行政院國家科學委員會專題研究計畫 成果報告
開發可應用於化妝品的抗皮膚老化之成份及研究其分子作 用機轉
計畫類別： 個別型計畫
計畫編號： NSC91-2626-B-041-001-
執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 嘉南藥理科技大學藥學系
計畫主持人： 施美份
報告類型： 精簡報告
處理方式： 本計畫可公開查詢
中 華 民 國 92 年 10 月 2 日
計劃名稱：開發可應用於化妝品的抗皮膚老化之成分及研究其分子作用機轉
NSC91-28626-B-041-001
執行期間：自民國 91 年 08 月 01 日起至民國 92 年 07 月 31 日 執行單位：嘉南藥理科技大學 藥學系
計劃主持人：施美份 助理教授
研究計畫之背景及目的
皮膚及眼睛是人類唯一暴露到日光紫外線的部位。日光紫外線的暴露引起的皮膚 細胞的傷害，進而影響到皮膚的緊縮性及彈性，並且誘發皮膚提前老化現象：包 括有皮膚粗糙、皺紋、斑點、皮膚鬆弛、及色澤暗沉等（Gilchrest &Yaar, 1992）。
紫外線的波長可分四段：UVC (<290nm), UVB (290-320nm), UVA2 (320-340nm), and UVA1 (340-400nm)。其中 UVB 所引起的皮膚機能的傷害主要是皮膚老化及 癌症，UVA2 的穿透能力較 UV 強因此也是引起皮膚老化的主要波長之一。陽光 紫外線中的 UVC 及大部分的 UVB 在穿過大氣層時，會被臭氧層所吸收，因此
抵達地球表面的量有限（除在臭氧層有破洞的南半球）。
正常的皮膚是靠 extracellular matrix (ECM)生合成及分解的平衡維持，而 Matrix metalloproteinases (MMPs)則在維護 ECM remodeling 上扮演著重要角色。
Interstitial collagenase (MMP-1) 是一種 Zinc-dependent 蛋 白 質 水 解 ，可分解含 type I 及 III 膠原蛋白(collagens)的細胞外纖維。而 MMP-3 (stromelysin-1)則是負 責分解醣蛋白 proteoglycans、fibronectin 和 type III 膠原(Giambernardi et al, 1998;
Kuroda & Shinkai 1997)，可以與 MMPs 的活性相抗衡的物質則是 tissue inhibitor of metalloproteinase (TIMPs)。膠原蛋白約為真皮乾重的 70﹪之多。其中 type I 膠原 約佔 85﹪，type III 膠原只佔 10﹪。因此任何的因素會造成 MMPs 的活性或 TIMPs 的活性不足皆會引起皮膚的變化，如失去彈性、皺紋的產生(Uitto & Bernstein, 1998)。實驗證實抽煙也會促進皮膚的老化，其發生原因也與紫外線引起的皮膚 改變相似。當皮膚細胞的培養液中添加有煙草的萃取物時，即可發現 MMP-1 及 MMP-3 的產量及其 mRNA 會比沒有添加煙草萃取物的細胞多(Yin et al., 2000)。
但相同的處理卻對 TIMP-1 及 TIMP-3 mRNA 無影響，顯示皮膚的細胞中的 MMPs 及 TIMPs 的活性只要失去了之間的平衡，皮膚老化現象即可被引發出來。
體外試驗發現：皮膚細胞來源若來自於提前老化的細胞其生命期（lifespan）則 比同年齡未有提前老化的細胞的短(Hayflick 1965)，而這種現象則與皮膚細胞來 源事來自於年紀較老的皮膚細胞相似(Gilchrest & Yaar, 1992)。紫外線引起的皮膚 改變，已知是一種氧化性傷害進而去活化及增加細胞內的 PKC 的活性，PKC 的 活性增加的結果便促進 MMPs 生合及造成膠原蛋白的分解，此一現象可藉由 Vitamin E 的抗氧化作用而達到改善(Berneburg et al, 1999)。其他的研究團體則是 證實出單一次的紫外線照射便會增加 MMP 基因的表現，而且這種現象可持續長 達 24 小時（Fisher et al., 1999），但 48-72 小時之後即可恢復至基本值。如果進行 多次的紫外線照射則會引起持續性的增加 MMP 基因的表現（此種增加可長達至
7 天之久）。紫外線照射雖然也同時增加了 TIMPs 的產量，但是增加的比率比
MMPs 的產量少而不足以阻遏 MMPs 的活性。因此，造成長期的紫外線照射易 促進皮膚老化的現象。
綠藻是一種淡水單胞藻,在日本及台灣食用已有多年的歷史。綠藻含有多種營養 成分，如多種胺基酸，礦物質及膳食纖維等(Borowitzka, 1988; Shubert, 1988)。目 前已有許多有關綠藻的生理功能的研究，如改善糖尿病鼠的高血糖情形
( Rodriguez-Lopez M. & Lopez-Quijada 1971; 李宏圖等人 1977)，降低血脂(Sano &
Tanaka, 1987；Okuda et al., 1975；Sano 等人 1988；Yang et al., 2001)提高免疫功 能(Singh et al., 1998; Tanaka et al., 1984; Tanaka et al., 1998; Konishi et al., 1996)，
抑制腫瘤生長 (Singh et al., 1999; Noda et al., 1996)，預防因壓力引起的潰瘍 (Tanak et al., 1997)，促進動物的生長速率(Ishibashi, 1972) ，及促進因 ethionine 誘發的肝傷害的修復速度(Wang et al., 1979)。
運用綠藻的萃取物當作緩和皮膚老化在上市的化妝品已有先例，雖然產品本身標 榜具有此一功效卻無任何相關的研究報告可查證。經我們初步的實驗結果證實綠 藻粗萃取物與 MMP inducers 同時加入培養液中可減少 MMP-1 的產量（請見圖
一）。 因此，我們將利用已知的促皮膚老化的方式，即以 UVA2 照射人的皮膚的
細胞(human skin fibroblasts)或 MMPs 的誘發藥物（如 IL-1β + PDGF-BB 或 PMA）
去促進 MMPs 的生成的方式。再比較以純化後的綠藻萃取物處理後的皮膚的細 胞的 MMPs 產量來當作初步的證實。
研究方法 Materials:
1. Normal skin fibroblast— 966SK (76 years old female skin), 1059SK (20 years old female skin), 1090SK (46 years old female skin)
2. MMP-1 ELISA assay kits 3. TIMP-1 ELISA assay kits
chemicals: phorbol 12 myristate, IL-1β,PDGF-BB, Vit C, Vit E, GM6001, Isolation of extract fraction of Chlorella
UV irradiation: UVA 2 (320-340nm) radiation
Process
先利用不同年齡的皮膚細胞株先定量其之間 MMP-1, MMP-3, TIMP 的差異，再 以不同的 MMP inducers：PKC activator (e.g. 100nM phorbol 12 myristate)或 IL-1β (2ng) + PDGF-BB (10 ng)去誘發 MMP-1 及 MMP-3。定量的方法：以 enzyme-linked immunosorbent assay (ELISA) kits 定量 MMP-1, MMP-3, TIMP。經證實此方法的 再現性後，再進行綠藻粗萃取物的有效性的實驗，即綠藻粗萃取物可阻止 MMP inducers 對 MMP-1 及 MMP-3 的誘發的最佳劑量。此可實驗結果與 MMP inhibitors:
vitamin E (25 µM)或 C (as positive control)或 GM6001 (0.4nM)對 MMPs 的抑制能 力相比較。目前已有測量 MMP-1，MMP-3，TIMP-1 及 TIMP-3 的 ELISA kits
（Chemicon Internation）。
接著利用 UV irradiation: UVA 2 (320-340nm) radiation 定量不同年齡的皮膚細胞 株所引發的 MMP-1, MMP-3, TIMP 的差異，再進行綠藻粗萃取物的有效性的實 驗，即綠藻粗萃取物可阻止 MMP inducers 對 MMP-1 及 MMP-3 的誘發的最佳劑 量。因為不同的 MMP inducers 及 UV irradiation 所引導的 MMP-1, MMP-3, TIMP 的差異性可能不同，因此可能導致所需的綠藻粗萃取物阻止 MMP 的誘發的最佳 劑量也不相同。
同時一併進行的尚有使用更精緻、純化的綠藻萃取物來篩選可能的成分及所 需的最佳劑量。綠藻精（1 Kg），加 95 %酒精 3 公升冷浸，過濾後所得的殘楂，
再用甲醇-氯仿溶液溶出，酒精濾液與甲醇-氯仿溶液溶出部分分別用減壓濃縮得 粗萃取物 Fr. A 與 Fr. B。粗萃取物 Fr. A 與 Fr. B 再分別進行減少 MMP 的產量實 驗。實驗顯示 Fr. B 的減少 MMP 的產量達近 100 %。Fr. B 將進行各種色層分析，
每次色層分析所得的各個分畫便進行減少 MMP 的產量實驗，以實驗的結果來當 作實驗的指標，期待分離精製到真正有減少 MMP 的產量的化合物。如 Scheme1 所示，由於粗萃取物 Fr. B 均為高極性物質，故先用 RP-18 open column
(H2O-MeOH 等)分離得到數個分畫後，再進行減少 MMP 的產量實驗，有作用的
分畫再用其他的色層分析分法，反覆分離精製，等分離到單一化合物，再利用各 種光譜分析法，如 NMR, IR, UV, MS 來決定其構造。
結果：
Figure 1: Effects of MMP1 level of Extract of Chlorella after 5 hr incubation with MMP activators
Il-1b+PDGF-BB CGF 0.2+IL-b CGF 0.15+IL-b PMA CGF 0.2+PMA CGF 0.15+PMA 0
10 20 30 40 50 60 70 80 90
Effects of hot water extract of Chlorella pyrenoidosa on production of MMP-1 in human fibroblast SK966 cell line
Incubation for 5 hours
*
*
*
*p<0.05, t-test
MMP Level (ng/mL)
MMP1 is a type of secreted proteins and its levels in cell culture medium were measured induced after incubating with its promoters (10ng PDGF-BB/2ng Il-1b and PMA). The increase was prevented by co-incubation of 0.15mg and 0.2 mg of
Chlorella extract (named CGF) 5 hours after the incubation.
Figure 2: Effects of MMP1 level of Extract of Chlorella compared to Vit C after 5 hr incubation with PDGF-BB/Il1b
Il-1b+PDGF-BB CGF 0.15+IL-b GM6001 GM+IL-b Vit C Vit C+IL-b Basal MMP 0
10 20 30 40 50 60
5 hr incubation
MMP Level (ng/mL)
Effects of CGF and vit C and GM6001 (a specific MMP inhibitor) on induced MMP1 levels were compared. Vit C (125µM) had a less effect compared to 0.15 mg of CGF or GM6001 (0.4nM). GM6001 and Vit C themselves did not affect basal MMP1 level after 5 hr incubation.
Figure 3: Effects of MMP level of Extract of Chlorella compared to Vit C after 5 hr incubation with PMA
PMA CGF 0.2+PMA CGF 0.15+PMA GM6001 GM+PMA Vit C Vit C+PMA Basal MMP 0
10 20 30 40 50 60 70 80 90
MMP Level (ng/mL)
MMP1 is a type of secreted proteins and its levels in cell culture medium were
measured induced after incubating with PMA (100nM). The increase was prevented
by co-incubation of 0.15mg and 0.2 mg of CGF 5 hours after the incubation.
GM6001 also inhibited increased MMP1 under the same stimulation, whereas Vit C had a less effect on PMA-induced MMP1 level.
Figure 4: Effects of extract of Chlorella on basal TIMP1 level
con/4h 0.2CGF/4h 0.15CGF/4h con/6h 0.2CGF/6h 0.15CGF/6h
0 5 10 15 20 25 30
Effects of acute hot water extract of Chlorella pyrenoidosa on basal TIMP 1 levels in human skin fibroblast 966SK cells
*
*p<0.05, t-test
TIMP (ng/mL)
mg/well mg/well
Basal TIMP1 levels were significantly higher in the cells co-culture with 0.2mg CGF for 6 hours compared to controls.
討論：
The extract of Chlorella (CGF) showed the capacities in preventing PDGF/Il-1b and PMA-induced MMP1 production in skin fibroblast (see figure 1). The preventing effects were compared with GM6001, an inhibitor of MMP, and Vit C, an anti-oxidant and a new component in cosmetic product. CGF had a similar effect as GM6001 in preventing MMP production, whereas Vit C showed a less effect. Neither CGF nor GM 6001 affected basal MMP1 levels. In addition, CGF increased basal TIMP-1 production after 6 hr incubation (see figure 4). The results show that CGF may provide a new and useful ingredient in preventing skin ageing in cosmetic industrial.
參考文獻
Bende K, Blattner C, Knebel A, Iordanov M, Herrlich P and Rahmsdorf HL (1997) UV-induced signal transduction. J. Photochem. Photobiol. 37:1-17
Berneburg M, Grether-Beck S, Kurten V, Ruzicka T, Briviba K, Sies H, Krutmann J (1999) Singlet oxygen mediates the UVA-induced generation of the
photoaging-associated mitochondrial common deletion. J. Biol. Chem.
274:15345-15349
Claret FX, Hibi M, Dhut S, Toda T, & Karin M (1996) A new group of converted coactivators that increase the specificity of AP-1 transcription factor. Nature 383:453-457
Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, Miki T, & Gutkind JS (1995) The small GTP-binding proteins Racl & Cdc42 regulate the activity of the JNK/SAPK signaling pathway Cell 81: 1137-1146
Davis RJ (1993) The mitogen-activated protein kinase signal transduction pathway.
J. Biol. Chem. 268:14553-14556
Demhardt DT (1996) Signal-transducting protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signaling.
Biochem. J. 318:729-747
Fisher GJ, Datta SC, Talwar HS, Wang ZQ, Varani J, Kang S, & Voorhees JJ (1996) Molecular basis of sun-induced premature skin aging and retinoid antagonists.
Nature 379:335-339
Fisher GJ, Talwar HS, Lin J & Voorhees JJ (1999) Molecular mechanisms of photoaging in human skin in vivo and their prevention by all-trans retinoic acid.
Photochemistry & Photobiology 69:154-157
Giambernardi TA, grant GM, Taylor GP, Hay RJ, Maher VM, McCormick JJ, Klebe RJ (1998) Overview of matrix metalloproteinase expression in cultured human cells. Matrix Biol. 16:483-496
Gibson MA, Sandberg LB, Grosso LE, Cleary EG (1991) Complementary DNA cloning establishes microfibril-associated glycoprotein (MAGP) to be a discrete component of the elastin-associated microfibrils. J. Biol. Chem. 266:7596-7601 Gibson MA, Hatzinikolas JS, Sandberg LB, Nicholl JK, Sutherland G,
KumaratilakeGR, Cleary EG (1996) Further characterization of proteins associated with elastic fiber microfibrils including the molecular cloning of MAGP-2 (MP 25). J. Biol. Chem. 271:1096-1103
Gilchrest BA & Yaar M (1992) Aging and photoaging of the skin:observation at the cellular & molecular levels. Br. J. Dermatol. 127 (suppl 41):25-35
Hayflick L. (1965) The limited in vitro lifetime of human diploid cell strains. Exp.
Cell Res. 37:614-636
Hollister DW (1991) Linkage of marfan syndrome and a phenotypically related disorder to two different fibrillin genes. Nature 352:330-334
Huang RP, Wu JX, Fan Y, and Adamson ED (1996) UV activates growth factor receptors via reactive oxygen intermediates. J. Cell Biol. 133:211-220
Karin M & Hunter T (1995) The mitogen-activated protein kinase signal transduction
pathway. J. Biol. Chem 268:14553-14556
Kay PA (1991) Microalgae as food and supplement. Crit. Rev. Food Sci & Nutr. 30 (6):
555-573
Kobayashi R, Tashima Y, Masuda H, Shozawa T, Numata Y, Miyauchi K-I, Hayakawa T (1989) Isolation and characterization of a new 36 kDa microfibrils associated glycoprotein from porcine aorta. J. Biol. Chem. 264:17437-17444 Kuroda K & Shinkai H (1997) Gene expression of type I and III collagne, decorin,
matrix metalloproteinases and tissue inhibitor of metaloproteinases in skin fibroblasts from patients with systemic sclerosis. Arch Dermatol. Res.
289:567-572
Lavker RM, Veres DA, Iewin CJ, and Kaidbey KH (1995) Quantitative assessment of cumulative damage from repetitive exposures to suberythemogenic doses of UVA in human skin. J. Photochem. Photobiol.62: 348-352
Lee B, Godfrey M, Vitale E, Hori H, Mattei M, Sarfarazi M, Tsipouras P, Ramirez F, Maslwn CL, Corson GM, Maddox BK, Glanville RW, Sakai LY (1991) Patial
sequence of a candidate gene for the Marfan syndrome. Nature 352:330-334 Minden A, Lin A, Claret FX, Abo A, & Karin M (1995) Selective activation of the
JNK signaling cascade and c-jun transcriptional activity by the small GTPases Rac and csc42. Cell 81:1147-1157
Noda, K., Ohno, N., Tanaka, K., Kamiya, N., Okuda, M., Yadomac, T., Nomoto, K., Shoyama, Y., (1996) A water-soluble antitumor glycoprotein from Chlorella vulgaris. Planta Med. 62(5):423-426
Okuda M, Hasegawa T., Sonoda M , Okabe T., & Tanaka Y (1975) The effects of Chlorella on the levels of cholesterol in serum and liver. Jap. J. Nutr. 33:3-8 Rodriguez-Lopez M. & Lopez-Quijada C. (1971) Plasma glucose and plasma insulin
in normal and alloxanized rats treated with Chlorella. Life Science 10:57-60 Rosette C & Marin M (1996) Ultraviolet light and osmotic stress: activation of the
JNK cascade through multiple growth factor and cytokine receptors. Science 274:1194-1197
Sachsenmaier C, Radler-Pohl, A, Zinck R, Nordheim A, Herrlich P and Rahmsdorf HJ (1994) Involvement of growth factor receptors in the mammalian UVC response Cell 78:963-972
Sano, T. & Tanaka, Y. (1987) effects of driedm powdered Chlorella vulgaris on experimental atherosclerosis and alimentary hypercholemia in cholesterol-fed rabbit. Artery14:76-84
Sano, T., Kumamoto Y., Kamiya N., Okuda M., & Tanaka Y. (1988) effect of lipophilic extract on Chlorella vulgaris on alimentary hyperlipidemia in cholesterol-fed rats. Artery 15:217-224
Shea R & Parrish JA (1991) Nonionizing radiation and the skin. In physiology,
Biochemistry, and Molecular Biology of the skin. Vol. II, LA Glodsmith, editor.
Oxford University Press, New York 910-927
Shubert LE (1988) The use of Spirulina (Cyanophycaea) and Chlorella
(Chlorophyceae) as food resource for animals and humans. In: progressin physiological research (Round and Chapman, eds) p.p.237. Biopress Ltd
Singh, A., Singh SP., & Bamazai, R. (1998) Perinatal influence of Chlorella vulgaris on hepatic drug metabolizing enzyme and lipids. Anticancer Res.
18:1509-1514
Singh, A., Singh SP., & Bamazai, R. (1999) Inhibitory potential of Chlorella vulgaris (E-25) on mouse skin papillomagenesis and xenobiotic detoxication system.
Anticancer Res. 19:1887-1891
Uitto J & Bernstein EF (1998) Molecular mechanisms of cutaneous ageing:
connective alterations in the dermis. J. investing. Dermatol. Symp. Proc.
3:41-44
Warmuth I, Harth Y, Matsui MS, Wang N, and Deleo (1994) Ultraviolet radiation induces phosphorylation on the epidermal growth factor receptors. Cancer Res.
54:374-376
Whitmarsh AJ & Davis RJ (1994) Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol. Med.
74:589-607
Yang S-C., Huang T-I., Huang C-C., Shieh, M-J., Chiu W-C., Cheng C-J., & Chen J-R. (2001) The effects of Chlorella on lipid metabolism in rats fed with high fat and high cholesterol diet. Nutr. Sci. J. 26:22-31
Yin, L, Morita A, Tshuji T (2000) Alterations of extracellular matrix induced by tabacco smoke extract. Arch Dermatol. Res. 292:188-194
李宏圖,賴精二,董一致(1977)綠藻之降血糖作用。臺灣醫誌 76:272-276
