Association of human gut microbiota composition and metabolic functions with *Ficus hirta* Vahl dietary supplementation

The study investigates how a tonic diet incorporating Ficus hirta Vahl (FHVD) affects the human gut microbiome’s composition and metabolic function. While FHV is traditionally used in southern China and has reported antioxidant and anti-inflammatory properties (primarily in vitro and animal studies), its effects on the human gut microbiota remain unclear. Given the established links between diet, gut microbiota, and host health, the authors aimed to determine whether FHVD modulates microbial taxa, functional pathways, and fecal metabolites in healthy adults, and whether such changes indicate a shift toward a healthier gut ecosystem.

Prior work shows diet is a major driver of gut microbiome composition and function. Vegetarian and Western diets differentially affect key taxa (e.g., Bacteroides, Faecalibacterium, Clostridium cluster XIVa, Roseburia, Eubacterium rectale, Ruminococcus bromii). Multi-omics comparisons suggest diet may influence microbial metabolism more than taxonomy, with microbial metabolites (vitamins, amino acids, SCFAs) linked to host health. Although FHV possesses antioxidant and anti-inflammatory activities, evidence largely stems from in vitro or animal studies, leaving a gap regarding its human gut microbiome impacts. The authors position their work within personalized nutrition and microbiome–diet interactions, addressing the lack of human multi-omics data on FHVD.

- Design and participants: Randomized dietary intervention in healthy adults. Initially recruited 48 participants (25 men, 23 women) without hyperglycemia, hyperlipemia, gastrointestinal disease, or antibiotic use in prior 6 months. Random allocation to FHVD (n=29) or NFHVD control (n=19). After protocol violations and incomplete records, 43 individuals remained (FHVD n=25; NFHVD n=18). Intervention duration: 8 weeks following a 2-week baseline. - Diets: Both provided as soups following a Cantonese recipe; NFHVD identical but without FHV. Participants consumed 250 mL per day, four random weekdays per week. Dietary intake was normalized by having meals at a designated dining hall and completing 3-day food records at sampling weeks; macronutrient intake showed no significant differences between groups or time points; FHV intake differed significantly between groups. - Sample collection: Fecal samples collected at week 0 (Pre) and week 8 (Post), total 86 samples. - 16S rRNA gene sequencing: V4 region amplified (primers 515F/806R). Sequenced on Illumina HiSeq 2500 (2×250 bp). Quality control and OTU clustering at 97% similarity via QIIME2 (deblur), taxonomy via Greengenes. Alpha/beta diversity and taxonomic analyses performed in R (vegan), with Wilcoxon matched-pairs for within-group pre/post and Mann–Whitney for between-group comparisons. - Co-occurrence networks: Constructed at genus level from 16S data to assess positive/negative correlations and core nodes pre/post within each diet group. - Shotgun metagenomics: MicroPITA used to select 32 representative samples (8 per group: FHVD-Pre, FHVD-Post, NFHVD-Pre, NFHVD-Post). Sequenced on MGISEQ-2000 (~10 GB per sample). Gene catalog generated; KEGG Orthologs profiled and mapped to pathway modules. Reporter score used to detect significantly altered modules (|RS| > 1.65). PCA based on Bray–Curtis distance of KO profiles. - Widely targeted metabolomics: LC-ESI-MS/MS (UPLC ExionLC AD; QTRAP) on lyophilized feces. Two chromatographic methods (C18 and BEH Amide) and positive/negative ion modes. VIP and statistical tests used to identify significantly changed metabolites. - SCFA quantification: GC-FID (Agilent 7820A, DB-FFAP column). Fecal SCFAs (acetate, propionate, butyrate) measured as µmol/g dry feces. Key gene abundances for SCFA pathways from metagenomes: acetate (fhs, ack), propionate (lcd, pduP, mmd), butyrate (but, buk). - FHV extract characterization and in vitro fermentation: Prepared water extract of FHV; measured total sugar, protein, flavonoids, polyphenols. In vitro anaerobic fermentation with human fecal inoculum in BNM medium comparing FHVE vs control over 0–24 h; SCFAs measured. - Statistics: Wilcoxon matched-pairs signed-rank tests for paired microbiome/metagenomics/metabolomics, Mann–Whitney for unpaired comparisons, Student’s t-test for in vitro assays, Spearman correlations for tripartite associations among species, pathway modules, and metabolites. Significance at P<0.05.

- Cohort and diversity: - 43 participants completed (FHVD n=25; NFHVD n=18); dietary macronutrient intake similar between groups/time points; FHV intake differed (P<0.001). - 16S: Total 2085 OTUs; slight but significant increase in Shannon index after FHVD; beta diversity changes mainly driven by individual variation, not group-level separation. - Taxonomic shifts (genus level): - Faecalibacterium: decreased in NFHVD (P=0.0268) but preserved/recovered with FHVD (horizontal comparison P=0.0389). - FHVD-specific increases: Dialister (P=0.0499), Veillonella (P=0.0163), unclassified Lachnospiraceae (P=0.0001); trend toward reduction of Shigella and significant reduction of Desulfovibrio (P=0.0163). - Coprococcus increased in both diets but significant only in NFHVD (P=0.0385). Holdemania enriched by both diets. - Bacteroidetes/Firmicutes (B/F) ratio: significantly increased after FHVD (P=0.0105); decreased (n.s.) after NFHVD (P=0.0814). - Co-occurrence networks: - FHVD: nodes from 35→41; positive edges from 28→29; negative from 16→22; core taxa shifted with disappearance of Shigella, Sutterella, Desulfovibrio and appearance of Parabacteroides post-FHVD, suggesting healthier network structure. - NFHVD: positive correlations decreased and negative correlations increased post-intervention. - Metagenomic functions (32 samples): - Genes mapped to 7256 KOs and 259 modules. Significant module changes: FHVD 69 (34 enriched), NFHVD 58 (29 enriched). - FHVD-enriched pathways: vitamin/cofactor synthesis (B1/thiamine RS=1.932; B5/pantothenate RS=2.313; K2/menaquinone RS=1.938), NAD biosynthesis (RS=2.463), citrate cycle (RS=2.345), glyoxylate cycle (RS=2.275), essential amino acid biosynthesis (threonine RS=2.615; methionine RS=2.879; tryptophan RS=3.551; lysine RS=2.583; isoleucine RS=1.840). Pentose phosphate pathway decreased. NFHVD notably enriched K2 synthesis (RS=3.843) and some amino acid pathways (tryptophan RS=2.693; lysine RS=1.843). - Metabolomics (fecal): - FHVD: 98 metabolites upregulated and 27 downregulated; key increases included L-serine (P=0.0122), L-methionine (P=0.0489), L-glutamine (P=0.0228), gamma-aminobutyric acid (GABA, P=0.0082/0.0083), tryptamine (P=0.0461), among others. NFHVD showed minimal changes (2 increased, 5 decreased). - SCFAs and pathway genes: - Fecal SCFAs increased after FHVD: acetate (P=0.0122), propionate (P=0.0122), butyrate (P=0.0122); no significant changes after NFHVD. - Butyrate synthesis gene abundance (but/buk) increased with FHVD (P=0.0391); no significant changes in acetate and propionate pathway gene abundance. - FHV water extract rich in carbohydrates; in vitro fermentation with FHV extract significantly increased acetate, propionate, and butyrate vs control at multiple time points. - Tripartite correlations (FHVD): - Essential amino acids positively associated with enriched species (e.g., Streptococcus spp., Blautia spp., Lactobacillus kalixensis) more than with specific pathways. - GABA positively associated with Blautia spp., Lactobacillus spp., Acidaminococcus spp. - Citrate cycle and vitamin synthesis pathways positively correlated with Bacteroides dorei, Ruminococcus bromii, Blautia obeum.

The findings demonstrate that adding Ficus hirta Vahl to a standard soup-based diet in healthy adults modulates the gut microbiome toward features associated with gut health: preservation of Faecalibacterium, enrichment of SCFA-producing taxa (Dialister, Veillonella, Lachnospiraceae), increased B/F ratio, and reduced potentially proinflammatory Proteobacteria (e.g., Desulfovibrio). Functionally, FHVD enhanced microbial pathways for B-vitamin and cofactor biosynthesis, central energy metabolism (citrate and glyoxylate cycles), and essential amino acid synthesis, coinciding with higher fecal levels of amino acids (serine, methionine, glutamine), neurotransmitter-related metabolites (GABA, tryptamine), and increased SCFAs. The in vitro fermentation and gene abundance data suggest SCFA elevations result from both increased substrate availability (carbohydrates in FHV) and higher abundance of key enzyme genes/species. Network analyses indicate a shift toward healthier co-occurrence structures with loss of pathogenic core nodes and emergence of potentially beneficial taxa. Collectively, results support the hypothesis that FHVD beneficially reshapes the gut microbial community and its metabolic outputs, potentially contributing to host health via nutrient, energy, and neuroactive metabolite pathways.

This exploratory randomized dietary intervention shows that FHVD optimizes gut microbial community structure and metabolic function in healthy adults. Key contributions include: preservation/enrichment of beneficial and SCFA-producing taxa, enhancement of B-vitamin and amino acid biosynthesis and central metabolic pathways, increased fecal SCFAs and neuroactive metabolites, and healthier microbial interaction networks. These multi-omics results suggest FHVD may promote gut homeostasis and metabolic health. Future work should involve larger, longer-duration clinical trials with diverse populations, controlled mechanistic studies (including animal models), targeted isolation/characterization of active FHV components (especially carbohydrates), and linkage of microbiome/metabolite changes to specific clinical phenotypes and host outcomes.

- Exploratory study with modest sample size (n=43 analyzed) and healthy population, limiting generalizability and power for clinical endpoints. - Inter-individual variation dominated beta diversity patterns; no significant changes observed in blood biochemical markers. - Metabolomics from feces cannot definitively attribute metabolite origin (host vs microbial) without tracer or isotopic studies. - Metagenomic functional inferences based on gene abundance; enzyme activities and actual fluxes were not directly measured. - In vitro fermentation provides supportive mechanistic insight but may not fully recapitulate in vivo gut conditions. - Short intervention duration (8 weeks) and frequency of intake may limit observation of longer-term or systemic effects.