Influenza A virus (IAV), particularly the H1N1 subtype, poses a significant global health threat due to its high mutation rate and potential for severe respiratory infections. Current antiviral treatments face challenges due to drug resistance, highlighting the need for novel antiviral compounds. Medicinal plants offer a promising source of such compounds, with several studies demonstrating the antiviral activity of various plant extracts against IAV. The Scutellaria genus, known for its traditional use in treating respiratory infections, has shown promise in preclinical studies. This study aims to evaluate the anti-influenza potential of various Iranian Scutellaria species, focusing on identifying the active compounds responsible for their antiviral effects. The research is grounded in the understanding that flavonoids, a major class of plant secondary metabolites, possess diverse biological activities, including antiviral properties. The abundance of flavonoids in Scutellaria species makes it a prime candidate for investigating antiviral potential against IAV. This study will build upon existing literature demonstrating the antiviral activity of plants and specifically focus on the detailed analysis of flavonoids within Scutellaria species found in Iran.

Several studies have highlighted the antiviral activity of various plants against influenza viruses. Plants like Sambucus nigra, Kaempferia parviflora, Curcuma longa, Ephedra sinica, Eleutherococcus senticosus, Glycyrrhiza glabra, and Brassica juncea have demonstrated significant antiviral effects through different mechanisms. Scutellaria baicalensis, another member of the Scutellaria genus, has been shown to possess anti-H1N1 activity, attributed to its flavonoid content. Previous research has established the diverse pharmacological activities of the Scutellaria genus, including anti-tumor, antioxidant, anti-inflammatory, antibacterial, and antiviral effects, all potentially linked to its high flavonoid concentration. Flavonoids themselves are known to possess a wide array of biological activities, including antioxidant, anti-inflammatory, and antiviral properties. The specific flavonoids investigated in this paper are luteolin, quercetin, kaempferol and apigenin, each with a substantial body of literature supporting their biological activity.

The study involved the collection and identification of several Iranian Scutellaria species: S. pinnatifida (with mucida, viridis, and alpina subspecies), S. tournefortii, S. tomentosa, and S. persica. These were extracted and fractionated using methanol and chloroform. Total phenol and flavonoid content were assessed using the Folin-Ciocalteu and aluminum-flavonoid complex methods respectively. Cytotoxicity (CC50) on MDCK cells was determined using the MTT assay, establishing non-cytotoxic concentrations (NCTC). Antiviral activity was evaluated using pre-, post-, and co-penetration treatments against IAV (A/Puerto Rico/8/1934 (H1N1)). Cell protection and virus titer reduction were measured using MTT and hemagglutination assays. The most effective species, S. pinnatifida subsp. viridis, underwent phytochemical analysis using various chromatographic techniques (Diaion HP-20 resin, reversed-phase silica gel, Sephadex LH-20) to isolate and purify compounds. Compound identification was achieved using UV, 1H-NMR, and 13C-NMR spectroscopy. Statistical analysis employed one-way ANOVA and Fisher's LSD post hoc test to compare treatment groups. The study meticulously documented all steps, ensuring reproducibility and rigor in its scientific approach.

The methanol fraction of S. pinnatifida subsp. viridis exhibited the highest flavonoid content and the strongest antiviral activity against IAV in all treatment groups (co-, pre-, and post-penetration). It significantly reduced viral titer by 5 logs (100,000-fold reduction) without displaying cytotoxicity. Five flavonoids were successfully isolated and identified from this species: kaempferol-3-O-glucoside, quercetin-3-O-glucoside, apigenin-4′-methoxy-7-O-glucoside, luteolin, and luteolin-7-O-glucoside. S. tomentosa also showed potent antiviral effects, warranting further investigation. Statistical analyses (one-way ANOVA and Fisher's LSD post hoc test) revealed significant differences between treatment groups and controls for both cell viability (MTT assay) and virus titer reduction (hemagglutination assay) (P < 0.05). The study also documented total phenol and flavonoid content for each Scutellaria species, indicating considerable variation among different species and fractions. Cytotoxicity assays provided CC50 and NCTC values for each fraction, guiding the selection of appropriate non-toxic concentrations for antiviral testing.

The results demonstrate that S. pinnatifida subsp. viridis is a promising source of natural flavonoids with potent anti-influenza activity. The identification of specific flavonoids responsible for this activity opens avenues for further research into their mechanisms of action against IAV. The study confirms the traditional medicinal use of Scutellaria species for respiratory infections, providing scientific validation for their efficacy against IAV. The significant reduction in viral titer observed in this study highlights the potential of these flavonoids as antiviral agents, potentially serving as lead compounds for the development of novel anti-influenza drugs or as supplements to enhance immunity and reduce the severity of influenza infections. The significant activity of S. tomentosa suggests further investigations into its antiviral properties and the identification of its bioactive compounds.

This study successfully identified S. pinnatifida subsp. viridis as a rich source of flavonoids with potent anti-influenza activity. Five flavonoids were isolated and identified, demonstrating their efficacy against IAV. S. tomentosa also exhibits significant potential and requires further investigation. These findings support the traditional use of Scutellaria in treating respiratory infections and highlight the potential for developing novel anti-influenza drugs or supplements from these plants. Future research should focus on elucidating the precise mechanisms of action of these flavonoids and evaluating their efficacy in in vivo models.

The study's findings are limited to in vitro studies using a specific IAV strain. Further in vivo studies are necessary to confirm the efficacy and safety of these flavonoids. The study focused on a limited number of Scutellaria species; a broader investigation across diverse species and geographic locations would enhance the generalizability of findings. The purification and identification of bioactive compounds were limited to the most potent species; similar investigations on other promising species would provide more comprehensive knowledge.