条斑紫菜R-藻红蛋白酶解物的制备及其抗氧化和肿瘤细胞增殖抑制活性

doi:10.3864/j.issn.0578-1752.2012.15.026

摘要/Abstract

摘要： 【目的】研究木瓜蛋白酶对条斑紫菜R-藻红蛋白的抗氧化性和肿瘤增殖抑制活性的影响。【方法】采用超声波破壁法提取条斑紫菜R-藻红蛋白，用DEAE柱层析法纯化后进行木瓜蛋白酶酶解，通过正交试验设计，以还原力A700为考察指标确定酶解反应的最佳工艺参数，进一步测定获得的R-藻红蛋白及其酶解物的还原力、清除羟自由基能力和对人肉瘤细胞U2O及人肝癌细胞HepG-2的肿瘤增殖抑制活性。【结果】R-藻红蛋白的最佳酶解工艺条件为：木瓜蛋白酶添加量25 000 U•g-1，pH 7.0，温度50℃，酶解时间4 h。在此条件下，R-藻红蛋白酶解物还原力为0.573，较未酶解的R-藻红蛋白提高了2.35倍；清除羟自由基能力为51.03%，较未酶解的R-藻红蛋白活性提高了3.22倍。随着浓度的增加，R-藻红蛋白及其酶解物对人肉瘤细胞U2O和人肝癌细胞HepG-2抑制生长作用增强。R-藻红蛋白对人肉瘤细胞U2O抑制作用的IC50值为2 431.32 μg•mL-1，其酶解物IC50值降低为1 271.46 μg•mL-1。R-藻红蛋白对肝癌细胞HepG-2抑制作用的IC50值为1 593.61 μg•mL-1，其酶解物IC50值降低为512.05 μg•mL-1。【结论】经木瓜蛋白酶酶解后，R-藻红蛋白酶解物具有较强的抗氧化和抑瘤活性。酶解技术可作为进一步提高R-藻红蛋白生物活性的有效手段。

关键词: 条斑紫菜, 藻红蛋白, 木瓜蛋白酶, 酶解物, 抗氧化, 抑瘤活性

Abstract: 【Objective】The effect of enzymatic hydrolysis by papain on antioxidant and tumor cell proliferation inhibiting activities of R-phycoerythrin from Porphyra yezoensis was investigated. 【Method】R-phycoerythrin of Porphyra yezoensis was extracted by ultrasonic cell wall breaking, and then purified by DEAE column chromatography. Orthogonal design was employed to obtain the optimal condition of enzymatic hydrolysis by determination of reducing powder A700 of their enzymatic hydrolysates. Subsequently, hydroxyl radical-scavenging ability and proliferation inhibiting activities of human sarcoma cancer cell U2O and liver cancer cell HepG-2 was compared as well. 【Result】 The optimal enzymatic hydrolysis of R-phycoerythrin was selected at an amount of papain of 25 000 U•g-1, pH 7.0, a temperature of 50℃ and at a hydrolysis time of 4 h. Under the optimal treatment, the reducing powder of the R-phycoerythrin was raised up to 0.573, which was 2.35 times higher than that of untreated group. Hydroxyl radical-scavenging ability was 51.03%, which was 3.22 times higher than that of untreated group. With the concentrations increasing, the growth inhibition of R-phycoerythrin and their enzymatic hydrolysates against human sarcoma cancer cell U2O and liver cancer cell HepG-2 increased. The inhibition IC50 values of R-phycoerythrin against human sarcoma cancer cell U2O was 2 431.32 μg•mL-1, which was decresed to 1 271.46 μg•mL-1 by enzymatic hydrolysates. The inhibition IC50 values of R-phycoerythrin against liver cancer cell HepG-2 was 1 593.61 μg•mL-1, which was decresed to 512.05 μg•mL-1 by enzymatic hydrolysates. 【Conclusion】The enzymatic hydrolysate of R-phycoerythrin by papain exhibited excellent antioxidant and antitumor activities, which proved that enzymatic hydrolysis could be an effective technology for further increasing the bioactivities of R-phycoerythrin.

Key words: Porphyra yezoensis, phycoerythrin, papain, enzymatic hydrolysates, antioxidant, antitumor

方勇, 杨方美, 赵殿峰, 杨文建, 赵立艳, 辛志宏, 马宁, 施瑛, 胡秋辉. 条斑紫菜R-藻红蛋白酶解物的制备及其抗氧化和肿瘤细胞增殖抑制活性[J]. 中国农业科学, 2012, 45(15): 3222-3230.

FANG Yong, YANG Fang-Mei, ZHAO Dian-Feng, YANG Wen-Jian, ZHAO Li-Yan, XIN Zhi-Hong, MA Ning, SHI Ying, HU Qiu-Hui. Preparation of Enzymatic Hydrolysate of R-phycoerythrin from Porphyra yezoensis and Its Antioxidant and Tumor Cell Proliferation Inhibiting Activities[J]. Scientia Agricultura Sinica, 2012, 45(15): 3222-3230.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.15.026

https://www.chinaagrisci.com/CN/Y2012/V45/I15/3222

参考文献

[1]Cai C, Wu L, Li C, He P, Li J, Zhou J. Purification, crystallization and preliminary X-ray analysis of phycocyanin and phycoerythrin from Porphyra yezoensis Ueda. Acta Crystallographica Section F, 2011, 67(5): 579-583.

[2]Tang J, Liu Z, Kang J, Zhang Y. Determination of salbutamol using R-phycoerythrin immobilized on eggshell membrane surface as a fluorescence probe. Analytical and Bioanalytical Chemistry, 2010, 397(7): 3015-3022.

[3]Niu J F, Chen Z F, Wang G C, Zhou B C. Purification of phycoerythrin from Porphyra yezoensis Ueda (Bangiales, Rhodophyta) using expanded bed absorption. Journal of Applied Phycology, 2010, 22(1): 25-31.

[4]Soni B, Kalavadia B, Trivedi U, Madamwar D. Extraction, purification and characterization of phycocyanin from Oscillatoria quadripunctulata-Isolated from the rocky shores of Bet-Dwarka, Gujarat, India. Process Biochemistry, 2006, 41(9): 2017-2023.

[5]Silveira S T, Burkert J F M, Costa J A V, Burkert C A V, Kalil S J. Optimization of phycocyanin extraction from Spirulina platensis using factorial design. Bioresource Technology, 2007, 98(8): 1629-1634.

[6]Benavides J, Rito-Palomares M. Simplified two-stage method to B-phycoerythrin recovery from Porphyridium cruentum. Journal of Chromatography B, 2006, 844: 39-44.

[7]马海乐, 肖海芳, 骆琳. 条斑紫菜藻红蛋白的脉冲超声辅助提取. 农业工程学报, 2007, 23(1): 207-211.

Ma H L, Xiao H F, Luo L. Optimization of the technology for pulse ultrasonic assistant extraction of phycoerythrin from Porphyra yazoensis. Transactions of the Chinese Society of Agricultural Engineering, 2007, 23(1): 207-211. (in Chinese)

[8]Sun L, Wang S M, Gong X Q, Zhao M R, Fu X J, Wang L. Isolation, purification and characteristics of R-phycoerythrin from a marine macroalga Heterosiphonia japonica. Protein Expression and Purification, 2009, 64(2): 146-154.

[9]Minkova K M, Toshkova R A, Gardeva E G, Tchorbadjieva M I, Ivanova N J, Yossifova L S, Gigova L G. Antitumor activity of B-phycoerythrin from Porphyridium cruentum. Journal of Pharmacy Research, 2011, 4(5): 1480-1482.

[10]时旭, 蔡春尔, 李春霞, 王源, 贾睿, 吴维宁, 何培民. 条斑紫菜藻胆蛋白对小鼠免疫功能及抗氧化活性的影响. 中国海洋药物杂志, 2011, 30(2): 44-48.

Shi X, Cai C E, Li C X, Wang Y, Jia R, Wu W N, He P M. Effects of phycobiliprotein from Porphyra yezoensis on the mice immunity and anti-oxidation activity. Chinese Journal of Marine Drugs, 2011, 30(2): 44-48. (in Chinese)

[11]陈美珍, 葛安山, 余杰, 杨文杰. 龙须菜藻红蛋白抗肿瘤活性的研究. 中国海洋药物, 2007, 26(4): 27-31.

Chen M Z, Ge A S, Yu J, Yang W J. Studies on antitumor activities of phycoerythrin from Gracilaria lemaneiftheormis. Chinese Journal of Marine Drugs, 2007, 26(4): 27-31. (in Chinese)

[12]Huang B, Wang G C, Zeng C K, Li Z G. The experimental research of R-phycoerythrin subunits on cancer treatment: A new photosensitizer in PDP. Cancer Biother Radiopharm, 2002, 17(1): 35-42.

[13]Isailovic D, Li H W, Yeung E S. Isolation and characterization of R-phycoerythrin subunits and enzymatic digests. Journal of Chromatography A, 2004, 1051(1):119-130.

[14]Glazer A N, Craig S H. Characterization of R-PC: chromophore content of R-phycocyanin and C-phycoerythrin. The Journal of Biological Chemistry, 1975, 250(14): 5487-5495.

[15]朱晓君, 安辛欣, 顾丽, 胡秋辉. 超声辅助同时提取条斑紫菜多糖及藻胆蛋白工艺的优化. 食品科学, 2008, 29(5): 241-244.

Zhu X J, An X X, Gu L, Hu Q H. Optimization technique of synchronous ultrasonic-assisted extraction of polysaccharide and phycobiliprotein from Porphyra yezoensis. Food Science, 2008, 29(5): 241-244. (in Chinese)

[16]Oyaizn M. Antioxidative activities of browning reaction products prepared from glucosamine. Japanese Journal of Nutrition, 1986, 44(6): 307-315.

[17]Yen G C, Duh P D. Scavenging effect of methanolic extracts of peanut hulls on free-radical and active-oxygen species. Journal of Agricultural and Food Chemistry, 1994, 42(3): 629-632.

[18]Bank U, Reinhold D, Ansorge S. Measurement of cellular activity with the MTT test: optimization of the method. Allergie und Immunologie (Leipz), 1991, 37(3/4): 119-123.

[19]Chen Y L, Lin S Z, Chang J Y, Cheng Y L, Tsai N M, Chen S P, Chang W L, Harn H J. In vitro and in vivo studies of a novel potential anticancer agent of isochaihulactone on human lung cancer A549 cell. Biochemical Pharmacology, 2006, 72(3): 308-319.

[20]蔡春尔, 何培民. 硫酸铵三步盐析对藻胆蛋白纯化的影响. 生物技术通报, 2006(4): 121-125.

Cai C E, He P M. Effects of thrice ammonium sulphate precipitation method on purification of crude phycobiliprotein. Biotechnology Bulletin, 2006(4): 121-125. (in Chinese)

[21]高洪峰, 曹文达, 纪明侯. 坛紫菜中藻胆蛋白的性质与化学组成研究. 海洋与湖沼, 1993, 24(40): 350-353.

Gao H F, Cao W D, Ji M H. Studies on chemical properties and components of phycobiliproteins from Porphyra haitanensis. Oceanologia Et Limnologia Sinica, 1993, 24(40): 350-353. (in Chinese)

[22]Romana R B, Alvaez-Pezb J M, Fernandez F G A, Grima E M. Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum. Journal of Biotechnology, 2002, 93(1): 73-85.

[23]Rossano R B, Ungaro N, D’Ambrosio A, Liuzzi G M, Riccio P. Extracting and purifying R-phycoerythrin from Mediterranean red algae Corallina elongata Ellis & Solander. Journal of Biotechnology, 2003, 101: 289-293.

[24]Omwamba M, Hu Q. Antioxidant activity in barley (Hordeum vulgare L.) grains roasted in a microwave oven under conditions optimized using response surface methodology. Journal of Food Science, 75(1): C66-C73.

[25]Willson R L. Hydroxyl radicals and biological damage in vitro: what relevance in vivo?//Ciba Foundation Symposium 65 - Oxygen Free Radicals and Tissue Damage. John Wiley & Sons, Ltd., 2008: 19-42.

[26]陈美珍, 葛安山, 崔鹏举, 廖灶辉. 龙须菜藻红蛋白对Hela细胞增殖抑制及其机制的研究. 食品科学, 2007, 28(9): 549-552.

Chen M Z, Ge A S, Cui P J, Liao Z H. Study on inhibition effects of phycoerythrin from Gracilaria lemaneiformis on Hela cells and its mechanism. Food Science, 2007, 28(9): 549-552. (in Chinese)

[27]马海乐, 肖海芳, 骆琳. 条斑紫菜藻红蛋白纯化方法的研究. 农业工程学报, 2007, 23(2): 249-253.

Ma H L, Xiao H F, Luo L. Technology for purifying phycoerythrin extracted from Porphyra yazoensis. Transactions of the Chinese Society of Agricultural Engineering, 2007, 23(2): 249-253. (in Chinese)

[28]Wang J S, Zhao M M, Zhao Q Z, Jiang Y M. Antioxidant properties of papain hydrolysates of wheat gluten in different oxidation systems. Food Chemistry, 2007, 101(4): 1658-1663.

[29]吴丹, 李铁晶, 赵新淮. 酪蛋白水解物的酶法修饰优化与抗氧化活性改善. 农业机械学报, 2010, 41(1): 139-145.

Wu D, Li T J, Zhao X H. Enzymatic modification of casein hydrolysates by plaste in reaction and improvement in antioxidant activity. Transactions of the Chinese Society for Agricultural Machinery, 2010, 41(1): 139-145. (in Chinese)

[30]刁静静, 孔保华, 刁新平, 陈洪生. 骨蛋白水解物抗氧化活性及其作用模式. 中国农业科学, 2009, 42(1): 238-244.

Diao J J, Kong B H, Diao X H, Chen H S. Antioxidant activity and the possible mode of action of bone protein hydrolysates. Scientia Agricultura Sinica, 2009, 42(1): 238-244. (in Chinese)

[31]季宇彬, 刘日嘉, 高世勇. 藻红蛋白诱导人乳腺癌MCF-7细胞衰老研究. 药物评价研究, 2009, 32(1):13-18.

Ji Y B, Liu R J, Gao S Y. Study on the senescence induced by phycoerythrin in human breast cancer MCF-7. Drug Evaluation Research, 2009, 32(1):13-18. (in Chinese)