Anti-viral and Anti-inflammatory Isoflavonoids from Ukrainian Iris aphylla Rhizomes: Structure-Activity Relationship Coupled with ChemGPS-NP Analysis.

Dried Iris rhizomes have been used in Chinese and European traditional medicine for the treatment of various diseases such as bacterial infections, cancer, and inflammation, as well as for being astringent, laxative, and diuretic agents. Eighteen phenolic compounds including some rare secondary metabolites, such as irisolidone, kikkalidone, irigenin, irisolone, germanaism B, kaempferol, and xanthone mangiferin, were isolated for the first time from Iris aphylla rhizomes. The hydroethanolic Iris aphylla extract and some of its isolated constituents showed protective effects against influenza H1N1 and enterovirus D68 and anti-inflammatory activity in human neutrophils. The promising anti-influenza effect of apigenin (13: , almost 100% inhibition at 50 µM), kaempferol (14: , 92%), and quercetin (15: , 48%) were further confirmed by neuraminidase inhibitory assay. Irisolidone (1: , almost 100% inhibition at 50 µM), kikkalidone (5: , 93%), and kaempferol (14: , 83%) showed promising anti-enterovirus D68 activity in vitro. The identified compounds were plotted using ChemGPS-NP to correlate the observed activity of the isolated phenolic compounds with the in-house database of anti-influenza and anti-enterovirus agents. Our results indicated that the hydroethanolic Iris aphylla extract and Iris phenolics hold the potential to be developed for the management of seasonal pandemics of influenza and enterovirus infections.

3,4-Dicaffeoylquinic Acid from the Medicinal Plant Ilex kaushue Disrupts the Interaction Between the Five-Fold Axis of Enterovirus A-71 and the Heparan Sulfate Receptor.

While infections by enterovirus A71 (EV-A71) are generally self-limiting, they can occasionally lead to serious neurological complications and death. No licensed therapies against EV-A71 currently exist. Using anti-virus-induced cytopathic effect assays, 3,4-dicaffeoylquinic acid (3,4-DCQA) from Ilex kaushue extracts was found to exert significant anti-EV-A71 activity, with a broad inhibitory spectrum against different EV-A71 genotypes. Time-of-drug-addition assays revealed that 3,4-DCQA affects the initial phase (entry step) of EV-A71 infection by directly targeting viral particles and disrupting viral attachment to host cells. Using resistant virus selection experiments, we found that 3,4-DCQA targets the glutamic acid residue at position 98 (E98) and the proline residue at position 246 (P246) in the 5-fold axis located within the VP1 structural protein. Recombinant viruses harboring the two mutations were resistant to 3,4-DCQA-elicited inhibition of virus attachment and penetration into human rhabdomyosarcoma (RD) cells. Finally, we showed that 3,4-DCQA specifically inhibited the attachment of EV-A71 to the host receptor heparan sulfate (HS), but not to the scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL1). Molecular docking analysis confirmed that 3,4-DCQA targets the 5-fold axis to form a stable structure with the E98 and P246 residues through noncovalent and van der Waals interactions. The targeting of E98 and P246 by 3,4-DCQA was found to be specific; accordingly, HS binding of viruses carrying the K242A or K244A mutations in the 5-fold axis was successfully inhibited by 3,4-DCQA.The clinical utility of 3,4-DCQA in the prevention or treatment of EV-A71 infections warrants further scrutiny. IMPORTANCE The canyon region and the 5-fold axis of the EV-A71 viral particle located within the VP1 protein mediate the interaction of the virus with host surface receptors. The three most extensively investigated cellular receptors for EV-A71 include SCARB2, PSGL1, and cell surface heparan sulfate. In the current study, a RD cell-based anti-cytopathic effect assay was used to investigate the potential broad spectrum inhibitory activity of 3,4-DCQA against different EV-A71 strains. Mechanistically, we demonstrate that 3,4-DCQA disrupts the interaction between the 5-fold axis of EV-A71 and its heparan sulfate receptor; however, no effect was seen on the SCARB2 or PSGL1 receptors. Taken together, our findings show that this natural product may pave the way to novel anti-EV-A71 therapeutic strategies.

Bio-guided bioactive profiling and HPLC-DAD fingerprinting of Ukrainian saffron (Crocus sativus stigmas): moving from correlation toward causation.

BACKGROUND: Saffron or stigmas of Crocus sativus L. is one of the most valuable food products with interesting health-promoting properties. C. sativus has been widely used as a coloring and flavoring agent. Stigmas secondary metabolites showed potent cytotoxic effects in previous reports. METHODS: The present study investigated the chemical composition and the cytotoxic effect of Ukrainian saffron crude extracts and individual compounds against melanoma IGR39, triple-negative breast cancer MDA-MB-231, and glioblastoma U-87 cell lines in vitro using MTT assay. Several bioactivity in vitro assays were performed. The chemical profile of the water and hydroethanolic (70%, v/v) crude extracts of saffron stigmas was elucidated by HPLC-DAD analysis. RESULTS: Seven compounds were identified including crocin, picrocrocin, safranal, rutin, apigenin, caffeic acid, ferulic acid. Crocin, picrocrocin, safranal, rutin, and apigenin were the major active constituents of Ukrainian C. sativus stigmas. The hydroethanolic extract significantly reduced the viability of MDA-MB-231 and IGR39 cells and the effect was more potent in comparison with the water extract. However, the water extract was almost 5.6 times more active against the U-87 cell line (EC50 of the water extract against U-87 was 0.15 ± 0.02 mg/mL, and EC50 of the hydroethanolic extract was 0.83 ± 0.03 mg/mL). The pure compounds, apigenin, and caffeic acid also showed high cytotoxic activity against breast cancer, melanoma, and glioblastoma cell lines. The screening of the biological activities of stigmas water extract (up to 100 µg/mL) including anti-allergic, anti-virus, anti-neuraminidase, and anti-inflammatory effects revealed its inhibitory activity against neuraminidase enzyme by 41%. CONCLUSIONS: The presented results revealed the qualitative and quantitative chemical composition and biological activity of Crocus sativus stigmas from Ukraine as a source of natural anticancer and neuraminidase inhibitory agents. The results of the extracts' bioactivity suggested future potential applications of saffron as a natural remedy against several cancers.

Perilla (Perilla frutescens) leaf extract inhibits SARS-CoV-2 via direct virus inactivation.

BACKGROUND: While severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents with mild or no symptoms in most cases, a significant number of patients become critically ill. Remdesivir has been approved for the treatment of coronavirus disease 2019 (COVID-19) in several countries, but its use as monotherapy has not substantially lowered mortality rates. Because agents from traditional Chinese medicine (TCM) have been successfully utilized to treat pandemic and endemic diseases, we designed the current study to identify novel anti-SARS-CoV-2 agents from TCM. METHODS: We initially used an antivirus-induced cell death assay to screen a panel of herbal extracts. The inhibition of the viral infection step was investigated through a time-of-drug-addition assay, whereas a plaque reduction assay was carried out to validate the antiviral activity. Direct interaction of the candidate TCM compound with viral particles was assessed using a viral inactivation assay. Finally, the potential synergistic efficacy of remdesivir and the TCM compound was examined with a combination assay. RESULTS: The herbal medicine Perilla leaf extract (PLE, approval number 022427 issued by the Ministry of Health and Welfare, Taiwan) had EC50 of 0.12 ± 0.06 mg/mL against SARS-CoV-2 in Vero E6 cells - with a selectivity index of 40.65. Non-cytotoxic PLE concentrations were capable of blocking viral RNA and protein synthesis. In addition, they significantly decreased virus-induced cytokine release and viral protein/RNA levels in the human lung epithelial cell line Calu-3. PLE inhibited viral replication by inactivating the virion and showed additive-to-synergistic efficacy against SARS-CoV-2 when used in combination with remdesivir. CONCLUSION: Our results demonstrate for the first time that PLE is capable of inhibiting SARS-CoV-2 replication by inactivating the virion. Our data may prompt additional investigation on the clinical usefulness of PLE for preventing or treating COVID-19.

Pharmacological Potential and Chemical Composition of Crocus sativus Leaf Extracts.

Crocus sativus L. (saffron) has been traditionally used as a food coloring or flavoring agent, but recent research has shown its potent pharmacological activity to tackle several health-related conditions. Crocus sp. leaves, and petals are the by-products of saffron production and are not usually used in the medicine or food industries. The present study was designed to determine the chemical composition of the water and ethanolic extracts of C. sativus leaves and test their cytotoxic activity against melanoma (IGR39) and triple-negative breast cancer (MDA-MB-231) cell lines by MTT assay. We also determined their anti-allergic, anti-inflammatory, and anti-viral activities. HPLC fingerprint analysis showed the presence of 16 compounds, including hydroxycinnamic acids, xanthones, flavonoids, and isoflavonoids, which could contribute to the extracts' biological activities. For the first time, compounds such as tectoridin, iristectorigenin B, nigricin, and irigenin were identified in Crocus leaf extracts. The results showed that mangiferin (up to 2 mg/g dry weight) and isoorientin (8.5 mg/g dry weight) were the major active ingredients in the leaf extracts. The ethanolic extract reduced the viability of IGR39 and MDA-MB-231 cancer cells with EC50 = 410 ± 100 and 330 ± 40 µg/mL, respectively. It was more active than the aqueous extract. Kaempferol and quercetin were identified as the most active compounds. Our results showed that Crocus leaves contain secondary metabolites with potent cytotoxic and antioxidant activities.

Rosmarinic acid exhibits broad anti-enterovirus A71 activity by inhibiting the interaction between the five-fold axis of capsid VP1 and cognate sulfated receptors.

Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.

Design, synthesis & structure-activity relationships of a new class of antihuman enterovirus D68 & A71 agents.

AIM: No antiviral medications are currently approved to treat enterovirus (EV)-associated disease or prevent EV infection. METHODS: In this study, a series of probenecid derivatives were designed via a rational strategy and synthesized to obtain more potent anti-EV agents. RESULTS: Compounds 8 and 24 exhibited the most potent activity against EV D68 and A71, with half maximal effective concentration (EC50) values of 2.49/2.09 and 2.59/2.41 µM, respectively, and revealed a broad inhibition spectrum toward other EV strains, with high selectivity indices. Additionally, compounds 8 and 24 showed good stability in rat serum, with half-lives of 48.39 and 60.26 min, respectively. CONCLUSION: Compounds 8 and 24 are the promising candidates for the development of new agents against EV D68 and A71 viruses.

An extract from Taxodium distichum targets hemagglutinin- and neuraminidase-related activities of influenza virus in vitro.

Influenza virus remains an emerging virus and causes pandemics with high levels of fatality. After screening different plant extracts with potential anti-influenza activity, a water extract of Taxodium distichum stems (TDSWex) showed excellent activity against influenza viruses. The EC50 of TDSWex was 0.051 ± 0.024 mg/mL against influenza virus A/WSN/33. TDSWex had excellent antiviral efficacy against various strains of human influenza A and B viruses, particularly oseltamivir-resistant clinical isolates and a swine-origin influenza strain. We observed that the synthesis of viral RNA and protein were inhibited in the presence of TDSWex. The results of the time-of-addition assay suggested that TDSWex inhibited viral entry and budding. In the hemagglutination inhibition assay, TDSWex inhibited the hemagglutination of red blood cells, implying that the extract targeted hemagglutin-related functions such as viral entry. In the attachment and penetration assay, TDSWex showed antiviral activity with EC50s of 0.045 ± 0.026 and 0.012 ± 0.003 mg/mL, respectively. In addition, TDSWex blocked neuraminidase activity. We conclude that TDSWex has bimodal activities against both hemagglutinin and neuraminidase during viral replication.

In vitro and in vivo studies of a potent capsid-binding inhibitor of enterovirus 71.

OBJECTIVES: Enterovirus 71 (EV-A71) is an important pathogen that can cause severe neurological symptoms and even death. Our aim was to identify potent anti-EV-A71 compounds and study their underlying mechanisms and in vivo activity. METHODS: We identified a potent imidazolidinone derivative (abbreviated to PR66) as an inhibitor of EV-A71 infection from the screening of compounds and subsequent structure-based modification. Time-course treatments and resistant virus selection of PR66 were employed to study the mode of mechanism of PR66. In vivo activity of PR66 was tested in the ICR strain of new-born mice challenged with EV-A71/4643/MP4. RESULTS: PR66 could impede the uncoating process during viral infection via interaction with capsid protein VP1, as shown by a resistant virus selection assay. Using site-directed mutagenesis, we confirmed that a change from valine to phenylalanine in the 179th amino acid residue of the cDNA-derived resistant virus resulted in resistance to PR66. PR66 increased the virion stability of WT viruses, but not the PR66-resistant mutant, in a particle stability thermal release assay. We further showed that PR66 had excellent anti-EV-A71 activity in an in vivo mouse model of disease, with a dose-dependent increase in survival rate and in protection against virus-induced hind-limb paralysis following oral or intraperitoneal administration. This was associated with reductions of viral titres in brain and muscle tissues. CONCLUSIONS: We demonstrated here for the first time that an imidazolidinone derivative (PR66) could protect against EV-A71-induced neurological symptoms in vivo by suppressing EV-A71 replication. This involved binding to and restricting viral uncoating.

Characterization of the anti-influenza activity of the Chinese herbal plant Paeonia lactiflora.

Bai Shao (BS, the root of Paeonia lactiflora Pall.), a common Chinese herb in many recipes used to treat viral infection and liver diseases, is recognized for its ability to nourish menstruation, its Yin convergence, and as an antiperspirant. However, the mechanism and components for its antiviral function remain to be elucidated. In this study, an ethanolic extract of BS was further partitioned into aqueous and organic parts (EAex) for in vitro functional study and in vivo efficacy testing. EAex exhibited an IC50 of 0.016 ± 0.005 mg/mL against influenza virus A/WSN/33 (H1N1), with broad-spectrum inhibitory activity against different strains of human influenza A viruses, including clinical oseltamivir-resistant isolates and an H1N1pdm strain. The synthesis of both viral RNA and protein was profoundly inhibited when the cells were treated with EAex. A time-of-addition assay demonstrated that EAex exerted its antiviral activity at various stages of the virus replication cycle. We addressed its antiviral activity at virus entry and demonstrated that EAex inhibits viral hemagglutination and viral binding to and penetration into host cells. In vivo animal testing showed that 200 mg/kg/d of EAex offered significant protection against viral infection. We conclude that BS possesses antiviral activity and has the potential for development as an anti-influenza agent.

Ching-fang-pai-tu-san inhibits the release of influenza virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Ching-fang-pai-tu-san (CFPTS) is a Chinese herbal decoction that is used as a cure for the common cold, fever, headache, and poor circulation. However, no previous studies have investigated the mode of action of CFPTS against influenza virus infections. To investigate the antiviral mechanism of CFPTS, we examined viral entry, transcription, translation, viral glycoprotein hemagglutinin (HA) transport, and budding of the influenza virus. MATERIALS AND METHODS: The antiviral activity of nontoxic concentrations of CFPTS against influenza virus A/WSN/33 was examined by assaying (neutralization assay) its inhibition of the virus-induced cytopathic effects. The mode of CFPTS action was first examined with a time-of-addition assay of synchronized infections, followed by monitoring HA transport by immunofluorescence microscopy. Viral endocytosis was evaluated with attachment and penetration assays. The inhibition of viral replication was measured by quantitative real-time PCR, immunoblotting, and immunofluorescence microscopy. We also performed assays related to the inhibition of viral entry, such as neuraminidase activity and hemagglutinin activity assays. RESULTS: Based on the inhibition of the virus-induced cytopathic effect in Madin-Darby canine kidney cells, the EC(50) of CFPTS was about 1.44 ± 0.22 mg/mL against influenza virus A/WSN/33. CFPTS displayed a broad spectrum of inhibitory activities against different strains of influenza A virus, as well as some enteroviruses. However, this extract proved less effective against clinical oseltamivir-resistant strains and influenza B viruses. CFPTS did not suppress viral RNA or protein synthesis. According to a time-of-addition assay, the antiviral mechanism of CFPTS may involve viral budding or intracellular viral glycoprotein transport. A plaque reduction assay showed that CFPTS reduced both the plaque size and plaque quantity. The intracellular transport of viral glycoprotein hemagglutinin was blocked by CFPTS by immunofluorescence microscopic analysis. Thus, it is possible that the antiviral mechanism of CFPTS might inhibit the assembly of progeny virions and/or their subsequent release. CONCLUSIONS: Our results give scientific support to the use of CFPTS in the treatment of influenza virus infections. CFPTS has potential utility in the management of seasonal pandemics of influenza virus infections, like other clinically available drugs.

Mechanism by which ma-xing-shi-gan-tang inhibits the entry of influenza virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Ma-xing-shi-gan-tang (MXSGT, aka maxing shigan powder), a Chinese herbal decoction, has been used for the treatment of the common cold, fever, and influenza virus infections. However, the underlying mechanisms of its activity against the influenza virus are not fully understood. In this study, we examined the antiviral effects of MXSGT in influenza-virus-infected MDCK cells and their underlying mechanisms, including the damage of the viral surface ultrastructure and the consequent inhibition of viral entry. MATERIALS AND METHODS: The antiviral activity of nontoxic concentrations of MXSGT against influenza virus A/WSN/33 was examined by assaying (neutralization assay) its inhibition of the virus-induced cytopathic effects. The mode of MXSGT action was first examined with a time-of-addition assay of synchronized infections, followed by viral attachment and penetration assays. Viral endocytosis was evaluated with attachment and penetration assays. We also performed assays related to the inhibition of viral entry, such as neuraminidase activity, hemagglutinin activity, and phosphoinositide-3-kinase (PI3K)/AKT phosphorylation assays. The inhibition of viral replication was demonstrated by quantitative real-time PCR, immunoblotting, and immunofluorescence microscopy. The surface ultrastructure of the MXSGT-treated virus was revealed by atomic force microscopy. RESULTS: MXSGT exhibited an EC(50) of 0.83±0.41mg/ml against influenza virus A/WSN/33 (H1N1), with broad-spectrum inhibitory activity against different strains of human influenza A viruses, including clinical oseltamivir-resistant isolates and an H1N1pdm strain. The synthesis of both viral RNA and protein was profoundly inhibited when the cells were treated with MXSGT. The time-of-addition assay demonstrated that MXSGT blocks the virus entry phase. This was confirmed with attachment and penetration assays, in which MXSGT showed similar inhibitory potencies (IC(50) of 0.58±0.07 and 0.47±0.08mg/ml). High-resolution images and quantitative measurements made with atomic force microscopy confirmed that the viral surface structure was disrupted by MXSGT. We also established that viral entry, regulated by the PI3K/AKT signaling pathway, was abolished by MXSGT. CONCLUSIONS: Our results give scientific support to the use of MXSGT in the treatment of influenza virus infections. MXSGT has potential utility in the management of seasonal pandemics of influenza virus infections, like other clinically available drugs.

Mechanism of action of the suppression of influenza virus replication by Ko-Ken Tang through inhibition of the phosphatidylinositol 3-kinase/Akt signaling pathway and viral RNP nuclear export.

AIMS OF THE STUDY: Ko-Ken Tang (KKT, aka kakkon-to), a conventional Chinese herbal medicine, has been used for the treatment of the common cold, fever and influenza virus infection. However, the underlying mechanism of its activity against influenza virus infection remains elusive. In this study, the antiviral effect and its underlying mechanism was evaluated, including the investigation of anti-influenza virus activity of KKT on MDCK cells and corresponding mechanism related to phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and its consecutive viral RNP nuclear export. MATERIALS AND METHODS: The antiviral activity of non-toxic concentration of KKT was examined against various strains of influenza virus and enterovirus 71 by neutralization assay. PI3K/Akt signaling activated by influenza virus was inspected in A549 cells by western blot. Inhibition of influenza polymerase activity by KKT was measured with plasmid-based reverse genetics using primer extension assay and luciferase reporter assay. Inhibition of viral vRNP nuclear export was demonstrated by laser confocal microscopy and interspecies heterokaryon assay. RESULTS: KKT inhibits influenza virus replication but not entry, and it exhibits a broad spectrum inhibitory activity against human influenza A viruses and enterovirus 71. KKT does not inhibit viral polymerase activity but directly blocks the virus-induced phosphatidylinositol 3-kinase/Akt signaling pathway, which in turns causes retention of viral nucleoprotein in the nucleus, thereby interfering with virus propagation. The inhibition by KKT of the nuclear export of viral protein was further confirmed by heterokaryon assay. CONCLUSIONS: The results obtained in this study give scientific support to KKT for the treatment of influenza virus infection. KKT could be of potential use in the management of seasonal pandemic influenza virus infection in addition to other clinically available drugs.