
Table 7. Change of total phenolic contents (TPC) of C. osmophloeum leaves from UV-B irradiation with different distances..................................................... 29
表8、不同光照順序對土肉桂葉子中總黃酮類化合物含量（Total flavonoid contents, TFC）影響
Table 8. Changes of total flavonoid contents (TFC) of C. osmophloeum from irradiation with different order of lights........................................................ 31
表9、不同光照順序對土肉桂葉子中總酚類化合物含量（Total phenolic contents, TPC）影響
Table 9. Changes of total phenolic contents (TPC) of C. osmophloeum leaves from irradiation with different order of lights........................................................ 32

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