蛹虫草MF27高抗氧化活性提取物筛选及保肝作用研究
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摘要
本研究对一株优质蛹虫草菌株MF27不同提取物进行体外抗氧化活性比较,筛选得到高抗氧化活性提取物,并进一步探究该提取物对CCl_4诱导的小鼠肝损伤的修复作用。以DPPH自由基和羟自由基的清除率为抗氧化评价指标,从菌丝体发酵液、菌丝体水提物/醇提物、以及子实体水提物/醇提物中筛选菌株MF27的高抗氧化活性提取物;以CCl_4致小鼠急性肝损伤为模型,通过检测血清生化指标、肝功指标的变化,来评价该高活性提取物的体内抗氧化保肝效果。体外抗氧化实验结果表明,MF27的不同提取物均具有较好的体外抗氧化活性,但对清除DPPH和OH自由基能力最好的提取物是子实体水提物,其对DPPH自由基的半数有效浓度(EC50)为0.096mg/mL,对羟自由基的半数有效浓度(EC50)为0.196mg/mL,当在1mg/mL时对DPPH自由基的清除率为94.94%,对羟自由基的清除率为70.17%;体内抗氧化保肝结果显示,给药组(子实体水提物)相比模型组而言,小鼠血清中ALT、AST以及肝脏中MDA水平极显著降低(P<0.01)、SOD水平极显著升高(P<0.01),表明子实体水提物能有效改善氧化性肝损伤,同时与阳性对照(联苯双酯)对比,给药组在肝脏指数上表现出相当的作用(P>0.05)。本研究表明菌株MF27的最有效抗氧化活性提取物是子实体水提物,它对体内氧化性肝损伤有一定的修复能力,揭示MF27子实体水提物具有成为抗氧化性肝损伤功能产品的潜力。
A comparison of antioxidant activity in vitro between different extract of Cordyceps militaris MF27 was made, and an extract with high antioxidant activity was obtained. The repair function of the extract to liver injury induced by CCl_4 in mice was further studied. Using extracorporeal scavenging rate of DPPH free radical and hydroxyl free radical as antioxidant evaluation index, the extract with high antioxidant activity was selected from fermentation broth, aqueous extract and alcohol extract of mycelia, and aqueous extract and alcohol extract of fruiting body. An acute liver injury model in mice by using CCl_4 was established, and evaluation of internal antioxidant and liver protection effect of the extract with high antioxidant activity was made by testing the change of serum biochemical indices and liver function index. The result showed that all the extracts had good antioxidant activity in vitro, but the aqueous extract of fruiting body was possessed of the best ability of scavenging free radicals. The half effective concentration(EC50) of the aqueous extract of fruiting body is 0.096 mg/mL for DPPH free radicals and 0.196 mg/mL for hydroxyl free radical. At concentration of 1 mg/mL, the clearance rate is 94.94% to DPPH free radicals and 70.17% to hydroxyl free radical. In comparison with the model experimental group, the levels of ALT and AST in serum and MDA in liver of the mice in the administration group treated with aqueous extract of fruiting body significantly decreased(P<0.01)as a result of in vivo antioxidation and improvement of liver function. The level of SOD in liver significantly increased(P<0.01), indicating that aqueous extract of the fruiting body can improve oxidative liver damage effectively. Compared with positive control group treated with biphenyl diester, the administration group manifested a great drug efficacy(P>0.05) in the liver index. It is proved that the aqueous extract of fruiting body of C. militaris MF27 has a certain repairing ability in vivo to oxidative liver injury, and possesses potential becoming of antioxidant damage functional product.
A comparison of antioxidant activity in vitro between different extract of Cordyceps militaris MF27 was made, and an extract with high antioxidant activity was obtained. The repair function of the extract to liver injury induced by CCl_4 in mice was further studied. Using extracorporeal scavenging rate of DPPH free radical and hydroxyl free radical as antioxidant evaluation index, the extract with high antioxidant activity was selected from fermentation broth, aqueous extract and alcohol extract of mycelia, and aqueous extract and alcohol extract of fruiting body. An acute liver injury model in mice by using CCl_4 was established, and evaluation of internal antioxidant and liver protection effect of the extract with high antioxidant activity was made by testing the change of serum biochemical indices and liver function index. The result showed that all the extracts had good antioxidant activity in vitro, but the aqueous extract of fruiting body was possessed of the best ability of scavenging free radicals. The half effective concentration(EC50) of the aqueous extract of fruiting body is 0.096 mg/mL for DPPH free radicals and 0.196 mg/mL for hydroxyl free radical. At concentration of 1 mg/mL, the clearance rate is 94.94% to DPPH free radicals and 70.17% to hydroxyl free radical. In comparison with the model experimental group, the levels of ALT and AST in serum and MDA in liver of the mice in the administration group treated with aqueous extract of fruiting body significantly decreased(P<0.01)as a result of in vivo antioxidation and improvement of liver function. The level of SOD in liver significantly increased(P<0.01), indicating that aqueous extract of the fruiting body can improve oxidative liver damage effectively. Compared with positive control group treated with biphenyl diester, the administration group manifested a great drug efficacy(P>0.05) in the liver index. It is proved that the aqueous extract of fruiting body of C. militaris MF27 has a certain repairing ability in vivo to oxidative liver injury, and possesses potential becoming of antioxidant damage functional product.
引文
Bai L,Jiang BH,Shi JS,Shi YX,Gao Y,2015.The curative effect study of bifendate used for anti-tuberculosis drug-induced liver injury.China Tropical Medicine,15(7):869-871(in Chinese)
Brand-Williams W,Cuvelier ME,Berset C,1995.Use of a free radical method to evaluate antioxidant activity.Food Science and Technology,28(1):25-30
Dai YC,Li Y,2011.Notes on the nomenclature of six important medicinal fungi in China.Mycosystema,30(4):515-518(in Chinese)
Deng QF,Zhou X,Chen HG,2016.Advance in study on hepatoprotective effects and its mechanism of polysaccharides.China Journal of Chinese Materia Medica,41(16):2958-2966(in Chinese)
Du XJ,Zhang JS,Jia W,Liu YF,Zhou S,2012.Comparative study on the antitumor effect and antiviral activity of ethanol extracts from codyceps militaris mycelia in vitro.Food Industry,184(1):127-129(in Chinese)
Guo T,Wei L,Sun J,Hou CL,Fan L,2011.Antioxidant activities of extract and fractions from Tuber indicum Cooke&Massee.Food Chemistry,127(4):1634-1640
Guo YH,2014.Analysis of adverse reactions and related factors caused by tiopronin.Chinese Journal of Clinical Rational Drug Use,7(7):92-93(in Chinese)
Hou JX,Zhang JX,Zhao XJ,Duan SN,Liu HX,Guan ZL,Wang LA,2014.Anti-oxidative activity and hepatoprotective effect of lyophilized powder of liquid fermentation products from Cordyceps militaris.Drug Evaluation Research,37(1):25-29(in Chinese)
Liang HJ,Jiao CW,Yang YS,Zhang ML,Xie YZ,2015.Antioxidant activity of water extraction from four rare edible mushroom species.Journal of Microbiology,35(4):106-109(in Chinese)
Liu YF,2011.Oxidative stress and hepatic protection.Chinese Journal of Gastroenterology and Hepatology,20(7):594-597(in Chinese)
Liu JB,Qi M,Du YR,Fu JS,Hu KH,2018.Genetic relationship analysis of 22 Cordyceps militaris based on split network and preliminary screening of high-yield cordycepin strains.Biotechnology Bulletin,34(4):133-138(in Chinese)
Li Y,Kong LQ,Gao H,Yan YL,2008.Progress in free radicals and diseases.Progress in Veterinary Medicine,29(4):85-87(in Chinese)
Li YK,Jin RM,2006.Experimental methodology of TCMpharmacology.Shanghai Science and Technology Press,Shanghai.515-529(in Chinese)
Li ZH,Hu HB,Du GH,2008.Comparison of the antioxidant activities of natural and cultured Cordyceps.China Medical Herald,5(16):13-16(in Chinese)
Lu CH,Guo XM,Mou DH,2016.Research of composition and antioxidant activity between water and alcohol extracts of black Termitomyces albuminosus.Food Engineering,3:35-40(in Chinese)
LüGY,Pan HJ,Wu YZ,Fan LF,2009.Evaluation of the antioxidant activity of extracts from anamorphic mycelia of Cordyceps militaris in vitro.Mycosystema,28(4):597-602(in Chinese)
Moukette BM,Pieme CA,Biapa PC,Moor VJA,Berinyuy E,Ngogang JY,2015.Afrostyrax lepidophyllus extracts exhibit in vitro free radical scavenging,antioxidant potential and protective properties against liver enzymes ion mediated oxidative damage.BMC Research Notes,8(334):1304-1315
Sagar BK,Singh RP,2011.Genesis and development of DPPHmethod of antioxidant assay.Journal of Food Science and Technology,48(4):412-422
Shao Y,Chen AH,Zhang CL,Zhou Y,2015.Isolation and purification of antioxidant components in fruiting body of Cordyceps militaris.Food Science,36(8):175-180(in Chinese)
Sun JD,Hou J,Yang Y,Wang H,2012.In vitro evaluation of antioxidant activities of aqueous extracts from selenium-enriched cultured mycelia and fruit body of Cordyceps militaris.Journal of Shenyang Agricultural University,43(2):179-183(in Chinese)
Wen TC,Zha LS,Kang JC,Kevin DH,2017.Problems and prospects of research and development of Cordyceps militaris.Mycosystema,36(1):14-27(in Chinese)
Wu N,Cai GM,He Q,2008.Oxidative stress and hepatic injury.World Chinese Journal of Digestology,16(29):3310-3315(in Chinese)
Xin XL,2001.The effect of rats′liver index and MDA content made by auoxan.Journal of Qinghai Normal University(Natural Science Edition),16(1):53-54(in Chinese)
Yin LH,Yu H,Peng JY,2015.Molecular mechanism and intervention of Chinese medicine of carbon tetrachloride induced liver injury.Chinese Journal of Modern Applied Pharmacy,32(9):1147-1155(in Chinese)
Yuan JF,Zhang ZQ,Fan ZC,Yang JX,2008.Antioxidant effects and cytotoxicity of three purified polysaccharides from Ligusticum chuanxiong Hort.Carbohydrate Polymers,74(4):822-827
Zhang XW,Zhao B,Zhang GC,Yue ZR,Zhang GZ,Zou CS,2017.Analysis of active components and antioxidant activity of albino Cordyceps militaris.Journal of Jilin Agricultural University,39(5):585-589(in Chinese)
Zhao WX,Jin MH,Li T,Wang YJ,Quan JS,2013.Intervention effect of aqueous fractions from Boschniakia rossica on hepatic oxidative stress in mice with liver injury induced by carbon tetrachloride.China Journal of Chinese Materia Medica,38(6):875-878(in Chinese)
白靓,姜本贺,石俊仕,史衍席,高燕,2015.联苯双酯治疗抗痨药物性肝损害的疗效研究.中国热带医学,15(7):869-871
戴玉成,李玉,2011.中国六种重要药用真菌名称的说明.菌物学报,30(4):515-518
邓青芳,周欣,陈华国,2016.多糖抗肝损伤作用及其机制研究进展.中国中药杂志,41(16):2958-2966
杜秀菊,张劲松,贾薇,刘艳芳,周帅,2012.北虫草醇提物体外抗肿瘤和抗病毒活性的比较研究.食品工业,184(1):127-129
郭燕华,2014.硫普罗宁所致不良反应特点及相关因素分析.临床合理用药杂志,7(7):92-93
侯金鑫,张金秀,赵晓静,段硕楠,刘红霞,管振龙,王立安,2014.蛹虫草液体发酵产物冻干粉的抗氧化活性及保肝作用.药物评价研究,37(1):25-29
李勇,孔令青,高洪,严玉霖,2008.自由基与疾病研究进展.动物医学进展,29(4):85-87
李仪奎,金若敏,2006.中药药理实验方法学.上海:上海科学技术出版社.515-529
梁慧嘉,焦春伟,杨咏善,张命龙,谢意珍,2015.4种珍稀食用菌水提物的抗氧化活性研究.微生物学杂志,35(4):106-109
刘亚锋,2011.氧化应激与肝脏保护.胃肠病学和肝病学杂志,20(7):594-597
刘建兵,戚梦,杜苑如,傅俊生,胡开辉,2018.拆分网络分析22株蛹虫草亲缘关系及高产虫草素菌株初筛.生物技术通报,34(4):133-138
卢彩会,郭晓萌,牟德华,2016.黑皮鸡枞菌水提物和醇提物的主要成分及抗氧化性研究.食品工程,3:35-40
吕国英,潘慧娟,吴永志,范雷法,2009.蛹虫草无性型菌丝体提取液体外抗氧化活性研究.菌物学报,28(4):597-602
邵颖,陈安徽,张传丽,周扬,2015.蛹虫草子实体中抗氧化活性化合物的分离纯化.食品科学,36(8):175-180
孙军德,侯静,杨逸,王鹤,2012.富硒蛹虫草水浸提物的体外抗氧化活性研究.沈阳农业大学学报,43(2):179-183
文庭池,查岭生,康冀川,Kevin D.HYDE,2017.蛹虫草研究和开发过程中的一些问题和展望.菌物学报,36(1):14-27
吴娜,蔡光明,何群,2008.氧化应激与肝脏损伤.世界华人消化杂志,16(29):3310-3315
辛晓林,2001.四氧嘧啶对小鼠肝指数和MDA含量的影响.青海师范大学学报(自然科学版),16(1):53-54
尹连红,于浩,彭金咏,2015.四氯化碳诱导肝损伤的分子机制及中药干预的研究进展.中国现代应用药学,32(9):1147-1155
张曦文,赵博,张国财,岳峥嵘,张国珍,邹传山,2017.白化型蛹虫草活性成分分析及抗氧化活性测定.吉林农业大学学报,39(5):585-589
赵文玺,金梅花,李天,王玉娇,全吉淑,2013.草苁蓉水萃取物对四氯化碳致肝损伤小鼠肝脏氧化应激的干预作用.中国中药杂志,38(6):875-878
Brand-Williams W,Cuvelier ME,Berset C,1995.Use of a free radical method to evaluate antioxidant activity.Food Science and Technology,28(1):25-30
Dai YC,Li Y,2011.Notes on the nomenclature of six important medicinal fungi in China.Mycosystema,30(4):515-518(in Chinese)
Deng QF,Zhou X,Chen HG,2016.Advance in study on hepatoprotective effects and its mechanism of polysaccharides.China Journal of Chinese Materia Medica,41(16):2958-2966(in Chinese)
Du XJ,Zhang JS,Jia W,Liu YF,Zhou S,2012.Comparative study on the antitumor effect and antiviral activity of ethanol extracts from codyceps militaris mycelia in vitro.Food Industry,184(1):127-129(in Chinese)
Guo T,Wei L,Sun J,Hou CL,Fan L,2011.Antioxidant activities of extract and fractions from Tuber indicum Cooke&Massee.Food Chemistry,127(4):1634-1640
Guo YH,2014.Analysis of adverse reactions and related factors caused by tiopronin.Chinese Journal of Clinical Rational Drug Use,7(7):92-93(in Chinese)
Hou JX,Zhang JX,Zhao XJ,Duan SN,Liu HX,Guan ZL,Wang LA,2014.Anti-oxidative activity and hepatoprotective effect of lyophilized powder of liquid fermentation products from Cordyceps militaris.Drug Evaluation Research,37(1):25-29(in Chinese)
Liang HJ,Jiao CW,Yang YS,Zhang ML,Xie YZ,2015.Antioxidant activity of water extraction from four rare edible mushroom species.Journal of Microbiology,35(4):106-109(in Chinese)
Liu YF,2011.Oxidative stress and hepatic protection.Chinese Journal of Gastroenterology and Hepatology,20(7):594-597(in Chinese)
Liu JB,Qi M,Du YR,Fu JS,Hu KH,2018.Genetic relationship analysis of 22 Cordyceps militaris based on split network and preliminary screening of high-yield cordycepin strains.Biotechnology Bulletin,34(4):133-138(in Chinese)
Li Y,Kong LQ,Gao H,Yan YL,2008.Progress in free radicals and diseases.Progress in Veterinary Medicine,29(4):85-87(in Chinese)
Li YK,Jin RM,2006.Experimental methodology of TCMpharmacology.Shanghai Science and Technology Press,Shanghai.515-529(in Chinese)
Li ZH,Hu HB,Du GH,2008.Comparison of the antioxidant activities of natural and cultured Cordyceps.China Medical Herald,5(16):13-16(in Chinese)
Lu CH,Guo XM,Mou DH,2016.Research of composition and antioxidant activity between water and alcohol extracts of black Termitomyces albuminosus.Food Engineering,3:35-40(in Chinese)
LüGY,Pan HJ,Wu YZ,Fan LF,2009.Evaluation of the antioxidant activity of extracts from anamorphic mycelia of Cordyceps militaris in vitro.Mycosystema,28(4):597-602(in Chinese)
Moukette BM,Pieme CA,Biapa PC,Moor VJA,Berinyuy E,Ngogang JY,2015.Afrostyrax lepidophyllus extracts exhibit in vitro free radical scavenging,antioxidant potential and protective properties against liver enzymes ion mediated oxidative damage.BMC Research Notes,8(334):1304-1315
Sagar BK,Singh RP,2011.Genesis and development of DPPHmethod of antioxidant assay.Journal of Food Science and Technology,48(4):412-422
Shao Y,Chen AH,Zhang CL,Zhou Y,2015.Isolation and purification of antioxidant components in fruiting body of Cordyceps militaris.Food Science,36(8):175-180(in Chinese)
Sun JD,Hou J,Yang Y,Wang H,2012.In vitro evaluation of antioxidant activities of aqueous extracts from selenium-enriched cultured mycelia and fruit body of Cordyceps militaris.Journal of Shenyang Agricultural University,43(2):179-183(in Chinese)
Wen TC,Zha LS,Kang JC,Kevin DH,2017.Problems and prospects of research and development of Cordyceps militaris.Mycosystema,36(1):14-27(in Chinese)
Wu N,Cai GM,He Q,2008.Oxidative stress and hepatic injury.World Chinese Journal of Digestology,16(29):3310-3315(in Chinese)
Xin XL,2001.The effect of rats′liver index and MDA content made by auoxan.Journal of Qinghai Normal University(Natural Science Edition),16(1):53-54(in Chinese)
Yin LH,Yu H,Peng JY,2015.Molecular mechanism and intervention of Chinese medicine of carbon tetrachloride induced liver injury.Chinese Journal of Modern Applied Pharmacy,32(9):1147-1155(in Chinese)
Yuan JF,Zhang ZQ,Fan ZC,Yang JX,2008.Antioxidant effects and cytotoxicity of three purified polysaccharides from Ligusticum chuanxiong Hort.Carbohydrate Polymers,74(4):822-827
Zhang XW,Zhao B,Zhang GC,Yue ZR,Zhang GZ,Zou CS,2017.Analysis of active components and antioxidant activity of albino Cordyceps militaris.Journal of Jilin Agricultural University,39(5):585-589(in Chinese)
Zhao WX,Jin MH,Li T,Wang YJ,Quan JS,2013.Intervention effect of aqueous fractions from Boschniakia rossica on hepatic oxidative stress in mice with liver injury induced by carbon tetrachloride.China Journal of Chinese Materia Medica,38(6):875-878(in Chinese)
白靓,姜本贺,石俊仕,史衍席,高燕,2015.联苯双酯治疗抗痨药物性肝损害的疗效研究.中国热带医学,15(7):869-871
戴玉成,李玉,2011.中国六种重要药用真菌名称的说明.菌物学报,30(4):515-518
邓青芳,周欣,陈华国,2016.多糖抗肝损伤作用及其机制研究进展.中国中药杂志,41(16):2958-2966
杜秀菊,张劲松,贾薇,刘艳芳,周帅,2012.北虫草醇提物体外抗肿瘤和抗病毒活性的比较研究.食品工业,184(1):127-129
郭燕华,2014.硫普罗宁所致不良反应特点及相关因素分析.临床合理用药杂志,7(7):92-93
侯金鑫,张金秀,赵晓静,段硕楠,刘红霞,管振龙,王立安,2014.蛹虫草液体发酵产物冻干粉的抗氧化活性及保肝作用.药物评价研究,37(1):25-29
李勇,孔令青,高洪,严玉霖,2008.自由基与疾病研究进展.动物医学进展,29(4):85-87
李仪奎,金若敏,2006.中药药理实验方法学.上海:上海科学技术出版社.515-529
梁慧嘉,焦春伟,杨咏善,张命龙,谢意珍,2015.4种珍稀食用菌水提物的抗氧化活性研究.微生物学杂志,35(4):106-109
刘亚锋,2011.氧化应激与肝脏保护.胃肠病学和肝病学杂志,20(7):594-597
刘建兵,戚梦,杜苑如,傅俊生,胡开辉,2018.拆分网络分析22株蛹虫草亲缘关系及高产虫草素菌株初筛.生物技术通报,34(4):133-138
卢彩会,郭晓萌,牟德华,2016.黑皮鸡枞菌水提物和醇提物的主要成分及抗氧化性研究.食品工程,3:35-40
吕国英,潘慧娟,吴永志,范雷法,2009.蛹虫草无性型菌丝体提取液体外抗氧化活性研究.菌物学报,28(4):597-602
邵颖,陈安徽,张传丽,周扬,2015.蛹虫草子实体中抗氧化活性化合物的分离纯化.食品科学,36(8):175-180
孙军德,侯静,杨逸,王鹤,2012.富硒蛹虫草水浸提物的体外抗氧化活性研究.沈阳农业大学学报,43(2):179-183
文庭池,查岭生,康冀川,Kevin D.HYDE,2017.蛹虫草研究和开发过程中的一些问题和展望.菌物学报,36(1):14-27
吴娜,蔡光明,何群,2008.氧化应激与肝脏损伤.世界华人消化杂志,16(29):3310-3315
辛晓林,2001.四氧嘧啶对小鼠肝指数和MDA含量的影响.青海师范大学学报(自然科学版),16(1):53-54
尹连红,于浩,彭金咏,2015.四氯化碳诱导肝损伤的分子机制及中药干预的研究进展.中国现代应用药学,32(9):1147-1155
张曦文,赵博,张国财,岳峥嵘,张国珍,邹传山,2017.白化型蛹虫草活性成分分析及抗氧化活性测定.吉林农业大学学报,39(5):585-589
赵文玺,金梅花,李天,王玉娇,全吉淑,2013.草苁蓉水萃取物对四氯化碳致肝损伤小鼠肝脏氧化应激的干预作用.中国中药杂志,38(6):875-878