板蓝根微粉水提物抗大肠杆菌活性及其机制的探究

Study on the Anti-Escherichia coli Activity and Mechanism of Water Extract of Radix isatidis Powder

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D600 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。

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.