以螺旋藻萃取之C-藻藍素對細菌懸浮細胞與生物膜之 光動力抑制作用

Abstract

光動力治療(Photodynamic therapy, PDT) 是一種使用特定波長 之激發光將光感物質(photosensitizer) 激發成高能量激發態，經由能 量或電子傳遞，將細胞周圍氧分子轉變成有細胞毒性的單態氧 (1O2)，或與環境分子作用產生自由基，造成細胞死亡的方法，目前已 應用於癌症治療及微生物防治(Photodynamic inactivation, PDI)。研究 顯示，將光動力治療應用在微生物防治上已有部分成效，而積極開發 新光源及光感物質，使光動力治療能更有效發揮，為目前研究的新趨 勢。藻藍素(phycocyanin, PC) 為螺旋藻(Spirulina platensis) 中的一 種水溶性螢光蛋白，可吸收光能並進行能量及電子傳遞，具有光動力 治療之潛力。本實驗由螺旋藻Spirulina platensis 中萃取藻藍素作為光 感物質，並以發光二極體(Light emitting diodes，LED) 為光源，對 革蘭氏陽性及陰性菌進行光動力抑制，並進一步探討菌體在懸浮狀態 與生物膜狀態下，光動力殺菌效果是否不同。實驗結果顯示由螺旋藻 中萃取並純化出藻藍素，回收率可達56.1%，純度(A620nm/280nm) 為 4.56，其品質與商品化之藻藍素相當。而以藻藍素進行光動力治療， 在藻藍素濃度100 µg ml-1，照光強度360 J cm-2 之條件下，可將濃度 107 CFU ml-1 之革蘭氏陽性菌S. aureus 及S. epidermis 懸浮細胞完全 抑制，但對革蘭氏陰性菌E. coli 及P. aeruginosa 之效果則不顯著； 而以藻藍素對S. aureus 及S. epidermis 生物膜進行光動力抑制，在藻 藍素濃度900 µg ml-1，照光強度360 J cm-2 之條件下。亦可將108 CFU cm-2 之生物膜菌體完全殺滅。此研究顯示由螺旋藻中萃取之天然光感 II 物質藻藍素對革蘭氏陽性菌之懸浮菌體及生物膜細胞皆有良好之光 動力抑制效果，在未來的應用上極具潛力。 關鍵詞：光動力抑制、螺旋藻、藻藍素、生物膜

Photodynamic inactivation (PDI) utilized photosensitizers and light with appropriate wave length to give a phototoxic response, normally via oxidative damage. Although PDI might be an effective approach in antimicrobial treatment, no photosensitizer is suitable for all possible applications. Therefore, the development of new photosensitizers became important to overcome the shortage of PDI. Phycocyanin (PC), a water-soluble non-toxic biliprotein, is one of the major constituents of Spirulina platensis. The photobiological properties of PC suggested its possibility to be a photosensitizer in photodynamic therapy. The goal of this study was to investigate the effect of the PC extracted from Sp. platensis on photodynamic inactivation against bacterial planktonic and biofilm cells. The extraction and purification of PC was accomplished by fractional precipitation with ammonium sulfate precipitation, following by ion-exchange chromagraphy on Macro-Prep DEAE Support system. The purity of PC was examined by absorbance and fluorescence spectrometry. Both Gram-positive and Gram-negative bacteria were tested for PC-PDI. Pure PC was finally obtained from Sp. platensis with purity ratio (A620nm/280nm) 4.56 and recovery rate 56.1%. After PC-PDI treatment, which carried with 100 µg ml-1 of PC and irradiation light dose of 360 J cm-2, no viable cell of the Gram-positive Staphylococcus aureus and S. epidermis planktonic cells were detected. However, the Gram-negative Escherichia coli and Pseudomonas aeruginosa were resistant to the PC-PDI treatment. Experiments with PC-PDI against S. aureus and S. epidermis biofilms showed that both species of the tested biofilms were sensitive to the PC-PDI treatment, with a decrease of about 8-log of viable cells. This study showed that the PC from Sp. platensis was a potential photosensitizer to inactivated Gram-positive bacteria planktonic cells and biofilms. Keyword: Photodynamic Inactivation, Spirulina platensis, Phycocyanin, Biofilm