Antibacterial potential of ellagic acid and gallic acid against IBD bacterial isolates and cytotoxicity against colorectal cancer

Abstract Ellagic acid and Gallic acid are polyphenols which have shown beneficial effects in animal models of colitis. In the present study Ellagic acid and Gallic acid were evaluated for antibacterial potential against clinical IBD isolates. (HM95, HM233, HM251, HM615). Cytotoxicity was determined against human colorectal adenocarcinoma cells (Caco2, COLO.205, HT.29), whereas, cytocompatibility against normal rat intestinal epithelial (IEC-6) using MTT assay. Ellagic acid showed the lowest MIC and MBC value of 2.5 and 5 mg/mL respectively against HM251 and HM233. Gallic acid exhibited the lowest MIC and MBC value of 1.25 and 2.5 mg/mL respectively against HM251 and HM615. Cytotoxicity assay resulted in reduction of percent cell viability when tested at concentrations ranging from 400–12.5 µg/mL. The polyphenols presented a concentration-dependent deduction in percent cell viability after 48 h exposure It is likely that these polyphenols are good anti-colitic agents. However, further investigations are required. Graphical Abstract


Introduction
Inflammatory Bowel Disease (IBD) is a group of chronic inflammatory disorders of the gastrointestinal tract which includes Ulcerative Colitis (UC) and Crohn's Disease (CD) (Chang et al. 2018). The incidence of IBD is gradually rising in Western countries, however this rise is rapid in newly industrialized countries (Kaplan and Ng 2017). Developing countries are estimated to have a high global disease burden with few resources to manage it (Kedia and Ahuja 2017). Drug therapies for IBD include amino salicylates and immunomodulators, However, they are associated with serious side effects. Recently, naturally occurring Polyphenols have gained attention as an alternative for IBD therapy working through various mechanisms (Arya et al. 2020).
IBD patients are at a greater risk of colorectal cancer due to prolonged exposure to chronic inflammation and immunosuppressive therapies (Axelrad et al. 2016). The colonic mucosa of CD and mucosa of colon cancer specimens contain relatively abundant E. coli, compared to normal mucosa.
Ellagic acid (EA) is a polyphenol which is synthesized in plants as a secondary metabolite and has a vital impact on human health through various mechanisms (Prabha et al. 2021). EA demonstrated anti-inflammatory effects in animal colitis model proving its role in dietary prevention of intestinal inflammation and related cancer development (Mar ın et al. 2013). It is reported for antimicrobial activity against various bacteria (Al-Mugdadi et al. 2019). Gallic acid (GA) another polyphenol maintains gut health by impacting the intestinal microbiome and modifying immune response (Yang et al. 2020). It possesses antimicrobial activity against various disease-causing bacteria and also potentiates the effectiveness of antimicrobials (Rajamanickam et al. 2018).
We have earlier reported the activity of Terminalia arjuna which has these phytoconstituents to be beneficial in management of IBD (Cota et al. 2019;. This study aims at evaluating antibacterial activity of Ellagic acid and gallic acid against isolated four E.coli strains from inflammatory bowel disease patients and estimate cytotoxicity of these polyphenols against 3 colorectal cancer cell lines.

Antibacterial study
The results of MIC, MBC and well diffusion assay of polyphenols against the four IBD clinical isolates are listed in Tables S1-S3 respectively and Figure S1. Ellagic acid showed the lowest MIC and MBC value of 2.5 and 5 mg/mL respectively against HM251 and HM233. Gallic acid exhibited the lowest MIC and MBC value of 1.25 and 2.5 mg/mL respectively against HM251 and HM615. MIC of Gallic acid was lower than Ellagic acid. The highest inhibitory zone value for Ellagic acid was 23 mm against HM615 and the lowest was 20 mm against HM251 and HM233. The highest value of zone of inhibition of Gallic acid was 26 mm against HM233 and lowest was 24 mm against HM95 and HM251.
E. coli is a common human intestinal bacterium which is mostly considered nonpathogenic. However, some E.coli strains, without virulence factors, have the capacity to adhere to the intestinal cells which further invade the infected cells and replicate there to cause intestinal diseases (Bertuccini et al. 2014). E. coli is linked to CD since an abundance of specific adherent-invasive E. coli (AIEC) was found in ileum from patients with CD. Similarly, E. coli are very predominant in inflamed mucosa of patients with UC (Petersen et al. 2011). There is increasing evidence that the mucosaassociated microbiota, may be essential in the pathogenesis of the inflammatory bowel diseases: ulcerative colitis, and Crohn's Disease (Martin et al. 2004) Our study investigated the antimicrobial effect of Ellagic acid and Gallic acid against AIEC strains. Previously Ellagic acid demonstrated growth inhibition of 55 H. pylori (De et al. 2018), Staphylococcus epidermis, Bacillus cereus, Klebsiella pneumonia and Salmonella typhi (Ghudhaib et al. 2010). Gallic acid also displayed activity against the E.coli O157:H7 biofilm (Cossu et al. 2016). These studies support our findings further confirming the antibacterial effects of polyphenols.

In-vitro anticancer and cytocompatibility assay
Cytotoxicity assay resulted in reduction of percent cell viability when tested at concentrations ranging from 400-12.5 mg/mL. The polyphenols presented a concentrationdependent deduction in percent cell viability after 48 h exposure ( Figures S2, S3). The IC 50 values are depicted in Table S4. Ellagitannins and ellagic acid are also known to be transformed by the gut microbiota to produce bioavailable metabolites that can exert anti-inflammatory and anticarcinogenic effects and can reach high concentrations in both normal and tumor human colonic tissues (Tom as-Barber an et al. 2014).
GA displayed chemo preventive effect on 1,2-dimethyl hydrazine induced colon carcinogenesis (Giftson et al. 2010). This study findings are in agreement with previous claims regarding cytotoxic nature of these polyphenols.

Experimental
Experimental details related to this article are available online (see supplementary data).

Conclusion
Ellagic acid and Gallic acid exhibited antibacterial efficacy against the CD and UC bacterial isolates used in the study. Cytotoxicity was observed against colorectal cancer cells with minimal effect on normal cells. However, further investigations are required to understand its precise mechanism.