壳聚糖对泥鳅毒性及其引发急性死亡分析
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摘要
壳聚糖对动物的安全性是公认的,但壳聚糖能造成泥鳅急性死亡,其死亡原因值得探索,因此本实验研究了不同种类、不同质量浓度的壳聚糖对泥鳅死亡的影响,并观察泥鳅死亡症状,测定半致死剂量(half lethal concentration,LC_(50))、呼吸强度、细胞膜透性、红细胞核异常情况等。结果发现不同壳聚糖均能引起泥鳅快速死亡,且质量浓度和温度越高,泥鳅死亡速度越快,壳聚糖对泥鳅的LC_(50)为16 mg/L。壳聚糖可引起泥鳅体表黏液大量脱落,导致液体浑浊;破坏泥鳅皮肤结构,影响鱼鳃呼吸;刺激泥鳅早期快速游动;显著增加泥鳅呼吸强度,导致水中溶解氧质量浓度低于极限值;使泥鳅耗尽自身糖原,导致后期无力浮出水面进行肠呼吸;促进泥鳅体内的氯、钠、钾离子和氨大量渗出,破坏细胞正常功能。壳聚糖引发泥鳅急性死亡主要与壳聚糖损害泥鳅呼吸系统有关,但壳聚糖一般不会进入人类呼吸系统,因此壳聚糖按照食品添加剂标准使用还是安全的。
It is well known that chitosan has no toxicity to animals. Surprisingly, however, chitosan has an acute lethal effect on loach(Misgurnus anguillicaudatus). The underlying mechanism deserves to be explored. In this study, we evaluated the effect of different types and concentrations of chitosan on the death of loach, and this study also observed symptoms during the dying process and measured half lethal concentration(LC_(50)), respiration rate, cell membrane permeability anderythrocyte cell nucleus abnormality. Results showed that all types of chitosan caused loach to rapidly die. Higher chitosan concentration or higher temperature accelerated the dying process. The LC_(50) of chitosan for loach was determined as 16 mg/L. In the presence of chitosan, large quantities of mucilage fell off the surface of loach skin, thus making the water appear turbid. Moreover, chitosan destroyed the structure of loach skin, affected gill respiration, stimulated loach to rapidly swim at the early stage of the experiment, and significantly increase respiration rate, resulting in a dissolved oxygen concentration below the safe limit. After using up all the glycogen, loach could not come up to the water surface and have to respire through intestine at the later stage of the experiment. Chitosan also led to exudation of large amounts of chlorine, sodium, potassium ion and ammonium from loach and destroyed cell functions. In conclusion, chitosan exerts its lethal effect on loach mainly by destroying therespiratory system. Since chitosan normally does not enter the human respiratory system, it can be used safely as a food additive.
It is well known that chitosan has no toxicity to animals. Surprisingly, however, chitosan has an acute lethal effect on loach(Misgurnus anguillicaudatus). The underlying mechanism deserves to be explored. In this study, we evaluated the effect of different types and concentrations of chitosan on the death of loach, and this study also observed symptoms during the dying process and measured half lethal concentration(LC_(50)), respiration rate, cell membrane permeability anderythrocyte cell nucleus abnormality. Results showed that all types of chitosan caused loach to rapidly die. Higher chitosan concentration or higher temperature accelerated the dying process. The LC_(50) of chitosan for loach was determined as 16 mg/L. In the presence of chitosan, large quantities of mucilage fell off the surface of loach skin, thus making the water appear turbid. Moreover, chitosan destroyed the structure of loach skin, affected gill respiration, stimulated loach to rapidly swim at the early stage of the experiment, and significantly increase respiration rate, resulting in a dissolved oxygen concentration below the safe limit. After using up all the glycogen, loach could not come up to the water surface and have to respire through intestine at the later stage of the experiment. Chitosan also led to exudation of large amounts of chlorine, sodium, potassium ion and ammonium from loach and destroyed cell functions. In conclusion, chitosan exerts its lethal effect on loach mainly by destroying therespiratory system. Since chitosan normally does not enter the human respiratory system, it can be used safely as a food additive.
引文
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[6]刘安军,詹伟,朱振元.水溶性低分子质量壳聚糖致鼠脱毛的毒理性研究[J].天津科技大学学报,2007,22(4):20-23.DOI:10.3969/j.issn.1672-6510.2007.04.005.
[7]赵传絪,吴贤均,杨海宝.鱼类的急性毒性试验[J].上海实验动物科学,1986,6(3):171-172.
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[11]顾双.青菜衰老和短期逆境处理的早期检测指标研究[D].杭州:浙江工商大学,2017:42-46.
[12]VARDAR G,ALTIKATO?LU M,ORTA?D,et al.Measuring calcium,potassium,and nitrate in plant nutrient solutions using ionselective electrodes in hydroponic greenhouse of some vegetables[J].Biotechnology and Applied Biochemistry,2015,62(5):663-668.DOI:10.1002/bab.1317.
[13]ZHOU Li,BOYD C E.Comparison of Nessler,phenate,salicylate and ion selective electrode procedures for determination of total ammonia nitrogen in aquaculture[J].Aquaculture,2016,450:187-193.DOI:10.1016/j.aquaculture.2015.07.022.
[14]ZUBAREVA A,SHAGDAROVA B,VARLAMOV V,et al.Penetration and toxicity of chitosan and its derivatives[J].European Polymer Journal,2017,93:743-749.DOI:10.1016/j.eurpolymj.2017.04.021.
[15]田丰,杨健,陈世谦,等.壳聚糖醋酸溶液对凝血作用的研究[J].生物医学工程学杂志,2005,22(5):999-1003.
[16]潘炎.壳聚糖对几种生物的作用差异研究[D].杭州:浙江工商大学,2014:21-26.DOI:10.7666/d.Y2531639.
[17]王晓芹,李晓辉,王贵波,等.壳聚糖胶原生物敷料的安全性研究[J].中国临床康复,2002,6(7):1030-1031.DOI:10.3321/j.issn:1673-8225.2002.07.090.
[18]石玲,胡利平,浦锦宝,等.壳聚糖的安全性研究[J].中国海洋药物,2000,19(1):25-29.DOI:10.3969/j.issn.1002-3461.2000.01.008.
[19]ZHAO YANG,HAN Baoqin,FU Dawei,et al.Acute toxicity of high dosage carboxymethyl chitosan and its effect on the blood parameters in rats[J].Journal of Materials Science:Materials in Medicine,2012,23(2):457-462.DOI:10.1007/s10856-011-4467-4.
[20]郭丰红,汪之和,陈必文,等.分子氨和亚硝态氮对鳜鱼成鱼的急性毒性试验[J].食品科学,2009,30(23):397-400.DOI:10.3321/j.issn:1002-6630.2009.23.089.
[21]刘德明,陶鑫峰,王一飞.泥鳅体表粘液流变行为的研究[J].高分子学报,2010,10(4):468-474.
[22]刘德明.鱼类体表粘液流变行为的研究[D].杭州:浙江大学,2011:14-17;51-54.
[23]WANG T,HOU J,SU C,et al.Hyaluronic acid-coated chitosan nanoparticles induce ROS-mediated tumor cell apoptosis and enhance antitumor efficiency by targeted drug delivery via CD44[J].Journal of Nanobiotechnology,2017,15(7):1-12.DOI:10.1186/s12951-016-0245-2.
[24]ABULKALAM M.The potential application of hyaluronic acid coated chitosan nanoparticles in ocular delivery of dexamethasone[J].International Journal of Biological Macromolecules,2016,89:559-568.DOI:10.1016/j.ijbiomac.2016.05.016.
[25]冯小强,李小芳,杨声,等.壳聚糖对细菌细胞膜的影响[J].食品科学,2009,30(7):63-67.DOI:10.3321/j.issn:1002-6630.2009.07.014.
[26]WIMARDHANI Y S,SUNIARTI D F,FREISLEBEN H J,et al.Chitosan exerts anticancer activity through induction of apoptosis and cell cycle arrest in oral cancer cells[J].Journal of Oral Science,2014,56(2):119-126.DOI:10.2334/josnusd.56.119.
[27]YALPANI M,PANTALEONE D.An examination of the unusual susceptibilities of aminoglycans to enzymalic hydrolysis[J].Carbohydrate Research,1994,256(1):159-175.DOI:10.1016/0008-6215(94)84235-3.
[28]MUZZARELLI R,TOMASETTI M,JARI P.Depolymerization of chitosan with the aid of papain[J].Enzyme&Microbial Technology,1994,16(2):110-114.
[29]PANTALEONE D,YALPANI M,SCOLLAR M.Unusual susceptibility of chitosan to enzymalic hydrolysis[J].Cheminform,1992,237:318-320.DOI:10.1016/S0008-6215(92)84256-R.
[30]刘珍利.微生物发酵生产壳聚糖及酶法降解壳聚糖研究[D].天津:天津科技大学,2010:50.DOI:10.7666/d.y2083048.
[31]王奎兰.生物酶降解壳聚糖及其应用研究[D].广州:广东工业大学,2004:52.DOI:10.7666/d.y597164.
[32]李专.酶法降解壳聚糖工艺的研究[D].武汉:华中农业大学,2007:39.DOI:10.7666/d.Y1198598.
[2]国家卫生和计划生育委员会.食品安全国家标准食品添加剂使用标准:GB 2760-2014[S].北京:中国标准出版社,2014.
[3]李冬,陈志田,谢庆阁.吸咐口蹄疫病毒的壳聚糖微球对动物的安全性试验[J].中国兽医科技,1998,28(10):28-29.DOI:10.16656/j.issn.1673-4696.1998.10.015.
[4]张荣,梁崇礼,张荣平,等.松毛虫中壳聚糖(CDG)的安全性实验研究[J].实验动物科学与管理,2001,18(2):21-25.DOI:10.3969/j.issn.1006-6179.2001.02.007.
[5]汪珍春,姚焱,廖娟,等.利用泥鳅红细胞核的遗传损伤监测广州大学内河水质[J].广州大学学报(自然科学版),2011,10(3):91-94.DOI:10.3969/j.issn.1671-4229.2011.03.018.
[6]刘安军,詹伟,朱振元.水溶性低分子质量壳聚糖致鼠脱毛的毒理性研究[J].天津科技大学学报,2007,22(4):20-23.DOI:10.3969/j.issn.1672-6510.2007.04.005.
[7]赵传絪,吴贤均,杨海宝.鱼类的急性毒性试验[J].上海实验动物科学,1986,6(3):171-172.
[8]李翠萍,吴民耀,王宏元.3种半数致死浓度计算方法之比较[J].动物医学进展,2012,33(9):89-92.DOI:10.3969/j.issn.1007-5038.2012.09.021.
[9]陈细香,卢昌义,蔡金聪.漂染废水对泥鳅红细胞核异常的诱导[J].厦门大学学报(自然科学版),2007,46(4):555-558.DOI:10.3321/j.issn:0438-0479.2007.04.026.
[10]FAN X T,SOKORAI K J B.Assessment of radiation sensitivity of fresh-cut vegetables using electrolyte leakage measurement[J].Postharvest Biology and Technology,2005,36(2):191-197.DOI:10.1016/j.postharvbio.2004.12.004.
[11]顾双.青菜衰老和短期逆境处理的早期检测指标研究[D].杭州:浙江工商大学,2017:42-46.
[12]VARDAR G,ALTIKATO?LU M,ORTA?D,et al.Measuring calcium,potassium,and nitrate in plant nutrient solutions using ionselective electrodes in hydroponic greenhouse of some vegetables[J].Biotechnology and Applied Biochemistry,2015,62(5):663-668.DOI:10.1002/bab.1317.
[13]ZHOU Li,BOYD C E.Comparison of Nessler,phenate,salicylate and ion selective electrode procedures for determination of total ammonia nitrogen in aquaculture[J].Aquaculture,2016,450:187-193.DOI:10.1016/j.aquaculture.2015.07.022.
[14]ZUBAREVA A,SHAGDAROVA B,VARLAMOV V,et al.Penetration and toxicity of chitosan and its derivatives[J].European Polymer Journal,2017,93:743-749.DOI:10.1016/j.eurpolymj.2017.04.021.
[15]田丰,杨健,陈世谦,等.壳聚糖醋酸溶液对凝血作用的研究[J].生物医学工程学杂志,2005,22(5):999-1003.
[16]潘炎.壳聚糖对几种生物的作用差异研究[D].杭州:浙江工商大学,2014:21-26.DOI:10.7666/d.Y2531639.
[17]王晓芹,李晓辉,王贵波,等.壳聚糖胶原生物敷料的安全性研究[J].中国临床康复,2002,6(7):1030-1031.DOI:10.3321/j.issn:1673-8225.2002.07.090.
[18]石玲,胡利平,浦锦宝,等.壳聚糖的安全性研究[J].中国海洋药物,2000,19(1):25-29.DOI:10.3969/j.issn.1002-3461.2000.01.008.
[19]ZHAO YANG,HAN Baoqin,FU Dawei,et al.Acute toxicity of high dosage carboxymethyl chitosan and its effect on the blood parameters in rats[J].Journal of Materials Science:Materials in Medicine,2012,23(2):457-462.DOI:10.1007/s10856-011-4467-4.
[20]郭丰红,汪之和,陈必文,等.分子氨和亚硝态氮对鳜鱼成鱼的急性毒性试验[J].食品科学,2009,30(23):397-400.DOI:10.3321/j.issn:1002-6630.2009.23.089.
[21]刘德明,陶鑫峰,王一飞.泥鳅体表粘液流变行为的研究[J].高分子学报,2010,10(4):468-474.
[22]刘德明.鱼类体表粘液流变行为的研究[D].杭州:浙江大学,2011:14-17;51-54.
[23]WANG T,HOU J,SU C,et al.Hyaluronic acid-coated chitosan nanoparticles induce ROS-mediated tumor cell apoptosis and enhance antitumor efficiency by targeted drug delivery via CD44[J].Journal of Nanobiotechnology,2017,15(7):1-12.DOI:10.1186/s12951-016-0245-2.
[24]ABULKALAM M.The potential application of hyaluronic acid coated chitosan nanoparticles in ocular delivery of dexamethasone[J].International Journal of Biological Macromolecules,2016,89:559-568.DOI:10.1016/j.ijbiomac.2016.05.016.
[25]冯小强,李小芳,杨声,等.壳聚糖对细菌细胞膜的影响[J].食品科学,2009,30(7):63-67.DOI:10.3321/j.issn:1002-6630.2009.07.014.
[26]WIMARDHANI Y S,SUNIARTI D F,FREISLEBEN H J,et al.Chitosan exerts anticancer activity through induction of apoptosis and cell cycle arrest in oral cancer cells[J].Journal of Oral Science,2014,56(2):119-126.DOI:10.2334/josnusd.56.119.
[27]YALPANI M,PANTALEONE D.An examination of the unusual susceptibilities of aminoglycans to enzymalic hydrolysis[J].Carbohydrate Research,1994,256(1):159-175.DOI:10.1016/0008-6215(94)84235-3.
[28]MUZZARELLI R,TOMASETTI M,JARI P.Depolymerization of chitosan with the aid of papain[J].Enzyme&Microbial Technology,1994,16(2):110-114.
[29]PANTALEONE D,YALPANI M,SCOLLAR M.Unusual susceptibility of chitosan to enzymalic hydrolysis[J].Cheminform,1992,237:318-320.DOI:10.1016/S0008-6215(92)84256-R.
[30]刘珍利.微生物发酵生产壳聚糖及酶法降解壳聚糖研究[D].天津:天津科技大学,2010:50.DOI:10.7666/d.y2083048.
[31]王奎兰.生物酶降解壳聚糖及其应用研究[D].广州:广东工业大学,2004:52.DOI:10.7666/d.y597164.
[32]李专.酶法降解壳聚糖工艺的研究[D].武汉:华中农业大学,2007:39.DOI:10.7666/d.Y1198598.
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