Differential in vitro and in vivo responses of Akkermansia muciniphila to Odontosoria biflora (Kaulf.) C.Chr. [Lindsaeaceae] hexane extract in diet- and alloxan-induced BALB/c mice

<i>Akkermansia muciniphila</i> is a mucin-degrading gut bacterium linked to metabolic health, yet culture-based growth stimulation may not translate to sustained enrichment <i>in vivo</i>. <i>Odontosoria biflora</i> ("tubho") is traditionally consumed in the Philippines as a herbal tea, but its extract-level activity toward <i>A. muciniphila</i> remains poorly characterized. <i>O. biflora</i> was sequentially extracted (hexane, ethyl acetate, methanol, aqueous) and screened for growth-supporting activity toward <i>A. muciniphila</i> in modified BHI under anaerobic conditions. The most active fraction (hexane; OBE HEX) was evaluated for acute oral tolerability in BALB/c mice according to OECD Tesy No. 423 (up to 2000 mg/kg) and subsequently assessed in a high-fat/high-sugar diet plus alloxan-induced diabetic model. Fecal <i>A. muciniphila-</i>specific qPCR signal was monitored at weeks 0, 1, 2, and 4 using a modified 2^-ΔΔCt approach with external ATCC genomic DNA as a reference. OBE-HEX produced the strongest <i>in vitro</i> growth-supporting effect (56.43% at 250 mg/L, p < 0.05; 85.62% at 500 mg/L, p < 0.001) and showed no observable toxicity <i>in vivo</i>. In contrast, <i>in vivo</i> analysis revealed only transient changes in fecal <i>A. muciniphila</i> detection following OBE-HEX administration, whereas sustained elevation was observed only in metformin-treated mice. Untargeted UPLC-ESI-QTOF-MS analysis of OBE-HEX yielded putative identification of 2-O-rhamnosylvitexin and 7-methoxy-9,10-dihydrophenanthrene-2,5-diol. Overall, these findings demonstrate that while <i>O. biflora</i> hexane extract exhibits direct growth-supporting activity toward <i>A. muciniphila in vitro</i> and is orally tolerable, such effects do not translate into sustained <i>in vivo</i> enrichment under diabetic conditions, underscoring the limitations of extrapolating culture-based microbiota screening results to host-associated systems.