Integrated Metabolomics and Network Pharmacology to Decipher the Antioxidant and Anti-inflammatory Potential of Gymnostachyum febrifugum (Benth) and an In Vitro Validation

Gymnostachyum febrifugum is an endemic species of the Western Ghats of India and is well-mentioned in ethnomedicine to treat fever, ulcers, cough, and metrorrhagia. The present study focuses on the GC-MS/MS analysis to identify the phytochemicals with antioxidant and anti-inflammatory properties followed by integrated metabolomics and network pharmacology approaches to determine the molecular targets of antioxidant and anti-inflammatory potential of the phytochemicals in G. febrifugum. Further, the antioxidant and anti-inflammatory activities were validated using in vitro experimental methods. A total of 10 antioxidant and anti-inflammatory metabolites were identified by GC-MS/MS analysis. Based on the network pharmacology, ALB, HSP90AA1, PPARG, SOD2, and CASP3 as antioxidant protein targets, and ALB, CD4, MMP9, HMOX1, and EGFR as anti-inflammatory protein targets were identified. Molecular docking also confirmed binding affinities of the detected phytochemicals with these protein targets. The in vitro studies validated the in-silico results on the antioxidant and anti-inflammatory activities by the G. febrifugum. The in vitro results showed the potent antioxidant activity of ethyl acetate extract of G. febrifugum with SC50 values for 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), and hydrogen peroxide scavenging assay values of 0.17 ± 0.0 mg/mL, 0.23 ± 0.03 mg/mL and 0.110 ± 0.03 mg/mL respectively. The Ferric-Reducing Ability of Plasma (FRAP) assay showed an FRAP value of 0.2108 ± 0.01 mg GAE/mL. The antioxidant activity of gallic acid with SC50 for DPPH and ABTS was 0.0028 ± 0.10 and 0.004 ± 0.7, respectively. Similarly, the anti-inflammatory potential was confirmed with the inhibition of heat-induced protein denaturation and proteinase enzyme activity with IC50 values for 0.0410 ± 0.003 mg/mL and 0.0721 ± 0.073 mg/mL, respectively. Therefore, the study concludes that integrating in silico analysis with in vitro results validates the antioxidant and anti-inflammatory potential of G. febrifugum. This will further confirm the ethnomedicinal claim of G. febrifugum to treat fever, ulcer, cough, and snake bites. Further work on the isolation of individual phytochemicals with antioxidant and anti-inflammatory activities will be beneficial to develop suitable health-benefiting antioxidant and anti-inflammatory agents.