复方双黄连制剂体外抗菌抗病毒活性研究

doi:10.16431/j.cnki.1671-7236.2022.12.031

摘要/Abstract

摘要： 【目的】探究复方双黄连制剂体外抗菌抗病毒活性，为复方双黄连的深度开发及家禽、家畜合理选择用药提供科学依据。【方法】将金银花、黄芩、连翘颗粒分别制成浸膏，再将药物浸膏及穿心莲颗粒按照比例制成穿心莲、双黄连（金银花：黄芩：连翘=1：1：2）和复方双黄连（金银花：黄芩：连翘：穿心莲=1：1：2：2）口服液，调节pH为7.0，生药浓度为100%。用大肠杆菌、沙门氏菌、金黄色葡萄球菌的12个菌株进行细菌敏感性检测，采用牛津杯结合试管二倍稀释法筛选出敏感菌株，根据药物的最小抑菌浓度（MIC）探讨其体外抗菌活性；采用二倍稀释法稀释药物，培养恒河猴胚肾细胞（MA-104）、鸡胚成纤维细胞（DF-1），用CCK-8法检测细胞活力，结合细胞病变（CPE）情况确定3种药物安全浓度，将最大药物安全浓度以二倍稀释法稀释3个梯度，用新城疫病毒、轮状病毒对MA-104和DF-1进行药物+病毒互作、先加药后攻毒和先攻毒后加药3种方式的处理，用CCK-8法检测细胞活力，并结合CPE探讨药物的体外抗病毒活性。【结果】复方双黄连的抗菌效果优于穿心莲和双黄连，其对大肠杆菌、沙门氏菌和金黄色葡萄球菌的MIC分别为0.20~1.56、0.20~1.56和0.78~1.56 mg/mL。CPE和细胞活力的测定结果表明，双黄连、穿心莲、复方双黄连对DF-1细胞的安全浓度范围分别为0~6.25、0~0.78和0~0.78 mg/mL，对MA-104细胞的安全浓度范围分别为0~3.13、0~0.78和0~0.78 mg/mL。新城疫病毒17E株对DF-1细胞的TCID₅₀为10^-6.19/100 μL；牦牛轮状病毒G6P[1]型SDA2株对MA-104细胞的TCID₅₀为10^-6.24/100 μL。复方双黄连通过直接灭活病毒（药物+病毒互作）、阻断病毒吸附（先加药后攻毒）及干扰病毒复制（先攻毒后加药）途径对新城疫病毒的有效抑制率分别为28.82%、55.92%、42.45%，对轮状病毒的有效抑制率分别为48.84%、26.52%、15.40%。【结论】复方双黄连的抗菌抗病毒作用均强于双黄连和穿心莲，且其对大肠杆菌、沙门氏菌的抗菌效果优于金黄色葡萄球菌，研究结果可为复方双黄连在兽医临床应用和深入开发提供科学依据。

关键词: 复方双黄连; 抗菌活性; 抗病毒活性

Abstract: 【Objective】 The aim of this study was to investigate the in vitro antibacterial and antiviral activity of the compound Shuanghuanglian preparation, and provide a scientific basis for the in-depth development of compound Shuanghuanglian and the rational selection of drugs for poultry and livestock.【Method】 The granules of honeysuckle, scutellaria and forsythia was made into infusion, respectively, and then the drug infusion and Andrographis paniculata were used to make the oral solution of Andrographis paniculata, double forsythia (honeysuckle:scutellaria:forsythia was 1:1:2) and compound double forsythia (honeysuckle:scutellaria:forsythia: Andrographis paniculata was 1:1:2:2) according to the ratio, and the raw drug concentration was 100%, pH was adjusted as 7.0.12 strains of Escherichia coli, Salmonella and Staphylococcus aureus were used for bacterial susceptibility testing, and the sensitive strains were screened by Oxford cup combined with test tube twofold dilution method.The in vitro antibacterial activity was investigated based on the minimum inhibitory concentration (MIC) of the drugs.The drugs were diluted by the twofold dilution method, rhesus monkey embryonic kidney cells (MA-104) and chicken embryonic fibroblasts (DF-1) were cultured, cell viability was detected by CCK-8 method, and the safe concentrations of the three drugs were determined in combination with cytopathic lesions (CPE).The maximum safe concentration of the drug was diluted in three gradients by twofold dilution, Newcastle disease virus (NDV) and Rotavirus were used to test MA-104 and DF-1 in three ways: Drug+virus interactions, drug addition followed by attack, and attack followed by drug addition.The cell viability was detected by CCK-8 method, and the in vitro antiviral activity of the drugs was explored in combination with CPE.【Result】 The antibacterial effect of compound Shuanghuanglian was better than that of Andrographis paniculata and Shuanghuanglian, and its MICs against Escherichia coli, Salmonella and Staphylococcus aureus were 0.20-1.56, 0.20-1.56 and 0.78-1.56 mg/mL, respectively.CPE and cell viability results showed that the safe concentration ranges of Shuanghuanglian, Andrographis paniculata and compound Shuanghuanglian against DF-1 cells were 0-6.25, 0-0.78 and 0-0.78 mg/mL, respectively, and the safe concentration ranges for MA-104 cells were 0-3.13, 0-0.78 and 0-0.78 mg/mL, respectively.The TCID₅₀ of Newcastle disease virus strain 17E on DF-1 cells was 10^-6.19/100 μL, and the TCID₅₀ of yak Rotavirus G6P[1] type SDA2 strain on MA-104 cells was 10^-6.24/100 μL.The effective inhibition rates of compound Shuanghuanglian against Newcastle disease virus by directly inactivating virus (drug+virus interactions), blocking virus adsorption (drug addition followed by attack) and interfering virus replication (attack followed by drug addition) pathways were 28.82%, 55.92% and 42.45%, respectively, and the corresponding effective inhibition rates of Rotavirus were 48.84%, 26.52% and 15.40%, respectively.【Conclusion】 The antibacterial and antiviral effects of the compound Shuanghuanglian were stronger than those of Shuanghuanglian and Andrographis paniculata, and its antibacterial effect on Escherichia coli and Salmonella was better than that on Staphylococcus aureus.The results could provide scientific basis for the clinical application and further development of compound Shuanghuanglian in veterinary medicine.

Key words: compound Shuanghuanglian; antibacterial activity; antiviral activity

中图分类号:

R285

郭彬, 史晗, 封海波, 刘群. 复方双黄连制剂体外抗菌抗病毒活性研究[J]. 中国畜牧兽医, 2022, 49(12): 4832-4842.

GUO Bin, SHI Han, FENG Haibo, LIU Qun. Study on in vitro Antibacterial and Antiviral Activity of Compound Shuanghuanglian Preparation[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(12): 4832-4842.

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