臺灣博碩士論文加值系統

本研究的目的是在探討萃取自牛至（Origanum vulgare L.）的粗迷迭香酸（Crude rosmarinic acid）的抗氧化特性及應用於抗光老化化妝品的評估。本研究分為三部份：1.探討粗迷迭香酸抗氧化能力評估包含清除DPPH、還原力的測定、清除過氧化氫能力，以求出化妝品的最適濃度。2.細胞培養方法觀察粗迷迭香酸對於人類皮膚纖維母細胞CCD-966SK存活率的影響、與細胞照射UVA後而有保護及修護的能力及促進膠原蛋白合成。3.探討添加於化妝中測量產品的色度及安定性，應用在人體的安全性與有效性，包含保水性、彈性、粗糙度、鱗片度、平滑度、皺紋度的評估。
抗氧化能力評估：不同濃度（0.625、1.25、2.5、5、10 mg/mL）的粗迷迭香酸之抗氧化能力隨著濃度增加而提高。清除DPPH自由基能力，以濃度1.0%清除效果為87.49±1.46%為最佳。至於清除過氧化氫能力，濃度高於0.25%，清除率超過90%。而濃度為0.5%和1%具有最佳的還原能力。
細胞實驗：粗迷迭香酸於濃度3.13-100 μg/ml 不會影響人類皮膚纖維母細胞CCD-966SK的存活率；細胞給予UVA（10 J/cm2）照射後，將近半數細胞受到UVA的傷害而死滅，將不同濃度RA（3.13-50 μg/ml）萃取物加入與細胞一起照射UVA，結果顯示隨著濃度增加，細胞存活率也隨之上升，濃度25 - 50 μg/ml細胞存活率與未照射UVA的控制組存活率無顯著差異，顯示具有保護的作用；細胞受到UVA照射後，給予不同濃度粗迷迭香酸培養24小時，結果顯示濃度12.5-50 μg/ml具有修護受傷CCD-966SK細胞的能力。粗迷迭香酸抗氧化能力可以清除由UVA引起的自由基，保護細胞不受到傷害。隨著濃度（6.25-50 μg/ml）增加，細胞生成膠原蛋白量也隨之上升。
含或不含0.5% 粗迷迭香酸乳霜之色度測定，經過L*a*b*值分析結果發現不含粗迷迭香酸的控制組白度較高，與目測結果相符。乳霜安定性測試當中，經過四個月不同溫度條件下（低溫、室溫、高溫、變溫）儲存，其外觀、顏色、味道、發霉情形皆無明顯的變化；離心機分離及超音波震盪後，再儲存於室溫，超過四個月均無分離的情形，顯示出該產品具有良好的安定性。使用含0.5% 粗迷迭香酸乳霜皮膚pH介於6.01±0.17，且無紅腫的現象發生。進行為期六星期的人體膚質試驗，含0.5% 粗迷迭香酸乳霜對於皮膚的保水力、皮膚彈性值R2和皮膚平滑度指標在實驗六週有顯著的增加，連續塗抹六週後皮膚的粗糙度、鱗片度、皺紋度指標皆有顯著降低。

The objective of the study are to explore the efficacy of an anti- photoaging skincare that containing the crude rosmarinic acid of Origanum vulgare L.. The study includes 1. To evaluate the efficacy of antioxidation of crude rosmarinic acid by observation the scavenging capacity of DPPH radical, reducing power, scavenging effects on hydrogen peroxide, 2. Determining cells viability, photoprotection and restoration, collagen formation of the cultured fibroblast. 3. To prepare and characterize the cream containing 0.5% crude rosmarinic acid. The characteristics of cream studied including storage stability, the safety of the products, water holding capacity, the skin elasticity, skin roughness, skin scaliness, skin smoothnes, and skin wrinkles index.
Scavenging capacity of DPPH radical, reducing power, scavenging effects on hydrogen peroxide increased with increasing the concentration of crude rosmarinic acid. The 1% crude rosmarinic acid had the highest (87.49±1.46%) DPPH scavenging effect. The concentration of crude rosmarinic acid greater than 0.25% showed more than 80 % scavenging effects on hydrogen peroxide. Crude rosmarinic acid concentration ranged between 0.5-1% showed the highest reducing power.
Cell viability results showed crude rosmarinic acid concentration rangad between 3.13-100 μg/ml has no effect on CCD-966SK viability. The optimum concentration of crude rosmarinic acid that showed protect and restore capacities on photo-damaging by UVA was 25-50 μg/ml promoted the cell proliferation significant. Crude rosmarinic acid concentration ranged between 6.25-50 μg/ml enhanced higher collagen formation of CCD-966SK than that of the control and collagen increased with increasing crude rosmarinic acid concentration after growth for 24 hr.
Cream product containing without crude rosmarinic acid was higher in whiteness than that containing 0.5% crude rosmarinic acid. The stabilities of both cream were stable for more than four months storage, at various temperature environments; after sonication, or centrifuging treatment. The pH of cream products did not change significantly. These results showed that the stability of cream products were very good. The safety test resulted in no erythema based on the Daize score test. The skin appearance such as water holding capacity, the skin elasticity (R2), and the skin smoothnes index significantly increased for applying sample containing 0.5% crude rosmarinic acid during six weeks test period. The skin roughness, skin scaliness and skin wrinkles index significantly decreased over time during six weeks test period.

目錄……………………………………………………………………… i
圖目錄…………………………………………………………………… vi
表目錄…………………………………………………………………viii
附圖表目錄………………………………………………………………ix
中文摘要………………………………………………………………… x
英文摘要...……………………………………………………………xii
一、前言……………………………………………………………………1
二、文獻整理………………………………………………………………3
1. 皮膚構造與功能……………………………………………………3
1.1 表皮層…………………………………………………………3
1.2 真皮層…………………………………………………………5
1.3 皮下組織………………………………………………………6
2. 紫外光與皮膚的關係……………………………………………… 6
2.1 陽光中的紫外線………………………………………………6
2.2 短期紫外線對皮膚的影響……………………………………7
2.3 長期紫外線對皮膚的影響……………………………………10
3. 迷迭香酸 ……………………………………………………………14
3.1 迷迭香酸的簡介………………………………………………14
3.2 迷迭香酸生合成途徑…………………………………………15
3.3 迷迭香酸的生理活性…………………………………………15
三、實驗設計………………………………………………………………19
四、實驗材料………………………………………………………………20
1. 藥材…………………………………………………………………20
2. 細胞株………………………………………………………………20
3. 藥品…………………………………………………………………20
3.1 抗氧化能力評估………………………………………………20
3.2 細胞培養藥品…………………………………………………21
3.3 促進膠原蛋白合成……………………………………………21
3.4 粗迷迭香酸乳霜製備…………………………………………21
4. 設備…………………………………………………………………22
五、實驗方法………………………………………………………………23
1. 抗氧化能力測定……………………………………………………23
1.1清除DPPH自由基能力測定……………………………………23
1.2還原力測定……………………………………………………23
1.3清除過氧化氫能力測定………………………………………24
2. 光保護能力測定……………………………………………………24
2.1細胞培養………………………………………………………24
2.1.1人類皮膚纖維母細胞培養………………………………24
2.1.2人類皮膚纖維母細胞繼代培養…………………………26
2.1.3細胞冷凍保存……………………………………………25
2.1.4細胞解凍…………………………………………………26
2.2光保護及修護能力測定…………………………………………26
2.2.1人類纖維母細胞存活能力…………………………………26
2.2.2人類纖維母細胞照射UVA的保護能力……………………27
2.2.3人類纖維母細胞照射UVA後的修護能力…………………27
3.抗老化能力………………………………………………………………28
3.1促進膠原蛋白合成的作用…………………………………………28
4. 含粗迷迭香酸乳霜製備…………………………………………………29
4.1含粗迷迭香酸乳霜配方……………………………………………29
4.2含粗迷迭香酸乳霜製作方法………………………………………30
5. 乳霜品質測定……………………………………………………………30
5.1 乳霜色度測定……………………………………………………30
5.2 乳霜安定性測試…………………………………………………30
5.3 人體皮膚安全性測試……………………………………………32
6. 人體皮膚有效性試驗…………………………………………………32
6.1保水力測定………………………………………………………32
6.2酸鹼值之測定………………………………………………………33
6.3彈性效果之測定…………………………………………………33
6.4粗糙度、鱗片度、平滑度、皺紋度指標之測定………………34
六、統計分析.………………………………………………………………35
七、結果與討論………………………………………………………………36
1. 抗氧化能力測定………………………………………………………36
1.1清除DPPH自由基能力測定………………………………………36
1.2還原力測定………………………………………………………36
1.3清除過氧化氫能力測定…………………………………………37
2. 光保護能力測定………………………………………………………38
2.1人類纖維母細胞存活能力………………………………………38
2.2人類纖維母細胞照射UVA的保護能力…………………………38
2.3人類纖維母細胞照射UVA後的修護能力………………………39
3. 抗老化能力……………………………………………………………40
3.1促進膠原蛋白合成的效果………………………………………40
4. 粗迷迭香酸乳霜製備……………………………………………………41
5. 含粗迷迭香酸乳霜品質測定…………………………………………41
5.1乳霜色度測定……………………………………………………41
5.2乳霜安定性測試……………………………………………………41
5.3人體皮膚安全性測試……………………………………………42
6. 人體皮膚有效性試驗…………………………………………………42
6.1保水力測定………………………………………………………43
6.2酸鹼值之測定……………………………………………………44
6.3彈性效果之測定…………………………………………………44
6.4粗糙度、鱗片度、平滑度、皺紋度指標之測定………………44
八、結論……………………………………………………………………46
參考文獻……………………………………………………………………48
圖表…………………………………………………………………………55
附錄…………………………………………………………………………79

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