# 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

## TL;DR

This study explores how a plant extract affects the growth of a gut bacterium linked to metabolic health in both lab and mouse models.

## Contribution

The study identifies the hexane extract of Odontosoria biflora as a growth promoter for Akkermansia muciniphila in vitro but shows limited in vivo efficacy in diabetic mice.

## Key findings

- O. biflora hexane extract significantly stimulated A. muciniphila growth in vitro at 250 and 500 mg/L.
- The extract showed no toxicity in mice but failed to sustainably increase A. muciniphila in fecal samples.
- Metformin-treated mice showed sustained A. muciniphila elevation, unlike OBE-HEX-treated mice.

## Abstract

Akkermansia muciniphila is a mucin-degrading gut bacterium linked to metabolic health, yet culture-based growth stimulation may not translate to sustained enrichment in vivo. Odontosoria biflora (“tubho”) is traditionally consumed in the Philippines as a herbal tea, but its extract-level activity toward A. muciniphila remains poorly characterized. O. biflora was sequentially extracted (hexane, ethyl acetate, methanol, aqueous) and screened for growth-supporting activity toward A. muciniphila 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 A. muciniphila-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 in vitro 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 in vivo. In contrast, in vivo analysis revealed only transient changes in fecal A. muciniphila 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 O. biflora hexane extract exhibits direct growth-supporting activity toward A. muciniphila in vitro and is orally tolerable, such effects do not translate into sustained in vivo enrichment under diabetic conditions, underscoring the limitations of extrapolating culture-based microbiota screening results to host-associated systems.