Elucidation of flavonoids from potent Iranian Scutellaria species against Influenza A (H1N1) virus

Influenza A virus (IAV), particularly the H1N1 subtype, causes significant morbidity and mortality due to frequent antigenic drift and shift, leading to resistance to current antivirals (M2 blockers and neuraminidase inhibitors). There is a need for new anti-influenza agents. Scutellaria (Lamiaceae), known as Skullcap, includes over 350 species and is used traditionally for various ailments including respiratory infections. Iranian flora hosts over 20 Scutellaria species. Flavonoids, major phenolic secondary metabolites abundant in Scutellaria, are associated with diverse bioactivities including antiviral effects. This study investigated phenolic and flavonoid contents and anti-influenza activity of several Iranian Scutellaria species and aimed to isolate and identify active constituents from the most potent species.

Medicinal plants have yielded numerous antiviral leads against diverse viruses. Several botanicals (e.g., Sambucus nigra, Kaempferia parviflora, Curcuma longa, Ephedra sinica, Eleutherococcus senticosus, Glycyrrhiza glabra, Brassica juncea, Scutellaria baicalensis) exhibit anti-influenza activity via multiple pathways. Scutellaria has extensive ethnomedicinal use in East Asia and beyond, with modern pharmacology validating anti-tumor, antioxidant, anti-inflammatory, antibacterial, antiviral, cardiovascular, hepatoprotective, and neuroprotective actions, largely attributed to flavonoids (e.g., baicalin, baicalein, wogonin, oroxylin A, scutellarin). Phenolics, particularly flavonoids, contribute to plant defense and human health (anti-atherosclerotic, antiviral, antimicrobial, antioxidant, etc.). Prior literature documents antiviral activities of key flavonoids such as quercetin, luteolin, kaempferol, and apigenin, including effects on influenza and coronaviruses, supporting the rationale to examine Iranian Scutellaria species as antiviral sources.

- Plant materials: Four Scutellaria species collected across Iran, including S. pinnatifida (subsp. mucida, viridis, alpina), S. tournefortii, S. tomentosa, S. persica. Species identification by botanists (voucher specimens deposited at Tehran University of Medical Sciences Herbarium and Iranian Institute of Medicinal Plants Herbarium). - Extraction and fractionation: Shade-dried, pulverized plants extracted by maceration in methanol:water (8:2) to yield total extracts, then partitioned with chloroform to yield chloroform fractions and methanol residues (methanol fractions). - Total phenol assay: Folin–Ciocalteu method on samples at 500 μg/ml in methanol; 1.5 ml diluted Folin reagent added, followed by 1.5 ml sodium bicarbonate; absorbance at 725 nm after 90 min in dark. Gallic acid (0–100 mg/ml) for calibration; results as mg gallic acid equivalents per g fraction. - Total flavonoid assay: Aluminum chloride colorimetric method. Samples 100 μg/ml processed with NaNO2, AlCl3, NaOH; absorbance at 510 nm after 30 min. Catechin standards (25–500 μg/ml); results as mg catechin equivalents per g fraction. - Cell culture and virus: MDCK cells in DMEM + 10% FBS + pen/strep. IAV A/Puerto Rico/8/1934 (H1N1) (ATCC VR-1469). Infectivity (CCID50) by Kärber method. - Cytotoxicity (MTT): MDCK exposed to serial concentrations (3.125–0.003 mg/ml) of fractions for 48 h (37°C, 5% CO2). MTT 1X incubation 3–4 h, formazan dissolved in DMSO; OD at 540 nm. CC50 defined; NCTC determined; Selectivity Index (SI) calculated as CC50/NCTC. - Antiviral assays: Co-, pre-, and post-penetration treatments using 100 TCID50 virus and NCTCs. After adsorption/incubation, unabsorbed virus replaced by TPCK trypsin medium (1 μg/ml); 48 h incubation. MTT measured cell protection. Concurrent HA assay on supernatants to determine virus titers. - Hemagglutination (HA) and hemagglutination inhibition (HI): Two-fold serial dilutions with 1% chicken RBCs; HA titer as reciprocal of highest dilution with agglutination. HI assessed physical interaction with viral HA by pre-incubating virus with serial dilutions of fractions. - Statistics: One-way ANOVA with Fisher’s LSD post hoc (P<0.05) for cell protection and HA log reductions. - Phytochemical separation/identification (for most potent species S. pinnatifida subsp. viridis): Polyphenol enrichment on Diaion HP-20; subsequent separations on reversed-phase silica gel and Sephadex LH-20 using water–methanol gradients. Isolation of five compounds. Structural elucidation by UV, 1H-NMR, and 13C-NMR.

- The methanol fraction of Scutellaria pinnatifida subsp. viridis exhibited the highest flavonoid content and strongest anti-influenza activity across treatment modes, reducing virus titer by 5 logs without detectable cytotoxicity. - Isolated compounds from S. pinnatifida subsp. viridis methanol fraction: kaempferol-3-O-glucoside, quercetin-3-O-glucoside, apigenin-4′-methoxy-7-O-glucoside (tilianin), luteolin, luteolin-7-O-glucoside. - HA titer reductions: • Co-penetration: Methanol fraction of S. pinnatifida subsp. viridis (Zarabad) and S. tomentosa (Kashan) each reduced virus titer by 5.00±0.82 logs; amantadine reduced by 7 logs. • Pre-penetration: Methanol fraction of S. pinnatifida subsp. viridis (Bahram abad) and S. persica (Takab) reduced titer by 4.5 logs. • Post-penetration: Methanol fraction of S. persica (Takab) and chloroform fraction of S. pinnatifida subsp. viridis (Zarabad) reduced titer by 5 logs. Oseltamivir reduced titers by 8 logs across treatments. - Cytotoxicity (CC50) ranges: • Chloroform fractions: Highest CC50 S. tournefortii (Gorgan) 771.74±2.89 μg/ml; lowest CC50 S. pinnatifida subsp. mucida (Barajin) 37.07±0.71 μg/ml. • Methanol fractions: Highest CC50 S. tournefortii (Ramsar) 6157.12±1.02 μg/ml; lowest CC50 S. tournefortii (Gorgan) 3.07±0.19 μg/ml. - Cell protection: Candidates were defined by ≥40% protection and ≥4.0 log HA reduction with SI ≥2. Most samples showed adequate optical densities indicating protection in various treatment modes, with noted exceptions (e.g., methanol fraction of S. pinnatifida subsp. viridis (Zarabad) 17.36±3.72% in co-penetration; chloroform fraction of S. persica (Takab) 23.16±6.29%). Oseltamivir showed OD >1.00 in all treatments. - Statistical significance: Pre-, co-, and post-penetration treatment groups showed significant improvements versus controls in both cell viability and HA log decrement (P<0.05).

The study addressed the need for new anti-influenza agents by demonstrating that Iranian Scutellaria species, particularly S. pinnatifida subsp. viridis, possess strong anti-H1N1 activity. Methanol fractions, enriched in polar flavonoids, consistently outperformed chloroform fractions, aligning with the hypothesis that flavonoids drive antiviral effects. Significant reductions in HA titers and increased cell protection suggest mechanisms including direct interaction with viral hemagglutinin and potential interference with viral entry/replication. Isolation of luteolin, quercetin, kaempferol, and apigenin derivatives supports literature reports of these flavonoids’ antiviral properties. Comparative activity across pre-, co-, and post-penetration treatments indicates both prophylactic and therapeutic potential. The findings are relevant for developing plant-derived antivirals or adjunct therapies and for guiding bioassay-guided isolation of active constituents.

Methanol fractions of S. pinnatifida subsp. viridis and S. tomentosa showed strong anti-influenza A (H1N1) activity, with up to 5-log reductions in viral titer and favorable cytotoxicity profiles. Five flavonoids (luteolin, luteolin-7-O-glucoside, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, apigenin-4′-methoxy-7-O-glucoside) were isolated from S. pinnatifida subsp. viridis, highlighting this species as a rich source of antiviral flavonoids. These findings support Scutellaria, especially S. pinnatifida subsp. viridis, as a promising candidate for anti-influenza therapeutics or preventive supplements. Future work will focus on purifying active compounds responsible for anti-IAV activity and elucidating their mechanisms of action, potentially integrating these agents with existing antivirals.