博碩士論文 etd-0715111-130900 詳細資訊


論文識別碼 etd-0715111-130900
統計 本論文已被瀏覽 1290 次,被下載 1 次
中文姓名 唐啟元
英文姓名 Chi-yuan Tang
電子信箱 不公開
系所名稱(中) 化學生物系
系所名稱(英) Department of Chemical Biology
學年度 99
學期 2
學位(中) 碩士
學位(英) Master
論文種類 碩士論文
論文語文別 中文
口試日期 2011-06-07
論文名稱(中) 槐花複方超臨界流體分離純化物之生物活性篩選
論文名稱(英) Bioactivities screening of multiple extracts of separation and purification from Sophora japonica.
頁數 157
關鍵字(中)
  • 酪胺酸酶
  • 分離純化
  • 抗氧化活性
  • 超臨界流體
  • 關鍵字(英)
  • Antioxidant
  • Tyrosinase
  • Supercritical fluid
  • Separation and Purification
  • 摘要(中) 超臨界流體可藉由改變溫度及壓力而控制流體之密度及溶解能力,選擇性地分離物質之成分,再以減壓方式即可將溶劑與萃取物分離,適合天然物活性成分萃取。此法所得之萃取物純度高、不需經有機溶劑分離純化、無溶劑殘留及不汙染環境等多項優點。本研究係以超臨界二氧化碳流體 (Supercritical Fluid Carbone
    Dioxide, SFE-CO2),萃取中草藥活性成分,藉由改變超臨界流體之溫度、壓力與修飾劑,與傳統溶劑萃取比較:酪胺酸酶抑制率及抗氧化力,並以 HPLC 分析萃取物之美白及抗氧化成分含量,評估超臨界流體於分離純化有效成分之應用及天然美白與抗氧化劑之開發。
      研究結果顯示,體外活性試驗中,七種中草藥酪胺酸酶抑制力分別為:槐花88.5%>肉桂 75.1%>蒲公英 71.6%>丁香 73. 8% ≒ 荷葉 73.8%>菊花60.6%>麥門冬 49.9%,其中槐花為同濃度抗壞血酸的 93.9%、BHT 的96.8%;清除DPPH之能力為: 丁香 82.3%>肉桂73.9%>荷葉 71.5%>蒲公英67.1%>菊花 64.2%>槐花 63.5%>麥門冬 54.2%,其中丁香在 1mg/mL 濃度下為同濃度抗壞血酸的 94.1%,BHT的96.8%;螯合鐵離子方面:菊花 74.1%>槐花 71.2
    %(無修飾劑)>荷葉 64.7% (無修飾劑)>蒲公英63.8%>麥門冬57.1%>丁香 38.7%>肉桂 37.9%;而還原能力則為:丁香 2.77>肉桂 1.67>菊花 1.03>荷葉 1.20>麥門冬 1.01>蒲公英 0.93,其中,丁香與肉桂於1mg/mL 分別為同濃度下抗壞血酸的 91.4%與 84.3% ; 另分析萃取溫度與壓力對活性成份之影響,實驗發現其中溫度50℃、壓力3000psi,為最適化萃取條件,而添2mL乙醇為
    修飾劑,有助於萃取率之提升,最高可提升至 27.5 倍。另配置四種不同比例之槐花複方萃取液 , 於濃度1mg/mL下酪胺酸酶抑制率89.32% 、IC 為0.4(mg/mL),抗氧化能力方面:自由基清除率84.55%、其 IC50為0.48(mg/mL)、螯合鐵離子能力為75.11%、EC50為0.57(mg/mL)、還原力方面為同濃度抗壞血酸的76.5%、EC50
    則為0.73(mg/mL) ; 體外活性實驗顯示七種中草藥皆較溶劑萃取有高的活性表現 ;其中以槐花複方具有較單方高的酪胺酸酶抑制力及抗氧化活性。比較七種中草藥之抗氧化成份含量:總酚含量以丁香853 (μg/g)居多,總類黃酮含量測定為槐花195(μg/g)較多 、 總花青素含量測定以荷葉14.7(μg/g)較高 、總醣含量測定以麥門冬 82.08(ug/g)最高。以 HPLC 分析美白成分中抗壞血酸及熊果素中:槐花 108(μg/mL)具有最高之抗壞血酸含量,而丁香 87(μg/mL)
    具有較高之熊果素含量;由HPLC 分析得知以槐花為主之複方萃取液,具有較單方高的抗壞血酸及熊果素含量,分別為:115.02(μg/g)、86.12(86.12μg/g) 。觀察含複方萃取液之乳液檢品,70日內檢品之 pH值、黏度、一般物性、及應力測試,結果顯示乳液樣品皆不隨時間而改變,呈現穩定狀態。
      綜合上述結果,比較不同萃取方法,超臨界流體萃取有高的活性成分萃取率,具有較傳統溶劑萃取高的活性表現, SFE 性質穩定,不會污染萃取物,且純度高,適合於化妝品有效成分之開發與應用。
    摘要(英) Supercritical fluid can dissociate contents selective- ly by controlling the temperature and pressure, and be moved by decreasing the pressure in the final step. It
    can be wellused in extraction of natural product, according to high concentration of extracts,non-organic-solvent purifying, no solvent remained and no environmental pollution.In this study, the supercritical fluid extracting (SFE) method is used to extract active
    components of Chinese herb medicine, and the tyrosinase inhibite and the antioxida-ting capacity of the extract will be examined. The high-performance liquid chromate-
    graphy (HPLC) will be introduced to analyze components of whitening and antioxidating. The experiment of test avtivities in vitro shows that the ability of anti- tyrosinase in seven Chinese herbal medicines were :Sophora Japonica 88.5%>Cinnamomum cassia 75.1% > Taraxacum mongolicum 71.6% > Eugeniacaryopyhllata 73. 8% ≒Nelumbo- nucifera 73.8%>Chrysanthemum morifoliu 60.6% > Ophiopogon japonicus 49.9% besides, the inhibition of Sophora japonica is 93.86%、96.76% to ascorbic acid and BHT. The ability for scavenging DPPH free radical show that: Eugenia caryopyhllata 82.3%>Cinnamomum cassia 73.8%>Nelumbo nucifera 71.5% > Taraxacum mongolicum 67.1% > Chrysanthemum morifolium 64.2%>Sophora japonica 63.5%>Ophiopogon japonicus 54.2%, the Eugenia caryopyhllata show higest scavenging ability at concentration 1mg/mL is 94.1%、96.8% to ascorbic acid and BHT, while for the capacity of chelating ferrous iron were: Sophora Japonica 71.2 % ( without modifier ) > Taraxacum
    mongolicum63.8%>Eugenia caryopyhllata38.7%>Cinnamomum cassia37.9% and the Chrysanthemum morifolium is 75.6% to EDTA. And the reducing power show that
    Eugenia caryopyhllata 2.77 > Cinnamomum cassia 1.67 > Chrysanthemum morifolium 1.03>Nelumbo nucifera 1.20>Ophiopogon japonicus 1.01>Taraxacum mongolicum 0.933, in the concentration of 1mg/mL Eugenia caryopyhllata 、
    Cinnamomum cassia show reducing power 91.4%、84.3% to ascorbic acid respectively. Analysis of the effect of extraction temperature and pressure on activity
    ingredients shows that the optimal condition of extraction is under 50℃、3000psi, If added 2ml ethanol to be the modifier, it would improve the extraction to 27 times at most. Analysis of multiple extracts of Sophora japonica shows the ability of inhibit tyrosinase under the concentration of 1mg/ml is 89.32%、IC50: 0.4(mg/mL). About the capacity of antioxidant: the ability of scavenging is 84.55%、IC50 : 0.48(mg/mL), while the chelating ability is 75.11%、EC50 : 0.57(mg/mL) and the reducing power was 76.5% to ascorbic acid、EC50 : 0.73 (mg/mL). In vitro experiments showed the multiple extracts from Sophora japonica show higher abilities of anti-tyrosinase and antioxidant activities than the single Chinese herbal medicine. Contrast the content of antioxidant to seven Chinese herbal medicine shows that about total polyphenols of Eugenia caryopyhllata 853(μg/g) is highest, total content of flavonoids, Sophora
    japonica 195(μg/g) is the highest, while the anthocyanidins was 0.651 (μmole/g) from Taraxacum mongolicum, and total content of sugar, Ophiopogon japonica 82.08(ug/g)is the highest. Analyzing whitening and antioxidant components by HPLC
    shows that Sophora japonica 107(μg/mL) possess the highest content of ascorbic acid and Eugenia caryopyhllata 86.79 (μg/mL) possess more content of eugenol than the single Chinese herbal medicines. Observing the pH value 、viscosity 、general
    physical properties and the stress test of the cream examines in 70 days which including the multiple extraction shows that the samples were not change by the time and is in stable status. Comprehensive mentioned above, supercritical fluid extraction of active ingredients has higher extraction yield and activities ability than traditional solvent extraction, the extracts of stable. Supercritical fluid extraction was suit to extract the active ingredient to develop natural product for cosmetics.
    論文目次 謝誌……………………………………………………………………I
    中文摘要………………………………………………………………II
    英文摘要………………………………………………………………V
    目錄……………………………………………………………………VI
    表目錄…………………………………………………………………IX
    圖目錄…………………………………………………………………XI
    第一章、緒論……………………………………………………………1
    1-1.研究動機……………………………………………………………1
    1-2.研究目的……………………………………………………………1
    第二章、文獻回顧………………………………………………………2
    2-1.超臨界流體簡介……………………………………………………2
    2-1-1.超臨界流體簡介…………………………………………………2
    2-1-2.超臨界流體之性質………………………………………………3
    2-1-3.超臨界流體萃取機制……………………………………………3
    2-1-4.超臨界二氧化碳流體……………………………………………3
    2-1-5.改變超臨界二氧化碳之物理參數………………………………4
    2-1-6.超臨界二氧化碳流體萃取中草藥………………………………5
    2-2.中草藥簡介…………………………………………………………7
    2-3.黑色素生成與酪胺酸酶抑制……………………………………10
    2-3-1.酪胺酸酶與黑色素生成………………………………………10
    2-3-2.抑制黑色素生成機制…………………………………………12
    2-4.自由基簡介………………………………………………………14
    2-4-1.自由基與活性氧………………………………………………14
    2-4-2.自由基的種類…………………………………………………14
    2-4-3.自由基、活性氧與疾病的關連………………………………17
    2-4-4.自由基與皮膚老化……………………………………………21
    2-5.天然抗氧化劑……………………………………………………24
    2-5-1.抗氧化劑及其作用機制………………………………………24
    2-5-2.天然抗氧化物質………………………………………………26
    第三章、實驗材料與方法……………………………………………35
    3-1.實驗藥材與樣品…………………………………………………35
    3-1-1.實驗驗器………………………………………………………36
    3-1-2.實驗藥材………………………………………………………36
    3-1-3.實驗藥品………………………………………………………36
    3-1-4.標準品…………………………………………………………37
    3-1-5.溶液配製………………………………………………………39
    3-2.實驗設計及流程…………………………………………………40
    3-2-1.實驗設計………………………………………………………40
    3-2-2.實驗流程圖……………………………………………………41
    3-3.實驗步驟…………………………………………………………42
    3-3-1.藥材篩選與樣品製備…………………………………………42
    3-3-2.超臨界二氧化碳萃取…………………………………………43
    3-4.體外活性實驗……………………………………………………44
    3-4-1.酪胺酸酶活性抑制實驗………………………………………44
    3-4-2.抗氧活性實驗…………………………………………………45
    3-4-3.紫外光吸收實驗………………………………………………46
    3-5.成分含量測定……………………………………………………47
    3-5-1.總酚含量測定…………………………………………………47
    3-5-2.總類黃酮含量測定……………………………………………47
    3-5-3.總花青素含量測定……………………………………………47
    3-5-4.總醣含量測定…………………………………………………48
    3-5-5.中草藥指標成分分析…………………………………………48
    3-5-6.美白成分含量分析……………………………………………49
    3-6.中草藥之乳霜製作………………………………………………50
    3-6-1.乳液製作………………………………………………………50
    3-7.產品安全性實驗…………………………………………………51
    3-7-1.貼布測試法……………………………………………………51
    3-8.產品安定性實驗…………………………………………………51
    3-8-1.高溫恆溫試驗…………………………………………………51
    3-8-2.運動狀態試驗…………………………………………………51
    3-8-3.超音波震盪實驗………………………………………………51
    3-8-4.膏霜儲藏 PH 值變化…………………………………………51
    3-9.產品有效性實驗…………………………………………………51
    3-9-1.人體臨床實驗皮膚保水力測試………………………………51
    3-9-2.人體臨床實驗皮膚之彈性效果………………………………51
    3-9-3.人體臨床實驗皮膚之美白效果………………………………52
    第四章、結果與討論…………………………………………………53
    4-1.體外活性試驗結果………………………………………………53
    4-1-1.酪胺酸酶抑制力試驗…………………………………………53
    4-1-2.清除DPPH自由基試驗…………………………………………53
    4-1-3.螯合鐵離子試驗………………………………………………54
    4-1-4.還原力試驗……………………………………………………55
    4-1-5.紫外光吸收實驗………………………………………………55
    4-2.萃取溫度、壓力與修飾劑對活性之影響………………………56
    4-2-1.萃取溫度對活性之影響………………………………………56
    4-2-2.萃取壓力對活性之影響………………………………………57
    4-2-3.修飾劑對活性之影響…………………………………………57
    4-3.成分含量分析……………………………………………………58
    4-3-1.抗氧化成分含量測定…………………………………………58
    4-3-2.美白成分含量測定……………………………………………59
    4-3-3.指標成分含量測定……………………………………………60
    4-3-4.複方萃取液IC50/EC50 評估…………………………………60
    4-4.含中草藥萃取物之乳霜品質分析………………………………61
    4-4-1.乳霜品質安定性試驗…………………………………………62
    4-4-2.乳霜品質有效性試驗…………………………………………63
    第五章、討論…………………………………………………………65
    參考文獻………………………………………………………………66
    表目錄
    頁次
    表一、常見超臨界流體及其臨界參數………………………………6
    表二、與自由基相關的臨床疾病……………………………………19
    表三、類黃酮種類與食物來源及總抗氧化力之比較………………33
    表四、超臨界流體之萃取變因與實驗中之變因……………………43
    表五、超臨界流體萃取流程表………………………………………140
    表六、含 SFE 複方萃取物美白抗皺霜(o/w 型)之成份配方……52
    表七、皮膚反應性等級………………………………………………52
    表八、皮膚刺激性等級………………………………………………53
    表九、傳統溶劑萃取之體外活性試驗數據表………………………82
    表十、超臨界流體萃取七種中草藥未添加修飾劑條件下之酪胺酸酶
     抑制率…………………………………………………………………83
    表十一、超臨界流體萃取七種中草藥添加(2mL Ethenol)修飾劑條件
     下之酪胺酸酶抑率致…………………………………………………84
    表十二、超臨界流體萃取七種中草藥未添加修飾劑條件下之 DPPH
     自由基清除率……………………………………………………………85
    表十三、超臨界流體萃取七種中草藥於添加修飾劑(2mL Ethenol)條件
     下之DPPH自由基清除率 …………………………………………86
    表十四、超臨界流體萃取七種中草藥未添加修飾劑條件下之螯和鐵
     離子 …………………………………………………………………87
    表十五、超臨界流體萃取七種中草藥於添加修飾劑(2mL Ethenol)條
     件下之螯和鐵離子能力 ……………………………………………88
    表十六、超臨界流體萃取七種中草藥未添加修飾劑條件下之還原力
     ……………………………………………………………………89
    表十七、超臨界流體萃取七種中草藥於添加修飾劑(2mL Ethenol)
     條件下之還原力…………………………………………………90
    表十八、七種中草藥之抗氧化成分含量分析 ……………………91
    表十九、七種中草藥之總醣含量分析 ……………………………92
    表二十、 七種中草藥之美白成分含量分析………………………93
    表二十一、槐花、丁香、蒲公英之指標成分與含量分析 ………94
    表二十二、複方中草藥之超臨界流體萃取物抑制酪胺酸酶之IC50
     ………………………………………………………………95
    表二十三、複方中草藥之超臨界流體萃取物清除 DPPH自由基之
     IC50 …………………………………………………………………96
    表二十四、複方中草藥之超臨界流體萃取物螯合亞鐵離子 EC50
     …………………………………………………………………………97
    表二十五、複方中草藥之超臨界流體萃取物還原力之 IC50……98
    表二十六、乳霜檢品於 4℃、70 日內一般物性檢測……………99
    表二十七、乳霜檢品室溫、70 日內一般物性測試………………100
    表二十八、乳霜檢品於 37℃、70日內一般物性測試……………101
    表二十九 、乳霜檢品於4℃下70日內之應力測試 ………………102
    表三十、 乳霜檢品於室溫下 70 日內之應力測試………………103
    表三十一 、乳霜檢品於37℃下70日內之應力測試 ………………104
    圖目錄
    圖一、二氧化碳相圖……………………………………………………2
    圖二、由氣液相至超臨界之相變化圖…………………………………3
    圖三、酪胺酸酶之化學結構式 ………………………………………10
    圖四、麥拉寧之生合成路徑 …………………………………………11
    圖五、酪胺酸酶合成及抑制圖 ………………………………………12
    圖六、常見之酪胺酸酶抑制劑之化學結構 …………………………13
    圖七、ROS 與癌症的相關性 …………………………………………20
    圖八、氧化應力與帕金森氏症之影響可能作用位置及機制 ………20
    圖九、UV 刺激導致光老化之途徑……………………………………23
    圖十、自由基清除作用機制 …………………………………………25
    圖十一、酚類化合物之化學結構 ……………………………………27
    圖十二、類黃酮基本結構…………………………………………… 28
    圖十三、黃酮類化合物之化學結構………………………………… 32
    圖十四、清除自由基之類黃酮結構………………………………… 34
    圖十五、單方萃取液未添加修飾劑之UV光譜圖,於280nm-310nm
     區間有較大吸收值樣品濃度為 2μM/mL…………………………… 105
    圖十六、單方萃取液添加修飾劑之UV光譜圖,於260-280nm
     與310nm 區間有較大吸收值,樣品濃度為2μM/mL…………………105
    圖十七、複方萃取液之 UV光譜圖,於260-280nm 與350-360nm
     區間有較大吸收值,樣品濃度為2μM/mL;A10:Rutin 標準品……106
    圖十八、七種中草藥於定溫下:(a)40℃(b)50℃(c)60℃,
     改變萃取壓力(2000、3000、4000 psi),對酪胺酸酶抑制率之影響
     …………………………………………………………………………107
    圖十九、七種中草藥於定溫下:(a)40℃(b)50℃(c)60℃,
     改變萃取壓力(2000、3000、4000 psi),對清除 DPPH自由基
     之影響…………………………………………………………………108
    圖二十、七種中草藥於定溫下:(a)40℃(b)50℃(c)60℃,
     改變萃取壓力(2000、3000、4000 psi),對螯和鐵離子之影響……109
    圖二十一、七種中草藥於定溫下:(a)40℃(b)50℃(c)60℃,
     改變萃取壓力(2000、3000、4000 psi),對還原力之影響…………110
    圖二十二、七種中草藥於定壓下(a)2000psi(b)3000psi(c)4000psi,
     改變萃取溫度(40、50、60 ℃ ),對酪胺酸酶抑制率之影響………111
    圖二十三、七種中草藥於定壓下(a)2000psi(b)3000psi(c)4000psi,
     改變萃取溫度(40、50、60 ℃ ),對清除DPPH自由基之影響……112
    圖二十四、七種中草藥於定溫下(a)2000 psi(b)3000 psi(c)4000 psi,
     改變萃取溫度(40、50、60),對螯和鐵離子之影響………………113
    圖二十五、七種中草藥於定溫下(a)2000psi(b)3000psi(c)4000psi,
     改變萃取溫度(40、50、60℃),對還原力之影響………………114
    圖二十六、比較槐花於不同萃取條件下,修飾劑的添加對萃
     取率的影響…………………………………………………………115
    圖二十七、菊花於不同萃取條件下,修飾劑的添加對萃
     取率的影響…………………………………………………………115
    圖二十八、丁香於不同萃取條件下,修飾劑的添加對萃
     取率的影響…………………………………………………………116
    圖二十九、肉桂於不同萃取條件下,修飾劑的添加對萃
     取率的影響…………………………………………………………116
    圖三十 、肉桂於不同萃取條件下,修飾劑的添加對萃
     取率的影響…………………………………………………………117
    圖三十一、荷葉於不同萃取條件下,修飾劑的添加對萃
     取率的影響…………………………………………………………117
    圖三十二、蒲公英於不同萃取條件下,修飾劑的添加對萃
     取率的影響…………………………………………………………118
    圖三十三、HPLC 分析美白成分之圖譜. (a) 熊果素標準品(b)
     抗壞血酸標準……………………………………………………119
    圖三十四、HPLC 分析槐花抗壞血酸與熊果素含量之圖譜………120
    圖三十五、HPLC 分析菊花抗壞血酸與熊果素含量之圖譜………120
    圖三十六、HPLC 分析丁香抗壞血酸與熊果素含量之圖譜………121
    圖三十七、HPLC 分析肉桂抗壞血酸與熊果素含量之圖譜………121
    圖三十八、HPLC 分析麥門冬抗壞血酸與熊果素含量之圖譜………122
    圖三十九、HPLC 分析荷葉抗壞血酸與熊果素含量之圖譜………122
    圖四十、 HPLC 分析蒲公英抗壞血酸與熊果素含量之圖譜………123
    圖四十一、HPLC 分析美白成分之圖譜(a)熊果素標準品(b)
     抗壞血酸標準品………………………………………………… 124
    圖四十二、HPLC 分析複方萃取液美白、抗氧化成分之圖譜
     (a)複方 1(b)複方 2 ……………………………………………… 125
    圖四十三、HPLC 分析複方萃取液美白、抗氧化成分之圖譜
     (a) 複方3 (b)複方4 ……………………………………………… 126
    圖四十四、丁香指標成分分析之 HPLC 圖譜(a) Eugenol 標準品
     (b)丁香萃取物……………………………………………………… 127
    圖四十五、槐花指標成分分析之 HPLC 圖譜(a) Rutin 標準品
     (b)槐花萃取物……………………………………………………… 128
    圖四十六、蒲公英指標成分槲皮素分析之 HPLC 圖譜.
     (a) 槲皮素標準品(b)蒲公英萃取物……………………………… 129
    圖四十七、複方中草藥超臨界流體萃取液之抑制酪胺酸酶力測定130
    圖四十八、複方中草藥超臨界流體萃取液之清除DPPH自由基測定131
    圖四十九、複方中草藥超臨界流體萃取液之螯和亞鐵離子測定…132
    圖五十、 複方中草藥超臨界流體萃取液之還原力力測定……… 133
    圖五十一、乳霜檢品於 4℃下 70 日內酸鹼值變化曲線圖………134
    圖五十二、乳霜檢品於室溫下 70 日內酸鹼值變化曲線圖………134
    圖五十三、乳霜檢品於37℃下70日內酸鹼值變化曲線圖…………135
    圖五十四、乳霜檢品於 4℃下 70 日內黏度變化曲線圖…………135
    圖五十五、乳霜檢品於 37℃下 70 日內黏度變化曲線圖……… 136
    圖五十六、乳霜檢品於 37℃下 70 日內黏度變化曲線圖……… 136
    圖五十七、乳霜對皮膚彈性效能隨時間變化之情形………………137
    圖五十八、乳霜對皮膚美白效能隨時間變化之情形………………138
    圖五十九、乳霜對皮膚保濕效能隨時間變化之情形………………139
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    口試委員
  • 陳榮輝教授 - 主席
  • 李佳穎教授 - 委員
  • 黃子瑜教授 - 指導教授
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