苎麻叶萃取物体外抗甲型流感病毒(H1N1)和抗氧化活性测定
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摘要
目的:研究苎麻叶不同溶剂萃取物对甲型流感病毒(H1N1)的体外抑制作用和抗氧化活性,以扩大苎麻植物的药用部位并开发天然抗病毒、抗氧化药物。方法:取苎麻叶以95%乙醇提取,分别用水加热溶解醇提物浸膏,再以不同溶剂分别萃取,制得苎麻叶石油醚相、三氯甲烷相、乙酸乙酯相、正丁醇相、水相萃取物。考察50～400μg/mL的苎麻叶水相萃取物对犬肾细胞(MDCK)的毒性作用;以利巴韦林为阳性对照,采用甲型流感病毒(H1N1)攻击MDCK细胞,并采用Western blotting法检测相同质量浓度的苎麻叶石油醚相、三氯甲烷相、乙酸乙酯相、正丁醇相、水相萃取物溶液(均为100μg/mL),不同质量浓度的苎麻叶水相萃取物溶液(50、100、200、400μg/mL)以及不同质量浓度的利巴韦林溶液(0.31、0.63、1.25μg/mL)对病毒感染的细胞中核蛋白(NP)表达水平。以维生素C为阳性对照,采用羟基自由基(·OH)清除试验、DPPH自由基清除试验和还原试验考察各相萃取物的体外抗氧化活性。结果:苎麻叶水相萃取物质量浓度在400μg/mL以内时对MDCK细胞无毒性;苎麻叶石油醚相、三氯甲烷相、乙酸乙酯相、水相萃取物质量浓度在100μg/mL时均能显著降低甲型流感病毒(H1N1)感染的细胞中NP蛋白表达水平(P<0.01);而不同质量浓度(50～400μg/mL)的水相萃取物均能显著降低NP蛋白表达水平(P<0.01),且呈一定浓度依赖趋势;其石油醚相和乙酸乙酯相萃取物的抗氧化活性与维生素C较为接近。结论:苎麻叶水相萃取物具有较好的体外抗甲型流感病毒(H1N1)作用;其石油醚相和乙酸乙酯相萃取物表现出较好的体外抗氧化活性。
OBJECTIVE:To study the in vitro inhibitory effects and antioxidant activity of different solvent extracts of Boehmeria nivea leaves against influenza A virus(H1N1),and to expand the medicinal parts of B. nivea and develop natural antiviral and antioxidant drugs. METHODS:The leaves of B. nivea were extracted with 95% ethanol. The ethanol extract was dissolved by water heating,and extracted with different solvents to obtain petroleum ether phase,trichloromethane phase,ethyl acetate phase,n-butanol phase and aqueous phase extracts of B. nivea leaves. The toxicity of aqueous extract of B. nivea leaves(50-400 μg/mL)on Madin-Darby canine kidney(MDCK)cell line was investigated. Using ribavirin as positive control,MDCK cells were attacked by influenza A virus(H1N1). Western blotting assay was used to detect the expression of nucleoproteins(NP)in viral infected cells after treated with same concentrations of petroleum ether phase,trichloromethane phase,ethyl acetate phase,n-butanol phase and aqueous phase extracts of B. nivea leaves(100 μg/mL),different concentrations of aqueous phase extract solution of B. nivea leaves(50,100,200,400 μg/m L)and different concentrations of ribavirin solution(0.31,0.63,1.25 μg/m L).Using vitamin C as a positive control,hydroxyl radical(·OH)scavenging test,DPPH radical scavenging test and reduction test were used to investigate in vitro antioxidant activity of the extracts. RESULTS:Aqueous phase extract of B. nivea leaves with concentration less than 400 μg/mL was nontoxic to MDCK cells. The petroleum ether phase,trichloromethane phase,ethyl acetate phase and aqueous phase extracts at 100 g/mL could significantly reduce the expression of NP protein in influenza A virus(H1N1)infected cells(P<0.01). Different concentrations(50-400 μg/mL) of aqueous extract could significantly reduce the protein expression of NP(P<0.01)in concentration-dependent manner. The in vitro antioxidant activity of petroleum ether phase and ethyl acetate phase was similar to that of vitamin C. CONCLUSIONS:B. nivea leaves extract have better anti-influenza A virus(H1N1)effects in vitro,and the extracts of petroleum ether phase and ethyl acetate phase show good antioxidant activity in vitro.
OBJECTIVE:To study the in vitro inhibitory effects and antioxidant activity of different solvent extracts of Boehmeria nivea leaves against influenza A virus(H1N1),and to expand the medicinal parts of B. nivea and develop natural antiviral and antioxidant drugs. METHODS:The leaves of B. nivea were extracted with 95% ethanol. The ethanol extract was dissolved by water heating,and extracted with different solvents to obtain petroleum ether phase,trichloromethane phase,ethyl acetate phase,n-butanol phase and aqueous phase extracts of B. nivea leaves. The toxicity of aqueous extract of B. nivea leaves(50-400 μg/mL)on Madin-Darby canine kidney(MDCK)cell line was investigated. Using ribavirin as positive control,MDCK cells were attacked by influenza A virus(H1N1). Western blotting assay was used to detect the expression of nucleoproteins(NP)in viral infected cells after treated with same concentrations of petroleum ether phase,trichloromethane phase,ethyl acetate phase,n-butanol phase and aqueous phase extracts of B. nivea leaves(100 μg/mL),different concentrations of aqueous phase extract solution of B. nivea leaves(50,100,200,400 μg/m L)and different concentrations of ribavirin solution(0.31,0.63,1.25 μg/m L).Using vitamin C as a positive control,hydroxyl radical(·OH)scavenging test,DPPH radical scavenging test and reduction test were used to investigate in vitro antioxidant activity of the extracts. RESULTS:Aqueous phase extract of B. nivea leaves with concentration less than 400 μg/mL was nontoxic to MDCK cells. The petroleum ether phase,trichloromethane phase,ethyl acetate phase and aqueous phase extracts at 100 g/mL could significantly reduce the expression of NP protein in influenza A virus(H1N1)infected cells(P<0.01). Different concentrations(50-400 μg/mL) of aqueous extract could significantly reduce the protein expression of NP(P<0.01)in concentration-dependent manner. The in vitro antioxidant activity of petroleum ether phase and ethyl acetate phase was similar to that of vitamin C. CONCLUSIONS:B. nivea leaves extract have better anti-influenza A virus(H1N1)effects in vitro,and the extracts of petroleum ether phase and ethyl acetate phase show good antioxidant activity in vitro.
引文
[1]廖丽萍,肖爱平,冷鹃,等.苎麻根、叶化学成分及药用研究概况[J].中国麻业科学,2013(3):163-166.
[2]陈保锋,陈建华,母波,等.苎麻药用研究进展[J].中国麻业科学,2016(5):237-241.
[3]HUANG KL,LAI YK,LIN CC,et al.Involvement of GRP78 in inhibition of HBV secretion by Boehmeria nivea extract in human HepG2.2.15 cells[J].J Viral Hepatitis,2009,16(5):367-375.
[4]闵勇,张薇,王洪.水苎麻叶挥发性成分分析及其抗菌活性研究[J].食品工业科技,2011,32(7):86-88.
[5]张贤,陈悟,周文聪,等.苎麻根抗氧化活性部位研究[J].时珍国医国药,2011,22(4):896-898.
[6]张宏琦,邹冲,汪签植,等.苎麻叶抗炎活性部位研究[J].中国民族医药杂志,2009,15(4):37-39.
[7]WEI J,LIN L,SU X,et al.Anti-hepatitis B virus activity of Boehmeria nivea leaf extracts in human HepG.2.2.15cells[J].Biomed Rep,2014,2(1):147-151.
[8]ZHANG WQ,SONG SX,WANG TZ.Advances in the structure and function of pandemic A/H1N1/2009 influenza virus HA protein[J].Chin J Virol,2012,28(4):444-452.
[9]SEO YJ,PRITZL CJ,VIJAYAN M,et al.Sphingosine kinase 1 serves as a pro-viral factor by regulating viral RNAsynthesis and nuclear export of viral ribonucleoprotein complex upon influenza virus infection[J].PLoS One,2013.DOI:10.1371/journal.pone.0075005.
[10]PROKUDINA EN,SEMENOVA N,CHUMAKOV V,et al.An antigenic epitope of influenza virus nucleoprotein(NP)associated with polymeric forms of NP[J].Virol J,2008.DOI:10.1186/1743-422X-5-37.
[11]ROVITO HA,OBLONG JE.Nicotinamide preferentially protects glycolysis in dermal fibroblasts under oxidative stress conditions[J].Br J Dermatol,2013.DOI:10.1111/bjd.12365.
[12]LIN CC,YEN MH,LO TS,et al.Evaluation of the hepatoprotective and antioxidant activity of Boehmeria nivea,var.nivea,and B.nivea,var.tenacissima[J].J Ethnopharmacol,1998,60(1):9-17.
[13]关文达,杨子峰,刘妮,等.白藜芦醇对多种呼吸道病毒作用体外实验研究[J].中药材,2008,31(9):1388-1390.
[14]NENCIONI L,DE CG,SGARBANTI R,et al.Bcl-2 expression and p38MAPK activity in cells infected with influenza A virus:impact on virally induced apoptosis and viral replication[J].J Biol Chem,2009,284(23):16004-16015.
[15]张贤.苎麻根黄酮类成分分离纯化及抗氧化活性研究[D].武汉:武汉纺织大学,2011.
[16]贺波.苎麻叶黄酮的提取、分离纯化、结构及抗氧化活性的研究[D].武汉:华中农业大学,2010.
[17]CHEN R,LIU Z,ZHAO J,et al.Antioxidant and immunobiological activity of water-soluble polysaccharide fractions purified from Acanthopanax senticosu[J].Food Chem,2011,127(2):434-440.
[18]BOULO S,AKARSU H,RUIGROK RW,et al.Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes[J].Virus Res,2007,124(1/2):12-21.
[19]许本善,雷宁,吕亚丽,等.雪灵芝醇提物不同极性萃取部分的体外抗氧化活性[J].中国实验方剂学杂志,2015,21(2):61-64.
[20]张彦,郭增军,张寒,等.八种植物挥发油的抗氧化活性比较研究[J].中国食品添加剂,2017(8):49-54.
[2]陈保锋,陈建华,母波,等.苎麻药用研究进展[J].中国麻业科学,2016(5):237-241.
[3]HUANG KL,LAI YK,LIN CC,et al.Involvement of GRP78 in inhibition of HBV secretion by Boehmeria nivea extract in human HepG2.2.15 cells[J].J Viral Hepatitis,2009,16(5):367-375.
[4]闵勇,张薇,王洪.水苎麻叶挥发性成分分析及其抗菌活性研究[J].食品工业科技,2011,32(7):86-88.
[5]张贤,陈悟,周文聪,等.苎麻根抗氧化活性部位研究[J].时珍国医国药,2011,22(4):896-898.
[6]张宏琦,邹冲,汪签植,等.苎麻叶抗炎活性部位研究[J].中国民族医药杂志,2009,15(4):37-39.
[7]WEI J,LIN L,SU X,et al.Anti-hepatitis B virus activity of Boehmeria nivea leaf extracts in human HepG.2.2.15cells[J].Biomed Rep,2014,2(1):147-151.
[8]ZHANG WQ,SONG SX,WANG TZ.Advances in the structure and function of pandemic A/H1N1/2009 influenza virus HA protein[J].Chin J Virol,2012,28(4):444-452.
[9]SEO YJ,PRITZL CJ,VIJAYAN M,et al.Sphingosine kinase 1 serves as a pro-viral factor by regulating viral RNAsynthesis and nuclear export of viral ribonucleoprotein complex upon influenza virus infection[J].PLoS One,2013.DOI:10.1371/journal.pone.0075005.
[10]PROKUDINA EN,SEMENOVA N,CHUMAKOV V,et al.An antigenic epitope of influenza virus nucleoprotein(NP)associated with polymeric forms of NP[J].Virol J,2008.DOI:10.1186/1743-422X-5-37.
[11]ROVITO HA,OBLONG JE.Nicotinamide preferentially protects glycolysis in dermal fibroblasts under oxidative stress conditions[J].Br J Dermatol,2013.DOI:10.1111/bjd.12365.
[12]LIN CC,YEN MH,LO TS,et al.Evaluation of the hepatoprotective and antioxidant activity of Boehmeria nivea,var.nivea,and B.nivea,var.tenacissima[J].J Ethnopharmacol,1998,60(1):9-17.
[13]关文达,杨子峰,刘妮,等.白藜芦醇对多种呼吸道病毒作用体外实验研究[J].中药材,2008,31(9):1388-1390.
[14]NENCIONI L,DE CG,SGARBANTI R,et al.Bcl-2 expression and p38MAPK activity in cells infected with influenza A virus:impact on virally induced apoptosis and viral replication[J].J Biol Chem,2009,284(23):16004-16015.
[15]张贤.苎麻根黄酮类成分分离纯化及抗氧化活性研究[D].武汉:武汉纺织大学,2011.
[16]贺波.苎麻叶黄酮的提取、分离纯化、结构及抗氧化活性的研究[D].武汉:华中农业大学,2010.
[17]CHEN R,LIU Z,ZHAO J,et al.Antioxidant and immunobiological activity of water-soluble polysaccharide fractions purified from Acanthopanax senticosu[J].Food Chem,2011,127(2):434-440.
[18]BOULO S,AKARSU H,RUIGROK RW,et al.Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes[J].Virus Res,2007,124(1/2):12-21.
[19]许本善,雷宁,吕亚丽,等.雪灵芝醇提物不同极性萃取部分的体外抗氧化活性[J].中国实验方剂学杂志,2015,21(2):61-64.
[20]张彦,郭增军,张寒,等.八种植物挥发油的抗氧化活性比较研究[J].中国食品添加剂,2017(8):49-54.