背景:鳞翅目的根茎(鳞翅目科,RDC),一种传统的东亚草药,具有广泛的药用特性,包括消炎药,抗癌,抗菌,和抗病毒活性。
目的:本研究调查了RDC的30%乙醇提取物对人冠状病毒OC43(HCoV-OC43)的抗病毒潜力,严重急性呼吸道综合征冠状病毒2(SARS-CoV-2),及其变种感染。
方法:RDC或其成分的30%乙醇提取物,用人冠状病毒感染(HCoV-OC43,SARS-CoV-2及其变体)治疗了丝状酸ABA(PubChemCID:15081408)和干crassinABBA(PubChemCID:3082025)。使用UPLC-Q/TOFMass评估RDC的基峰色谱图,以鉴定RDC,使用LC-MS/MS进行RDC化合物的定量分析。细胞病变效应(CPE)减少试验,westernblot和免疫荧光染色病毒蛋白表达,进行定量病毒RNA拷贝数的qRT-PCR以确定抗病毒活性。添加时间测定,病毒附件,穿透力,和杀病毒试验,SARS-CoV-2Mpro和PLpro活性测定用于阐明作用方式。
结果:RDC表现出剂量依赖性抑制HCoV-OC43诱导的细胞病变效应,降低病毒RNA拷贝数和病毒蛋白水平。添加时间测定表明,RDC靶向HCoV-OC43生命周期的早期阶段,抑制病毒体附着和渗透具有杀病毒活性。值得注意的是,丝状酸ABA和干蛋白酶ABBA,RDC的组成部分,降低HCoV-OC43病毒RNA载量。此外,RDC在假型化慢病毒检测中有效阻断病毒进入,涉及SARS-CoV-2Deltaplus和南非变体的刺突蛋白,以及表达水泡性口炎病毒糖蛋白G的对照慢病毒颗粒。RDC证明通过靶向病毒蛋白酶抑制SARS-CoV-2感染及其变体,即主要蛋白酶(Mpro)和木瓜蛋白酶(PLpro)。
结论:这些发现强调了RDC通过阻止病毒进入和抑制病毒蛋白酶活性来靶向病毒感染的多阶段方法。因此,RDC作为一种强有力的承诺,广谱抗冠状病毒治疗剂。
BACKGROUND: The rhizome of Dryopteris crassirhizoma Nakai (Dryopteridaceae, RDC), a traditional East Asian herbal medicine, possesses a broad spectrum of medicinal properties, including anti-inflammatory, anticancer, antibacterial, and antiviral activities.
OBJECTIVE: This study investigates the 30% ethanolic extract of RDC\'s antiviral potential against human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and its variants infections.
METHODS: A 30% ethanolic extract of RDC or its components, filixic acid ABA (PubChem CID: 15081408) and dryocrassin ABBA (PubChem CID: 3082025) were treated with Human Coronavirus infection (HCoV-OC43, SARS-CoV-2 and its variants). The base peak chromatogram of RDC was evaluated using UPLC-Q/TOF Mass to identify the RDC, and the quantitative analysis of RDC compounds was performed using LC-MS/MS. A cytopathic effect (CPE) reduction assay, Western blot, immunofluorescence staining of viral protein expression, and qRT-PCR were performed to quantify the viral RNA copy numbers and determine the antiviral activity. The time-of-addition assay, the virus attachment, penetration, and virucidal assays, and SARS-CoV-2 Mpro and PLpro activity assay were used to elucidate the mode of action.
RESULTS: RDC exhibited dose-dependent inhibition of HCoV-OC43-induced cytopathic effects, reducing viral RNA copy numbers and viral protein levels. Time-of-addition assays indicated that RDC targets the early stages of the HCoV-OC43 life cycle, inhibiting virion attachment and penetration with virucidal activity. Notably, filixic acid ABA and dryocrassin ABBA, constituents of RDC, reduced HCoV-OC43 viral RNA loads. Furthermore, RDC effectively blocked viral entry in pseudotyped lentivirus assays, involving spike proteins of SARS-CoV-2 Delta plus and South Africa variants, as well as control lentiviral particles expressing vesicular stomatitis virus glycoprotein G. Additionally, RDC demonstrated inhibition of SARS-CoV-2 infection and its variants by targeting viral proteases, namely main protease (Mpro) and papain-like protease (PLpro).
CONCLUSIONS: These findings underscore RDC\'s multistage approach to targeting viral infections by impeding virus entry and inhibiting viral protease activity. Therefore, RDC holds promise as a potent, broad-spectrum anticoronaviral therapeutic agent.