Material basis, herbal contribution, and potential quality markers of Kanggan Granules against influenza revealed by UPLC-HRMS, network pharmacology, and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Kanggan Granules (KGG) is a classical traditional Chinese medicine formulation recorded in the Chinese Pharmacopoeia. Historically, KGG has been used for wind-heat colds and related respiratory infections. Although KGG is widely used, its efficacy against influenza A virus infection, as well as its underlying mechanisms and material basis, has not been systematically elucidated.

AIM OF THE STUDY: This study aimed to evaluate the therapeutic effects of KGG against influenza virus pneumonia (IVP), elucidate the contribution of its constituent herbs, and clarify its pharmacological substance basis and potential quality markers.

MATERIALS AND METHODS: Integrated in vitro and in vivo models were used to assess the anti-influenza activity of KGG and explore its underlying mechanisms. UPLC-HRMS was used to characterize the chemical constituents of KGG and serum-absorbed components in mice. Network pharmacology based on absorbed constituents was used to predict potential bioactive compounds, targets, and pathways, while proteomics and Western blot analysis were performed to validate these predictions at the system and protein levels, respectively. The antiviral activities of selected candidate active constituents were verified in vitro, and major active constituents of KGG were quantified by HPLC.

RESULTS: In vitro, KGG suppressed influenza A virus (A/WSN/33) replication with high selectivity, reduced viral plaques, blocked hemagglutination and neuraminidase activity, and attenuated pro-inflammatory cytokine/chemokine upregulation. In vivo, KGG improved the survival of H1N1-infected mice, lowered lung index, relieved hematological abnormalities, mitigated lung tissue damage, and modulated cytokine levels in lung homogenates. UPLC-HRMS characterized 472 constituents in KGG, of which 122 were detected in mouse serum. Network pharmacology analysis suggested potential bioactive constituents of KGG and implicated TLR4/TLR7-MyD88- NF-κB and NLRP3-related pathways in its anti-influenza effects, which were further supported by proteomics and validated by Western blot analysis. Among the tested candidate constituents, syringin (109) and sweroside (244) showed the strongest antiviral activities in vitro. Twelve major constituents were quantified in KGG, including nine serum-absorbed components. Based on comprehensive comparative analysis, seven compounds, namely gallic acid (106), morroniside (194), chlorogenic acid (203), sweroside (244), secoxyloganin (272), paeoniflorin (278), and 3,4-O-dicaffeoylquinic acid (323), may serve as potential quality markers linked to the anti-influenza efficacy of KGG.

CONCLUSION: KGG exerts significant anti-influenza effects both in vitro and in vivo. Its activity is associated with the regulation of the TLR/MyD88/NF-κB and NLRP3 signaling pathways, while chlorogenic acid (203) and paeoniflorin (278) were identified as key bioactive constituents. In addition, seven absorbed components were proposed as potential anti-influenza quality markers for KGG, providing a reference for elucidating its pharmacological substance basis and quality control.