Flavonoids are natural biomolecules that are known to be effective antivirals. These biomolecules can act at different stages of viral infection, particularly at the molecular level to inhibit viral growth. Enterovirus A71 (EV-A71), a non-enveloped RNA virus, is one of the causative agents of hand, foot and mouth disease (HFMD), which is prevalent in Asia. Despite much effort, no clinically approved antiviral treatment is available for children suffering from HFMD. Flavonoids from plants serve as a vast reservoir of therapeutically active constituents that have been explored as potential antiviral candidates against RNA and DNA viruses. Here, we reviewed flavonoids as evidence-based natural sources of antivirals against non-picornaviruses and picornaviruses. The detailed molecular mechanisms involved in the inhibition of EV-A71 infections are discussed.

Background: The prevalence of infertility and vitamin D deficiency is common in Pakistan. Therefore, our study aims were to assess and compare Vitamin D; 25-hydroxyvitamin (25OHD) and reproductive hormone levels in male fertile and infertile subjects with normal and abnormal sperm parameters. Furthermore, the study is aimed to explore the association of 25OHD levels with these sperm parameters in a selected population of Karachi, Pakistan. Methods: The cross-sectional study was carried out from August 2016 till December 2017, 313 study subjects were recruited from an Infertile Clinic from Islamabad, Pakistan, and the general population. First, we took the couples' history of parenting and then carried out a semen analysis and infertile and fertile male subjects were then subgrouped into “normal” and “altered sperm parameter/s.” Forward linear regression was done for selection of 25OHD as a significant predictor of sperm parameters. Results: The median values of the total count, motility, morphology as well as serum 25OHD were significantly higher in the group with “normal” (186) as compared to subjects (127) in “abnormal sperm parameters” group. The 25OHD levels were significantly high in males with “normal sperm parameters”; 80.90 ± 23.33 nmol/L vs. “altered sperm parameter/s,” 64.68 ± 24.21 nmol/L (mean ± SD) with p < 0.001. Serum testosterone level had a significant positive correlation with 25OHD while LH had a significant negative correlation with 25OHD (p < 0.001), and FSH level had a non-significant negative correlation with 25OHD. Results of regression model showed one unit increase of motility would give 0.15-unit positive significant impact on 25OHD; 20% variation in 25OHD was explained by the total count, motility, and morphology, while the model was adjusted for BMI. Conclusion: The impact of 25OHD levels on sperm parameters can be emphasized on the basis of detection of its high serum levels in “normal” subjects in both fertile as well as infertile males in comparison to subjects that had altered sperm parameters; total sperm count, motility, and normal morphology. The considerably positive association between 25OHD, testosterone, total count, motility, and morphology further accentuates its impact on normal spermatogenesis and the male reproductive functions required for acquiring fertility.

Background: The hand, foot and mouth disease (HFMD) is a febrile and exanthematous childhood disease mainly caused by Enterovirus 71 (EV-A71). In severe HFMD, virulent EV-A71 strains can cause acute flaccid paralysis and cardiopulmonary edema leading to death. Currently, no FDA approved antiviral treatment or vaccine is available for EV-A71. Flavonoids such as silymarin and baicalein are known to possess in vitro antiviral properties against viruses. In this study, the cytotoxicity and antiviral activity of silymarin, baicalein and baicalin were investigated. Methods: The cytotoxic effects of three flavonoids towards rhabdomyosarcoma (RD) cells were first examined using cell proliferation MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Compounds found to be non-cytotoxic in RD cells were evaluated for their in vitro antiviral properties against the EV-A71 subgenotype B4 strain 41 (5865/SIN/000009) using antiviral assays. Viral infectivity was determined by reduction of the formation of plaques in RD cells. For the measurement of RNA copy number, the real time quantitative reverse transcription PCR (qRT-PCR) was used. The most potent compound was further evaluated to determine the mode of action of inhibition by time course, virus attachment and entry assays in Vero cells. Results: Silymarin was shown to exert direct extracellular virucidal effects against EV-A71 at 50% inhibitory concentration (IC₅₀) of 15.2 ± 3.53 μg/mL with SI of 10.53. Similarly, baicalein exhibited direct extracellular virucidal effects against EV-A71 at a higher IC₅₀ value of 30.88 ± 5.50 μg/mL with SI of 13.64. Besides virucidal activity, silymarin was shown to block both viral attachment and entry of EV-A71 to inhibit infection in Vero cells. Conclusions: Silymarin has a stronger inhibition activity against EV-A71 in comparison to baicalein. It could serve as a promising antiviral drug to treat EV-A71 infections.