【作者】 马军成；

【导师】 李宁；

【作者基本信息】 安徽医科大学 ， 药物化学， 2023， 硕士

【摘要】 2021年新型冠状病毒（SARS-CoV-2）仍在全球范围蔓延,疫情防控及相关药物研发刻不容缓。针对新型肺炎（COVID-19）尚无特效抗冠状病毒药物,指南推荐的药物仍具有一定局限性,效果欠佳。百蕊草（Thesium Chinense Turcz.）为檀香科（Santalaceae）植物百蕊草的干燥全草。约有30属,400余种,分布广泛。中国现已确定有35种,6个变种,各地均存在大量产出。百蕊草载于《图经本草》,具有“清热解毒”和“通顺气脉”之功效,百蕊草本身性味平缓、微苦、涩、寒,素有“植物抗生素”之美称。百蕊草含有黄酮、有机酸、生物碱、甾醇,酚类、挥发油等多种化学组分。百蕊草全草入药,具有抗菌消炎、清热解毒、补肾涩精之功效,为一种具有较好应用前景的广谱抗菌中草药。根据文献报导桑黄具有抗甲型流感病毒A（H1N1和H3N2）和乙型流感病毒的作用。故实验设计上使用桑黄和百蕊草不同比例配伍组成复合液,充分考虑到了百蕊草较强的抗病毒作用以及桑黄较强的抗炎作用。目的:本研究旨在研究百蕊草提取物及其复合物抗新冠病毒作用及其抗炎作用,进一步补充现在传统中药对新冠病毒的研究。方法:初步筛选了百蕊草不同极性部位（乙醇、石油醚、乙酸乙酯、正丁醇和水提取物）、桑黄（乙醇和水提取物）对于抑制SARS-CoV-2复制的能力。以非洲绿猿猴肾上皮细胞（Vero）为宿主细胞,通过实时定量PCR检测病毒中核衣壳蛋白（NP）的表达水平。为了进一步研究百蕊草乙醇提取物和桑黄水提取物对SARS-CoV-2病毒的抗进入作用,通过实时定量PCR和Western blotting检测NP的表达水平。评估了不同极性部位的百蕊草和桑黄提取物（乙醇、石油醚、乙酸乙酯、正丁醇和水提取物）对脂多糖（LPS）刺激的小鼠单核巨噬细胞白血病细胞（RAW264.7）细胞中炎症细胞因子产生的抑制活性。利用ELISA和PCR检测肿瘤坏死因子（TNF-α）和白介素-6（IL-6）的表达水平。最后对于复合物（百蕊草乙醇提取物和桑黄水提物依照比例1:1、1:2、2:1）抑制SARS-CoV-2复制和进入的能力,进行PCR检测NP的表达水平。利用ELISA和PCR检测对LPS刺激的RAW264.7细胞中TNF-α和IL-6的抑制活性。结果:百蕊草乙醇提取物显著抑制在Vero细胞中SARS-CoV-2的复制(EC₅₀:259.3μg/mL)和进入(EC₅₀:364.2μg/mL),并显著降低LPS刺激的RAW264.7细胞产生的IL-6和TNF-α水平。石油醚(EC₅₀:163.6μg/mL)、乙酸乙酯(EC₅₀:22.92μg/mL)和正丁醇(EC₅₀:56.80μg/mL)提取物对Vero细胞中SARS-CoV-2复制的抑制作用较弱,而水提取物则完全没有活性。在抗炎作用中,百蕊草石油醚部分、乙酸乙酯部分和正丁醇部分在特定药物浓度范围内表现出明显的浓度依赖性,并且随着浓度的增加,LPS诱导的IL-6和TNF-α的表达水平逐渐降低。水提取物仅在160μg/mL的低浓度下对LPS诱导的TNF-α表达有轻微的抑制作用。桑黄乙醇提取物(EC₅₀:690.3μg/mL)和水提物具有一定的抑制SARS-CoV-2的复制作用,但无抗病毒进入作用。桑黄各部位均呈现较好的抗炎效果,并且随着浓度的增加有明显的浓度依赖性,其中桑黄水提取物呈现出极显著的抗炎作用。复合物1(百蕊草:桑黄=1:1,EC₅₀:500.6μg/mL),复合物2(百蕊草:桑黄=1:2,EC₅₀:522.9μg/mL),复合物3(百蕊草:桑黄=2:1,EC₅₀:410.8μg/mL)均能较好抑制SARS-CoV-2的复制,针对SARS-CoV-2的进入阶段复合物1(EC₅₀:593.9μg/mL)抑制病毒作用较优于复合物2和复合物3。三种复合物均对细胞炎症因子TNF-α、IL-6的释放量的有极显著抑制的作用,且呈现明显趋势。结论:百蕊草乙醇提取物及其复合物1能显著抑制在Vero细胞中SARS-CoV-2的复制和进入,并显著降低LPS刺激的RAW264.7细胞产生的IL-6和TNF-α水平。更多还原

【Abstract】 In 2021,the novel coronavirus（SARS-CoV-2）is still spreading around the world,and the prevention and control of the epidemic and related drug research and development are urgent.For the novel pneumonia（COVID-19）,there are no specific anti-coronavirus drugs,and the drugs recommended by the guidelines are still limited and ineffective.Thesium Chinense Turcz is a dried whole plant of the Santalaceae plant.There are about30 genera and more than 400 species,widely distributed.In China,35 species and 6varieties have been identified,with substantial production occurring everywhere.T.chinense is listed in the book of"Tujing Herbal Medicine".It has the functions of"clearing heat and detoxifying"and"regulating qi".The taste of T.chinense is gentle,slightly bitter,astringent and cold,so it is known as"plant antibiotic".T.chinense contains flavonoids,organic acids,alkaloids,sterols,phenols,volatile oils and other chemical components.As a kind of broad-spectrum antibacterial herbal medicine with good application prospects,the whole herb has the functions of antibacterial and anti-inflammatory,clearing heat and detoxifying,tonifying kidney and astringent essence.It has been reported that Sanghuangprous vaninii has anti-influenza A（H1N1and H3N2）and influenza B viruses.Therefore,in the experimental design,the combination of S.vaninii and T.chinense in different proportions was used to form the composite solution,which fully took into account the strong antiviral effect of T.chinense and the strong anti-inflammatory effect of S.vaninii.Objective:The purpose of this study is to study the inhibition of SARS-CoV-2 and inflammation by the extract of T.chinense and its complex,and further complement the current research on the novel coronavirus of traditional Chinese medicine.Methods:The inhibitory effects of different polar fractions（ethanol,petroleum ether,ethyl acetate,n-butanol and water extract）T.chinense and S.vaninii（ethanol and water extract）on the replication of SARS-CoV-2 were initially screened.Using African green ape kidney epithelial cells（Vero）as host cells,nucleocapsid protein（NP）expression was detected by real-time quantitative PCR.In order to further study the anti-entry effects of the ethanol extract of T.chinense and the water extract of S.vaninii on SARS-CoV-2 virus,the expression level of NP was detected by real-time quantitative PCR and Western blotting.We evaluated the inhibitory activity of different polar extracts（ethanol,petroleum ether,ethyl acetate,n-butanol,and water）of T.chinense and S.vaninii on the production of inflammatory cytokines in lipopolysaccharide（LPS）-stimulated mouse monocyte macrophage leukemia cells（RAW264.7）.The expression levels of tumor necrosis factor（TNF-α）and interleukin-6（IL-6）were detected by ELISA and PCR.Finally,the expression level of NP was detected by PCR for the inhibition of SARS-CoV-2 replication and entry ability of the complex（the ethanol extract of T.chinense and the water extract of S.vaninii in the ratio of 1:1,1:2and 2:1）.The inhibitory activity of TNF-αand IL-6 in RAW264.7 cells stimulated by LPS was determined by ELISA and PCR.Results:The ehanol extract of T.chinense inhibited the replication(EC₅₀:259.3μg/mL)and entry(EC₅₀:364.2μg/mL)of SARS-CoV-2 in Vero cells and significantly reduced the levels of IL-6 and TNF-αproduced by LPS-stimulated RAW264.7 cells.The petroleum ether(EC₅₀:163.6μg/mL),ethyl acetate(EC₅₀:22.92μg/mL)and n-butanol(EC₅₀:56.80μg/mL)extracts showed a weak inhibitory effect on the replication of SARS-CoV-2 in Vero cells,while the water extract showed no activity at all.In the anti-inflammatory effect,the petroleum ether fraction,ethyl acetate fraction and n-butanol fraction showed significant concentration dependence in the specific drug concentration range,and the expression levels of IL-6 and TNF-αinduced by LPS gradually decreased with the increase of concentration.The aqueous extract only slightly inhibited the expression of TNF-αinduced by LPS at the low concentration of160μg/mL.Ethanol extract(EC₅₀:690.3μg/mL)and water extract of S.vaninii had certain inhibitory effect on the replication of SARS-CoV-2,but they had no antiviral entry effect.All parts of S.vaninii showed good anti-inflammatory effect,and there was a significant concentration dependence with the increase of concentration,and the water extract of S.vaninii showed a very significant anti-inflammatory effect.Complex 1(T.chinense:S.vaninii=1:1,EC₅₀:500.6μg/mL),complex 2(T.chinense:S.vaninii=1:2,EC₅₀:522.9μg/mL),complex 3(T.chinense:S.vaninii=2:1,EC₅₀:410.8μg/mL)inhibited the replication of SARS-CoV-2,and the entry phase complex 1(EC₅₀.593.9μg/mL)against SARS-CoV-2 inhibited the virus better than complex 2 and complex 3.All the three compounds had extremely significant inhibitory effects on the release of cytokines TNF-αand IL-6,and showed an obvious trend.Conclusion:The ethanol extract of T.chinense and its complex 1 significantly inhibited the replication and entry of SARS-CoV-2 in Vero cells,and significantly reduced the levels of IL-6 and TNF-αproduced by RAW264.7 cells stimulated by LPS.更多还原