Title: 
Proanthocyanidin polymeric tannins from Stryphnodendron adstringens are effective against Candida spp. and treat vaginal candidiasis

Objectives:
Stryphnodendron adstringens, the Brazilian plant popularly used as a cicatrizing and anti-inflammatory agent, has been studied in order to determine its antifungal potential. In this study, the antifungal activity of proanthocyanidin polymeric
tannins from the S.adstringens steam bark (presents into the fraction F2 and semi-purified subfraction F2.4) was evaluated against planktonic cells, biofilm sessile cells and biofilm dispersion cells from Candida albicans and Candida non-albicans (CNA), including fluconazole-resistant isolates. In order to determine in vivo activity, we tested the efficacy of a gel formulation containing hexameric tannins of proanthocyanidins (F2 fraction) in the control of vaginal candidiasis in murine model.

Methods:
The evaluation of antifungal activity of tannins (F2 and sub-fraction F2.4) from S. adstringens stem barks against planktonic and dispersion cells from Candida biofilms was performed by broth microdilution assay while the antifungal activity on sessile cells from biofilms was evaluated by XTT reduction assay. For in vivo antifungal activity analysis, BALB/c mice were infected by C.albicans ATCC 10231 or C.glabrata ATCC 2001 and treated with a topical gel containing 2.5% or 5% of F2 (alone or combined with oral fluconazole – 20 mg/kg) and vaginal fungal burden (CFU/g) and
histopathology were analyzed.

Results:
F2 and F2.4 inhibited the proliferation of planktonic cells of Candida spp., including fluconazole- and/or amphotericin B-resistant isolates. Our results show that both fraction F2 and subfraction F2.4 were more effective to inhibit fungal growth of dispersion cells that detached from pre-formed biofilms than planktonic cells for all Candida isolates tested, represented by the lower minimal inhibitory concentration values on dispersion cells (<0.48 – 31.25 µg/mL) compared with the minimal inhibitory concentration on planktonic cells (0.98 - 250 µg/mL) (Figure 1). In vivo experiments
demonstrated that vaginal gel containing fraction F2 from S. adstringens significantly decreased the fungal burden in the vaginal tissue of the mice infected by C. albicans (concentration of 5%, p<0.001) and by C. glabrata (concentration of 2.5% and 5%, p<0.01 and p<0.001, respectively) compared with untreated mice (untreated group). Moreover, oral FCZ combined with vaginal gel containing 5% of F2 drastically reduced the fungal burden of C. albicans and C. glabrata (p<0.001) (Figure 1). Histopathological analysis showed that the treatment with the vaginal gel containing 5% F2 reduced the fungal burden in the vaginal tissue and vaginal canal of infected animals and caused no histological alterations in the uninfected vaginal mucosa.

Conclusion:
Our data suggest that the inhibitory effects of both F2 and F2.4 could be important to control the dissemination and re-infection by dispersion cells from Candida spp. biofilms. Furthermore, it was seen that these hexameric tannins of proanthocyanidins have potential use in the treatment of vaginal candidiasis. once that they were efficient to control the fungal infection by both C. albicans and C. glabrata using a murine model of vaginal candidiasis with no noticeable toxicity to vaginal tissue. Our findings were important to prove the efficacy of tannins extracted from S. adstringens on fungal vaginitis caused by Candida spp. corroborating with its popular use.