向日葵茎髓提取物的抑菌活性及机制
|
摘要
以向日葵茎髓为原料,制得3种不同极性向日葵茎髓提取物。采用琼脂扩散法测定3种不同极性向日葵茎髓提取物对4种受试菌的抑菌活性;并以其中的金黄色葡萄球菌和大肠杆菌为研究对象,探讨了乙酸乙酯萃取部位(EE)的抑菌机制。结果显示:不同极性萃取部位对4种受试菌均有一定抑制作用。其中,EE处理金黄色葡萄球菌和大肠杆菌的最低抑菌质量浓度(MIC)均为0.78 g/L,半数抑制质量浓度(IC50)分别为(1.11?0.01)和(1.59?0.03)g/L;抑菌作用与其总酚、总黄酮含量呈显著正相关性(P<0.05)。通过SEM分析发现,EE处理引起菌体形态变化,破坏菌体的细胞膜结构;EE可增加金黄色葡萄球菌和大肠杆菌细胞膜结构的通透性,使菌体内的电解质、蛋白质、碱性磷酸酶向胞外泄露;应用4?,6-二脒基-2-苯基吲哚(DAPI)染色和荧光检测发现,EE能干扰细菌遗传物质(DNA)的复制,最终抑制受试菌增殖。
Three different polarity extracts were prepared from sunflower stalk pith. The agar diffusion assay was used to investigate the antibacterial activities of the extracts against four tested bacteria, and the action mechanism of the ethyl acetate extract(EE) against Staphylococcus aureus and Escherichia coli was discussed. The results showed that all extracts had certain bacteriostatic effects on the four tested bacteria.The minimum inhibitory concentration(MIC) values of both Staphylococcus aureus and Escherichia coli treated by EE were 0.78 g/L, and the corresponding IC50 values were(1.11±0.01) and(1.59±0.03) g/L,respectively. There was positive correlation between the antibacterial activities and the content of total phenols and total flavonoids(P<0.05). The damaged membrane structures induced by EE were observed by SEM. EE also increased the permeability of the cell wall and cell membrane of the tested bacterial strains with the release of the electrolytes, proteins and alkline phosphatase(AKP) in the cell. Finally, the results of4?,6-diamidino-2-phenylindole(DAPI) staining assay revealed that EE could inhibit the replication of bacterial genetic material(DNA) and ultimately inhibit bacterial reproduction.
Three different polarity extracts were prepared from sunflower stalk pith. The agar diffusion assay was used to investigate the antibacterial activities of the extracts against four tested bacteria, and the action mechanism of the ethyl acetate extract(EE) against Staphylococcus aureus and Escherichia coli was discussed. The results showed that all extracts had certain bacteriostatic effects on the four tested bacteria.The minimum inhibitory concentration(MIC) values of both Staphylococcus aureus and Escherichia coli treated by EE were 0.78 g/L, and the corresponding IC50 values were(1.11±0.01) and(1.59±0.03) g/L,respectively. There was positive correlation between the antibacterial activities and the content of total phenols and total flavonoids(P<0.05). The damaged membrane structures induced by EE were observed by SEM. EE also increased the permeability of the cell wall and cell membrane of the tested bacterial strains with the release of the electrolytes, proteins and alkline phosphatase(AKP) in the cell. Finally, the results of4?,6-diamidino-2-phenylindole(DAPI) staining assay revealed that EE could inhibit the replication of bacterial genetic material(DNA) and ultimately inhibit bacterial reproduction.
引文
[1]Flora of China Editorial Committee,Chinese Academy of Sciences(中国科学院中国植物志编委会).Flora of China[M].Beijing:Science Press(科学出版社),1979:357.
[2]Liu Haimin(刘海敏).Development trend of edible sunflower[J].Modern Agriculture(现代农业),2018,2:56.
[3]Nanjing University of Chinese Medicine(南京中医药大学).Chinese medicine dictionary(中药大辞典第二版)[M].Shanghai:Shanghai Scientific&Technical Publishers(上海科学技术出版社),2010:1287-1288.
[4]Martelli G,Giacomini D.Antibacterial and antioxidant activities for natural and synthetic dual-activecompounds[J].European Journal of Medicinal Chemistry,2018,158:91-105.
[5]Newman D J,Cragg G M.Natural products as sources of new drugs from 1981 to 2014[J].Journal of Natural Products,2016,79(3):629-661.
[6]Silva L N,Rigon Zimmer K R,Macedo A J,et al.Plant natural product stargeting bacterial virulence factors[J].Chemical Reviews,2016,116(16):9162-9236.
[7]Rossiter S E,Fletcher M H,Wuest W M,et al.Natural products as platforms to overcome antibiotic resistance[J].Chemical Reviews,2017,117(19):12415-12474.
[8]Ben Yakoub A R,Abdehedi O,Jridi M,et al.Flavonoids,phenols,antioxidant,and antimicrobial activities in various extracts from Tossa jute leave(Corchorus olitorus L.)[J].Industrial Crops and Products,2018,118:206-213.
[9]Pinheiro P F,Parreira Menini L A,Bernardes P C,et al.Semisynthetic phenol derivatives obtained from natural phenols:antimicrobial activity and molecular properties[J].Journal of Agricultural and Food Chemistry,2018,66(1):323-330.
[10]Bartmanska A,Walecka-Zacharska E,Tronina T,et al.Antimicrobial properties of spent hops extracts,flavonoids isolated therefrom,and their derivatives[J].Molecules,2018,23(8):2059-2071
[11]Seshimoto F,Hong S W,Nakajyo H,et al.New antibacterial polyacetylenes from sunflower(Helianthus annuus L.)seedlings[J].Heterocycles,2011,83(5):1067-1076.
[12]Sankaranarayanan S,Bama P,Deccaraman M,et al.Isolation and characterization of bioactive and antibacterial compound from Helianthus annuus Linn[J].Indian Journal of Experimental Biology,2008,46(12):831-835.
[13]Zhang He(张鹤),Zhang Ying(张莹),Yang Fengjian(杨逢建),et al.Correlation between total phenols and total flavonoids contents and antioxidant activity of different fractions from ethanol extract of sunflower stalk pith[J].Food Science(食品科学),2018,39(15):54-59.
[14]She Zhiyun(佘之蕴),Huang Baoying(黄宝莹),Liu Haiqing(刘海卿),et al.Antibacterial activity of foods additives against five strains of probiotics by oxford plate assay system[J].Food Industry(食品工业),2016,(1):171-174.
[15]Wang Hongyan(王红岩),Wang Lu(汪璐),Xie Kunpeng(谢鲲鹏),et al.The antimicrobial and antioxidant activity of the powder of Ganoderma lucidum[J].Chinese Journal of Biochemical Pharmaceutics(中国生化药物杂志),2017,37(3):52-54.
[16]Zhou Benhong(周本宏),Song Changqing(松长青),Jiang Shan(姜姗),et al.Antibacterial activity of sanguisorba tannin extract and mechanism of antibacterial effect on Staphylococcus aureus[J].China Pharmacist(中国药师),2016,19(3):464-469.
[17]Liang Jingnan(梁静南),Liu Yiwei(刘一苇),Xie Jiayi(谢家仪).The improvement of bacteria sample preparation method for scanning electron microscope observation[J].Journal of Chinese Electron Microscopy Society(电子显微学报),2013,32(3):276-278.
[18]Lee H,Choi G,Cho K.Correlation of lipid peroxidation in Botrytis cinerea caused by dicarboximide fungicides with their fungicidal activity[J].Journal of Agricultural and Food Chemistry,1998,46(2):737-741.
[19]Maruyama F,Yamaguchi N,Kenzaka T,et al.Simplified sample preparation using frame spotting method for direct counting of total bacteria by fluorescence microscopy[J].Journal of Microbiological Methods,2004,59(3):427-431.
[20]Bhartia R,Salas E,Hug W,et al.Label-free bacterial imaging with Deep-UV-Laser-Induced native fluorescence[J].Applied&Environmental Microbiology,2010,76(21):7231-7237.
[21]Tian Y,Puganen A,Alakomi H L,et al.Antioxidative and antibacterial activities of aqueous ethanol extracts of berries,leaves,and branches of berry plants[J].Food Research International,2018,106:291-303.
[22]SunX,Zhou T,Wei C,et al.Antibacterial effect and mechanism of anthocyanin rich Chinese wild blueberry extract on various food borne pathogens[J].Food Control,2018,94:155-161.
[23]Zhu Junli(朱军莉),Wang Huimin(王慧敏),Wang Guiyang(王桂洋),et al.Inhibition activity of tea polyphenols and grape seed extract on biofilm formation of pseudomonas[J].Journal of Chinese Institute of Food Science and Technology(中国食品学报),2016,16(1):84-90.
[24]Zheng Cuiping(郑翠萍),Quan Meiping(权美平),Kang Lina(康莉娜),et al.Study on the antibacterial activity and its mechanism of acetone extract from Rubia cordifolia[J].Science and Technology of Food Industry(食品工业科技),2015,36(9):116-119.
[25]Fatrcova-Sramkova K,Nozkova J,Mariassyova M,et al.Biologically active antimicrobial and antioxidant substances in the Helianthus annuus L.bee pollen[J].Journal of Environmental Science and Health Part B-Pesticides Food Contaminants and Agricultural Wastes,2016,51(3):176-181.
[26]Duan Lindong(段林东),Wang Xiuqun(王秀群).Extraction and antibiotic effect of chlorogenic acid from sunflower shell mea[J].Journal of Southwest Agricultural University(Natural Science)(西南农业大学学报:自然科学版),2006,28(1):124-126,130.
[27]Kumar V D,Verma P R P,Singh S K.Morphological and in vitro antibacterial efficacy of quercetin loaded nanoparticles against food-borne microorganisms[J].LWT-Food Science and Technology,2016,66:638-650.
[28]Fu Dan(付丹),Jin Yongguo(金永国),Han Mengqi(韩梦琪),et al.Studies on antibacterial activity and antibacterial mechanism of ovomucin[J].Journal of Chinese Institute of Food Science and Technology(中国食品学报),2016,16(10):28-35.
[29]Teissie J,Eynard N,Gabriel B,et al.Electropermeabilization of the cell membranes[J].Advanced Drug Delivery Reviews,1999,35:3-19.
[30]Lu Haixia(陆海霞),Huang Xiaoming(黄小鸣),Zhu Junli(朱军莉).Effect of ultrahigh pressure on cell membrane destruction and death of Listeria monocytogenes[J].Acta Microbiologica Sinica(微生物学报),2014,54(7):746-753.
[31]Li Tingting(李婷婷),Yang Bing(杨兵),Zhang Huifang(张慧芳),et al.The antimicrobial effect and the action mechanism of catfish epidermal mucus extracts[J].Journal of Chinese Institute of Food Science and Technology(中国食品学报),2017,17(4):21-29.
[32]Wang Yonggang(王永刚),Yang Guangrui(杨光瑞),Ma Xueqing(马雪青),et al.Antibacterial mechanisms of endophytic fungi Alternaria alternata of ethyl acetateextract on Escherichia coli[J].Chinese Traditional and Herbal Drugs(中草药),2018,49(2):374-381.
[33]Cho H,Uehara T,Bernhardt T.Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery[J].Cell,2014,159(6):1300-1311.
[34]Goggins S,Naz C,Marsh B,et al.Ratiometric electrochemical detection of alkaline phosphatase[J].Chemical Communications,2015,51(3):561-564.
[35]Huo Jiancong(霍健聪),Deng Shanggui(邓尚贵),Li Jianrong(励建荣).Preparation of flavonoids of Houttuyniacordata Thunb.and the antibacterial mechanismon Bacillus subtilis[J].Journal of Chinese Institute of Food Science and Technology(中国食品学报),2017,17(9):82-89.
[36]The Uniprot Consortium.UniProt:a hub for protein information[J].Nucleic Acids Research,2015,43(Database issue):D204-D212.
[37]Yuan Zhongwei(袁中伟),Chen Zhiying(陈志英),Gan Yingying(甘盈盈),et al.Antibacterial mechanism of thymol to methicillinresistant Staphylococcus aureus[J].Journal of South China Agricultural University(华南农业大学学报),2018,39(6):18-23.
[38]Li Changqin(李昌勤),Zhao Lin(赵琳),Xue Zhiping(薛志平),et al.Antibacterial mechanism of cryptotanshinone[J].Chinese Pharmaceutical Journal(中国药学杂志),2012,47(21):1706-1710.
[39]Matsuura M,Abe H,Tominaga T,et al.A novel method of DAPIstaining for differential diagnosis of renal amyloidosis[J].Journal of Medical Investigation,2017,64(3/4):217-221.
[40]Zhang Y,Feng R,Li L,et al.The antibacterial mechanism of terpinen-4-ol against Streptococcus agalactiae[J].Current Microbiology,2018,75(9):1214-1220.
[2]Liu Haimin(刘海敏).Development trend of edible sunflower[J].Modern Agriculture(现代农业),2018,2:56.
[3]Nanjing University of Chinese Medicine(南京中医药大学).Chinese medicine dictionary(中药大辞典第二版)[M].Shanghai:Shanghai Scientific&Technical Publishers(上海科学技术出版社),2010:1287-1288.
[4]Martelli G,Giacomini D.Antibacterial and antioxidant activities for natural and synthetic dual-activecompounds[J].European Journal of Medicinal Chemistry,2018,158:91-105.
[5]Newman D J,Cragg G M.Natural products as sources of new drugs from 1981 to 2014[J].Journal of Natural Products,2016,79(3):629-661.
[6]Silva L N,Rigon Zimmer K R,Macedo A J,et al.Plant natural product stargeting bacterial virulence factors[J].Chemical Reviews,2016,116(16):9162-9236.
[7]Rossiter S E,Fletcher M H,Wuest W M,et al.Natural products as platforms to overcome antibiotic resistance[J].Chemical Reviews,2017,117(19):12415-12474.
[8]Ben Yakoub A R,Abdehedi O,Jridi M,et al.Flavonoids,phenols,antioxidant,and antimicrobial activities in various extracts from Tossa jute leave(Corchorus olitorus L.)[J].Industrial Crops and Products,2018,118:206-213.
[9]Pinheiro P F,Parreira Menini L A,Bernardes P C,et al.Semisynthetic phenol derivatives obtained from natural phenols:antimicrobial activity and molecular properties[J].Journal of Agricultural and Food Chemistry,2018,66(1):323-330.
[10]Bartmanska A,Walecka-Zacharska E,Tronina T,et al.Antimicrobial properties of spent hops extracts,flavonoids isolated therefrom,and their derivatives[J].Molecules,2018,23(8):2059-2071
[11]Seshimoto F,Hong S W,Nakajyo H,et al.New antibacterial polyacetylenes from sunflower(Helianthus annuus L.)seedlings[J].Heterocycles,2011,83(5):1067-1076.
[12]Sankaranarayanan S,Bama P,Deccaraman M,et al.Isolation and characterization of bioactive and antibacterial compound from Helianthus annuus Linn[J].Indian Journal of Experimental Biology,2008,46(12):831-835.
[13]Zhang He(张鹤),Zhang Ying(张莹),Yang Fengjian(杨逢建),et al.Correlation between total phenols and total flavonoids contents and antioxidant activity of different fractions from ethanol extract of sunflower stalk pith[J].Food Science(食品科学),2018,39(15):54-59.
[14]She Zhiyun(佘之蕴),Huang Baoying(黄宝莹),Liu Haiqing(刘海卿),et al.Antibacterial activity of foods additives against five strains of probiotics by oxford plate assay system[J].Food Industry(食品工业),2016,(1):171-174.
[15]Wang Hongyan(王红岩),Wang Lu(汪璐),Xie Kunpeng(谢鲲鹏),et al.The antimicrobial and antioxidant activity of the powder of Ganoderma lucidum[J].Chinese Journal of Biochemical Pharmaceutics(中国生化药物杂志),2017,37(3):52-54.
[16]Zhou Benhong(周本宏),Song Changqing(松长青),Jiang Shan(姜姗),et al.Antibacterial activity of sanguisorba tannin extract and mechanism of antibacterial effect on Staphylococcus aureus[J].China Pharmacist(中国药师),2016,19(3):464-469.
[17]Liang Jingnan(梁静南),Liu Yiwei(刘一苇),Xie Jiayi(谢家仪).The improvement of bacteria sample preparation method for scanning electron microscope observation[J].Journal of Chinese Electron Microscopy Society(电子显微学报),2013,32(3):276-278.
[18]Lee H,Choi G,Cho K.Correlation of lipid peroxidation in Botrytis cinerea caused by dicarboximide fungicides with their fungicidal activity[J].Journal of Agricultural and Food Chemistry,1998,46(2):737-741.
[19]Maruyama F,Yamaguchi N,Kenzaka T,et al.Simplified sample preparation using frame spotting method for direct counting of total bacteria by fluorescence microscopy[J].Journal of Microbiological Methods,2004,59(3):427-431.
[20]Bhartia R,Salas E,Hug W,et al.Label-free bacterial imaging with Deep-UV-Laser-Induced native fluorescence[J].Applied&Environmental Microbiology,2010,76(21):7231-7237.
[21]Tian Y,Puganen A,Alakomi H L,et al.Antioxidative and antibacterial activities of aqueous ethanol extracts of berries,leaves,and branches of berry plants[J].Food Research International,2018,106:291-303.
[22]SunX,Zhou T,Wei C,et al.Antibacterial effect and mechanism of anthocyanin rich Chinese wild blueberry extract on various food borne pathogens[J].Food Control,2018,94:155-161.
[23]Zhu Junli(朱军莉),Wang Huimin(王慧敏),Wang Guiyang(王桂洋),et al.Inhibition activity of tea polyphenols and grape seed extract on biofilm formation of pseudomonas[J].Journal of Chinese Institute of Food Science and Technology(中国食品学报),2016,16(1):84-90.
[24]Zheng Cuiping(郑翠萍),Quan Meiping(权美平),Kang Lina(康莉娜),et al.Study on the antibacterial activity and its mechanism of acetone extract from Rubia cordifolia[J].Science and Technology of Food Industry(食品工业科技),2015,36(9):116-119.
[25]Fatrcova-Sramkova K,Nozkova J,Mariassyova M,et al.Biologically active antimicrobial and antioxidant substances in the Helianthus annuus L.bee pollen[J].Journal of Environmental Science and Health Part B-Pesticides Food Contaminants and Agricultural Wastes,2016,51(3):176-181.
[26]Duan Lindong(段林东),Wang Xiuqun(王秀群).Extraction and antibiotic effect of chlorogenic acid from sunflower shell mea[J].Journal of Southwest Agricultural University(Natural Science)(西南农业大学学报:自然科学版),2006,28(1):124-126,130.
[27]Kumar V D,Verma P R P,Singh S K.Morphological and in vitro antibacterial efficacy of quercetin loaded nanoparticles against food-borne microorganisms[J].LWT-Food Science and Technology,2016,66:638-650.
[28]Fu Dan(付丹),Jin Yongguo(金永国),Han Mengqi(韩梦琪),et al.Studies on antibacterial activity and antibacterial mechanism of ovomucin[J].Journal of Chinese Institute of Food Science and Technology(中国食品学报),2016,16(10):28-35.
[29]Teissie J,Eynard N,Gabriel B,et al.Electropermeabilization of the cell membranes[J].Advanced Drug Delivery Reviews,1999,35:3-19.
[30]Lu Haixia(陆海霞),Huang Xiaoming(黄小鸣),Zhu Junli(朱军莉).Effect of ultrahigh pressure on cell membrane destruction and death of Listeria monocytogenes[J].Acta Microbiologica Sinica(微生物学报),2014,54(7):746-753.
[31]Li Tingting(李婷婷),Yang Bing(杨兵),Zhang Huifang(张慧芳),et al.The antimicrobial effect and the action mechanism of catfish epidermal mucus extracts[J].Journal of Chinese Institute of Food Science and Technology(中国食品学报),2017,17(4):21-29.
[32]Wang Yonggang(王永刚),Yang Guangrui(杨光瑞),Ma Xueqing(马雪青),et al.Antibacterial mechanisms of endophytic fungi Alternaria alternata of ethyl acetateextract on Escherichia coli[J].Chinese Traditional and Herbal Drugs(中草药),2018,49(2):374-381.
[33]Cho H,Uehara T,Bernhardt T.Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery[J].Cell,2014,159(6):1300-1311.
[34]Goggins S,Naz C,Marsh B,et al.Ratiometric electrochemical detection of alkaline phosphatase[J].Chemical Communications,2015,51(3):561-564.
[35]Huo Jiancong(霍健聪),Deng Shanggui(邓尚贵),Li Jianrong(励建荣).Preparation of flavonoids of Houttuyniacordata Thunb.and the antibacterial mechanismon Bacillus subtilis[J].Journal of Chinese Institute of Food Science and Technology(中国食品学报),2017,17(9):82-89.
[36]The Uniprot Consortium.UniProt:a hub for protein information[J].Nucleic Acids Research,2015,43(Database issue):D204-D212.
[37]Yuan Zhongwei(袁中伟),Chen Zhiying(陈志英),Gan Yingying(甘盈盈),et al.Antibacterial mechanism of thymol to methicillinresistant Staphylococcus aureus[J].Journal of South China Agricultural University(华南农业大学学报),2018,39(6):18-23.
[38]Li Changqin(李昌勤),Zhao Lin(赵琳),Xue Zhiping(薛志平),et al.Antibacterial mechanism of cryptotanshinone[J].Chinese Pharmaceutical Journal(中国药学杂志),2012,47(21):1706-1710.
[39]Matsuura M,Abe H,Tominaga T,et al.A novel method of DAPIstaining for differential diagnosis of renal amyloidosis[J].Journal of Medical Investigation,2017,64(3/4):217-221.
[40]Zhang Y,Feng R,Li L,et al.The antibacterial mechanism of terpinen-4-ol against Streptococcus agalactiae[J].Current Microbiology,2018,75(9):1214-1220.