<p>Supporting materials for figures.</p>

Traditional herbal extracts are attracting attention in the context of animal disease control because of their low side effects, diverse bioactive compounds, and low antimicrobial resistance risk. However, the underlying mechanisms remain inadequately understood. To characterize the multifaceted biological activities that underlie their therapeutic potential, this study systematically evaluated the antiviral and complex immunomodulatory properties of three distinct herbal combinations (designated Extracts A, B, and C) in vitro. The antiviral activities of the extracts were tested against bovine rotavirus and bovine coronavirus, two major pathogens of neonatal calf diarrhea, and their regulation of inflammatory mediators was assessed in a murine macrophage model (RAW 264.7 cells) stimulated with lipopolysaccharide from Escherichia coli by measuring nitric oxide production and the gene expression of inflammation related enzymes. Immunomodulatory effects were investigated by analyzing the gene expression of T helper cell-associated cytokines in both RAW 264.7 cells and bovine peripheral blood mononuclear cells (PBMCs). All three extracts exhibited inhibitory activity against both viruses and reduced the expression of specific inflammatory mediators. Furthermore, the extracts demonstrated complex immunomodulatory effects in bovine PBMCs, with Extract C promoting T helper 1 and T helper 17 responses while suppressing the regulatory T-cell transcription factor Forkhead Box P3. In conclusion, this in vitro study demonstrates that these herbal extracts possess antiviral and immunomodulatory potential, providing a basis for future studies to determine their relevance in viral infections associated with calf diarrhea.在动物疫病防控的背景下，传统植物提取物因副作用低、生物活性成分多样且抗菌耐药风险低而受到广泛关注。然而其发挥治疗作用的潜在分子机制仍未得到充分阐明。为了阐明支撑其治疗潜力的多维度生物学活性，本研究通过体外实验系统评估了三种不同植物组合（分别命名为提取物A、B、C）的抗病毒活性与复杂免疫调节特性。本研究以引起新生犊牛腹泻的两大主要病原体——牛轮状病毒与牛冠状病毒为靶标，检测了各提取物的抗病毒活性；同时以大肠杆菌脂多糖刺激的小鼠巨噬细胞模型（RAW 264.7细胞）为对象，通过检测一氧化氮生成量与炎症相关酶的基因表达水平，评估了提取物对炎症介质的调控作用。此外，本研究通过分析RAW 264.7细胞与牛外周血单个核细胞（peripheral blood mononuclear cells，PBMCs）中辅助性T细胞（T helper cell）相关细胞因子的基因表达，探究了提取物的免疫调节效应。三种提取物均对上述两种病毒表现出抑制活性，且可降低特定炎症介质的基因表达水平。此外，三种提取物在牛外周血单个核细胞中展现出复杂的免疫调节活性，其中提取物C可促进辅助性T细胞1（T helper 1）与辅助性T细胞17（T helper 17）的应答，同时抑制调节性T细胞的转录因子叉头框P3（Forkhead Box P3）。综上，本体外研究证实这些植物提取物具备抗病毒与免疫调节潜力，为后续探究其在犊牛腹泻相关病毒感染中的应用价值提供了研究基础。