Potent In Vitro Activity of Citrus aurantium Essential Oil and Vitis vinifera Hydrolate Against Gut Yeast Isolates from Irritable Bowel Syndrome Patients-The Right Mix for Potential Therapeutic Use

Abstract

Background: Irritable bowel syndrome (IBS) is a functional disorder without any pathological alteration, in which the alterations of the Candida/Saccharomyces ratio of the gut microbiota, the balance of pro and anti-inflammatory cytokines and the brain-gut-microbiome axis are important for the development and progression of IBS. The aim of the study was to identify natural products, including essential oils or hydrolates, which were contextually harmless for the gut beneficial strains (e.g. Saccharomyces spp.) but inhibitory for the pathogenic ones (Candida spp.).

Methods: The effectiveness of 6 essential oils and 2 hydrolates was evaluated using microbiological tests, carried out on 50 clinical isolates (Candida, Saccharomyces and Galattomyces species) and 9 probiotic strains (Saccharomyces cerevisiae, Lactobacillus species, Akkermansia muciniphila and Faecalibacterium prausnitzii) and immunological and antioxidant assays.

Results: The study led to a mixture based on a 1/100 ratio of Citrus aurantium var. amara essential oil / Vitis vinifera cv Italia hydrolate able to contextually reduce, in a concentration-dependent manner, the ability of Candida species to form hyphal filaments and have an interesting immunomodulatory and anti-oxidant action. This mixture can potentially be useful in the IBS treatment promoting the restoration of the intestinal microbial and immunological balance.

Keywords: Akkermansia muciniphila; Candida species; Citrus aurantium var. amara essential oil; Saccharomyces cerevisiae; Vitis vinifera cv Italia hydrolate.

Figure 1

cfu/ml of Saccharomyces cerevisiae (filled symbol) and Candida albicans (empty symbol) in response to Citrus aurantium var. amara essential oil (EO) concentration. Solid line, exponential decay function (Equation (2)) describing S. cerevisiae abatement at increasing EO concentration. Dashed line, linear function (Equation (1)) describing C. albicans abatement at increasing EO concentration. ⁽⁺⁾ and *, significant at p ≤ 0.10 and p ≤ 0.05, respectively.

Figure 2

Variation of the amount of cytokines IL-10 and TNF-α in the medium culture of PBMCs treated with scalar dilutions of the V. vinifera cv Italia Hy alone or in combination with EO and/or LPS. The circle-shaped indicators refer to the samples treated with the Hy only; the squares are referred to the samples treated with the Hy plus LPS, while the triangles show the samples treated with Hy plus the EO of C. aurantium var. amara plus LPS. The white rhombus is the untreated control and the black rhombus is the control treated with LPS. Letters a, b, c and d indicate samples treated with scalar dilution of the Hy (50% v/v, 25% v/v, 12.50% v/v and 0.06% v/v respectively). Letters e, f, g and h are referred to samples treated with scalar dilution of the Hy (50% v/v, 25% v/v, 12.50% v/v and 0.06% v/v respectively) and LPS (1 μgr/mL). Letters i, l, m and n are referred to PBMCs treated with a 1:100 v/v mixture of the Hy, (50% v/v, 25% v/v, 12.50% v/v and 0.06% v/v respectively) and the EO (0.50% v/v, 0.25% v/v, 0.12% v/v and 0.06% v/v respectively) plus LPS.

Figure 3

Relative growth of the clinical isolates of C. albicans (3.1) and S. cerevisiae (14.3) when cultured in presence of scalar dilutions of both the EO of C. aurantium var amara and the Hy of V. vinifera cv Italia. The values are relative to the positive control (CTR+) and are expressed in percentage. The average of the two isolates’ positive controls is set at 100%.

Figure 4

Cytotoxicity tests. Cytotoxicity assays performed with a Cell Titer Blue viability assay on human gingival fibroblasts at 24 (A) and 48 h (B) upon cell synchronization (24 h). Cells were treated with scalar dilution of a 1:100 v/v mixtures of Citrus aurantium var. amara EO (0.50% v/v to 0.06% v/v) and Vitis vinifera cv Italia Hy (50% v/v to 6.25%).