Phytochemical Profiling and Bioactivity Evaluation of Ganoderma lucidum (Reishi Mushroom) Fractions: In Vitro Antioxidant, Antimicrobial, and Antidiabetic Activities

Background/Objectives: Ganoderma lucidum (Curtis) P. Karst. (commonly known as reishi mushroom), a well-characterized medicinal fungus, contains diverse bioactive metabolites. This study aimed to fractionate, characterize and identify the biologically active inhibitors present in G. lucidum and to evaluate their antioxidant, antimicrobial, and antidiabetic activities. Methods: The ethanol extract of G. lucidum was fractionated using column chromatography, yielding ten distinct fractions (designated as A, B, E, F, K, L, M, N, O, and P based on their elution order and visual characteristics). Liquid Chromatography–Mass Spectrometry (LC-MS) analysis identified 46 bioactive compounds, including terpenoids, alkaloids, flavonoids, and polysaccharides. Results: Among the fractions, Fraction L exhibited the strongest antioxidant activity, with an IC50 of 1.59 mg/mL. Fraction O displayed significant antibacterial activity against Escherichia coli ATCC 25922 (24.4 ± 0.238 mm), Klebsiella pneumoniae ATCC 13883 (20.5 ± 0.035 mm), Bacillus subtilis ATCC 6633 (8 ± 0.176 mm), and Staphylococcus warneri ATCC 10209 (20 ± 0.080 mm). Regarding antidiabetic activity, Fraction B demonstrated the strongest inhibition of α-amylase (IC50 1.69 ± 0.03 mg/mL), while Fraction E showed the strongest α-glucosidase inhibition (IC50 = 1.69 ± 0.02 mg/mL), demonstrating reciprocal selectivity between enzyme targets. Conclusions: These results establish that chromatographic fractionation concentrates specific bioactivities into distinct fractions, supporting its potential for the development of novel therapeutic agents with enhanced specificity and efficacy.