Interpretation of the anti-influenza active ingredients and potential mechanisms of Ge Gen Decoction based on spectrum-effect relationships and network analysis

Ge Gen Decoction (GGD) is a classic traditional Chinese medicine (TCM) prescription that originated in the ancient Chinese medical book "Treatise on Febrile Diseases". The prescription consists of 7 herbs: Pueraria lobata (Willd.) Ohwi, Ephedra sinica Stapf, Cinnamomum cassia (L.) J.Presl, Paeonia lactiflora Pall., Glycyrrhiza uralensis Fisch., Zingiber officinale Rosc., and Ziziphus jujuba Mill. It can alleviate high fever and soreness in the neck and shoulders caused by exogenous wind chill and is widely used in both China and Japan. Currently, GGD is primarily utilized for treating flu and the common cold. GGD has been reported to show significant anti-influenza A virus (IAV) activity both in vitro and in vivo. However, the active ingredients responsible for its anti-influenza properties have not been elucidated, and the mechanisms underlying its anti-influenza effects require further research.This study aims to investigate the active ingredients and molecular mechanisms of GGD in treating influenza.HPLC chromatograms were established for GGD water and different polar extracts. The effect of different GGD extracts on pulmonary virus titers and TNFα expression was assessed through RT-PCR analysis. Spectrum-effect relationships between chromatographic peaks of GGD and its virus inhibition rate and TNFα inhibition rate were investigated using partial least squares regression (PLSR) analysis. HPLC-Q-TOF-MS was utilized to identify the constituents absorbed into the blood after oral administration of GGD. Network analysis of the absorbed forms of active ingredients was conducted to predict the potential mechanisms of GGD. Subsequently, total SOD activity, CAT and HO-1 expression and Nrf2 nuclear translocation were then analyzed. Finally, the impact of interfering with HO-1 expression on the anti-IAV activity of GGD was examined.The study identified 11 anti-influenza active ingredients in GGD, which are daidzein, ononin, genistin, daidzin, 3'-methoxypuerarin, puerarin, pseudoephedrine, paeoniflorin, pormononetin-7-xylosyl-glucoside, penistein-7-O-apiosyl-glucoside, and ephedrine. Network analysis revealed various biological activities of GGD, including responses to ROS and oxidative stress. GGD also involves multiple antiviral pathways, such as hepatitis B, IAV, and Toll-like receptor pathways. Experimental assays demonstrated that GGD possesses independent antioxidant activity both in vitro and in vivo. In vitro, GGD inhibits the increase in intracellular ROS induced by IAV. In vivo, it reduces MDA levels and increases total pulmonary SOD activity. Applying siRNA and flow cytometry analysis revealed that GGD alleviates IAV-induced oxidative burst by promoting the expression of HO-1 and CAT. Western blot analysis revealed that GGD effectively promotes Nrf2 nuclear translocation and enhances Nrf2 expression. Furthermore, this study found that the enhancement of HO-1 expression by GGD contributed to its anti-IAV activity.The study identified the active ingredients of GGD against influenza and demonstrated the beneficial role of GGD's antioxidant activity in treating flu. The antioxidant activity of GGD is associated with the promotion of Nrf2 nuclear translocation and the upregulation of antioxidant enzymes such as SOD, HO-1, and CAT. Overall, this study provides evidence supporting the use of GGD as an adjunctive or complementary therapy for influenza.