Prebiotic-like and antioxidant activities of Aesculus hippocastanum L. ethanolic extract: Effects on probiotic lactobacilli and lipid peroxidation

Abstract

Background: Horse chestnut (Aesculus hippocastanum L.) seeds contain numerous biologically active constituents, particularly polyphenols, flavonoids, and saponins. Although extracts derived from this plant are widely recognized for their venotonic and anti-inflammatory properties, their interactions with probiotic microorganisms and their broader potential as functional food ingredients have received limited attention. The present study therefore examined the functional characteristics of horse chestnut ethanolic extract (HCEE), including its compatibility with selected probiotic bacteria and its antioxidant activity in vitro and in animal models. Objective: This study investigated whether HCEE inhibited or promoted the growth of selected probiotic Lactobacillus species. Its capacity to scavenge DPPH radicals and reduce lipid peroxidation in the liver and brain tissues of white rats was also assessed. Methods: Antimicrobial activity was evaluated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a two-fold serial dilution method. The total phenolic content of the extract was quantified using the Folin–Ciocalteu assay. The potential growth-promoting effects of HCEE on Lactiplantibacillus plantarum, Lactobacillus delbrueckii, and Lactobacillus helveticus were examined by measuring optical density at 600 nm and enumerating viable cells as CFU/mL. The free-radical-scavenging activity of HCEE was evaluated in vitro using the DPPH assay. For the in vivo assessment, the dried extract was administered orally at doses of 20 or 40 mg/kg, after which malondialdehyde (MDA) concentrations were measured in rat liver and brain tissues as indicators of lipid peroxidation. Results: HCEE exerted little or no inhibitory effect on most of the tested probiotic bacteria and on Candida albicans, while showing pronounced inhibitory activity against the tested Escherichia coli strain. The extract also supported the growth of L. plantarum when used as the sole nutrient component, resulting in an increase of more than 4 log₁₀ CFU/mL after 24 h of incubation. The total phenolic content was 121.5 ± 3.0 mg GAE/g dry extract, and the DPPH radical-scavenging activity reached 67.8 ± 2.4% at the tested concentration. In vivo, HCEE significantly reduced MDA levels in both tissues. The strongest effect was observed at 20 mg/kg, corresponding to reductions of 42.58% in the liver and 34.43% in the brain. The 40 mg/kg dose produced a less pronounced effect. Conclusion: HCEE demonstrated selective antimicrobial activity, compatibility with selected probiotic lactobacilli, and prebiotic-like growth-supporting effects, particularly toward L. plantarum. It also exhibited substantial DPPH radical-scavenging activity and reduced tissue MDA levels, with the greatest effect observed at 20 mg/kg. These findings indicate that HCEE may have potential as a bioactive ingredient for the development of functional foods and nutraceutical formulations intended to support beneficial microorganisms and provide antioxidant activity. Further studies are required to confirm its safety, characterize its active constituents, and evaluate its performance in food matrices and more complex biological systems. Novelty: The novelty of this study lies in demonstrating the prebiotic-like activity of horse chestnut extract and its differential effects on probiotic lactic acid bacteria. The magnitude of the response varied among the tested microorganisms, indicating a strain-dependent effect. Under the tested conditions, the strongest growth-supporting response was observed for L. plantarum. These findings are described as prebiotic-like activity rather than definitive prebiotic activity. Keywords: horse chestnut; ethanolic extract; probiotics; growth-promoting activity; functional food ingredient; antioxidant activity; lipid peroxidation; malondialdehyde