Bifidobacteria-Fermented Red Ginseng and Its Constituents Ginsenoside Rd and Protopanaxatriol Alleviate Anxiety/Depression in Mice by the Amelioration of Gut Dysbiosis.

Gut dysbiosis is closely connected with the outbreak of psychiatric disorders with colitis. Bifidobacteria-fermented red ginseng (fRG) increases the absorption of ginsenoside Rd and protopanxatriol into the blood in volunteers and mice. fRG and Rd alleviates 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice. Therefore, to understand the gut microbiota-mediated mechanism of fRG against anxiety/depression, we examined the effects of red ginseng (RG), fRG, ginsenoside Rd, and protopanaxatriol on the occurrence of anxiety/depression, colitis, and gut dysbiosis in mice. Mice with anxiety/depression were prepared by being exposed to two stressors, immobilization stress (IS) or Escherichia coli (EC). Treatment with RG and fRG significantly mitigated the stress-induced anxiety/depression-like behaviors in elevated plus maze, light-dark transition, forced swimming (FST), and tail suspension tasks (TST) and reduced corticosterone levels in the blood. Their treatments also suppressed the stress-induced NF-κB activation and NF-κB+/Iba1+ cell population in the hippocampus, while the brain-derived neurotrophic factor (BDNF) expression and BDNF+/NeuN+ cell population were increased. Furthermore, treatment with RG or fRG suppressed the stress-induced colitis: they suppressed myeloperoxidase activity, NF-κB activation, and NF-κB+/CD11c+ cell population in the colon. In particular, fRG suppressed the EC-induced depression-like behaviors in FST and TST and colitis more strongly than RG. fRG treatment also significantly alleviated the EC-induced NF-κB+/Iba1+ cell population and EC-suppressed BDNF+/NeuN+ cell population in the hippocampus more strongly than RG. RG and fRG alleviated EC-induced gut dysbiosis: they increased Bacteroidetes population and decreased Proteobacteria population. Rd and protopanaxatriol also alleviated EC-induced anxiety/depression and colitis. In conclusion, fRG and its constituents Rd and protopanaxatriol mitigated anxiety/depression and colitis by regulating NF-κB-mediated BDNF expression and gut dysbiosis.

[ Figure S2] Figure S2. Effects of RG and fRG on the infiltration of BDNF + /NeuN + (H) and NF-κB + /Iba1 + cells (I) into the CA1 region of hippocampus of Escherichia coli K1-exposed mice. Mice were exposed to Escherichia coli K1 (1×10 9 CFU/mouse/day) and test agents (Ec, vehicle [saline]; EPc, 1 mg/kg/day of buspirone; ERL, 10 mg/kg/day of RG; ERH, 25 mg/kg/day of RG; EFL, 10 mg/kg/day of fRG; EFH, 25 mg/kg/day of fRG) were gavaged (for vehicle, RG, and fRG) or intraperitoneally injected (for buspirone) daily for 5 days. Normal control group (Con), not exposed to EC, was treated with saline instead of test agents. Data values were indicated as mean ± SD (n = 6). ## p < 0.01 vs. Con group. ### p < 0.05 vs. Con group. * p < 0.05 vs. Ec group. *** p < 0.001 vs. Ec group. Effects of ginsenoside Rd and protopanaxatriol: NC (blue), Ec (red), ERd (green), and EPt (purple) groups. Test agents treated in each group are indicated in Supplement Tables S2 and  S4. The described strains were analyzed to the Linear Discriminant Analysis (LDA) along with effect size measurement (LEfSE) in Galaxy (http://huttenhower.sph.harvard.edu/galaxy/). It was used to discriminate significant differentially strains at each taxon level. The threshold logarithmic score set at 3.5 and ranked. Bacterial strains were described based on 16SrRNA sequencing data.
[ Table S4] mg/kg/day of ginsenoside Rd; EPt, 5 mg/kg/day of protopanaxatriol) were gavaged daily for 5 days. Normal control group (Con), not exposed to EC, was treated with saline instead of test agents. *p<0.05 vs. Con group, # p<0.05 vs Ec group.
[ Table S6] Pharmacokinetic study of protopanaxatriol in healthy volunteers orally treated with red

ginseng (RG) or Bifidobacteria-fermented RG (fRG) extracts
Pharmacokinetic study of protopanaxatriol in healthy volunteers orally treated with RG or FRG extracts was performed, as previously reported [1,2]. The healthy volunteers were orally given RG or fRG at a dose of 3 g/60kg/day and the blood samples were collected for 24 h.
Subsequently, the serum concentrations of protopanaxatrol were determined using LC-MS/MS analysis. The pharmacokinetic parameters are summarized in Table S6.