海洋聚球藻藻藍素的免疫抗癌及抗氧化活性

Abstract

藻藍素是藍綠藻中的主要成分，為一水溶性的蛋白，具有多種生理藥物活性特性。本論文實驗是探討海洋聚球藻藻藍素的免疫、抗癌及抗氧化活性，以評估未來應用的潛力。大量培養聚球藻後，以離心方式濃縮細胞，並予以冷凍乾燥備用。將乾燥的藻細胞以液態氮研磨後，溶於磷酸塩緩衝食鹽水，接著利用超音波將細胞壁打破。藻藍素粗萃取液在35-80% 硫酸銨飽和濃度下沉澱。本論文實驗採用雙管柱層析方法：陰離子交換層析管柱層析和石灰岩層析管柱，純度比值A620/A280為4.9，回收率為35%。所得之藻藍素以原態電泳和SDS-PAGE電泳分析其純度，再以Sephadex S-200層析管柱估算出藻藍素分子量為128 kDa。 將所得的藻藍素對老鼠巨噬細胞及人類癌細胞做體外活性檢測，以評估其對免疫能力及抑制腫瘤細胞的能力。藻藍素濃度為65 μg/ml時，可以刺激巨噬細胞生長提高增殖達38%。藻藍素也具有抑制癌細胞生長的活性，濃度為65 μg/ml時，可以抑制胰臟癌細胞及乳癌細胞生長達80%左右。另外，以DPPH法檢測藻藍素清除氧自由基的能力，在藻藍素78 μg/ml濃度時，經50 min後，清除率可達36%。因此，藻藍素不僅可以應用在醫學檢驗，更可以應用在促進免疫細胞增殖、抑制腫瘤細胞生長及清除自由基方面。由本論文實驗得知從海洋聚球藻所純化的藻藍素，具有活化多種生理機能，值得進一步研究，以期未來可被應用在保健及醫藥上。

Phycocyanin, one of the major biliproteins from blue green algae, has various biological and pharmacological properties. It is a water-soluble and non-toxic fluorescent protein pigment. The present study used two-step column chromatographic method to isolate and purify c-phycocyanin from a marine Synechococcus. The isolation involved a multi-step treatments of the crude extract by fractional precipitation with ammonium sulfate, then by Q-sepharose column and finally ceramic hydroxyapatite column, giving a purity ration A620/A280 of 4.9. The yield of c-phycocyanin was 35% from Synechococcus dried powder. Respectively, further the purity and homogeneity were confirmed by native and SDS-PAGE electrophoresis. The estimated molecular weight of the phycocyanin was 128 kDa as determined by Sephadex S-200 gel filtration and electrophoresis. Phycocyanin from the reported alga was assayed in vitro for proliferation stimulation in murine macrophages and for anti-tumor activity against human mammary and pancreas tumor cells, using MTT method. The results indicated that macrophage proliferation increased by 38% while treated with phycocyanin at the concentration of 65 μg/ml and the stimulation activity was a dose-dependent manner. Tumor cells used in the present study reduced markedly in growth rate in the presence of phycocyanin, i.e. reduced by 80% at concentration of 65 μg/ml. The obtained phycocyanin was also an antioxidant as determined by DPPH method. Its antioxidation activity, as expressed in % scavenge of oxygen radicals, reached 36% at concentration of 78 μg/ml. The above findings revealed that marine Synechococcus and the extracted phycocyanin can be a potent source for biomedical research, as well as therapeutic agent not only in immune stimulation and modulation, but also in tumor growth inhibition.