# Anti-Inflammatory Effect of Excretion-Secretion Products of Clinostomum marginatum (Digenea: Clinostomidae) and Its Effect over the Viability and Antioxidative Activity of a Mix of Lactobacillus and/or Bifidobacterium

## TL;DR

This study explores how excretory-secretory products from Clinostomum marginatum reduce inflammation and boost probiotic bacteria viability and antioxidant activity.

## Contribution

The novel contribution is identifying Clinostomum marginatum ESPs as anti-inflammatory and probiotic-enhancing agents.

## Key findings

- ESPs from C. marginatum significantly reduced nitric oxide and pro-inflammatory cytokine expression in macrophages.
- ESPs enhanced the growth and antioxidant activity of Lactobacillus and Bifidobacterium probiotic strains.
- Three major protein bands (47, 54, and 58 kDa) were identified in the ESPs of C. marginatum.

## Abstract

The trematode Clinostomum marginatum, secretes excretory-secretory products (ESPs) which have the potential to increase the viability and antioxidant activity of probiotic strains. The aim of this study was to identify the ESP profile of C. marginatum and to evaluate its anti-inflammatory activity in RAW 264.7 macrophages, as well as its effect on the viability and antioxidant activity of a consortium of bacteria comprising Lactobacillus and/or Bifidobacterium. C. marginatum was maintained in RPMI-1640 medium for ESP collection. Anti-inflammatory activity was assessed in LPS-stimulated RAW264.7 cells treated with 800 µg/mL of ESPs, measuring cell viability, nitric oxide production, and the relative expression of pro-inflammatory cytokines (IL-6, TNF-α, INF-γ) and the COX-2 gene by qPCR. The influence of ESPs (800–1600 µg/mL) on probiotic viability and antioxidant activity was determined using MTT, DPPH, hydroxyl, and superoxide radical scavenging assays. C. marginatum showed 74% survival in vitro, and SDS-PAGE analysis revealed three major protein bands in the ESPs (47, 54, and 58 kDa). ESP treatment significantly reduced nitric oxide and the mRNA expression of pro-inflammatory markers in LPS-activated macrophages. ESPs supplemented at 1200 µg/mL optimized the growth kinetics of Lactobacillus (specific growth rate μL = 1.12 h−1, doubling time td = 0.62 h) and Bifidobacterium (μB = 1.09 h−1, td = 0.63 h) compared to control conditions. In conclusion, ESPs from C. marginatum exhibited significant anti-inflammatory and antioxidant effects while enhancing bacterial viability, which positions them as promising candidates for biotherapeutics agents in the management of inflammatory control and gut microbiota modulation.