板蓝根微粉水提物抗大肠杆菌活性及其机制的探究

doi:10.16431/j.cnki.1671-7236.2020.09.032

摘要/Abstract

摘要： 为了探究板蓝根微粉水提物的抗菌活性及其抗菌机制，试验通过扫描电镜、透射电镜检测板蓝根微粉水提物对大肠杆菌形态和结构影响；酶标仪测定板蓝根微粉对大肠杆菌电导率、胞内物质总漏出率影响；测定大肠杆菌培养液中碱性磷酸酶含量以及大肠杆菌菌体内、外蛋白质含量；通过DAPI染色DNA、RNA，检测板蓝根微粉水提物对大肠杆菌核酸的影响；检测板蓝根微粉水提物对大肠杆菌菌体内、外谷丙转氨酶（ALT）、谷草转氨酶（AST）、丙酮酸以及三磷酸腺苷（ATP）等菌体内代谢的影响。结果显示，板蓝根微粉水提物作用大肠杆菌10 h后，扫描电镜检测可见菌体出现溢缩，菌体长度明显变短，断裂形成许多残体，有的中间凹陷，发生变形；透射电镜下可观察大肠杆菌的胞壁界限模糊不清，壁膜呈现锯齿状，弯弯曲曲，变形，有的菌体破碎。总漏出率、电导率以及碱性磷酸酶含量测定结果显示，板蓝根微粉水提物各组D_{600 nm}均高于空白对照组，且呈剂量依赖性。菌体外蛋白质含量从8 h开始与空白对照组差异极显著（P<0.01）；菌体内蛋白质含量从4 h开始与空白对照组差异极显著（P<0.01）。DNA含量在12 h前与空白对照组无显著差异（P>0.05），从16 h开始与空白对照组差异极显著（P<0.01）；RNA含量在8 h开始降低，在12 h时与空白对照组差异显著（P<0.05），从16 h开始差异极显著（P<0.01）。ALT和AST浓度测定无显著性差异（P>0.05）。培养液和菌体内的丙酮酸含量均高于空白对照组，且从4 h开始与空白对照组差异极显著（P<0.01）。培养液中的ATP含量与空白对照组差异极显著（P<0.01）；菌体内ATP含量从4 h开始与空白对照组差异极显著（P<0.01）。综上，板蓝根微粉水提物可以通过破坏细胞壁、细胞膜的完整性，抑制细菌遗传物质合成和代谢，影响丙酮酸和ATP含量从而实现抗大肠杆菌作用。

关键词: 板蓝根; 抗菌作用; 大肠杆菌; 抗菌机制

Abstract: In order to explore the antibacterial activity and mechanism of the Radix isatidis powder water extract,the effects of the Radix isatidis powder water extract on the morphology and structure of Escherichia coli (E.coli) were tested by scanning electron microscopy and transmission electron microscopy.The effects of the Radix isatidis powder water extract on the conductivity and total leakage rate of E.coli and the content of alkaline phosphatase in the culture medium of the content of protein,DNA and RNA were stained by DAPI to detect the effect of the Radix isatidis powder water extract on the nucleic acid of E.coli,and the effect of the Radix isatidis powder water extract on the metabolism of E.coli in vivo,in vitro,ALT,AST,pyruvic acid and ATP.The results showed that after the Radix isatidis powder water extract acted on E.coli for 10 h,it was observed by SEM that the bacteria appeared to overflow and shrink,the length of the bacteria became shorter obviously,many residues were formed due to breakage,some of them were sunken in the middle and deformed.Under TEM,it was observed that the boundary of the cell wall of E.coli was unclear,the wall membrane was zigzag,deformed and some of the bacteria were broken.The protein content outside the cell was significantly different from that in the blank control group from 8 h (P<0.01), and that in the cell from 4 h (P<0.01). DNA content had no significant difference with blank control group before 12 h (P>0.05), but had significant difference with blank control group from 16 h (P<0.01); RNA content began to decrease at 8 h, and was significantly different from blank control group at 12 h (P<0.05), and was extremely significant from 16 h (P<0.01). There was no significant difference between ALT and AST (P>0.05). The pyruvate content in culture medium and bacteria was higher than that in blank control group, and the difference was very significant from 4 h (P<0.01). The ATP content in the culture medium was significantly different from that in the blank control group (P<0.01), and the ATP content in the cell was significantly different from that in the blank control group from 4 h (P<0.01).In conclusion,the Radix isatidis powder water extract could inhibit the synthesis and metabolism of bacterial genetic material and the content of pyruvate and ATP by destroying the integrity of cell wall and cell membrane.

Key words: Radix isatidis; antimicrobial activity; Escherichia coli; antimicrobial mechanism

中图分类号:

S853

姜晓文, 燕鸽, 李叔洪, 于文会, 魏佳宇, 艾莉雯, 白文. 板蓝根微粉水提物抗大肠杆菌活性及其机制的探究[J]. 中国畜牧兽医, 2020, 47(9): 2968-2978.

JIANG Xiaowen, YAN Ge, LI Shuhong, YU Wenhui, WEI Jiayu, AI Liwen, BAI Wen. Study on the Anti-Escherichia coli Activity and Mechanism of Water Extract of Radix isatidis Powder[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(9): 2968-2978.

参考文献

[1] CHUNG Y C,TANG F Y,LIAO J W,et al.Isatis indigotica induces hepatocellular cancer cell death via caspase-independent apoptosis-inducing factor transloca-tion apoptotic pathway in vitro and in vivo[J].Integrative Cancer Therapies,2011,10(2):201.
[2] DU Z,LIU H,ZHANG Z,et al.Antioxidant and anti-inflammatory activities of Radix isatidis,poly-saccharide in murine alveolar macrophages[J].International Journal of Biological Macromolecules,2013,58:329.
[3] HO Y L,CHANG Y S.Studies on the antinociceptive,anti-inflammatory and anti pyretic effects of Isatis indigotica root[J].Phytomedicine,2002,9(5):419-424.
[4] 耿飞,王伟,周涛.乌梅提取液对李斯特菌的抑菌机理[J].食品科学,2011,15:88-93. GENG F,WANG W,ZHOU T.Antibacterial mechanism of Wumei extract on Listeria[J].Food Science,2011,15:88-93.(in Chinese)
[5] 王彦芳,张颖颖.大青叶、板蓝根、青黛不同提取物对4种临床常见耐药菌株抑菌活性研究[A].世界中医药学会联合会中医药抗病毒研究专业委员会学术年会[C].2015. WANG Y F,ZHANG Y Y.Study on the bacteriostatic activity of different extracts from Folium isatidis,Radix isatidis and Indigo naturalis against four common clinical drug-resistant strains[A].Academic Annual Meeting of the Chinese Medicine Antiviral Research Professional Committee of the World Federation of Chinese Medicine Societies[C].2015.(in Chinese)
[6] 肖莉春,孔祥峰,郭小权.中药抑菌成分及其在养猪生产中的应用前景[J].动物营养学报,2012,24(12):2311-2318. XIAO L C,KONG X F,GUO X Q.Antibacterial components of traditional Chinese medicine and its application prospect in pig production[J].Journal of Animal Nutrition,2012,24(12):2311-2318.(in Chinese)
[7] 农石生,龚子龙,周金花,等.板蓝根抗氧化成分及抗氧化性能研究[J].中国野生植物资源,2017,36(3):18-22. NONG S S,GONG Z L,ZHOU J H,et al.Study on antioxidant components and antioxidant properties of Radix isatidis[J].Chinese Wild Plant Resources,2017,36(3):18-22.(in Chinese)
[8] 彭爱红.板蓝根药理活性成分及临床应用进展[J].中国当代医药,2010,17(12):13-14. PENG A H.Progress in pharmacological active components and clinical application of isatis root[J].Chinese Contemporary Medicine,2010,17(12):13-14.(in Chinese)
[9] WU Y,ZHANG Z X,HU H,et al.Novel indole C-glycosides from Isatis indigotica and their potential cytotoxic activity[J].Fitoterapia,2011,82(2):288-292.
[10] 王唯霖.中草药提取物的抑菌机理及对大肠杆菌的耐药性研究[D].保定:河北农业大学,2015. WANG W L.Antibacterial mechanism of Chinese herbal extracts and their resistance to Escherichia coli[D].Baoding:Hebei Agricultural University,2015.(in Chinese)
[11] 王译鹤,丁一冉,陈海琴,等.丁香抑菌活性及机理的初步研究[J].中国科技论文在线,2017,2:1-9. WANG Y H,DING Y R,CHEN H Q,et al.Preliminary study on bacteriostatic activity and mechanism of Syringa[J].Chinese Scientific Papers Online,2017,2:1-9.(in Chinese)
[12] 刘盛,陈万生,乔传卓,等.不同种质板蓝根和大青叶抗甲型流感病毒作用[J].第二军医大学学报,2000,21(3):204-206. LIU S,CHEN W S,QIAO C Z,et al.Anti influenza a virus effects of different germplasm Radix isatidis and Folium isatidis[J].Journal of the Second Military Medical University,2000,21(3):204-206.(in Chinese)
[13] MILARDI G L,STRINGARO A,COLONE M,et al.The cell membrane is the main target of resveratrol as shown by interdisciplinary biomolecular/cellular and biophysical approaches[J].Journal of Membrane Biology,2014,247(1):1-8.
[14] VAIDYANATHAN S,ORR B G,BANASZNK HOLL M M.Role of cell membrane-vector interactions in successful gene delivery[J].Accounts of Chemical Research,2016,49(8):1486-1493.
[15] TROWSDALE J,MARTIN D,BICKNELL D,et al.Alkaline phosphatases[J].Biochemical Society Transac-tions,1990,18(2):178-180.
[16] HARA S,YAMAKAWA M.A novel antibacterial peptide family isolated from the silkworm,Bombyx mori[J].Biochemical Journal,1995,310(2):651-656.
[17] DUNKER A K,BROWN C J,LAWSON J D,et al.Intrinsic disorder and protein function[J].Bioche-mistry,2002,41(21):6573-6582.
[18] 孙燕杰.马齿苋提取物对产ESBLs大肠杆菌体外抑菌作用及抑菌机理的研究[D].南宁:广西大学,2016. SUN Y J.Study on antibacterial effect and mechanism of purslane extract on ESBLs producing Escherichia coli in vitro[D].Nanning:Guangxi University,2016.(in Chinese)
[19] EYNARD S E,WINDIG J J,HIEMSTRA S J,et al.Whole-genome sequence data uncover loss of genetic diversity due to selection[J].Genetics Selection Evolu-tion,2016,48(1):33.
[20] 董璐,代增英,韩晴,等.茶多酚对大肠杆菌抑菌机理的研究[J].生物学杂志,2015,1:72-75. DONG L,DAI Z Y,HAN Q,et al.Study on the antibacterial mechanism of tea polyphenols against Escherichia coli[J].Journal of Biology,2015,1:72-75.(in Chinese)
[21] SAUNIER E,BENELLI C,BORYOLI S.The pyruvate dehydrogenase complex in cancer:An old metabolic gatekeeper regulated by new pathways and pharmacological agents[J].International Journal of Cancer,2016,138(4):809-817.
[22] 王佶.大肠杆菌积累丙酮酸的研究[D].杭州:浙江大学,2008. WANG J.Study on the accumulation of pyruvate by Escherichia coli[D].Hangzhou:Zhejiang University,2008.(in Chinese)
[23] 罗艺晨,黄利明,杨颖,等.绿原酸抑制金黄色葡萄球菌机理研究[J].西南大学学报(自然科学版),2016,38(3):15-19. LUO Y C,HUANG L M,YANG Y,et al.Mechanism of chlorogenic acid inhibiting Staphylococcus aureus[J].Journal of Southwest University (Natural Science Edition),2016,38(3):15-19.(in Chinese)
[24] MIDZAK A S,CHEN H,AON M A,et al.ATP synthesis,mitochondrial function,and steroid biosynthesis in rodent primary and tumor leydig cells[J].Biology of Reproduction,2016,84(5):976-985.
[25] WITTING I,SCHAGGER H.Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes[J].Biochimica et Biophysica Acta Bioenergetics,2009,1787(6):672-680.
[26] 周本宏,松长青,姜姗,等.地榆鞣质提取物的抗菌活性及对金黄色葡萄球菌的抑菌机制研究[J].中国药师,2016,19(3):464-469. ZHOU B H,SONG C Q,JIANG S,et al.Antibacterial activity of Sanguisorba tannin extract and its mechanism against Staphylococcus aureus[J].Chinese Pharmacist,2016,19(3):464-469.(in Chinese)