高原鼢鼠组织中透明质酸提取工艺及分子表征

doi:10.13560/j.cnki.biotech.bull.1985.2017.03.022

摘要/Abstract

摘要： 旨在建立高原鼢鼠组织中透明质酸（Hyaluronic acid,HA）提取工艺。采用单因素试验确定粗提、酶解和除杂等工艺的最佳条件,通过响应面法进一步优化粗提和酶解工艺条件。通过红外分光光度法确定HA的分子结构,Bitter-Muir法确定其葡萄糖醛酸的含量,黏度法和体积排阻色谱法确定透明质酸的分子量。高原鼢鼠最佳粗取工艺∶液料比4 mL/g（水/动物组织,V/W）,提取温度26℃,提取时间1.6 h;最佳酶解工艺：酶解温度37℃,pH8.7,加酶量0.7%（1∶250 胰蛋白酶/动物组织,W/W）;最佳除杂工艺为：氯苯体积20%（氯苯/HA水溶液,V/V）,以3倍体积95%乙醇沉淀,以7%活性碳（活性碳/HA水溶液,W/V）除杂,透析时间3 h。在此工艺条件下,每500 g 高原鼢鼠组织可得到（144±3.61）mg HA粉末。平均回收率为72.73%。红外图谱显示提取物具有与Sigma公司的HA标准品一致的吸收峰,葡萄糖醛酸的质量分数为45.60%,蛋白质含量为0.11%,其分子量达到3×10⁶Da。本工艺提取的高原鼢鼠组织来源的HA回收率好,纯度高;高原鼢鼠组织中HA分子量大,含量高。

关键词: 高原鼢鼠, 透明质酸（HA）, 提取工艺, 表征方法

Abstract: We aim to establish an extraction process of hyaluronic acid（HA）from the tissues of plateau zokor（Myospalax baileyi）. One-factor experiments were used to determine the optimal conditions of crude extraction,enzymolysis,and edulcoration process,and response surface method was applied to further optimize the crude extraction and enzymolysis process. The molecular structure of the extracted HA was determined by infrared spectrophotometry,the content of glucuronic acid by the Bitter-Muir method,and the molecular weight by the method of viscosity and size exclusion chromatography. As results,the optimal conditions of crude extraction was: liquid/material ratio 4 mL/g（water/tissues,V/W）,extraction temperature 26℃,and extraction time 1.6 h. The optimal enzymolysis process was: temperature 37℃,pH 8.7,and 0.7% added enzyme（1:250 trypsasy/tissue,W/W）. The optimal edulcoration process was: 20% chlorobenzene（chlorobenzene/HA solution,V/V）,95% alcohol precipitation ratio in 3 times volume,7% activated carbon（activated carbon/HA solution,W/V）to do edulcoration,and dialysis for 3 h. Under above optimal conditions,144 ± 3.61 mg HA powder was obtained from 500 g tissues of plateau zokor,and the average recovery rate was 72.73%. Infrared spectrum displayed the same absorption peak of HA by standard samples from Sigma. The mass fraction of glucuronic acid was 45.60%,and the protein content was 0.11%. The molecular weight of hyaluronic acid reached 3×106 Da. Conclusively,the HA from plateau zokor’s tissues by this extraction method is of good recovery rate and high purity,and HA in the tissues of plateau zokor is of high molecular weight and high content.

Key words: plateau zokor, hyaluronic acid, extraction process, characterization methods

魏琳娜, 汪洋, 魏登邦, 魏莲, 王宁, 索有瑞. 高原鼢鼠组织中透明质酸提取工艺及分子表征[J]. 生物技术通报, 2017, 33(3): 151-161.

WEI Lin-na, WANG Yang, WEI Deng-bang, WEI Lian, WANG Ning, SUO You-rui. The Extraction Process and Molecular Characterization of Hyaluronic Acid in the Plateau Zokor Tissues[J]. Biotechnology Bulletin, 2017, 33(3): 151-161.

参考文献

[1] Rapport MM, Weissman B, Linker A, et al. Isolation of a crystalline disaccharide, hyalobiuronic acid fromhyaluronic acid[J]. Nature, 1951, 168：997-997.
[2] Saari H, Konttinen YT, Friman C, et al. Differential effects of reactive oxygen species on native synovial fluid and purified human umbilical cord hyaluronate[J]. Inflammation, 1993, 17（4）：403-415.
[3] Jeon O, Song SJ, Lee KJ, et al. Mechanical properties anddegradation behaviors of hyaluronic acid hydrogels crosslinkedat various cross-linking densities[J]. Carbohydrate Polymers, 2007, 10：251-257.
[4] Meyer K, Palmer JW. The polysaccharide of the vitreous humor[J]. Journal of Biological Chemistry, 1934, 107：629-634.
[5] Weissmann B, Meyer K. The structure of hyalobiuronic acid and of hyaluronic acid from umbilical Cord1, 2[J]. Journal of the American Chemical Society, 1954, 76（7）：1753-1757.
[6] 郭学平, 刘爱华, 凌沛学. 透明质酸在化妆品、健康食品和软组织填充剂中的应用[J]. 食品与药品, 2005, 7（1A）：20-23.
[7] Yamamoto H. Antiaging and cosmetic effects of dietary hyaluronic acid（extracellular matrix extract）[J]. New Food Ind, 1998, 40：33-41.
[8] 凌沛学, 贺艳丽, 张青. 透明质酸对骨关节炎的治疗作用[J]. 食品与药品, 2005, 7（1A）：1-3.
[9] Balazs EA, Miller D and Stegmann R. Viscosurgery and the use of Na-hyaluronate in intraocular lens implantation[J]. Lecture, Cannes, France, 1979, 5：56-59.
[10] Scott JE, Cummings C, Brass A, et al. Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing-electron microscopy and computer simulation. Hyaluronan is a very efficient network-forming polymer[J]. Biochemical Journal, 1991, 274（3）：699-705.
[11] Buffenstein R, Jarvis JUM. The nakedmole rat—a new record for the oldest living rodent[J]. Sci Aging Knowledge Environ, 2002（21）：pe7.
[12] Buffenstein R. Negligible senescence in the longest living rodent, the naked mole-rat：insights from a successfully aging species[J]. Journal of Comparative Physiology B, 2008, 178, 439-445.
[13] Tian X, Azpurua J, Hine C, et al. High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat[J]. Nature, 2013, 499（7458）：346-349.
[14] Holmes MW, Bayliss MT, Muir H. Hyaluronic acid in human articular cartilage. Age-related changes in content and size[J]. Biochemical Journal, 1988, 250, 435-441.
[15] Jiang D, Liang J, Noble PW. Hyaluronan in tissue injury and repair[J]. Annu Rev Cell Dev Biol, 2007, 23：435-461.
[16] 曾缙祥, 王祖望, 师治贤. 高山地区高原鼢鼠的代谢特点及若干生理指标的观察[J]. 高原生物学集刊, 1984, 3：163-171.
[17] 王晓君, 魏登邦, 魏莲, 等. 高原鼢鼠和高原鼠兔肺细叶结构特征[J]. 动物学报, 2008, 54（3）：531-539.
[18] 齐新章, 王晓君, 朱世海, 等. 高原鼢鼠和高原鼠兔心脏对低氧环境的适应[J]. 生理学报, 2008, 60（3）：348-354.
[19] 魏莲, 魏登邦, 王晓君, 等. 高原鼢鼠、鼠兔及大鼠心肌和骨骼肌乳酸脱氢酶活力及同工酶谱[J]. 四川动物, 2009, 28（1）：64-68.
[20] 朱世海, 齐新章, 王晓君, 等. 高原鼢鼠和高原鼠兔骨骼肌摄氧功能差异研究[J]. 生理学报, 2009, 61（4）：373-378.
[21] 郑亚宁, 朱瑞娟, 王多伟, 等. 高原鼢鼠血管内皮生长因子基因编码和mRNA的表达以及微血管密度：与其它鼠类的比较[J]. 生理学报, 2011, 63（2）：155-163.
[22] 朱瑞娟, 饶鑫峰, 魏登邦, 等. 高原鼢鼠和高原鼠兔肝脏苹果酸天冬氨酸穿梭系统的功能差异[J]. 生理学报, 2012, 64（2）：177-186.
[23] Wei DB, Wei L, Zhang JM. Blood-gas properties of plateau zokor（Myospalax baileyi）[J]. Comparative Biochemistry and Physiology Part A：Molecular & Integrative Physiology, 2006, 145：372-375.
[24] Wang DW, Wei L, Wei DB. Testiss-pecific lactate dehydrogenase is expressed in somatic tissues of plateau pikas[J]. FEBS Open Bio, 2013, 3：118-123.
[25] Sun SZ, Wei L, Wei DB, et al. Differences of glycolysis in skeletal muscle and lactate metabolism in the liver of plateau zokor（Myospalax baileyi）and plateau pika（Ochotona curzoniae）[J]. Acta Physiologica Sinica, 2013, 65（3）：276-284.
[26] Wei DB, Wang DW, Wei L, et al. Differences in physiological adaptive strategies to hypoxic environments in plateau zokor and plateau pika[J]. Science（suppl.）, 2012, 36.
[27] Bitter T, Muir HM. A modified uronic acid carbazole reaction[J]. Analytical Biochemistry, 1962, 4（4）：330-334.
[28] 谢音. 食品分析[M]. 北京：科学技术文献出版社, 2006. 79-80.
[29] 郭育涛, 陈斌, 江元汝, 等. 从牛眼玻璃体中分离纯化透明质酸的新方法[J]. 精细化工, 2008（3）：246-250.
[30] Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent[J]. J Biol Chem, 1951, 193（1）：265-275.
[31] 栾途. 透明质酸粘多糖的分子表征、流变学性质及其物理凝胶的研究[D]. 上海：上海交通大学, 2011.
[32] 卢佳芳, 杨佳玉, 王菁, 等. 鱿鱼眼透明质酸的酶法提取与分离纯化[J]. 中国食品学报, 2010（4）：88-93.
[33] 王大为, 张艳荣, 沙坤. 中国林蛙皮多糖类天然保湿因子的提取与鉴定[J]. 吉林农业大学学报, 2002, 24（6）：99-102.
[34] 王继波. 环胞苷二棕榈酸酯脂质体粒度分布及微黏度的测定[J]. 精细化工, 2006, 23（2）：154-156.
[35] 蔡明志, 王昆, 黄复生. 载药脂质体物理化学稳定性的研究进展[J]. 国际药学研究杂志, 2005, 32（6）：404-407.
[36] Arifin DR, Palmer AF. Physical properties and stability mechani-sms of poly（Ethylene Glycol）conjugated liposome encapsulated hemoglobin dispersions[J]. Artificial Cells Blood Substitutes & Biotechnology, 2005, 33（2）：137-62.
[37] 王健, 李明轩. 冷冻干燥对提高脂质体稳定性的研究概况[J]. 中国医药工业杂志, 2005, 36（9）：576-580.
[38] 崔媛, 段潜, 李艳辉. 透明质酸的研究进展[J]. 长春理工大学学报, 2011, 34（3）：101-106.
[39] 高玉琼, 刘建华, 霍昕, 等. 不同组织来源透明质酸的分离纯化及其鉴定[J]. 生物化学杂志, 1996, 12（2）：215-218.
[40] 黄岳山, 潘艺茗, 薛静. 不同相对分子量透明质酸功能及应用的研究[J]. 透析与人工器官, 2011（4）：22-25.