Phytochemical profiling, antioxidant activity, and structural elucidation of bioactive compounds from anaerobically digested cow dung slurry

Introduction
Anaerobic digestion (AD) is heralded as a cornerstone of sustainable waste management, converting organic residues into valuable biogas. While biogas production from AD is well-documented, the valorisation of anaerobic digestate, particularly for its pharmaceutical applications, remains underexplored. We analysed aqueous ethanolic (70%) and methanolic (50%) extracts from cow dung slurry (CSW) to characterise their bioactive compounds, antioxidant capacity, and structural properties.

Results
Qualitative and quantitative phytochemical analyses revealed a diverse array of secondary metabolites, including phenolics, flavonoids, tannins, alkaloids, terpenoids, glycosides, and saponins, whereas phlobatannins, volatile oils, and resins were undetectable. Notably, the ethanol extract exhibited significantly higher concentrations of total phenolics (~ 250 µg catechin equivalents [CE]/mL) and flavonoids (~ 220 µg CE/mL) than the methanol extract (p < 0.0001), while other classes, such as saponins, tannins, flavanols, and proanthocyanidins, were present at lower and comparable levels across solvents (p > 0.05). Fourier-transform infrared spectroscopy (FTIR) analysis indicated a higher abundance of phenolic (O-H stretching at 3450.53 cm− 1) and carboxyl (C = O at 1778.40 cm− 1) groups in the methanol extract, suggesting strong antioxidant potential. In contrast, the ethanol extract contained alkyl (2982.51 cm− 1), nitro (1588.02 cm− 1), and halogenated (652.35 cm− 1) functional groups. Nuclear magnetic resonance (NMR) spectroscopy further identified key secondary metabolites, including harmine, salicylic acid, anacardic acid, quercetin, and gallic acid. In vitro, free radical scavenging assays demonstrated concentration-dependent antioxidant activity, with the methanol extract consistently outperforming the ethanol extract (p < 0.05). At 800 µg/mL, the methanol extract exhibited near-comparable activity to gallic acid (p = 0.0927).

Conclusion
These findings suggest that CSW-derived methanol extract holds significant promise as a natural source of bioactive compounds with potential pharmaceutical and nutraceutical applications.