黃耆納豆菌發酵物對人類皮膚細胞生合成膠原蛋白及透明質酸之影響

Effect of Bacillus subtilis natto- fermented Radix astragali on collagen and hyaluronic acid biosynthesis in human skin cells

許美芳(Mei-Fang Hsu)

指導教授 ： 蔣丙煌

國立臺灣大學/生物資源暨農學院/食品科技研究所/博士(2008年)

https://doi.org/10.6342/NTU.2008.00178

摘要

老化皮膚在生化上的表現包括膠原蛋白及彈力蛋白等胞外基質成分(extracellular matrix components, ECM) 生成量減少，金屬蛋白酶 (matrix metalloproteinases, MMPs) 水解活性表現過度，使得膠原蛋白分解增加，因而造成乾燥、萎縮、皺紋等臨床外觀的變化。中草藥在化妝保養品上的應用已有五千年以上的歷史，其中黃耆 (Radix astragali, RA) 在外用美容中藥上是作為補氣生肌藥。利用微生物發酵中草藥使其成分進行生物轉化反應，為一種傳統之中草藥炮製方法。本研究第一部份為探討數十種中草藥發酵物之自由基清除能力，膠原蛋白與彈力蛋白分解酶活性抑制能力，以及促進人類皮膚纖維母細胞 (CCD-966SK) 生合成膠原蛋白等活性，以篩選出最具皮膚保健效果潛力之中草藥原料與菌株。抗氧化能力之試驗結果發現，紅花乳酸菌發酵樣品 (SAL) 在最終濃度1250 μg/mL較其它發酵樣品有較佳DPPH自由基清除能力，其DPPH清除率為60.33%；而總抗氧化力 (TEAC) 則是以紅花 (SAG) 及柿蒂 (PDG) 靈芝菌發酵樣品有最高的TEAC值，在濃度1000 μg/mL時，分別相當於Trolox標準品抗氧化力之1.09及0.90 mM。所有樣品以PDG具有最佳的膠原蛋白與彈力蛋白分解酶活性抑制能力，其IC50分別為170.85 µg/mL與369.59 µg/mL，然其細胞毒性高。在促進纖維母細胞 (CCD-966SK) 生合成膠原蛋白方面，以黃耆納豆菌發酵樣品 (HQB) 在濃度500 μg/mL下可顯著的促進纖維母細胞生合成第I型膠原蛋白 (type I procollagen)，為所有測試樣品之最高者。後續研究選擇具較低細胞毒性及可顯著促進纖維母細胞生合成膠原蛋白的黃耆納豆菌發酵樣品 (HQB)，探討其對人類皮膚細胞生合成膠原蛋白及透明質酸之影響。
在探討HQB對初代人類真皮纖維母細胞 (human dermal fibroblasts, HDF) 生合成膠原蛋白影響部分，黃耆未發酵物 (HQNB) 較黃耆納豆菌發酵物 (HQB)可顯著促進纖維母細胞生長。藉由酵素免疫分析 (ELISA) 與西方轉漬分析法 (Western blot analysis)，我們證明了HQB較HQNB可顯著的促進兩株分離自不同年齡 (22y and 81y) 女性皮膚的纖維母細胞生合成較多的type I procollagen。除此之外，即時定量反轉錄聚合酶鏈式反應 (real time RT-PCR) 結果指出，HQB較HQNB可更顯著的促進兩株纖維母細胞表現type I、type III procollagen 與TGF-β1 mRNA。同時，HQB促進纖維母細胞表現膠原蛋白基因與其蛋白質之間有一致相關性。推測HQB可能透過促進細胞生長以及誘導TGF-β1表現而調控人類纖維母細胞中原膠原蛋白的生合成。另外，在探討HQB對初代人類皮膚細胞生合成透明質酸 (hyaluronic acid, HA) 之影響部分，研究結果發現HQNB對人類表皮角質細胞 (human epidermal keratinocytes, HEK) 亦有顯著的增生效果，而HQB則對角質細胞的增生無顯著影響。然而HQB可顯著的促進人類皮膚角質細胞與纖維母細胞生合成透明質酸。再者，HQB亦可顯著的促進ＨaCaT角質細胞株及纖維母細胞分別表現透明質酸合成酶 (hyaluronan synthase, HAS) HAS3及HAS2 mRNA，顯示HQB促進皮膚細胞生合成透明質酸與促進透明質酸合成酶基因表現有密切關係。研究結果亦發現HQB中所含的異黃酮類成分 (HQB-I) 促進表皮角質細胞生合成透明質酸效果顯著較HQB樣品為佳，但HQB中所含的calycosin，formononetic與ononin等異黃酮類成分含量顯著較HQNB為少，推測黃耆因納豆菌的發酵，使得原異黃酮類成分轉換成不帶糖基型式或其代謝物 (如daidzein, equol等)，可能因而促進人類皮膚細胞生合成膠原蛋白與透明質酸。綜言之，HQB可預防因年齡增長所造成的膠原蛋白與透明質酸等胞外基質成分之減少，因此，HQB在預防皮膚老化上扮演一個具潛力的化妝保養品成分。

關鍵字

黃耆 ； 納豆菌 ； 發酵 ； 膠原蛋白 ； 透明質酸 ； 初代人類表皮角質細胞 ； 初代人類真皮纖維母細胞

並列摘要

The biochemical changes associated with skin aging include reduced expression of type I collagen messenger ribonucleic acid (mRNA), overexpression of matrix metalloproteinase (MMPs), and decreased synthesis of extracellular matrix (ECM). These changes result in increased collagen degradation and leads to skin laxity, atrophy, wrinkles, dryness, and other clinical manifestations of skin. Chinese herbs are of particular interests because they have been applied in skincare products for more than 5000 years. Radix astragali has traditionally been used for skin health, and microbial fermentation is one of the traditional methods for processing Chinese herbs. The first part of this study was to perform a screening test on the evaluation of free radical scavenging activities, collagenase and elastase inhibitory effects, and the stimulating effect on collagen production in human fibroblasts with fermented Chinese herbal preparations for potential cosmetic applications. The results revealed that the DPPH free radical scavenging effect of SAL (60.3%), at a concentration of 1250 μg/mL, was better than that of other preparations. One mg/ml of SAG and PDG were equivalent to 1.09 and 0.90 mM of Trolox in TEAC scavenging assay, respectively. In addition, 170.85 µg/mL and 369.59 µg/mL of PDG were needed for 50% collagenase and elastase inhibitory effect, respectively. The HQB treatment (500 μg/mL) increased type I procollagen production by 33% in fibroblasts (CCD-966SK) as compared with the untreated group. With relatively low toxicity and the stimulating effects on collagen production of HQB, the effect of B. subtilis natto– fermented R. astragali (HQB) on collagen and hyaluronic acid production in normal primary human skin cells were investigated in the last two parts of this research.
It was found that HQNB significantly improved cell growth and proliferation of human dermal fibroblasts (HDF). Conversely, the enzyme-linked immunosorbent assay (ELISA) and Western blot analysis demonstrated that HQB, but not HQNB, significantly stimulated the biosynthesis of type I procollagen in both aged (81y) and young (22y) HDF cells in dose-dependently manners. Real-time reverse transcription-polymerase chain reaction revealed that expression of type I, type III procollagen and transforming growth factor β1 (TGF-β1) mRNA was significantly induced in HQB-treated HDF cells than that of HQNB-treated and un-treated HDF cells, suggesting that there was a close correlation between the mRNA expression and protein production in the fibroblasts response to HQB stimulation. The results also suggested that HQB could stimulate the collagen biosynthesis in human dermal fibroblasts, which is, at least in part, associated with the regulation of procollagen biosynthesis resulting from HQB-induced TGF-β1 expression and the mitogenic activity in HDF cells. On the other hand, it was found that human epidermal keratinocytes (HEK) reacted to HQNB much more sensitive than to HQB on growth-stimulating effect. In contrast, HQB significantly and dose-dependently stimulated hyaluronic acid (HA) production in cultured primary human epidermal keratinocytes and human dermal fibroblasts. In addition, HQB markedly and dose-dependently increased the expression of hyaluronan synthase 3 (HAS3) and hyaluronan synthase 2 (HAS2) mRNA in HaCaT cells and human fibroblasts, respectively. These findings suggest that the enhancement of HA synthesis in HQB-treated skin cells does not mainly depend on the basis of proliferation effect, and correlates well with the higher expression of hyaluronan synthase genes. The isoflavonoids of HQB contained lower levels of calycosin, formononetin and ononin than that of HQNB, but had significantly higher enhancement effect on the production of HA in the cultures of human epidermal keratinocytes. It is suggested that isoflavonoid aglycones or other metabolites (e.g. daidzein, equol), converted from their primary isoflavones during the fermentation, might be responsible for the skincare functions found in this study. Taken together, HQB might act to reduce the age-dependent loss of extracellular matrix components (e.g. collagen and hyaluronan), and might play a promising role in antiaging cosmetic applications.

並列關鍵字

Radix astragali ； Bacillus subtilis natto ； Fermentation ； Collagen ； Hyaluronic acid ； Human epidermal keratinocytes ； Human dermal fibroblasts

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