106 ISSN 1120-1770 online, DOI 10.15586/ijfs.v35i1.2320 P U B L I C A T I O N S CODON Italian Journal of Food Science, 2023; 35 (1): 106–117 P U B L I C A T I O N S CODON Marjoram oil attenuates oxidative stress and improves colonic epithelial barrier function in dextran sulfate sodium-induced ulcerative colitis in Balb/c mice Bouayad DI1*, Hadria Grar1,2, Sadia Berzou3, Omar Kheroua1, Djamel Saidi1,4, Hanane Kaddouri1 1Laboratory of Physiology of Nutrition and Food Safety, Department of Biology, Faculty of Natural and Life Sciences, University Oran 1 Ahmed Ben Bella, Oran, Algeria; 2Department of Biology, Faculty of Natural and Life Sciences, University of Mostaganem, Algeria; 3Laboratory of Clinical and Metabolic Nutrition, Department of Biology, University Oran 1 Ahmed Ben Bella, Oran, Algeria; 4Higher School of Biological Sciences of Oran (ESSBO), Oran, Algeria *Corresponding Author: Ismahan Bouayad Debbagh, Laboratory of Physiology of Nutrition and Food Safety, Department of Biology, Faculty of Natural and Life Sciences, University Oran 1 Ahmed Ben Bella, BP 1524 EL M’Naouer, Oran, Algeria. Email: bouayadd.bio@gmail.com Received: 26 December 2022; Accepted: 7 February 2023; Published: 16 March 2023 © 2023 Codon Publications OPEN ACCESS ORIGINAL ARTICLE Abstract In this study, we explored the effects of marjoram oil (MO) on dextran sulfate sodium (DSS)-induced colitis in Balb/c mice. Marjoram oil was found to significantly reduce the severity of DSS-induced colonic inflammation, as reflected by improved disease activity index, prevented colon length shortening, lower histopathological score, decreased myeloperoxidase activity, and reduced interlukin 6 (IL-6) levels. Moreover, marjoram oil pretreat- ment enhanced the colonic epithelial integrity by decreasing paracellular permeability. Marjoram oil was found to clearly reduce the colonic levels of thiobarbituric acid reactive substances assay and enhance the activity of superoxide dismutase, catalase, and glutathione sulfhydryl content. Marjoram oil could exert a protective effect on ulcerative colitis through its anti-inflammatory and antioxidant properties. Keywords: dextran sulfate sodium, experimental colitis, Origanum majorana, oxidative stress, inflammation, Ussing chamber Introduction Inflammatory bowel disease (IBD) is a long-term gastro- intestinal disorder marked by recurrent inflammation and extensive mucosal destruction in the intestine (Mijan and Lim, 2018). Histologically, extensive ulcerated regions with significant neutrophil infiltration and epithelial cell necro- sis characterize ulcerative colitis (UC) (Wang et al., 2019). Although the cause of IBD is unknown, various factors are known to have a role in its development, including break- down of the epithelial barrier, a decrease in commensal bacteria tolerance, immunologic alterations, oxidative stress, and an increase in inflammatory mediators (Liu et al., 2017; Goyal et al., 2014). Immunological cell infiltra- tion into the gut’s lamina propria, as well as uncontrolled synthesis of pro-inflammatory mediators and reactive oxygen species (ROS) in colonic tissues, contributes to the breakdown of intestinal barrier integrity and an increased inflammation of the mucosa (Maloy and Powrie, 2011). An overproduction of ROS causes lipid peroxidation which reduces cellular antioxidant capacity and leads to severe colonic inflammation (Tahan et al., 2011). Endogenous antioxidants can ordinarily protect the intes- tinal mucosa from oxidative damage (Mandalari et al., 2011). Nevertheless, inflammation increases the demand for these antioxidants, resulting in a pro-oxidant– antioxidant imbalance, resulting in mucosal damage (Mandalari et al., 2011; Zhu and Li, 2012).  Myeloperoxidase (MPO) activity is another indicator of the severity of ulcerative colitis. In colitis, MPO is widely mailto:bouayadd.bio%40gmail.com?subject= Italian Journal of Food Science, 2023; 35 (1) 107 Protective effect of marjoram oil on ulcerative colitis in Balb/c mice through its anti-inflammatory and antioxidant properties oil of Origanum majorana suppressed the development of colon cancer cells through p38 mitogen-activated protein kinase (MAPK)-provoked apoptosis in vitro and in vivo (Athamneh et al., 2020). To date, no scientific data exist on the effect of Origanum majorana on intestinal inflammation. Therefore, the pur- pose of our study was to evaluate the possible protective effect of Origanum majorana on DSS-provoked colitis in Balb/c mice. Materials and Methods Chemicals Marjoram oil was obtained from commercial CAP Pharm (EL Captain Company, Egypt). Dextran sulfate sodium (40 kDa), fluorescein isothiocyanate conjugated dextran (FITC-Dextran; catalog number: FD4 [4 kDa]), and mouse interlukin 6 (IL-6) enzyme-linked-immunosorbent sero- logic assay (ELISA) kit were purchased from Sigma Aldrich (France). The remaining solvents and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Marjoram oil composition was analyzed using Perkin Elmer Clarus 500 gas chromatograph capillary column HP-5 (30 m × 0.25 mm ID, 0.25-µm film thickness), coupled to Clarus 500 mass spectrometer (GC-MS). More details about the chemical composition of mar- joram oil were reported earlier by Bouayad Debbagh et al. (2021). Experimental animals  Male Balb/c mice, 8-week old, were kept at the University of Oran’s animal facility with a 12-h light–dark cycle at 23 ± 2°C. Animals had free access to commercial stan- dard diet and water. All experimental procedures involv- ing animals were approved by the current Algerian legislation covering the protection of animals. Colitis induction and design of treatment Animals were divided into the following four groups, with eight mice in each group: control group (Ctrl) received tap water; DSS group (DSS) received DSS 4% (40 kDa; Sigma-Aldrich) in their drinking water ad libitum for 7 days; marjoram oil group (M) received 0.5- mL/kg body weight per os (by gavage) of marjoram oil for 14 day; and marjoram oil + DSS group (M+DSS), in which mice received MO (0.5 mL/kg body weight) orally for 14 day and were exposed to DSS 4% for 7 days (day 15–21) (Figure 1). used as a marker of oxidative stress and gastrointestinal inflammation (Klebanoff et al., 2005). A previous investi- gation demonstrated that inflammatory cytokine-caused apoptosis was found to have a deleterious impact on the structure of adherent junction and epithelial barrier functions (Su et al., 2013). Animal models of IBD are suitable for studying the pathogenesis of the disease and developing new drugs. Dextran sulfate sodium (DSS)-provoked acute colitis is among the most regularly adopted methods (Bang and Lichtenberger, 2016) because of its great homo- geneity, lesions’ reproducibility predominantly in the distal colon, and similarity to clinical IBD (Valatas et al., 2013). Currently, several therapies, such as nonsteroi- dal anti-inflammatory medications, corticosteroids, immune-suppressing medications, and biological agents are being investigated (Baakhtari et al., 2018; Yang et al., 2015). Nevertheless, homeostasis and main- tenance of intestinal barrier are rarely emphasized in conventional pharmacotherapy, and interest in integra- tive and complementary medicines is growing (Larussa et al., 2017). Patients with IBD frequently use comple- mentary and alternative medicines (CAMs) (Cheifetz et al., 2017; Lin and Cheifetz, 2018), which may include plants or herbal items, probiotics, and prebiotics (Cheifetz et al., 2017). Thus, in IBD patients, use of CAM, particularly herbal medicines, is increasing as a safe anti-oxidant/anti-inflammatory therapeutic option (Wan et al., 2014). Owing to their high phenolic content, plants belonging to family Lamiaceae are studied extensively as natural antioxidant sources (Trouillas et al., 2003). Marjoram (Origanum majorana) is the most important member of family Lamiaceae, which includes a wide variety of aro- matic herbs and shrubs and volatile oil plants (Paterson et al., 2006). Polyphenols, such as flavonoids, having several therapeu- tic properties are found in abundance in Origanum majo- rana (Goel and Vasudeva, 2015). Leaves of Origanum majorana are found to exhibit antioxidant (Duletic et al., 2018; Erenler et al., 2016), antimicrobial (Della Pepa et al., 2019; Guerra-Boone et al., 2015), anti-neurodegenerative (Della Pepa et al., 2019; Duletic et al., 2018; Erenler et al., 2016; Guerra-Boone et al., 2015), hepatoprotec- tive (Mossa et al., 2013), cardioprotective (Ramadan et al., 2013), antiulcer (Al-Howiriny et al., 2009), anti- inflammatory (Arranz et al., 2015), and anticancer prop- erties (Al Benhalilou et al., 2019 Dhaheri et al., 2013). Origanum majorana has also demonstrated positive effects in acute infectious diarrhoea (Makrane et al., 2018). Recent studies have demonstrated that essential 108 Italian Journal of Food Science, 2023; 35 (1) Bouayad DI et al. 500-μL buffer solution) were taken for 90 min at 30-min intervals for recording electrophysiological parameters. The concentration of FD-4 was determined using a flu- orescence microplate reader with a 520-nm emission wavelength and a 490-nm excitation wavelength (Perkin- Elmer, Waltham, MA, USA). The apparent permeabil- ity coefficient (Papp) was calculated for this period as follows: Papp (cm/s × 10−6) = dc/dt × (V/(A × C0)), where dc/dt is the change in serosal concentration during 60–90 min (mol/L/s) period, C0 is the initial marker con- centration in mucosal reservoir (mol/L), “V” is the vol- ume of serosal reservoir (cm3), and “A” is the exposed intestinal area in the chamber (cm2). Histological assessment of colitis Distal colon sections were taken and fixed in phosphate- buffered formalin (10%), and then dried and paraffin- embedded. For light microscopic examination, these samples were stained with hematoxylin and eosin (H&E). Histological damage was determined using the scoring system of Stillie and Stadnyk (2009). Measurement of IL-6 level Level of IL-6 in plasma samples was measured in dupli- cate using commercially mouse IL-6 ELISA kit (Sigma- Aldrich) according to manufacturer’s instructions. Myeloperoxidase activity assay The MPO activity is defined as the quantity of enzyme that degraded 1 μmol of H2O2 per minute. It was mea- sured in units per milligram of protein. In this study, the MPO activity was measured in proximal colon Clinical colitis severity assessment The disease activity index (DAI) was measured daily on the basis of clinical manifestations of colitis (Cooper et al., 1993) comprising body weight, faecal bleeding, stool consistency, and diarrhoea (Table 1). Analysis of colon inflammation The animals were sacrificed after 21 days of treatment. Blood was collected and the plasma was kept at −80°C for assays. The colon was excised and washed with saline solution. The length and weight of colon was measured. A 5-mm section of the distal colon was put in freshly pre- pared 10% (w/v) formaldehyde (pH 7.0), processed, and paraffin-embedded for histopathological evaluation. The mid-distal portion (30–60% region) was used for elec- trophysiological studies. The remaining colon tissue was kept at −80°C for additional biochemical analysis. Ex vivo experiments: Ussing chamber assay After sacrifice, distal colon was removed and excised through mesenteric border. Colonic tissue samples were immediately placed in Ussing chambers as sheets (Physiologic Instrument, San Diego, CA) with an exposed area of 0.2 cm². Both sides were filled with Ringer solu- tion (5 mL), oxygenated continuously (95% O2 and 5% CO2), and maintained at 37°C. The mucosal-to-serosal flux of FITC-Dextran with a 4-kDa mean molecular mass (FD4) was measured across colonic samples to determine paracellular permeability. Colonic paracellular permeability was determined by measuring mucosal-to-serosal flux of FITC-Dextran across colonic samples. After a stabilization period of 15–25 min, the mucosal reservoir was injected with FD4 at a concentration of 2.2 mg/mL. For measuring FD4, basolateral samples (500 μL replaced by appropriate Figure 1. Experimental protocol of the study. Tap water Control group DSS group M + DSS group Marjoram group Tap water Tap water Days 1 14 21 Marjoram oil Gavage (0.5 ml/kg) Marjoram oil Gavage (0.5 ml/kg) DSS (4%) DSS (4%) Italian Journal of Food Science, 2023; 35 (1) 109 Protective effect of marjoram oil on ulcerative colitis in Balb/c mice through its anti-inflammatory and antioxidant properties Catalase (CAT) activity was determined according to the method of Aebi (1984), in which 20-µL of supernatant was mixed with 1.8 mL of 100 mmol/L phosphate buffer (pH 7.4) and 200-µL H2O2 (50 mM). Following a decrease in absorbance at 240 nm, CAT activity was measured spectrophotometrically in units/milligram of protein, compared to a standard curve. Superoxide dismutase (SOD) was determined according to the method described by Marklund and Marklund (1974). The mixture included 970 µL of Tris-HCl (50 mM, pH 8.2) and 15 µL of supernatant. The reaction began by adding 15 µL of pyrogallol (12-mM pyrogallol in 1-mM HCl) to the mixture. A control was prepared from the same mixture by adding 985-µL Tris-HCl instead of 15-µL of pyrogallol. Change in absorbance was measured during a 5-min period at a wavelength of 325 nm, and variation in absorbance per minute was calculated and expressed per milligram of protein, corresponding to the quantity of enzyme that suppressed auto-oxidation of pyrogallol by 50%. Glutathione peroxidase was determined by Rodrigues et al. (2002) method, in which 200 µL of tissue homog- enate was added to a solution of Tris-HCl (0.4 M, pH 7), 0.1-mL sodium azide (10 mM), and 0.2-mL reduced glu- tathione (1 mM) followed by 0.1 mL of 0.2-mM hydro- gen peroxide. After a 10-min incubation period at room temperature, the mixture was centrifuged at 1,200×g for 10 min after adding 0.4 mL of 10% trichloroacetic acid. After this, 0.2 mL of supernatant and 0.1 mL of Ellman’s reagent were combined, and the absorbance was mea- sured at 340 nm. Reduced glutathione (GSH) levels in colon tissues were assessed according to the method described by Sedlak and Lindsay (1968). GSH reacts with Dithiobis- 2-nitrobenzoic acid (DTNB) to produce thio-2-ni- trobenzoic acid, which has a wavelength of 412 nm. For measuring it, 1 mL of tissue homogenate (100-mg sample diluted with 0.9 mL of 0.12 mmol/L phosphate buffer, pH 7.2) was combined with 800 μL of ice-cold distilled water and 200 μL of trichloroacetic acid (50%) and incubated for 15 min; 400 μL of supernatant was mixed with 800 μL of Tris buffer (0.4 mol/L, pH 8.9) and 20-μL DTNB reagent (0.01 mol/L) after centrifugation at 1,200×g for 15 min. After 5 min of incubation, the absorbance of the reaction mixture was measured at 412 nm against a blank reagent. Statistical analysis Data were presented as mean ± standard error of mean (SEM). Results were examined by one-way ANOVA fol- lowed by Fisher’s least significant difference (LSD) test tissue according to Lenoir et al. (2011). In a 50-mM phosphate buffer with hexadecyl trimethyl ammonium bromide (0.5%), a section of colon was homogenized in ice with a T25 Ultra-Turrax system (HTAB; IKA, Staufen, Germany). The homogenate was sonicated in ice for 30 s prior to centrifugation at 3,200 g for 10 min at 4°C. The supernatants were frozen and thawed thrice prior to being centrifuged for 10 min at 3,200×g at 4°C. In microcuves, 50 μL of supernatants were combined with 1 mL of phosphate buffer (10 mM), 500 μL of 0.22% guaiacol, and 10 μL of H2O2 (0.3 %). After 3 min, absor- bance was measured at every minute for 20 min at 470 nm. The study was performed in triplicate. In vivo antioxidant activity Homogenization of tissues and quantification of proteins In ice cold water, frozen tissue specimens were weighed (range 100 mg) and homogenized with 0.9-mL potassium phosphate buffer (0.12 mmol/L, pH = 7.4). The samples were centrifuged twice at 3,000×g for 20 min at 4ºC. Superoxide dismutase, catalase, glutathi- one peroxidase (Gpx) activities, and reduced glutathione level were determined in all supernatants. Protein con- tent was measured according to the method described by Lowry et al. (1951). Thiobarbituric acid reactive substances assay (TBARS) Lipid peroxidation was measured by the method of Salih et al. (1987), In which 2 g of colon was homogenized in 30 mL of trichloroacetic acid (10%) and 200 µL of butyl hydroxyl toluene. Samples were centrifuged for 10 min at 3,500 rpm. Thiobarbituric acid (0.02 M), 5 mL, was then added to 5 mL of supernatant and the mixture was heated for 30 min at 100°C. Absorbance of the superna- tant was read against blank specimen at 532 nm. TBARS was expressed in nanomole (nmol) of MDA (malondial- dehyde) per gram of tissue. Table 1. Disease activity index (DAI) score (Cooper et al., 1993) (n = 8). Score Weight loss Stool consistency Bleeding 0 None Normal No bleeding 1 1–5% – – 2 5–10% Loose stools Slight bleeding 3 10–15% – – 4 More than 15% Watery diarrhoea Gross bleeding 110 Italian Journal of Food Science, 2023; 35 (1) Bouayad DI et al. Figure 2. Marjoram oil attenuated clinical signs of dextran sulfate sodium (DSS)-induced colitis in mice. (A) Body weight changes *p < 0.05, **p < 0.01 J21 vs J14. (B) Scores of disease activity index (DAI) in marjoram pre-treated mice and colitic (DSS) mice monitored every day. Data are expressed as the mean ± SD (n = 6 mice/groups). *p < 0.05 compared with DSS. 32 30 28 26 24 22 20 0 1 2 3 4 5 6 7 85 10 15 Time (day) Days (after DSS) B od y w ei gh t ( g) 4 3.5 4.5 3 2 2.5 1.5 1 0.5 0D is ea se a ct iv ity in de x (D A I) 20 Control (A) (B) Marjoram M+DSS DSS Control Marjoram M+DSS DSS 25 Figure 3. (A) Colon length. (B) Colon weight. (C) Colon macroscopic analysis. Data are expressed as mean ± SD (n = 6 mice/ groups). ##p < 0.01, ###p < 0.001, compared with the control group; *p < 0.05, **p < 0.01, ***p < 0.001, compared with the DSS group. 0 1 2 3 4 5 6 7 8 9 10 11 12cm 5 10 15 C ol on le ng th ( cm ) 0.00 0.5 1.0 1.5 C ol on w ei gh t ( g) Co ntr ol Ma rjo ra m M+ DS S DS S Co ntr ol Ma rjo ra m M+ DS S DS S (A) (B) (C) (Statistica Stat Soft, Maisons-Alfort, France); p < 0.05 was considered as statistically significant. Results Marjoram oil improved DSS-induced colitis Mice exposed to DSS-induced colitis, as evidenced by severe anorexia, weight loss (Figure 2A), reduction in DAI score from day 5 until the last experimental day (Figure 2B), decreased colon weight (Figure 3B), and drab hair color. The length of colon in DSS-treated mice was significantly shorter by 43.69% (p < 0.001) than that in mice of both control and marjoram groups (Figure  3A), indicating the successful establishment of mice colitis. Pre-treatment of colitic mice with marjoram oil showed a reduction in the severity of colitis as evidenced by a decreased weight loss, prevented colon shortening, and reduced DAI score on day 8 (p < 0.05). The length of Italian Journal of Food Science, 2023; 35 (1) 111 Protective effect of marjoram oil on ulcerative colitis in Balb/c mice through its anti-inflammatory and antioxidant properties colon was significantly restored (27%) in the (M+DSS) group, compared to the DSS group (p < 0.001). Marjoram oil alleviated DSS-induced colonic cellular permeability After DSS treatment, FITC-dextran permeation was sig- nificantly higher, compared to both control and marjo- ram groups (p < 0.05). However, mice from the (M+DSS) group possessed a lower colonic permeability to FITC- dextran (p < 0.05; Figure 4A). To determine the func- tional viability and integrity of colonic epithelium under ex vivo conditions, transepithelial ion conductance mea- surements were done for 120 min. As illustrated in Figure 4B, transepithelial ion conductance in colonic tissues from DSS group was considerably (p < 0.05) higher than that of the control group, indicating that colonic perme- ability to ions was relatively increased in colitic group. In contrast, the (M+DSS) group showed a significant recov- ery (p < 0.01) in transepithelial ion conductance. These results suggested that marjoram oil could decrease para- cellular permeability by maintaining colonic epithelial integrity. Marjoram oil ameliorated DSS-induced histopathological damage Representative results and microscopic scores are illus- trated in Figure 5. The DSS-treated mice had severe epi- thelial damage, including important cellular invasion into the lamina propria and colon mucosa, goblet cell deple- tion, thickening of the mucosa, and total architecture alteration, leading to a high microscopic damage score (Figure 5B). However, pre-treatment of DSS-treated mice with marjoram oil reduced infiltration of inflammatory cells with a minimum decrease of epithelial cells, result- ing in a significantly reduced microscopic damage score when compared to colitic mice. These results suggested the effective protection rendered by marjoram oil to counter DSS-induced colitis. Marjoram oil decreased pro-inflammatory cytokine levels As shown in Figure 6, induction of colitis by DSS pro- duced a marked increase in IL-6 level (p < 0.01). However, pre-treatment with marjoram oil significantly reduced the IL-6 content in plasma (p < 0.05) when compared to the DSS group. Marjoram oil reduced colonic myeloperoxidase activity In the DSS group, colonic inflammation resulted in an increase in the activity of MPO in comparison to the control group (p < 0.001), reflecting the enhanced neutrophil infiltra- tion that characterized this inflammatory process. However, pre-treatment of DSS-treated group with marjoram oil sig- nificantly reduced the colonic activity of MPO, compared to colitic animals (p < 0.001, vs the DSS group; Figure 7). Marjoram oil decreased oxidative stress in DSS-induced colitis In the DSS group, substantial oxidative stress was generated in the colonic mucosa as evidenced by a marked increase in TBARS (Figure 8) levels and a sig- nificant decrease in SOD and CAT activity in the colon (Figures 9A and 9B). GSH content in the colon of the DSS-treated mice was relatively lesser than that in the control and marjoram oil groups (p < 0.01; Figure 9D). Interestingly, marjoram oil reversed the loss of SOD Figure 4. Analysis of basal gut permeability in male Balb/c mice. (A) Colonic permeability to FITC-dextran (FD4) was measured in Ussing chamber. (B) Transepithelial ion conductance was measured in the colon. Bars indicate mean and SD. n = 6/group. (*p < 0.05, **p < 0.01 compared to DSS group; #p < 0.05, ##p < 0.01 compared to control group). Co ntr ol Ma rjo ra m M+ DS S DS S 0 100 200 300 400 P er m ea nc e cl ea ra nc e (c m /s ec × 1 0– 6 ) 0 10 0 50 100 150 20 30 40 C on du ct an ce ( M S /c m 2 ) Control Marjoram M+DSS DSS Time (min) (A) (B) B 112 Italian Journal of Food Science, 2023; 35 (1) Bouayad DI et al. Figure 5. Effects of marjoram oil on histological damage in DSS-induced mice colitis. (A) Histological appearance of colonic tissue sections (H&E, G×10). (B) Changes in histological score. Data are expressed as the mean ± SD (n = 6). **p < 0.01 is significantly different from the DSS group, and ###p < 0.001 is significantly different from the control group. Control Marjoram M+DSSDSS C on tro l Ma rjo ra m M+ DS S DS S 5 10 15 H is to lo gi ca l s co re 0 (A) (B) Figure 6. Effect of marjoram oil on plasma levels of IL-6 in mice treated with 4% DSS. Statistical analysis was performed using one-way Anova. Values are mean ± SD (n = 6 for each group). *Indicates significant differences (*p < 0.05, **p < 0.01) compared with DSS group. ##p < 0.01 compared to control. Co ntr ol Ma rjo ra m M+ DS S DS S 0 100 200 300 400 IL -6 ( pg /m l) Co ntr ol Ma rjo ra m M+ DS S DS S 0 10 20 30 U /m g pr ot ei n Figure 7. Colonic myeloperoxidase activity. Data represent mean ± SD (n = 6 per group), ###p < 0.001 compared with control group; ***p < 0.001 compared with DSS group. and CAT activity (p < 0.01) and increased GSH content, compared to the DSS group. Furthermore, colonic lev- els of TBARS were reduced in colitic mice pre-treated with marjoram oil. However, no significant changes in glutathione peroxidase activity were observed in the colonic tissues of different groups (Figure 9C). Discussion One of the two main types of IBDs is ulcerative colitis, increasing rapidly in prevalence globally (Kokkinidis et al., 2017). Anti-inflammatory medications, includ- ing corticosteroids and 5-amino salicylic acid and its derivatives, are frequently used to treat IBD. In contrast, because of the development of additional oxidative stress, these medications are considered as a double-edged sword, as they cause hepatotoxicity and other adverse reactions (Uko et al., 2012). For this reason, natural prod- ucts have garnered substantial research interest and are being investigated extensively. Our study explored the preventive effects of marjoram oil as a therapeutic strat- egy for acute ulcerative colitis caused by DSS. In this study, a colitis model was established with mice being fed with 4% DSS solution for 7 days. DAI score was used as an indicator to evaluate the gravity of gas- trointestinal disease in experimental groups. Our results Italian Journal of Food Science, 2023; 35 (1) 113 Protective effect of marjoram oil on ulcerative colitis in Balb/c mice through its anti-inflammatory and antioxidant properties Figure 8. Effect of marjoram oil on the TBARS levels in colonic tissue of DSS-treated mice (n = 6). Values are expressed as mean ± SD and analyzed using one-way anal- ysis of variance followed by Anova. (##p < 0.01, compared to control; **p < 0.01 compared with DSS group). Co ntr ol Ma rjo ra m M+ DS S DS S 0 1 2 3 4 T B A R S ( nm ol /g ti ss ue ) Figure 9. Effect of marjoram oil on (A) catalase, (B) superoxide dismutase, (C) glutathione peroxidise, and (D) total glutathione activities in the colonic tissues of DSS-treated mice (n = 6). Values are expressed as mean ± SD and analyzed using one way analysis of variance followed by ANOVA. #p < 0.05, ##p < 0.01, ###p < 0.001, compared to control; *p < 0.05, **p < 0.01, ***p < 0.001, compared with the DSS group). 0.5 1.0 1.5 C AT ( U /m g pr ot ) 0.0 G px ( U /m g pr ot ) 0.00 0.05 0.10 0.15 0.25 0.20 Co ntr ol Ma rjo ra m M+ DS S DS S Co ntr ol Ma rjo ra m M+ DS S DS S Co ntr ol Ma rjo ra m M+ DS S DS S (A) (C) 2 4 6 8 S O D ( U /m g pr ot ) 0 Co ntr ol Ma rjo ra m M+ DS S DS S (B) 2 4 6 8 10 G S H ( U /m g pr ot ) 0 (D) weight as reported by Cooper et al. (1993). Higher DAI score was related to smaller colon length and elevated histopathological score. DSS is a heparin- like polysac- charide. It is considered to cause mucosal damage and inflammation initially through a direct toxic influence on intestinal epithelial cells by disrupting mucosal bar- rier to cause colitis (Perse and Cerar, 2012). DSS also causes indirect damage because of modifications in resident bacteria (Okayasu et al., 1990) followed by the mustering and activation of inflammatory cells and the upregulation of inflammatory mediators, resulting in severe colitis (Lewellyn et al., 2017). The clinical symptoms of IBD induced by DSS were less- ened by marjoram oil. Administration of marjoram oil to colitic mice resulted in a considerably reduced symp- toms of the disease, such as diarrhoea, bloody stools, and weight loss, which were evidenced by a lower DAI score. In accordance with previous results reported by Al Howiriny et al. (2009), histopathological damage of the gastric mucosa was significantly prevented by adminis- tering marjoram extract. IBD is characterized by inflammation that affects the integrity of epithelial barrier (Landy et al., 2016). Tissue showed that the DSS-treated mice displayed abnormal physiological conditions with an increased clinical score (DAI), indicating the earlier onset of colitis symptoms, such as diarrhoea, bloody stools, and reduced body 114 Italian Journal of Food Science, 2023; 35 (1) Bouayad DI et al. conductance or its reciprocal transepithelial electrical resistance is considered as a sensitive biomarker of intes- tinal barrier integrity and its function (Catanzaro et al., 2015). Changes in transcellular and paracellular ion transport through the epithelium of colonic mucosa, or a disruption in the functional integrity of intercellular tight junctions, could explain an increase in electrical conductance (Mlodzik-Danielewicz and Tyrakowski, 2005). In the present study, DSS was found to increase the electrical conductivity and cellular permeability to FITC-dextran. Our results were in line with the study conducted by Kitajima et al. (1999), which showed that DSS enhanced colonic mucosal permeability, resulting in the alteration of inflammatory reactions and mucosal barrier function. Indeed, loss of intestinal barrier func- tion in the DSS colitis model was related to changes in tight junction protein distribution (Uko et al., 2012). The impairment of intestinal barrier function induced by DSS in our study was clearly diminished by marjoram oil in colitic mice. This amelioration was related to a decrease in IL-6 level, which plays an integral role in developing IBD (Helieh et al., 2013; Mazur-Bialy et al., 2017). Our data supported the previously reported anti-inflam- matory properties of marjoram oil. Arranz et al. (2015) indicated that sabinene hydrate and terpineol in sweet marjoram essential oil inhibited the production of proin- flammatory cytokines. In the current study, the status of inflammation in colitic animals was confirmed by an increase in MPO activity, a specific biomarker of inflammation in colonic mucosa (Malle et al., 2007). However, pre-treatment with marjoram oil significantly reduced the colonic MPO activity. MPO is highly expressed in neutrophils, but less so in monocytes and certain macrophages (Malle et al., 2007). Its activity is linearly related to neutrophil infiltration (Iba et al., 2003). Bhattacharyya et al. (2014) showed that MPO was able to catalyse the generation of ROS. When ROS formation surpasses the antioxidant capa- bility of cells, cellular macromolecules are destroyed. This oxidative stress is hypothesized to have a role in the development of several human diseases, particu- larly ulcerative colitis (Mekkioui and Djerdjouri, 2012; Xu et al., 2009). It was reported that DSS-treated mice exhibited a significant increase in lipid peroxidation (Amirshahrokhi et al., 2011; Yan et al., 2015). This was in line with the results of our research, in which TBARS concentrations were higher in colitic mice than in other groups. Interestingly, pre-treatment with marjoram oil resulted in a considerable reduction of TBARS level, demonstrating the antioxidant activity of marjoram oil. This result confirmed the results of our recent study, in which marjoram oil demonstrated a strong in vitro anti- oxidant power (Bouayad Debbagh et al., 2021). Glutathione sulfhydryl is thought to be the first line of defense against oxidative stress and inflammatory cas- cades (Zhu and Li, 2012). GSH is the main reducing agent for colonic epithelial cells; it is frequently reported to diminish during severe DSS-induced colitis (Oz et al., 2005; Wardman et al., 2007). Hence, in agreement with the above findings, we discovered lower GSH levels in the colitic group. Enzymatic antioxidants are another line of oxidative defense system. In the current study, the activity of CAT and SOD was reduced in the colonic tissues of colitic group. Similar to what was observed in our study and in human IBD patients (Mueller et al., 2013), several stud- ies have revealed that when rodents are exposed to DSS, enzymatic antioxidants are depleted, suggesting enhanced oxidative stress-mediated colon injury (Nishiyama et al., 2012; Pandurangan et al., 2015; Xing et al., 2013). Conclusions Our data suggested that marjoram oil lessened DSS- induced experimental colitis as evidenced by reduced severity of ulcerative colitis, preventing oxidative dam- age, reducing neutrophil infiltration, and inhibiting IL-6 production. Importantly, marjoram oil preserved the intestinal barrier function from damage caused by inflammatory stimuli. Acknowledgments This research was supported by the Directorate General for Scientific Research and Technological Development (DGRSDT, MESRS), Algeria. 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