The study explores the impact of *Ficus hirta* Vahl (*FHV*), a traditional Chinese herbal ingredient popular in southern China, on the human gut microbiome. A "tonic diet" incorporating FHV (FHVD) is increasingly used for health benefits. While *FHV* possesses antioxidant and anti-inflammatory properties, its effects on the gut microbiome remain largely unknown. The gut microbiome, interacting directly with ingested nutrients, influences their efficacy and bioavailability, shaping host physiology. Dietary habits significantly affect gut microbiome composition; for example, vegetarian diets increase *Bacteroides* and *Faecalibacterium*, while Western diets reduce polysaccharide-metabolizing bacteria. Omics technologies reveal diet's impact on microbial metabolism more than taxonomy, highlighting the importance of metabolites like vitamins, amino acids, and short-chain fatty acids (SCFAs). This exploratory research uses 16S rRNA gene sequencing, shotgun metagenomic sequencing, and metabolomics to analyze the effect of FHVD on gut microbial taxonomy, function, and metabolic profiles, providing insights into the interaction between this tonic diet and gut microbiota.

The introduction cites several studies showcasing the relationship between diet and gut microbiome composition. Studies on vegetarian and Western diets illustrate the impact of diet on bacterial abundance. Metagenomic and metabolomic comparisons of Western and Mediterranean diets highlight diet's stronger influence on microbial metabolism than on specific taxa. The importance of microbial metabolites (vitamins, amino acids, SCFAs) in human health is emphasized. However, existing literature lacked information on the effects of *Ficus hirta* Vahl on the gut microbiome, motivating the current study.

This randomized controlled trial involved 43 healthy individuals (25 FHVD, 18 control). Participants consumed 250 ml of soup daily (FHVD or control) for eight weeks. Fecal samples were collected at baseline (week 0) and after intervention (week 8). 16S rRNA gene sequencing targeted the V4 region to assess gut microbiota composition. Shotgun metagenomic sequencing (32 samples selected using microPITA) analyzed microbial functions. Widely targeted metabolomics examined fecal metabolite profiles using LC-ESI-MS/MS. Statistical analyses included Wilcoxon matched-pairs signed-rank tests and Mann-Whitney tests. In-vitro fermentation of FHV extract with human gut microbiota was also conducted to examine SCFA production. Data analysis was performed using QIIME2, R, and GraphPad Prism.

Eight weeks of FHVD intervention led to significant changes in gut microbiota composition and function. Specifically: * **Increased Abundance of Specific Taxa:** The FHVD increased the relative abundance of *Dialister*, *Veillonella*, *Clostridium*, and an unclassified Lachnospiraceae organism, while preserving *Faecalibacterium*. *Coprococcus* was also increased, but significantly so only in the control group. *Shigella* and *Desulfovibrio* were decreased. * **Altered Metabolic Pathways:** Metagenomic analysis showed that the FHVD significantly enhanced various metabolic pathways, including those involved in amino acid synthesis (threonine, methionine, tryptophan, lysine, and isoleucine), vitamin B1, B5, and K2 synthesis, the citrate cycle, coenzyme synthesis, and the glyoxylate cycle. The pentose-phosphate pathway was decreased. * **Increased SCFAs and Other Metabolites:** The FHVD led to significantly higher levels of fecal acetate, propionate, and butyrate. Other increased metabolites included serine, glutamine, GABA, and tryptamine. In-vitro fermentation of FHV extract also showed increased SCFA production. * **Changes in Co-occurrence Networks:** The co-occurrence networks of gut microbial communities demonstrated altered patterns of interactions after both interventions, although the FHVD resulted in a more clearly defined beneficial shift. *Shigella*, *Sutterella*, and *Desulfovibrio* disappeared as core nodes in the FHVD group, and *Parabacteroides* appeared. * **Correlation Analysis:** Correlation analysis revealed positive associations between essential amino acids and FHVD-enriched species like *Streptococcus*, *Blautia*, and *Lactobacillus kalixensis*. GABA levels positively correlated with acid-producing bacteria. The citrate cycle and vitamin synthesis pathways showed positive correlations with *Bacteroides dorei*, *Ruminococcus bromii*, and *Blautia obeum*.

The study demonstrated that FHVD significantly altered the gut microbiome's composition and metabolic function in a beneficial manner. The increase in beneficial bacteria, such as *Faecalibacterium* and SCFA-producing bacteria, along with the reduction of potential pathogens, suggests a protective effect on gut health. The enhanced synthesis pathways for various amino acids and B vitamins highlight potential improvements in host metabolism and immunity. The increased levels of SCFAs, known for their anti-inflammatory and immunomodulatory effects, further support the positive impact of FHVD on gut health. The changes observed in co-occurrence networks imply a shift towards a healthier microbial ecosystem. The findings suggest that FHVD's health benefits could be associated with the enrichment of beneficial bacteria and the production of health-promoting metabolites.

This study provides comprehensive multi-omics insights into the effects of *Ficus hirta* Vahl dietary supplementation on the human gut microbiome. FHVD optimized gut microbial composition and metabolic function, resulting in increased levels of beneficial bacteria, SCFAs, and other metabolites. This indicates that FHVD could promote a healthier gut microbiome, potentially contributing to improved health outcomes. Future research should focus on larger clinical trials and animal studies to further explore the mechanisms and specific health benefits of FHVD in different disease contexts and investigate the specific roles of various *FHV* components.

The study's relatively small sample size and short intervention duration are limitations. The study focused on healthy individuals, limiting the generalizability to populations with existing health conditions. While the study identified correlations, it didn't establish direct causal relationships between specific *FHV* components and gut microbiome changes. Further research with a larger cohort, longer intervention, and more detailed analysis of *FHV*'s chemical composition is needed to solidify these findings.