Molecular networking-based mass spectral identification of Brucea javanica (L.) Merr. metabolites and their selective binding affinities for dengue virus enzymes.

Brucea javanica, a valued traditional medicinal plant in Malaysia, known for its fever-treating properties yet remains underexplored for its potential antiviral properties against dengue. This study aims to simultaneously identify chemical classes and metabolites within B. javanica using molecular networking (MN), by Global Natural Product Social (GNPS), and SIRIUS in silico annotation. Liquid chromatography-mass spectrometry (LC-MS2)-based MN explores chemical diversity across four plant parts (leaves, roots, fruits, and stem bark), revealing diverse metabolites such as tryptophan-derived alkaloids, terpenoids, and octadecadenoids. Simultaneous LC-MS2 and MN analyses reveal a discriminative capacity for individual plant components, with roots accumulating tryptophan alkaloids, fruits concentrating quassinoids, leaves containing fusidanes, and stem bark primarily characterised by simple indoles. Subsequently, extracts were evaluated for dengue antiviral activity using adenosine triphosphate (ATP) and plaque assays, indicates potent efficacy in the dichloromethane (DCM) extract from roots (EC50 = 0.3 µg/mL, SI = 10). Molecular docking analysis of two major compounds; canthin-6-one (264) and 1-hydroxy-11-methoxycanthin-6-one (275) showed potential binding interactions with active sites of NS5 RNA-dependent RNA polymerase (RdRp) of dengue virus (DENV) protein. Subsequent in vitro evaluation revealed compounds 264 and 275 had a promising dengue antiviral activity with SI value of 63 and 1.85. These identified metabolites emerge as potential candidates for further evaluation in dengue antiviral activities.

Arctigenin from Arctium lappa L. inhibits chikungunya virus by affecting its entry and replication.

BACKGROUND: Dengue and chikungunya, caused by dengue virus (DENV) and chikungunya virus (CHIKV) respectively, are the most common arthropod-borne viral diseases worldwide, for which there are no FDA-approved antivirals or effective vaccines. Arctigenin, a phenylpropanoid lignan from the seeds of Arctium lappa L. is known for its anti-inflammatory, anti-cancer, antibacterial, and immunomodulatory properties. Arctigenin's antimicrobial and immunomodulatory capabilities make it a promising candidate for investigating its potential as an anti-DENV and anti-CHIKV agent. PURPOSE: The aim of the study was to explore the anti-DENV and anti-CHIKV effects of arctigenin and identify the possible mechanisms of action. METHODS: The anti-DENV or anti-CHIKV effects of arctigenin was assessed using various in vitro and in silico approaches. Vero CCL-81 cells were infected with DENV or CHIKV and treated with arctigenin at different concentrations, temperature, and time points to ascertain the effect of the compound on virus entry or replication. In silico molecular docking was performed to identify the interactions of the compound with viral proteins. RESULTS: Arctigenin had no effects on DENV. Various time- and temperature-dependent assays revealed that arctigenin significantly reduced CHIKV RNA copy number and infectious virus particles and affected viral entry. Entry bypass assay revealed that arctigenin inhibited the initial steps of viral replication. In silico docking results revealed the high binding affinity of the compound with the E1 protein and the nsp3 macrodomain of CHIKV. CONCLUSION: This study demonstrates the in-vitro anti-CHIKV potential of arctigenin and suggests that the compound might affect CHIKV entry and replication. Further preclinical and clinical studies are needed to identify its safety and efficacy as an anti-CHIKV drug.

Four undescribed triterpenes from the aerial parts of Verbena officinalis.

Verbena officinalis is used as a Chinese folk medicine for the treatment of rheumatism and bronchitis. Herein, four undescribed triterpenes, officinalisoids A-D (1-4), together with thirty-three known compounds (5-37) were isolated from the aerial parts of V. officinalis. The chemical structures of the new compounds were determined by spectrometric data interpretation using NMR, HRESIMS, IR and UV spectroscopy. Biological evaluation results revealed that compound 30 exhibited potential anti-inflammatory activity with IC50 value of 6.07 µM (CC50 > 50 µM) and compound 12 showed moderate anti-dengue virus activity with the IC50 value of 24.55 µM (CC50 > 50 µM).

Liang-Ge-San inhibits dengue virus serotype 2 infection by reducing caveolin1-induced cytoplasmic heat shock protein 70 translocation into the plasma membrane.

BACKGROUND: Dengue virus (DENV) is a major public health threat. However, there are no specific therapeutic drugs for DENV. Many Chinese heat-cleaning formulas, such as Liang-Ge-San (LGS), have been frequently used in the virus-induced diseases. The antiviral effect of LGS has not been reported yet. PURPOSE: In this study, the effect of LGS on the inhibition of dengue virus serotype 2 (DENV-2) was investigated and the relevant mechanism was explored. METHODS: High-performance liquid chromatography was applied to analyze the chemical characterization of LGS. The in vitro antiviral activities of LGS against DENV-2 were evaluated by time-of-drug-addition assay. The binding of heat shock protein 70 (Hsp70) and envelope (E) protein or caveolin1 (Cav1) were analyzed by immunofluorescence and immunoprecipitation assays. Then the role of Cav1 in the anti-DENV-2 effects of LGS was further examined. DENV-2 infected Institute of Cancer Research suckling mice (n = 10) and AG129 mice (n = 8) were used to examine the protective effects of LGS. RESULTS: It was found that geniposide, liquiritin, forsythenside A, forsythin, baicalin, baicalein, rhein, and emodin maybe the characteristic components of LGS. LGS inhibited the early stage of DENV-2 infection, decreased the expression levels of viral E and non-structural protein 1 (NS1) proteins. LGS also reduced E protein and Hsp70 binding and attenuated the translocation of Hsp70 from cytoplasm to the cell membrane. Moreover, LGS decreased the binding of Hsp70 to Cav1. Further study showed that the overexpression of Cav1 reversed LGS-mediated E protein and Hsp70 inhibition in the plasma membrane. In the in vivo study, LGS was highly effective in prolonging the survival time, reducing viral loads. CONCLUSION: This work demonstrates for the first time that LGS exerts anti-DENV-2 activity in vitro and in vivo. LGS decreases DENV-2-stimulated cytoplasmic Hsp70 translocation into the plasma membrane by Cav1 inhibition, thereby inhibiting the early stage of virus infection. These findings indicate that LGS may be a candidate for the treatment of DENV.

Identification of an effective fraction from Ampelopsis Radix with anti-dengue virus activities in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Dengue virus (DENV) infection is a global public health issue without effective therapeutic interventions. Chinese medicine with heat-clearing and detoxifying properties has been frequently used in the treatment of viral infection. Ampelopsis Radix (AR) is a traditional Chinese medicine for clearing heat and detoxification that has been widely used in the prevention and treatment of infectious diseases. However, no studies on the effects of AR against viral infection have been reported, thus far. AIM OF THE STUDY: To explore the anti-DENV activities of the fraction (AR-1) obtained from AR both in vitro and in vivo. MATERIALS AND METHODS: The chemical composition of AR-1 was identified by liquid chromatography-tandem MS (LC‒MS/MS). The antiviral activities of AR-1 were studied in baby hamster kidney fibroblast BHK-21 cells, ICR suckling mice and induction of interferon α/ß (IFN-α/ß) and IFN-γ R-/- (AG129) mice. RESULTS: Based on LC‒MS/MS analysis, 60 compounds (including flavonoids, phenols, anthraquinones, alkaloids and other types) were tentatively characterized from AR-1. AR-1 inhibited the cytopathic effect, the production of progeny virus and the synthesis of viral RNA and proteins by blocking DENV-2 binding to BHK-21 cells. Moreover, AR-1 significantly attenuated weight loss, decreased clinical scores and prolonged the survival of DENV-infected ICR suckling mice. Critically, the viral load in blood, brain and kidney tissues and the pathological changes in brain were remarkably alleviated after AR-1 treatment. Further study on AG129 mice showed that AR-1 obviously improved the clinical manifestations and survival rate, reduced viremia, attenuated gastric distension and relieved the pathological lesions caused by DENV. CONCLUSIONS: In summary, this is the first report that AR-1 exhibits anti-DENV effects both in vitro and in vivo, which suggests that AR-1 may be developed as a therapeutic candidate against DENV infection.

Quassinoids from Eurycoma longifolia with antiviral activities by inhibiting dengue virus replication.

BACKGROUND: Dengue caused by dengue virus (DENV) spreads rapidly around the world. However, there are no worldwide licensed vaccines or specific antivirals to combat DENV infection. Quassinoids are the most characteristic components of Eurycoma longifolia, which have been reported to display a variety of biological activities. However, whether quassinoids exert anti-DENV activities remains unknown. PURPOSE: To test the quassinoids of E. longifolia for their activity against DENV and to clarify the potential mechanisms. METHODS: The quassinoids from E. longifolia were isolated by chromatography techniques, and their chemical structures were elucidated by spectroscopic analysis. The anti-DENV activities of quassinoids on baby hamster kidney cells BHK-21 were determined by lactate dehydrogenase (LDH) assay. The synthesis of progeny virus was measured by plaque assay. The expression levels of envelope protein (E) and non-structural protein 1 (NS1) were evaluated by qRT-PCR, Western blot and immunofluorescence assays. Molecular docking was used to screen the potential targets of the most active quassinoid against DENV-2, and surface plasmon resonance analysis was employed to confirm the direct binding between the most active quassinoid and potential target. RESULTS: Twenty-four quassinoids, including three new quassinoids (1 - 3), were isolated from the ethanol extract of E. longifolia. Quassinoids 4, 5, 9, 11, 12, 15, 16, 17, 19 and 20 significantly reduced the LDH release at the stages of viral binding and entry or intracellular replication. Among them, 19 (6α-hydroxyeurycomalactone, 6α-HEL) exhibited the best anti-DENV-2 activities with an EC50 value of 0.39 ± 0.02 µM. Further experiments suggested that 6α-HEL remarkably inhibited progeny virus synthesis and mRNA and protein expression levels of E and NS1 of DENV-2. Time-of-drug-addition assay suggested that 6α-HEL inhibited intracellular replication of DENV-2 at an early stage. Moreover, 6α-HEL was shown to interact with NS5-RdRp domain at a binding affinity of -8.15 kcal/mol. SPR assay further verified 6α-HEL bound to RdRp protein with an equilibrium dissociation constant of 1.49 × 10-7 M. CONCLUSION: Ten quassinoids from E. longifolia showed anti-DENV activities at processes of virus binding and entry or intracellular replication. The most active quassinoid 6α-HEL exerts the anti-DENV-2 activities at intracellular replication stage by directly targeting the NS5-RdRp protein. These results suggest that 6α-HEL could be a promising candidate for the treatment of DENV-2 infection.

Alocasia odora-mediated synthesis of silver nanoparticles, their cytotoxicity, and virucidal potential.

Silver nanoparticles (AgNPs) have various applications in the biomedical field and are considered excellent microbicidal agents. Moreover, biological synthesis of AgNPs using medicinal plants further improves the medicinal applicability of these plants. In this study, the aqueous extract of Alocasia odora rhizome (RE) and Alocasia odora stem (SE) were used to synthesize stem aqueous extract-AgNPs (SNP) and rhizome aqueous extract-AgNPs (RNP). Furthermore, RNP and SNP were evaluated for their virucidal potential. The synthesis of SNP and RNP was monitored using a UV spectrophotometer by observing their surface plasmon resonance peak. In addition, scanning electron microscopy (SEM) gave further insight into their morphology and particle size, whereas energy-dispersive X-ray spectroscopy (EDX) confirmed the presence of silver ions. Interestingly, Fourier-transform infrared spectroscopy (FTIR) analysis of AgNPs revealed that phytomolecules acted as capping and stabilizing agents for SNP and RNP. The in vitro cytotoxicity of SNP and RNP was further analyzed using MTT assay on the U87-MG human glioblastoma cancer cell line and SNP found to be the most cytotoxic (43.40 µg/ml) among all. Besides that, SNP has also found to show the maximum cytopathic effects (CPE) against dengue virus type 2 (DENV-2) on Huh-7 cell line. As a result of the observations, it can be concluded that they can become a promising antiviral drug candidate and thus merit further testing. KEY POINTS: • AgNPs were successfully synthesized through Alocasia odora aqueous extract. • AgNPs were more cytotoxic on the U87-MG cell line than the extract alone. • AgNPs have shown significant reduction in the dengue viral infection than the extract alone.

Vitamin D modulates inflammatory response of DENV-2-infected macrophages by inhibiting the expression of inflammatory-liked miRNAs.

Dengue disease caused by dengue virus (DENV) infection is the most common vector-borne viral disease worldwide. Currently, no treatment is available to fight dengue symptoms. We and others have demonstrated the antiviral and immunomodulatory properties of VitD3 as a possible therapy for DENV infection. MicroRNAs (miRNAs) are small non-coding RNAs responsible for the regulation of cell processes including antiviral defense. Previous transcriptomic analysis showed that VitD3 regulates the expression of genes involved in stress and immune response by inducing specific miRNAs. Here, we focus on the effects of VitD3 supplementation in the regulation of the expression of inflammatory-liked miR-182-5p, miR-130a-3p, miR125b-5p, miR146a-5p, and miR-155-5p during DENV-2 infection of monocyte-derived macrophages (MDMs). Further, we evaluated the effects of inhibition of these miRNAs in the innate immune response. Our results showed that supplementation with VitD3 differentially regulated the expression of these inflammatory miRNAs. We also observed that inhibition of miR-182-5p, miR-130a-3p, miR-125b-5p, and miR-155-5p, led to decreased production of TNF-α and TLR9 expression, while increased the expression of SOCS-1, IFN-ß, and OAS1, without affecting DENV replication. By contrast, over-expression of miR-182-5p, miR-130a-3p, miR-125b-5p, and miR-155-5p significantly decreased DENV-2 infection rates and also DENV-2 replication in MDMs. Our results suggest that VitD3 immunomodulatory effects involve regulation of inflammation-linked miRNAs expression, which might play a key role in the inflammatory response during DENV infection.

Medicinal Plants with Anti-dengue and Immunomodulatory Activity.

Dengue fever is a disease with a high mortality rate around the world, which is an important issue for the health authorities of many countries. As a result of this, the search for new drugs that are effective to combat this disease has become necessary. Medicinal plants have been used since ancient times to treat a wide list of diseases, including dengue fever. In this mini-review, 12 medicinal plants with known pharmacological properties are presented, which have been used in studies to evaluate their antiviral activity in vitro tests. Among the chemical agents involved in the antiviral response, found in the alcoholic extracts of these plants, are flavonoids, terpenes and alkaloids, which within the mechanism of action in blocking viral replication are considered entry inhibitors, fusion inhibitors, translation inhibitors and protease inhibitors. The present work shows whether these plants possess antiviral activity and the chemical compounds involved in this response.

Antiviral Effect of Stenocline ericoides DC. and Stenocline inuloides DC., Two Flavonoid-Rich Endemic Plants from Madagascar, against Dengue and Zika Viruses.

Dengue and Zika viruses are identified as the most medically important arthropod-borne viral pathogens. Over the past 20 years, the global dengue incidence has dramatically increased with epidemics of severe dengue where the case fatality rate can reach up to 20% in untreated patients. The association between Zika virus infection and severe congenital anomalies was first reported in 2015. Today no specific antiviral therapies are available for dengue and Zika virus infections, accentuating the need of adapted antiviral strategies based on medicinal plant drug discovery. Plants are a potential source of antiviral phytocompounds which act primarily by blocking virus entry in the host-cell. In the present study, we evaluated whether crude extracts from Stenocline ericoides DC. and Stenocline inuloides DC., two endemic plants from Madagascar, may have antiviral effects against dengue and Zika viruses. We showed that S. ericoides has virucidal action whereas S. inuloides inhibits the early steps of virus infection with a non-cytotoxic effect in human cells. The administration of S. ericoides and S. inuloides extracts in zebrafish had no effect on the behavior of animals at the active doses against dengue and Zika viruses, suggesting the absence of adverse effects at these doses. LC-HRMS2 and molecular networking analyses revealed the richness of these two plants in polyphenols and flavonoid with the presence of clusters of phytocompounds specific to each Stenocline species. Consequently, S. ericoides and S. inuloides represent potential sources for natural and safe antiviral phytocompounds against flaviviruses of medical concern.

Essential Oils from Colombian Plants: Antiviral Potential against Dengue Virus Based on Chemical Composition, In Vitro and In Silico Analyses.

Currently, there are no therapies to prevent severe dengue disease. Essential oils (EOs) can serve as primary sources for research and the discovery of phytomedicines for alternative therapy. Fourteen EOs samples were obtained by distillation from six plants used in Colombian folk medicine. GC/MS analysis identified 125 terpenes. Cytopathic effect (CPE) reduction assays revealed differences in antiviral activity. EOs of Lippia alba, citral chemotype and carvone-rich fraction; Lippia origanoides, phellandrene chemotype; and Turnera diffusa, exhibited strong antiviral activity (IC50: 29 to 82 µg/mL; SI: 5.5 to 14.3). EOs of Piper aduncum, Ocimum basilicum, and L. origanoides, carvacrol, and thymol chemotypes, exhibited weak antiviral activity (32 to 53% DENV-CPE reduction at 100 µg/mL; SI > 5.0). Cluster and one-way ANOVA analyses suggest that the strong antiviral activity of EOs could be attributed to increased amounts of non-phenolic oxygenated monoterpenes and sesquiterpene hydrocarbons. Docking analyses (AutoDock Vina) predicted binding affinity between the DENV-2 E protein and terpenes: twenty sesquiterpene hydrocarbons (−8.73 to −6.91 kcal/mol), eight oxygenated monoterpenes (−7.52 to −6.98 kcal/mol), and seven monoterpene hydrocarbons (−7.60 to −6.99 kcal/mol). This study reports for the first time differences in the antiviral activity of EOs against DENV, corresponding to their composition of monoterpenes and sesquiterpenes.

A Comprehensive Study to Unleash the Putative Inhibitors of Serotype2 of Dengue Virus: Insights from an In Silico Structure-Based Drug Discovery.

Dengue fever is a mosquito-borne disease that claims the lives of millions of people around the world. A number of factors like disease's non-specific symptoms, increased viral mutation, growing antiviral drug resistance due to reduced susceptibility, unavailability of an effective vaccine for dengue, weak immunity against the virus, and many more are involved. Dengue belongs to the Flaviviridae family of viruses. The two species of the vector transmitting dengue are Aedes aegypti and Aedes albopictus, with the former one being dominant. Serotypes 2 of dengue fever are spread to the human body and cause severe illness. Recently, dengue has imposed an aggressive effect synergistically with the COVID-19 pandemic. As a result, we concentrated our efforts on finding a potential therapeutic. For this, we chose natural compounds to fight dengue fever, which is currently regarded as successful among many drug therapies. Following this, we started the in silico experiment with 922 plant extracts as lead compounds to fight serotype 2. In this study, we used SwissADME for analyzing ligand drug-likeness, pkCSM for designing an ADMET profile, Autodock vina 4.2 and Swissdock tools for molecular docking, and finally Desmond for molecular dynamics simulation. Ultimately 45 were found effective against the 2'O methyltransferase protein of serotype 2. CHEMBL376820 was found as possible therapeutic candidates for inhibiting methyltransferase protein in this thorough analysis. Nevertheless, more in vitro and in vivo research are required to substantiate their potential therapeutic efficacy.

Novel Antiviral Efficacy of Hedyotis diffusa and Artemisia capillaris Extracts against Dengue Virus, Japanese Encephalitis Virus, and Zika Virus Infection and Immunoregulatory Cytokine Signatures.

Currently, there are no specific therapeutics for flavivirus infections, including dengue virus (DENV) and Zika virus (ZIKV). In this study, we evaluated extracts from the plants Hedyotis diffusa (HD) and Artemisia capillaris (AC) to determine the antiviral activity against DENV, ZIKV, and Japanese encephalitis virus (JEV). HD and AC demonstrated inhibitory activity against JEV, ZIKV, and DENV replication and reduced viral RNA levels in a dose-responsive manner, with non-cytotoxic concentration ranging from 0.1 to 10 mg/mL. HD and AC had low cytotoxicity to Vero cells, with CC50 values of 33.7 ± 1.6 and 30.3 ± 1.7 mg/mL (mean ± SD), respectively. The anti-flavivirus activity of HD and AC was also consistent in human cell lines, including human glioblastoma (T98G), human chronic myeloid leukemia (K562), and human embryonic kidney (HEK-293T) cells. Viral-infected, HD-treated cells demonstrated downregulation of cytokines including CCR1, CCL26, CCL15, CCL5, IL21, and IL17C. In contrast, CCR1, CCL26, and AIMP1 were elevated following AC treatment in viral-infected cells. Overall, HD and AC plant extracts demonstrated flavivirus replication inhibitory activity, and together with immunoregulatory cytokine signatures, these results suggest that HD and AC possess bioactive compounds that may further be refined as promising candidates for clinical applications.

Potential application of chitosan stabilized silver nanoparticles for simultaneous control of dengue virus and mosquito vectors.

Dengue fever pandemic caused by dengue virus has been claimed for many lives, however, no specific treatment is available. Prevention based on vector control posed to be the most effective measure so far. The application of chitosan stabilized silver nanoparticle (AgNPs) to control larvae and adult mosquitoes has recently discovered, but their ability to inhibit Dengue virus has scarcely discussed. In this study, chitosan stabilized AgNPs have been prepared and tested against dengue virus type 1. The results showed that chitosan stabilized AgNPs can inhibit 96.66% and 98.33% dengue virus type 1 at 12.50 ppm and 25.00 ppm, respectively, without any toxicity to BHKFcɣ cells. This study confirms that chitosan stabilized AgNPs have a highly antiviral ability against dengue virus in addition to its mosquitocidal properties. This allows one to control both virus and vector simultaneously. The study suggested that chitosan stabilized AgNPs could be an effective tool to prevent the dengue epidemic.

New insights into the antiviral activity of nordihydroguaiaretic acid: Inhibition of dengue virus serotype 1 replication.

BACKGROUND: Dengue virus (DENV) is considered one of the most important pathogens in the world causing 390 million infections each year. Currently, the development of vaccines against DENV presents some shortcomings and there is no antiviral therapy available for its infection. An important challenge is that both treatments and vaccines must be effective against all four DENV serotypes. Nordihydroguaiaretic acid (NDGA), isolated from Larrea divaricata Cav. (Zygophyllaceae) has shown a significant inhibitory effect on a broad spectrum of viruses, including DENV serotypes 2 and 4. PURPOSE: We evaluated the in vitro virucidal and antiviral activity of NDGA on DENV serotype 1 (DENV1), including the study of its mechanism of action, to provide more evidence on its antiviral activity. METHODS: The viability of viral particles was quantified by the plaque-forming unit reduction method. NDGA effects on DENV1 genome and viral proteins were evaluated by qPCR and immunofluorescence, respectively. Lysosomotropic activity was assayed using acridine orange and neutral red dyes. RESULTS: NDGA showed in vitro virucidal and antiviral activity against DENV1. The antiviral effect would be effective within the first 2 h after viral internalization, when the uncoating process takes place. In addition, we determined by qPCR that NDGA decreases the amount of intracellular RNA of DENV1 and, by immunofluorescence, the number of cells infected. These results indicate that the antiviral effect of NDGA would have an intracellular mechanism of action, which is consistent with its ability to be incorporated into host cells. Considering the inhibitory activity of NDGA on the cellular lipid metabolism, we compared the antiviral effect of two inhibitors acting on two different pathways of this type of metabolism: 1) resveratrol that inhibits the sterol regulatory element of binding proteins, and 2) caffeic acid that inhibits the 5-lipoxygenase (5-LOX) enzyme. Only caffeic acid produced an inhibitory effect on DENV1 infection. We studied the lysosomotropic activity of NDGA on host cells and found, for the first time, that this compound inhibited the acidification of cell vesicles which would prevent DENV1 uncoating process. CONCLUSION: The present work contributes to the knowledge of NDGA activity on DENV. We describe its activity on DENV1, a serotype different to those that have been already reported. Moreover, we provide evidence on which stage/s of the viral replication cycle NDGA exerts its effects. We suggest that the mechanism of action of NDGA on DENV1 is related to its lysosomotropic effect, which inhibits the viral uncoating process.

Chemical Composition and Antifungal, Anti-Inflammatory, Antiviral, and Larvicidal Activities of the Essential Oils of Zanthoxylum acanthopodium DC. from China and Myanmar.

Zanthoxylum acanthopodium DC. is a widely used traditional medicinal plant to treat fever, flu, stomachache, traumatic injury, and mosquito bite in tropical and subtropical Asia. This study aimed to investigate the antifungal, anti-inflammatory, antiviral, and larvicidal activities of its fruit essential oil. The essential oil sample from China (EOZC) was mainly composed of limonene (29.78%) and ß-myrcene (26.65%), while the sample from Myanmar (EOZM) was dominated by Terpinen-4-ol (43.35%). Both essential oils showed antifungal activity, with 90% minimum inhibitory concentration (MIC90) values ranging from 26.3 to 499 µg/mL. By obviously inhibiting nitric oxide (NO) in RAW 264.7 cells, EOZC (IC50, 16 µg/mL) showed comparable anti-inflammatory activity to the positive control L-NMMA (IC50, 12.2 µg/mL). EOZM showed significant antiviral activity against the dengue virus with an IC50 value of 13 µg/mL. Additionally, both EOZC and EOZM demonstrated dose-dependent larvicidal activity against Aedes albopictus, with LC50 and LC90 values ranging from 45.8 to 144.0 µg/mL. Our results contribute a theoretical foundation for the further application of Zanthoxylum acanthopodium DC. as an antifungal and anti-inflammatory ingredient in the pharmaceutical industry and further indicate that it has the potential to be developed as a new source of natural and eco-friendly medicine for the prevention and treatment of dengue virus.

In silico testing of flavonoids as potential inhibitors of protease and helicase domains of dengue and Zika viruses.

Background: Dengue and Zika are two major vector-borne diseases. Dengue causes up to 25,000 deaths and nearly a 100 million cases worldwide per year, while the incidence of Zika has increased in recent years. Although Zika has been associated to fetal microcephaly and Guillain-Barré syndrome both it and dengue have common clinical symptoms such as severe headache, retroocular pain, muscle and join pain, nausea, vomiting, and rash. Currently, vaccines have been designed and antivirals have been identified for these diseases but there still need for more options for treatment. Our group previously obtained some fractions from medicinal plants that blocked dengue virus (DENV) infection in vitro. In the present work, we explored the possible targets by molecular docking a group of molecules contained in the plant fractions against DENV and Zika virus (ZIKV) NS3-helicase (NS3-hel) and NS3-protease (NS3-pro) structures. Finally, the best ligands were evaluated by molecular dynamic simulations. Methods: To establish if these molecules could act as wide spectrum inhibitors, we used structures from four DENV serotypes and from ZIKV. ADFR 1.2 rc1 software was used for docking analysis; subsequently molecular dynamics analysis was carried out using AMBER20. Results: Docking suggested that 3,5-dicaffeoylquinic acid (DCA01), quercetin 3-rutinoside (QNR05) and quercetin 3,7-diglucoside (QND10) can tightly bind to both NS3-hel and NS3-pro. However, after a molecular dynamics analysis, tight binding was not maintained for NS3-hel. In contrast, NS3-pro from two dengue serotypes, DENV3 and DENV4, retained both QNR05 and QND10 which converged near the catalytic site. After the molecular dynamics analysis, both ligands presented a stable trajectory over time, in contrast to DCA01. These findings allowed us to work on the design of a molecule called MOD10, using the QND10 skeleton to improve the interaction in the active site of the NS3-pro domain, which was verified through molecular dynamics simulation, turning out to be better than QNR05 and QND10, both in interaction and in the trajectory. Discussion: Our results suggests that NS3-hel RNA empty binding site is not a good target for drug design as the binding site located through docking is too big. However, our results indicate that QNR05 and QND10 could block NS3-pro activity in DENV and ZIKV. In the interaction with these molecules, the sub-pocket-2 remained unoccupied in NS3-pro, leaving opportunity for improvement and drug design using the quercetin scaffold. The analysis of the NS3-pro in complex with MOD10 show a molecule that exerts contact with sub-pockets S1, S1', S2 and S3, increasing its affinity and apparent stability on NS3-pro.

Anti-Dengue Screening on Several Vietnamese Medicinal Plants: Experimental Evidences and Computational Analyses.

Worldwide, medicinal plants have been known for economic and geographical advantages, thus possibly holding potentiality against dengue hemorrhagic fever. The methanol/water extracts from different parts of fourteen Vietnam-based plant species were subjected for experimental screening on anti-dengue activity using baby hamster kidney cells (BHK21) and plaque reduction neutralisation test (PRNT). Firstly, the methanol/water extracts were tested against serotype dengue virus DENV-1. Seven out from nineteen extracts show the PRNT50 values less than 31.25 µg/mL. Four of the above extracts namely from Euphorbia hirta, Cordyline terminalis, Carica papaya, and Elaeagnus latifolia were chosen for testing against the serotype DENV-2. All of them exhibit good activity with the PRNT50 values less than 31.25 µg/ml, which were further fractionated to obtain hexane, ethyl acetate and butanol fractions. Anti-dengue virus activity of the fractions against four serotypes DENV-1, -2, -3 and -4 was evaluated. As results, the ethyl acetate fraction of Elaeagnus latifolia is highly active against all four serotype viruses. The structural formulae of its nine constituents were input for molecular docking simulation. The docking-based order for static inhibitability is 6-3L6P>7-3L6P>9-3L6P>2-3L6P>3-3L6P≈5-3L6P>9-3L6P>1-3L6P>8-3L6P; QSARIS-based analysis reveals the biocompatibility of the most promising ligands (4-7); ADMET-based analysis expects their pharmacological suitability. Exceptional finding on 2-3LKW hydrophilic interaction at Lys43 (with the associated Gibbs free energy of -10.3 kcal mol-1 ) raises an open explanation for inhibitory effects. The results encourage further investigations for more in-depth mechanisms and drug development, such as in vitro enzyme assays or in vitro clinical trials with natural substances from E. latifolia.

Molecular Characterization and Identification of Potential Inhibitors for 'E' Protein of Dengue Virus.

Dengue is an arthropod-borne acute febrile illness caused by Dengue Virus (DENV), a member of Flaviviridae. Severity of the infection ranges from mild self-limiting illness to severe life-threatening hemorrhagic fever (DHF) and dengue shock syndrome (DSS). To date, there is no specific antiviral therapy established to treat the infection. The current study reports the epidemiology of DENV infections and potential inhibitors of DENV 'E' protein. Among the various serotypes, DENV-2 serotype was observed more frequently, followed by DENV-4, DENV-1, and DENV-3. New variants of existing genotypes were observed in DENV-1, 2, and 4 serotypes. Predominantly, the severe form of dengue was attributable to DENV-2 infections, and the incidence was more common in males and pediatric populations. Both the incidence and the disease severity were more common among the residents of non-urban environments. Due to the predominantly self-limiting nature of primary dengue infection and folk medicine practices of non-urban populations, we observed a greater number of secondary dengue cases than primary dengue cases. Hemorrhagic manifestations were more in secondary dengue in particularly in the pediatric group. Through different computational methods, ligands RGBLD1, RGBLD2, RGBLD3, and RGBLD4 are proposed as potential inhibitors in silico against DENV-1, -2, -3, and -4 serotypes.

Cranberry Pomace Extract Exerts Antiviral Activity against Zika and Dengue Virus at Safe Doses for Adult Zebrafish.

Mosquito-borne dengue virus (DENV) and zika virus (ZIKV) infections constitute a global health emergency. Antivirals directly targeting the virus infectious cycle are still needed to prevent dengue hemorrhagic fever and congenital zika syndrome. In the present study, we demonstrated that Cranberry Pomace (CP) extract, a polyphenol-rich agrifood byproduct recovered following cranberry juice extraction, blocks DENV and ZIKV infection in human Huh7.5 and A549 cell lines, respectively, in non-cytotoxic concentrations. Our virological assays identified CP extract as a potential inhibitor of virus entry into the host-cell by acting directly on viral particles, thus preventing their attachment to the cell surface. At effective antiviral doses, CP extract proved safe and tolerable in a zebrafish model. In conclusion, polyphenol-rich agrifood byproducts such as berry extracts are a promising source of safe and naturally derived nutraceutical antivirals that target medically important pathogens.