臺灣博碩士論文加值系統

本研究乃針對五種臺灣特有青草植物：馬蹄金（Dichondra micrantha）、蠅翼草（Desmodium triflorum）、魚針草（Anisomeles indica）、大飛揚草（Euphorbia hirta）、艾草（Artemisia princeps. var. orientalis），其中艾草分為艾草梗及艾草葉。分別以熱迴流萃取獲得水萃物，再進行基質金屬蛋白酶（matrix metalloproteinases, MMPs）活性抑制分析、玻尿酸酶（hyaluronidase, HAase）活性抑制分析及抑菌能力試驗，藉以評估6種青草植物水萃物之化粧品功能性，並研發具有功效性之複方青草植物化粧品原料。從研究結果得知，複方青草植物水萃物組合A5（馬蹄金、艾草葉）、B4（蠅翼草、艾草葉）及C1（魚針草、大飛揚草），具抑制基質金屬蛋白酶（MMP-2與MMP-9）及玻尿酸酶活性之作用，可保護真皮層細胞外基質（Extracellular matrix, ECM）之膠原蛋白與玻尿酸不被分解，改善皮膚鬆弛、減少皺紋產生等老化徵兆以及具抗過敏作用。另外，根據貼布試驗（Patch test）結果，10位受測者敷貼A5、B4及C1複方乳霜48小時後，均無不良反應，再持續觀察24小時與48小時，其中一位受測者在A5與B4之受測位置出現輕微紅疹之現象。此結果顯示，C1複方乳霜為安全性之化粧品，不會造成皮膚刺激過敏。依據膚質檢測結果，15位受測者分別使用A5、B4、C1之複方乳霜，均勻塗抹全臉，連續使用30天，A5複方乳霜有2位受測者皺紋具有明顯的改善；B4複方乳霜有3位受測者皺紋具有明顯的改善；C1複方乳霜有3位受測者皺紋具有明顯的改善。
根據上述實驗發現，複方青草植物水萃物經體外試驗所得知結果，應用於人體試驗上，確實具有抗皺與抗發炎效果，在未來開發化粧品原料方面，複方青草植物水萃物可取代化學成分，提升化粧品應用於人體更趨安全與天然的準則。

In this study, five species of Taiwan herbal plants, including Dichondra micrantha, Desmodium triflorum, Euphorbia hirta, Anisomeles indica, Artemisia princeps var. orientalis, were extracted and used as cosmetic ingredients. Hara plants were harvested and divided into two parts, stems and leaves. Therefore, six samples of herbal plants were extracted by used the heat reflux extraction and then. The extracts were analyzed by anti-MMPs zymography for anti-aging investigation, anti-hyaluronidase zymography for anti-allergy test, and disc diffusion method for anti-bacterial assay.
According to the experimental results, the effective samples were selected and mixed into three combined extraction solutions (designated as A5, B4, and C1). Research data showed that three multiple herbal extraction combination (A5, B4, and C1) have marked functions of inhibiting both matrix metalloproteinases (MMP-9 or MMP-2) and hyaluronidase to prevent collagen fibers and hyaluronic acids, present in the dermal extracellular matrix, from their degradation. Finally, the best anti-aging and anti-allergy extract combinations were added to the anti-wrinkle creams further used for the safety and efficiency evaluation of cosmetics on the upper arm skin of volunteer subjects.
The patch testing results showed that one of ten volunteer subjects (10%) had a weak positive reaction (a rash), and the other nine volunteer subjects (90%) had negative irritant reactions for 96 h. These results confirmed that three multiple herbal extraction creams (A5, B4, and C1) were safe cosmetics that would not cause skin irritation. On the other hand, 15 volunteer subjects used multiple herbal extraction creams (A5, B4, and C1) and evenly applied to the face for one month. According to the anti-wrinkle efficiency evaluation testing results, the A5, B4, and C1 creams showed 40% (two of five volunteer subjects), 60% (three of five volunteer subjects), and 60% (three of five volunteer subjects) improvements, respectively.
In short, according to the results presented in this study, the multiple herbal extracts exhibited anti-aging and anti-allergy capacities in vitro, as well as these multiple herbal extraction creams with the anti-wrinkle effects were also proven effective in vivo.

中文摘要 I
Abstract III
致謝 V
青草植物編號 XX
第一章 緒論 1
1-1 前言 1
1-2 實驗架構 3
第二章 文獻探討 4
2-1. 青草植物介紹 4
2-1-1. 艾草 4
2-1-2. 馬蹄金 5
2-1-3. 蠅翼草 6
2-1-4. 魚針草 7
2-1-5. 大飛揚草 8
2-2. 皮膚老化之探討 9
2-2-1. 皮膚構造 9
2-2-2. 老化 13
2-3. 膠原蛋白 15
2-4. 基質金屬蛋白酶 17
2-4-1. MMP-2與MMP-9的特性 19
2-5. 玻尿酸 20
2-6. 玻尿酸酶其抗過敏作用的關聯性 22
第三章 材料與方法 23
3-1. 實驗儀器 23
3-2. 實驗藥品 24
3-3. 實驗配製 27
3-3-1. 樣品萃取 27
3-3-2. 細胞培養 27
3-3-3. 細菌培養 28
3-4. 實驗方法 30
3-4-1. 基質金屬蛋白酶活性抑制分析（anti-MMPs assay） 30
3-4-2. 玻尿酸酶活性抑制分析（anti-hyaluronidase assay） 34
3-4-3. 抑菌能力試驗（anti-bacterial assay） 38
3-4-4. 乳霜製作 39
3-4-5. 貼布試驗（Patch test） 40
3-4-6. 膚質檢測 41
3-5. 統計分析 42
第四章 結果與討論 43
4-1. 青草植物水萃物之基質金屬蛋白酶活性抑制分析 43
4-2. 青草植物水萃物之玻尿酸酶活性抑制分析 52
4-3. 青草植物水萃物之抑菌能力抑制分析 61
4-4. 複方青草植物水萃物之基質金屬蛋白酶活性抑制分析 69
4-5. 複方青草植物水萃物之玻尿酸酶活性抑制分析 84
4-6. 複方青草植物水萃物之抑菌能力抑制分析 98
4-7. 乳霜（含複方青草植物水萃物A5、B4、C1）之貼布試驗 102
4-8. 乳霜（含複方青草植物水萃物A5、B4、C1）之確效性評估 113
第五章 結論 132
第六章 參考文獻 135
附件 143

[1]中村敏郎（1992），醫學美容指南，台北市；青春出版社，p.12-17。
[2]水嵨昇（2005），天然草藥治百病，台北縣；暖流出版社，p.124。
[3]朱冠潔（2015），乳酸菌醱酵物應用於化粧品之功能性評估，嘉南藥理大學，台南市；化粧品應用與管理系，p.130。
[4]李玉蓮（2011），膠原蛋白的發展與展望，國立虎尾科技大學，雲林縣；生物科技研究所，p.104。
[5]李岡榮（2012），常見的臺灣青草藥第三冊，台北市；西北國際文化有限公司，p.96-161。
[6]李榮傑（2010），不同製程台灣產艾草當為傷口癒合製劑之研究，大仁科技大學，屏東縣；製藥科技研究所，p.149。
[7]林宜信、郭朝麟（2006），常用藥用植物圖鑑，台北市；行政院衛生署中藥委員會，p.15。
[8]島野紘一（1996），如何認識膠原蛋白，台北市；國際村文庫書店，p.27-28。
[9]徐玟靖（2011），蝦紅素及香茹、苦茶花萃取物之化粧品生物活性探討，嘉南藥理科技大學，台南市；化妝品科技研究所，p.116。
[10]郜嬌嬌、楊樹林（2017），基因工程技術優化透明質酸生產的研究進展，中國生物工程雜誌，37（8），p.72-77。
[11]高木村（2002），臺灣民間藥(2)，台北市；南天書局有限公司，p.85-156。
[12]高木敬次郎、木村正康、秋葉保忠（2007），漢方藥理學，台北市；文光圖書有限公司，p.279。
[13]康劍書、何為民（2009），明膠酶MMP-2和MMP-9及其组織抑制劑與眼科疾病，國際眼科雜誌，p.2154-2157。
[14]張容鵠、白洋、馮建成、溫珍昌（2010），鏈球菌合成透明質酸的研究進展，生物技術通報，4，p.63-68。
[15]張碧倫（2008），以酵素降解樟芝深層醱酵培養之多醣體應用於抗老化化粧品之研究，嘉南藥理科技大學，台南市；化妝品科技研究所，p.101。
[16]張憲昌（2003），臺灣自然觀察圖鑑藥草(二)，台北市；渡假出版社有限公司，p.13-44。
[17]張憲昌（2007），臺灣藥用植物圖鑑，台中市；晨星出版有限司，p.172-341。
[18]陳姿樺（2011），魚針草萃取物對口腔癌FaDu 細胞生長、移行與入侵之抑制效果，義守大學，高雄市；生物技術與化學工程研究所碩士班，p.90。
[19]陳建澍、王婧茜、易喻、龔海平、應國清（2015），透明質酸及其衍生物研究進展。
[20]黃育亭（2011），山苦瓜萃取物抑制黑色素生成及抗光老化效應的評估，國立臺灣師範大學，台北市；人類發展與家庭學系，p.153。
[21]黃冠中、黃世勳、洪心容（2009），彩色藥用植物圖鑑，台中市；文興出版事業有限公司，p.85-127。
[22]黃瑞齡、施慧娟、陳春香、潘孝萱（2011），艾草萃取物之抗 B 型肝炎病毒活性試驗。
[23]楊小鳳、仇佩虹、葉筱琴（2005），馬蹄金中黄酮成分分析，光譜實驗室，22（6），p.1211-1215。
[24]楊文乾（2005），現代神農草藥圖鑑1，台北市；新潮社文化事業有限公司，p.138-139。
[25]楊宜蓁（2015），啤酒花萃取物對人體皮膚細胞抗光老化之評估，國立臺灣大學，台北市；食品科技研究所，p.97。
[26]劉六郎（2014），民間草藥食用圖鑑，台南市；世一文化事業股份有限公司，p.39-170。
[27]蔡新茂、楊佳璋、劉家全、楊采秀（2008），美容醫學，台北縣；新文京開發出版股份有限公司，p.12-169。
[28]鄭元春、吳進錩、李勉民、金紹基、譚啟楨（1994），常見藥草圖說，香港；讀者文摘遠東有限公司，p.194-326。
[29]Agata, Jabłońska Trypuć, Marzena, Matejczyk and Stanisław, Rosochacki. (2016). Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs. Journal of Enzyme Inhibition and Medicinal Chemistry, 31(sup1), pp.177-183.
[30]Basma, Abu Arra, Zakaria, Zuraini, Latha, Lacimanan Yoga and Sasidharan, Sreenivasan. (2011). Antioxidant activity and phytochemical screening of the methanol extracts of Euphorbia hirta L. Asian Pacific Journal of Tropical Medicine, 4(5), pp.386-390.
[31]Belem Gonçalves, Silvia, Tsan, Pascale, Lancelin, Jean Marc, Alves, Tito l.M, Salim, Vera.M and Besson, Françoise. (2006). Interfacial behaviour of bovine testis hyaluronidase. Biochemical Journal, 398(Pt 3), pp.569-576.
[32]Bhosle, Vaibhav. (2013). Anticonvulsant and antioxidant activity of aqueous leaves extract of Desmodium triflorum in mice against pentylenetetrazole and maximal electroshock induced convulsion. Revista Brasileira de Farmacognosia, 23(4), pp.692-698.
[33]Borkakoti, N. (2000). Structural studies of matrix metalloproteinases. Journal of Molecular Medicine-Jmm, 78(5), pp.261-268.
[34]Bralley, Eve, Greenspan, Phillip, Hargrove, James.L and Hartle, Diane.K. (2007). Inhibition of Hyaluronidase Activity by Vitis rotundifolia. (Muscadine) Berry Seeds and Skins. Pharmaceutical Biology, 45(9), pp.667-673.
[35]Chen, Chung Shih, Wang, Mei Lin, Liu, Rosa Huang, Chen, Shih Pin, Lu, Tsong Ming, Tsai, Wei Yu, Huang, Chien Fu, Yang, Chi Chiang and Tzeng, Yew Min. (2016). Anti-fatigue effect of aqueous extract of Anisomeles indica (L) Kuntze in mice. Tropical Journal of Pharmaceutical Research, 15(3), pp.489-495.
[36]Chen, Li Hui, Xue, Jian Feng, Zheng, Zhi Yong, Shuhaidi, Muhammad, Thu, Hnin Ei and Hussain, Zahid. (2018). Hyaluronic acid, an efficient biomacromolecule for treatment of inflammatory skin and joint diseases: A review of recent developments and critical appraisal of preclinical and clinical investigations. International Journal of Biological Macromolecules, 116, pp.572-584.
[37]Choi, Soo Im, Park, So Ra and Heo, Tae Ryeon. (2006). Matrix degradation inhibitory effect of Schisandra fructus on human articular cartilage and chondrocytes. Journal of Ethnopharmacology, 106(2), pp.279-284.
[38]Esser, Philipp.R, Wölfle, Ute, Dürr, Christoph, Loewenich, Friederike.D, Schempp, Christoph.M, Freudenberg, Marina.A, Jakob, Thilo and Martin, Stefan.F. (2012). Contact Sensitizers Induce Skin Inflammation via ROS Production and Hyaluronic Acid Degradation. PLOS ONE, 7(7), p.e41340.
[39]Ghazanfari, Samaneh, Khademhosseini, Ali and Smit, Theodoor.H. (2016). Mechanisms of lamellar collagen formation in connective tissues. Biomaterials, 97, pp.74-84.
[40]Guntenhöner, Markus.W, Pogrel, M.Anthony and Stern, Robert. (1992). A substrate-gel assay for hyaluronidase activity. Matrix, 12(5), pp.388-396.
[41]Hsu, Hsia Fen, Chang, Shih Fan, Chen, Zi Hua, Yuan, Shyng Shiou.F, Tsai, Yu Duan, Wang, Chao Ping, Wang, Shih Wei, Fang, Li Wen and Houng, Jer Yiing. (2012). Cytotoxic effect of Anisomeles indica extract on human pharynx squamous cancer cells. source: Journal of Medicinal Plants Research, 6, pp.5002-5012.
[42]Huang, Linfang, Chen, Shilin and Yang, Meihua. (2012). Euphorbia hirta (Feiyangcao): A review on its ethnopharmacology, phytochemistry and pharmacology. Journal of Medicinal Plants Research, 6(39), pp.5176-5185.
[43]Jung, Hoe Yune, Shin, Jae Cheon, Park, Seon Min, Kim, Na Ri, Kwak, Wonjung and Choi, Bo Hwa. (2014). Pinus densiflora extract protects human skin fibroblasts against UVB-induced photoaging by inhibiting the expression of MMPs and increasing type I procollagen expression. Toxicology Reports, 1, pp.658-666.
[44]Jung, Yu Ra, Hwang, Chul, Ha, Jeong Min, Choi, Dae Kyoung, Sohn, Kyung Cheol, Lee, Young, Seo, Young Joon, Lee, Young Ho, Kim, Chang Deok, Lee, Jeung Hoon and Im, Myung. (2017). Hyaluronic Acid Decreases Lipid Synthesis in Sebaceous Glands. Journal of Investigative Dermatology, 137(6), pp.1215-1222.
[45]Kim, Sangmin, Kim, Yoonkyung, Lee, Youngae, Cho, Kwang Hyun, Kim, Kyu Han and Chung, Jin Ho. (2007). Cholesterol inhibits MMP-9 expression in human epidermal keratinocytes and HaCaT cells. FEBS Letters, 581(20), pp.3869-3874.
[46]Kumar, Sunil, Malhotra, Rashmi and Kumar, Dinesh. (2010). Euphorbia hirta: Its chemistry, traditional and medicinal uses, and pharmacological activities. Pharmacognosy Reviews, 4(7), pp.58-61.
[47]Kupai, K, Szucs, G, Cseh, S, Hajdu, I, Csonka, C, Csont, T and Ferdinandy, P. (2010). Matrix metalloproteinase activity assays: Importance of zymography. Journal of Pharmacological and Toxicological Methods, 61(2), pp.205-209.
[48]Löffek, S, Schilling, O and Franzke, C.W. (2011). Biological role of matrix metalloproteinases: a critical balance. European Respiratory Journal, 38(1), pp.191-208.
[49]Lai, S.C, Ho, Y.L, Huang, S.C, Huang, T.H, Lai, Z.R, Wu, C.R, Lian, K.Y and Chang, Y.S. (2010). Antioxidant and antiproliferative activities of Desmodium triflorum (L.) DC. Am J Chin Med, 38(2), pp.329-342.
[50]Madlener, Marianne, Parks, William.C and Werner, Sabine. (1998). Matrix Metalloproteinases (MMPs) and Their Physiological Inhibitors (TIMPs) Are Differentially Expressed during Excisional Skin Wound Repair. Experimental Cell Research, 242(1), pp.201-210.
[51]Maity, Niladri, Nema, Neelesh.K, Sarkar, Birendra.K and Mukherjee, Pulok.K. (2012). Standardized Clitoria ternatea leaf extract as hyaluronidase, elastase and matrix-metalloproteinase-1 inhibitor. Indian Journal of Pharmacology, 44(5), pp.584-587.
[52]Mamun or Rashid, A.N.M , Mahmud, Shohel, Towfique, Nayeem and Monokesh, Kumer Sen. (2013). A compendium ethnopharmaceutical review on euphorbia hirta L. Cite This Article A compendium ethnopharmaceutical review on Euphorbia hirta (Vol. 22).
[53]Marković Housley, Zora, Miglierini, Giuliana, Soldatova, Lyudmila, Rizkallah, Pierre.J, Müller, Ulrich and Schirmer, Tilman. (2000). Crystal Structure of Hyaluronidase, a Major Allergen of Bee Venom. Structure, 8(10), pp.1025-1035.
[54]Matsuda, Minoru, Hoshino, Tatsuya, Yamakawa, Naoki, Tahara, Kayoko, Adachi, Hiroaki, Sobue, Gen, Maji, Daisuke, Ihn, Hironobu and Mizushima, Tohru. (2013). Suppression of UV-Induced Wrinkle Formation by Induction of HSP70 Expression in Mice. Journal of Investigative Dermatology, 133(4), pp.919-928.
[55]Monfrecola, Giuseppe, Cacciapuoti, Sara, Capasso, Claudia, Delfino, Mario and Fabbrocini, Gabriella. (2016). Tolerability and camouflaging effect of corrective makeup for acne: results of a clinical study of a novel face compact cream. Clinical, Cosmetic and Investigational Dermatology, 9, pp.307-313.
[56]Nagase, Hideaki, Visse, Robert and Murphy, Gillian. (2006). Structure and function of matrix metalloproteinases and TIMPs. Cardiovascular Research, 69(3), pp.562-573.
[57]Naise, M.J and Bhadange, D.G. (2013). In-vitro antibacterial activity of Anisomeles indica (L) O. Ktze Lamiaceae plant from Melghat,(MS) India.
[58]Necas, J, Bartosikova, L, Brauner, P and Kolar, J. (2008). Hyaluronic acid (hyaluronan): a review. Veterinarni medicina, 53(8), pp.397-411.
[59]Ng, Keng Wooi and Lau, Wing Man. (2015). Skin Deep: The Basics of Human Skin Structure and Drug Penetration.
[60]Pandey, Abhay.K and Singh, Pooja. (2017). The Genus Artemisia: A 2012–2017 Literature Review on Chemical Composition, Antimicrobial, Insecticidal and Antioxidant Activities of Essential Oils. Medicines, 4(3), p.68.
[61]Papakonstantinou, Eleni, Roth, Michael and Karakiulakis, George. (2012). Hyaluronic acid: A key molecule in skin aging. Dermato-endocrinology, 4(3), pp.253-258.
[62]Qiu, J, Xu, B, Gong, Q, Pan, W, Liu, C, Huang, Z, Gu, X and Liang, G. (2016). Synthesis and Biological Evaluation of Matijin-Su Derivatives as Potential Antihepatitis B Virus and Anticancer Agents. Chem Biodivers, 13(11), pp.1584-1592.
[63]Rajeh, M.A, Zuraini, Z, Sasidharan, S, Latha, L.Y and Amutha, S. (2010). Assessment of Euphorbia hirta L. leaf, flower, stem and root extracts for their antibacterial and antifungal activity and brine shrimp lethality. Molecules, 15(9), pp.6008-6018.
[64]Rodríguez, David, Morrison, Charlotte.J and Overall, Christopher.M. (2010). Matrix metalloproteinases: What do they not do? New substrates and biological roles identified by murine models and proteomics. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1803(1), pp.39-54.
[65]Shoulders, Matthew.D and Raines, Ronald.T. (2009). COLLAGEN STRUCTURE AND STABILITY. Annual review of biochemistry, 78, pp.929-958.
[66]Stern, Robert and Jedrzejas, Mark.J. (2006). The Hyaluronidases: Their Genomics, Structures, and Mechanisms of Action. Chemical reviews, 106(3), pp.818-839.
[67]Sternlicht, Mark.D and Werb, Zena. (2001). How Matrix Metalloproteinases Regulate Cell Behavior. Annual Review of Cell and Developmental Biology, 17(1), pp.463-516.
[68]Thankachan, Anu.K, Chandran, Meena and Krishnakumar, K. (2017). Pharmacological activity of Desmodium triflorum-a review. Asian Journal of Phytomedicine and Clinical Research.
[69]Vedpal, Dhanabal, S.P, Dhamodaran, P, Chaitnya, M.V.N.L, Duraiswamy, B, Jayaram, Unni and Srivastava, Neha. (2016). Ethnopharmacological and Phytochemical profile of three potent Desmodium species: Desmodium gangeticum (L.) DC, Desmodium triflorum Linn and Desmodium triquetrum Linn (Vol. 2016).
[70]Visse, Robert and Nagase, Hideaki. (2003). Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases. Structure, Function, and Biochemistry, 92(8), pp.827-839.
[71]Xu, Bixue, Wang, Ning, Pan, Weidong, Qiu, Jingying, Cao, Peixue, Zhu, Meifen, Feng, Yibin and Liang, Guangyi. (2014). Synthesis and anti-tumor activity evaluation of Matijin-Su derivatives. Bioorganic Chemistry, 56, pp.34-40.
[72]Xu, Zhenghao and Chang, Le. (2017). Convolvulaceae Identification and Control of Common Weeds: Volume 3 (pp. 99-147). Singapore: Springer Singapore.
[73]Yu, Chia Jung, Ko, Chun Jung, Hsieh, Chang Hsun, Chien, Chiang Ting, Huang, Lien Hung, Lee, Chien Wei and Jiang, Ching Chuan. (2014). Proteomic analysis of osteoarthritic chondrocyte reveals the hyaluronic acid-regulated proteins involved in chondroprotective effect under oxidative stress. Journal of Proteomics, 99, pp.40-53.