Comparative Study of Sulfated Polysaccharides From Ulva Lactuca Grown in Tunisia and Morocco: in Vitro Antioxidant Activity and in Vivo Anti‐Inflammatory and Anti‐Ulcer Effects
Nourhene Kharrat, Yasmine Touhamia, Rihab Ben Abdallah kolsi, Khaled Hamden, Rafik Ben Said, Touria Ould Bel Lahcen, Hichem Ben Salah, Noureddine Allouche
TL;DR
This study compares sulfated polysaccharides from seaweed in Tunisia and Morocco, finding differences in their antioxidant and anti-inflammatory effects.
Contribution
The study reveals how geographic origin affects the structure and biological activity of Ulva lactuca polysaccharides.
Findings
PSUM showed stronger in vivo anti-inflammatory and gastroprotective effects than PSUT.
Both polysaccharides significantly reduced oxidative stress markers in a rat model.
Geographic origin influences the structural and functional properties of the polysaccharides.
Abstract
This study presents a comparative analysis of sulfated polysaccharides extracted from Ulva lactuca collected in Tunisia (PSUT) and Morocco (PSUM). FTIR confirmed the presence of sulfate groups, while GC‐MS identified diverse sugar components. Both polysaccharides showed strong antioxidant activity, with DPPH and ABTS radical scavenging rates exceeding 50% at the highest concentrations. In a rat model of gastric ulceration, PSUT and PSUM significantly reduced inflammation and lymphocytic infiltration and lowered the activities of 5‐lipoxygenase and myeloperoxidase. Treatment also produced clear antioxidant effects, decreasing hydrogen peroxide by 48% and 61% and reducing TBARS by 65% and 71%, respectively. In addition, both polysaccharides inhibited gastric H+/K+‐ATPase and pepsin, contributing to notable anti‐acidic effects and improved protection of the gastric mucosa. Mucin levels…
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FIGURE 11| No | Mineral elements | PSUT (g/L) | PSUM (g/L) |
|---|---|---|---|
| 1 | Soduim | 0.241 | 0.285 |
| 2 | Potassium | 0.224 | 0.440 |
| 3 | Magnesium | 0.096 | 0.322 |
| 4 | Calcuim | 0.054 | 0.129 |
| Peak | Retention time Tr (min) | Compounds | Area (%) | |
|---|---|---|---|---|
| PSUT | PSUM | |||
| 1 | 15.72 | D‐xylopyranose,1,2,3,4‐tetrakis‐O‐(trimethylsilyl) | 22.51 | 5.11 |
| 2 | 16.34 | D‐xylose, tetrakis(trimethylsilyl) | 18.88 | 10.08 |
| 3 | 16.55 | Glucose, pentakis‐O‐trimethylsilyl | 11.83 | 2.29 |
| 4 | 16.76 | D‐arabinose, tetrakis(trimethylsilyl) | 1.83 | nd |
| 5 | 16.91 | D‐ribose,2,3,4,5‐tetrakis‐O‐ethylsilyl) | 11.66 | nd |
| 6 | 17.82 | D‐mannopyranose,1,2,3,4,6‐pentakis‐O(trimethylsilyl) | 16.35 | 1.05 |
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Taxonomy
TopicsSeaweed-derived Bioactive Compounds · Phytochemical Studies and Bioactivities · Polysaccharides and Plant Cell Walls
Introduction
1
In recent years, there has been a substantial increase in the search for natural antioxidant and anti‐inflammatory molecules, as oxidative stress and inflammation play a critical role in the development of numerous chronic diseases. These conditions are major contributors to dysfunction in multiple organs, including the reproductive system, liver, and kidneys, and are implicated in diseases such as cancer, neurodegeneration (e.g., Alzheimer's disease), cardiovascular diseases, and chronic inflammatory conditions [1, 2]. Among these disorders, gastric ulcers stand out as a serious health concern caused by an imbalance between aggressive factors (e.g., hydrochloric acid, pepsin, reactive oxygen species) and the stomach's defense mechanisms. Two of the main contributing mechanisms to the development and progression of gastric ulcers are chronic inflammation and oxidative stress. Oxidative stress, defined as the accumulation of reactive oxygen species (ROS) beyond the capacity of the body's antioxidant defenses, leads to lipid peroxidation and cellular damage, ultimately compromising the integrity of the gastric mucosa. Concurrently, inflammation often triggered by infections or irritantspromotes leukocyte infiltration and the release of pro‐inflammatory mediators such as myeloperoxidase (MPO), cyclooxygenase‐2 (COX‐2), and 5‐lipoxygenase (5‐LO). These factors exacerbate tissue injury and delay healing. Therefore, simultaneously targeting oxidative stress and inflammation represents a promising therapeutic strategy for the prevention and treatment of gastric ulcers.
Sulfated polysaccharides are a complex group of macromolecules with numerous biologically significant properties. These anionic polymers are found in a wide range of organisms, including mammals and invertebrates [3, 4]. Marine algae are the primary nonanimal source of sulfated polysaccharides. The structure of algal sulfated polysaccharides differs depending on the algae species [5, 6]. As a result, each newly isolated sulfated polysaccharide from marine algae presents a unique structure and, therefore, potentially novel biological activities. Sulfated polysaccharides are found in different proportions across the three main groups of marine algae: Rhodophyta (red algae), Phaeophyta (brown algae), and Chlorophyta (green algae). In Rhodophyta, the primary sulfated polysaccharides are galactans, composed mainly of galactose or modified galactose units [7, 8]. In Phaeophyta, sulfated polysaccharides consist of fucans polydisperse molecules based on sulfated L‐fucose and their heterogenous forms are known as fucoidans [6, 9, 10, 11]. In Chlorophyta, polydisperse heteropolysaccharides are predominant, although homopolysaccharides may also be present [12, 13, 14]. Algal sulfated polysaccharides have been shown to possess a wide range of biological activities, including anticoagulant, antioxidant, antiproliferative, antitumoral, anticomplementary, anti‐inflammatory, antiviral, antiseptic, and antiadhesive effects [15, 16, 17]. Although the relationship between their structure and biological activity remains unclear [6], certain features such as sulfate groups arranged in clusters appear to be essential for interacting with cationic proteins and ensuring biological functionality [18]. Molecular weight is also thought to influence their bioactivity [19, 20]. In recent years, sulfated polysaccharides from marine algae have been increasingly recognized for their free radical scavenging and antioxidant properties, which can help prevent oxidative damage in living organisms. It is well established that the chemical composition and antioxidant potential of marine algae vary depending on their geographical origin (sea vs. ocean). However, to our knowledge, no comparative studies have explored the differences in chemical composition and biological activity between Ulva lactuca collected from Tunisian (Mediterranean Sea) and Moroccan (Atlantic Ocean) coasts.
Hence, the purpose of this research is to compare the chemical and biological properties of sulfated polysaccharides isolated from the green algae U. lactuca collected from the Mediterranean Sea (Tunisia) and the Atlantic Ocean (Morocco). While this study uses a single animal model and dose level, it provides valuable preliminary evidence supporting further mechanistic and translational research.
Results and Discussion
2
Chemical Analysis
2.1
On a wet‐weight basis, the extraction yield of sulfated polysaccharides from U. lactuca of the Tunisian extract (PSUT) was 19.70%, while that of sulfated polysaccharides from U. lactuca of the Moroccan extract (PSUM) was 14.7%. These values are consistent with previously reported yields for ulvan isolated from U. lactuca (13.06%) and other green algae (17.57%) [21, 22]. The total phenolic content (TPC) and total flavonoid content (TFC) of the sulfated polysaccharide extracts were expressed as mg GAE/g DE and mg QE/g DE, respectively, and the results are presented in Figure 1. Both PSUT and PSUM contained substantial levels of phenolic and flavonoid compounds. PSUT exhibited the highest concentrations, with TPC = 30.742 mg GAE/g DE and TFC = 71.037 mg QE/g DE (p < 0.05), whereas PSUM showed comparatively lower values, with TPC = 6.312 mg GAE/g DE and TFC = 47.050 mg QE/g DE.
Total phenols and falvonoids contents of sulfated polysaccharides of U. lactuca.
These findings are consistent with previous reports indicating substantial variability in the phenolic and flavonoid contents of U. lactuca extracts, largely influenced by geographic origin and extraction conditions. For example, methanolic extracts of U. lactuca collected from Egypt have been shown to contain high phenolic levels, reaching up to 45.69 mg GAE/g dry weight [23]. Other studies have similarly documented wide phenolic ranges, from approximately 4 to 51 mg GAE/g, depending on the extraction solvent and collection site [24]. Flavonoid contents also vary widely but generally correlate with antioxidant activity potentials. The elevated phenolic and flavonoid levels detected in the Tunisian extract suggest a strong antioxidant capacity, consistent with findings in other regional samples where high phenolic content was linked to enhanced free‐radical scavenging activity [25]. Such comparisons emphasize the influence of environmental factors, algal physiology, and extraction methods on bioactive compound yields in U. lactuca. These variations highlight the importance of regional characterization to identify promising sources for nutraceutical and pharmaceutical applications. Further studies focusing on detailed bioactivity assays and compound isolation will be essential to fully explore the therapeutic potential of these sulfated polysaccharide extracts [26].
Mineral Composition of Sulfated Polysaccharides
2.2
The concentrations of mineral elements are summarized in Table 1 and illustrated in Figure 2. Sodium (Na), potassium (K), magnesium (Mg), and calcium (Ca) in PSUT and PSUM were quantified using ion chromatography (IC). Analyses were carried out on a Thermo Scientific AQUION system equipped with Dionex Chromeleon 7.2 software, following the standard CATIONS DEF method. For each measurement, 25 µL of sample solution was injected, and all determinations were performed in triplicate to ensure analytical reproducibility. For PSUM, ion chromatographic analysis produced retention times of 5.51, 7.07, 9.62, and 11.90 min for Na, K, Mg, and Ca, corresponding to concentrations of 0.285 g/L, 0.440 g/L, 0.322 g/L, and 0.129 g/L, respectively. In comparison, PSUT displayed retention times of 5.54, 7.07, 9.89, and 12.07 min for the same elements, with concentrations of 0.241 g/L (Na), 0.224 g/L (K), 0.096 g/L (Mg), and 0.054 g/L (Ca).
Chromatogram of the mineral compostion of PSUT and PSUM.
According to these data, the (Na) was the predominant mineral in PSUT, followed by (K), (Mg), and (Ca). However, in PSUM, the predominant mineral was (K) followed by, (Mg), (Na), and (Ca). The high mineral concentration may be explained by the ability of marine plants to efficiently absorb and accumulate nutrients from the surrounding seawater [27]. The carboxyl, sulfate, and hydroxyl groups present in the polysaccharide structure can participate in ion‐exchange processes, thereby acting as active binding sites for metallic cation complexation [28]. These findings are consistent with previous studies showing that the mineral composition of marine algal polysaccharides varies considerably according to species, environmental conditions, and structural characteristics factors that collectively shape their functional properties, including antioxidant activity and metal‐chelation capacity. [29, 30].
Functional Group Analysis
2.3
The functional groups present in the structures of the sulfated polysaccharides were identified using a Fourier Transform Infrared (FTIR) spectrophotometer (Spectrum Two, PerkinElmer, USA). The sulfated polysaccharides were pressed into pellets and analyzed over a wavenumber range of 4000–370 cm^−^ ^1^.The IR spectra of the sulfated polysaccharides are presented in Figure 3. The peaks obtained at 845 cm^−1^ and 854 cm^−1^ show that PSUT and PSUM, respectively, contain compounds with the (C‐S‐O) group. Both polysaccharides also showed bands at 1424 cm^−1^ and 1473 cm^−1^ which indicate the presence of (S ═ O) group. This result confirms the presence of sulfate groups in PSUT and PSUM. Previous studies on the chemical composition of algal polysaccharides have shown that these biomolecules contain sulfate groups formed during polysaccharide synthesis within the Golgi apparatus of algal cells [31]. The presence of sulfate groups is well documented as a key contributor to the biological activities of algal polysaccharides [32, 33]. In the present study, characteristic peaks of similar intensity were observed at 1600 cm^−^ ^1^ for PSUM and 1636 cm^−^ ^1^ for PSUT, corresponding to the stretching vibrations of the C═O bond in carboxylate groups [34]. Additionally, the absorption bands detected at 3277 cm^−^ ^1^ and 3306 cm^−^ ^1^ fall within the typical range for O–H stretching, confirming the presence of hydroxyl groups in both polysaccharide samples.
FTIR spectra of PSUT and PSUM (U. lactuca).
Thermal Analysis
2.4
Differential scanning calorimetry (DSC) is the most widely used technology for studying the properties of thermal transitions in polysaccharides. It is commonly used in food research to study the thermodynamic and kinetic properties of polysaccharides in solution and solid forms [21, 22]. During thermal scans from −50 to 300°C (Figure 4), a significant endothermic peak was identified for the dried PSUM and PSUT at 100°C and 70°C, respectively, corresponding to the glass transition temperatures. This transition between the glassy and rubbery state conditions is usually signified by rapid changes in the physical, mechanical, electrical, and rheological properties of the finished product [35]. This glass transition temperature is significantly lower than that reported for U. armoricana (132.6°C) [36]. Ronkart et al. found that the glass transition temperatures of polymers change according to their chemical nature, origin (natural or synthetic) and composition [37]. Furthermore, the same reaserchers showed that the glass transition temperature increases with the polymerization degree. An exothermic peak appears in PSUM at around 230°C, which is absent in PSUT. A similar trend was noticed in the case of Ulvan, with an exothermic peak appearing around 230°C due to Ulvan degradation [38].
DSC thermogram of sulfated polysaccharides PSUT and PSUM.
Antioxidant Activity
2.5
Antioxidants have a significant role in the regulation of oxidative disease processes in humans. In this case, marine green algae were studied and found to have a wide range of strong antioxidant activity [39]. It is well known that U. lactuca contains several bioactive substances with possible health advantages. One important class of compounds in U. lactuca are polysaccharides, which have a variety of biological properties, including antioxidant activity [40].
DPPH Radical Scavenging Activity
2.5.1
Antioxidant efficacy is commonly assessed using multiple analytical approaches because oxidative stress encompasses a wide array of reactive metabolites and interconnected biochemical processes. As emphasized by Jindal et al. (2012) [41], no single assay is sufficient to accurately quantify antioxidant activity. Therefore, a comprehensive evaluation requires the use of several complementary methods and diverse substrate systems [42].
The present study assessed the antioxidant capacity of sulfated polysaccharide extracts PSUT and PSUM, using vitamin C as a reference. In order to evaluate the antioxidant activity of U. lactuca, four complementary methods were used: DPPH, TAC, FRAP, ABTS.
The DPPH radical‐scavenging activity of the sulfated polysaccharides is presented in Figure 5.A. Accordingly, PSUT and PSUM exhibited an interesting DPPH radical‐scavenging activity which was higher than 50% (inhibition percentage) at the highest concentration (1 mg/mL) with IC** 50 ** = 0.087 ± 0.005 and 0.080 ± 0.0001 mg/mL, respectively. This activity was comparable to that of vitamin C. Moreover, the comparative analysis of inhibition percentages demonstrated that the differences were statistically non‐significant between extracts (p > 0.05), which indicates that they have considerable DPPH scavenging activity.
DPPH (A), ABTS (B), TAC (C), and FRAP (D) of sulfated polysaccharides. (Values wrere obtained in triplicate and are expressed as means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001).
The DPPH radical scavenging activities of PSUT and PSUM differed notably from those reported for sulfated polysaccharides extracted from U. lactuca at Wild Coast Abalone in East London [43]. This variation is likely attributable to differences in environmental conditions, extraction techniques, and degrees of sulfation, all of which influence polysaccharide structure and bioactivity. These findings are consistent with previous studies demonstrating that the chemical composition and antioxidant properties of ulvan are strongly affected by geographic origin and methodological factors [25, 44]. Furthermore, other reports confirm that U. lactuca polysaccharides can activate antioxidant enzymes and reduce oxidative stress markers, supporting their effectiveness as natural antioxidants [45, 46]. Collectively, these observations highlight that both intrinsic factors, such as sulfation and polysaccharide structure, and extrinsic factors, such as environmental conditions, play crucial roles in determining the antioxidant potential of U. lactuca extracts.
ABTS•+ (2,2‐azinobis 3‐ethylbenzothiazoline‐6‐sulfonate radical) Assay
2.5.2
To validate the findings, a second test was carried out using the cationic radical ABTS^•+^'s proton‐trapping ability. PSUT and PSUM exhibited a good scavenging activity against ABTS radical as shown in Figure 5.B. Vitamin C showed higher scavenging activity (98.71%) than the polysaccharides at the highest concentration (1 mg/mL).
The percentage radical scavenging activity of PSUT (49.73–56.7%) and PSUM (59.39–72.82%) was higher than 50% at the highest concentration (1 mg/mL) with an IC_50_ = 0.108 ± 0.002 and 0,091 ± 0.002 mg/mL, respectively, which shows that they are potent scavengers of ABTS radical. Moreover, PSUM revealed higher radical scavenging activity than PSUT, with a percentage scavenging activity of 72.82% at the highest concentration (1 mg/mL). These results are consistent with previous research showing that sulfated polysaccharides from U. lactuca collected at Wild Coast Abalone in East London exhibit comparable radical scavenging activity, ranging from 39.30% to 78.34% at the highest concentration tested (333.33 µg/mL) [43]. Similar potent antioxidant activities have also been reported for marine polysaccharides in general, further supporting their efficacy as effective in vitro radical scavengers [47].
TAC Assay
2.5.3
The TAC method was applied to assess the antioxidant capacity of sulfated polysaccharide extracts using vitamin C as a standard (1 mg/mL). The results are provided in Figure 5.C. The findings showed that PSUT exhibits the strongest antioxidant power (285.33 mg GAE/g) than PSUM (28.08 mg GAE/g). Vitamin C presents the strongest antioxidant power (717.023 mg GAE/g). Furthermore, the comparative study demonstrated that the differences were statistically significant between the extracts and the reference (Vitamin C) p < 0,05. These results are in agreement with the finding of Qi et al., who showed that the degree of sulfation and molecular weight have an important effect on the antioxidant activity of sulfated polysaccharides from green seaweeds. This demonstrates the potential of U. lactuca's specially derived polysaccharides as promising natural antioxidants [48].
Additionally, sulfated polysaccharide derivatives from other sources, such as Auricularia auricular, have shown enhanced antioxidant activity compared to their non‐sulfated counterparts and vitamin C, supporting the role of sulfate groups in antioxidant efficacy [49].
Reducing Power FRAP
2.5.4
The ability of reductants or antioxidants to change ferric form (Fe^3+^) into ferrous form (Fe^2+^) was used to calculate the reducing power. As a result, prussian blue formation at 700 nm can be used to measure Fe^2+^. A greater absorbance value indicates a sample with a stronger reducing power [22]. The findings of the current investigation in Figure 5.D demonstrated that, in comparison with vitamin C, the reducing power activity of PSUT and PSUM extracts was not high. Additionally, the comparison of the extracts and the reference (vitamin C) revealed statistically significant differences (p < 0.05).
Previous studies have suggested that the molecular weight of algal polysaccharides is correlated with their reducing power. Additionally, sulfate content has been reported to influence this property. For example, Zhang et al. found that the reducing power of polysaccharides from different algal species was inversely proportional to their sulfate content [50]. However, other studies have reported no significant correlation between sulfate levels and reducing power [22]. The observed discrepancies between total antioxidant capacity (TAC), radical scavenging assays, and reducing power (FRAP) may reflect the diversity of antioxidant mechanisms and the different types of radicals involved. In some assays, a polysaccharide may exhibit activity, while in others it may not. This variability is likely related to the additive, synergistic, and antagonistic interactions among the various bioactive components within the extracts. [51].
Composition of Sulfated Polysaccharides
2.6
Ulvan is a polysaccharide whose composition changes according to the harvesting season and the taxonomic origin of the algal biomass [52]. Precisely defining the composition of the extracted ulvan proved challenging due to its complex structure and the presence of several types of sugars. The best way to depolymerize polysaccharides into monomeric units is by acid hydrolysis, which can then be identified using the appropriate chromatographic method [53].
The sugar composition of the polysaccharide samples was examined using GC‐MS. The results are depicted in Table 2 and Figure 6. PSUT exhibited a more complex and diverse composition, characterized by high proportions of xylose derivatives, followed by notable amounts of mannose, glucose, and ribose. In contrast, PSUM displayed a simpler profile with substantially lower overall sugar diversity and content, dominated mainly by xylose‐ and glucose‐derived residues, with only minor contributions from mannose. These differences indicate that PSUT possesses a richer and more heterogeneous polysaccharide structure compared to PSUM.The sugar composition of PSUT and PSUM was comparable to ulvan found in other studies. According to Olasehinde et al., ulvan from G. gracilis and U. lactuca is constituted of rhamnose and galactose, as well as ribose, arabinose, glucose, xylose, and mannose [43]. Furthermore, the study is similar to the monosaccharides of ulvan from the marine green algae U. conglobate. The study found that it contains rhamnose, glucose, xylose, fucose, galactose, mannose, and arabinose, with rhamnose > 60% and glucose > 13%, while other saccharides have minor contents [54]. Additionally, the results obtained in a previous investigation indicated the presence of various monosaccharides with relative abundances in the order: fucopyranose (22.09%), L‐rhamnose (18.17%), L‐fucose (17.46%), rhamnopyranose (14.29%), mannopyranose(8.59%), α‐D‐glactopyranose (7.64%), galactopyranose (6.14%) and β‐arabinopyranose (5.62%) [55]. Variations in sugar composition among ulvan samples are often attributed to differences in extraction methods as well as environmental factors, including season and habitat. Broad compositional ranges have been reported for rhamnose (5.0–92.2 mol%), glucose, and other monosaccharides across studies of Ulva species worldwide. Such diversity in monosaccharide composition can have significant implications for the functional properties and bioactivity of the polysaccharides [56].
GC‐MS chromatograms of sulfated polysaccharides PSUT and PSUM (Compounds 1–6 are identified as listed in Tables 2).
In Vivo Biological Activities of Sulfated Polysaccharides From U. lactuca
2.7
PSUT and PSUM Effects on Key Enzymes Involved in Lymphocyte Infiltration and Inflammation in Gastric Ulcers
2.7.1
The present study demonstrates that ethanol ingestion is strongly associated with immune cell infiltration, particularly lymphocytes, in the gastric juice. This immune activation is evidenced by a marked increase in myeloperoxidase (MPO) activity by 142% compared to healthy rats, reflecting an enhanced recruitment of neutrophils and lymphocytes into the gastric mucosa. Furthermore, the sustained inflammatory response was confirmed by a significant elevation in 5‐lipoxygenase (5‐LO) activity by 126%, indicating the persistence of leukotriene‐mediated inflammation. These alterations contributed to mucosal injury and ulceration. These findings are illustrated in Figure 7A–B.
*Effect of PSUT and PSUM on ethanol‐induced lymphocyte infiltration and inflammation in gastric juice. (A) Gastric juice Melyoperoxidase (GJ MPO) activity and (B) Gastric juice 5‐ Lypoxygenase (GJ5‐LO) activity. Abbreviations: Con = control; GU = gastric ulcer; U‐PSUT200 = ulcer + PSUT (200 mg/kg); U‐PSUM200 = ulcer + PSUM (200 mg/kg); U‐Omz = ulcer + omeprazole. Each value represents the mean ± SEM for each group (n = 6). Differences were considered statistically significant at p < 0.05. Statistical significance is indicated as follows: p < 0.05 vs normal rats; @p < 0.05 vs untreated gastric ulcer (GU) rats; ≠p < 0.05 vs GU rats treated with PSUT; &p < 0.05 vs GU rats treated with PSUM.
The results show that both polysaccharides exert significant anti‐inflammatory activity, with PSUM demonstrating the strongest effect. These compounds effectively alleviate the ulcerogenic effects of ethanol by reducing immune cell infiltration and inflammatory enzyme activity in the gastric environment.
Compared to untreated rats with gastric ulcers, the oral administration of PSUT and PSUM in rats with gastric ulcers markedly reduced lymphocyte infiltration and recruitment to the gastric system, demonstrated by a suppression of MPO activity by 46 and 57%, respectively. Consequently, inflammation was also reduced, as evidenced by a decrease in gastric juice 5‐LO activity by 38 and 47%, respectively. These results are consistent with previous studies showing that ethanol‐induced gastric ulcers are associated with severe inflammation and lymphocyte infiltration, which trigger a cascade of inflammatory responses leading to mucosal damage and ulceration [57, 58]. In contrast, the administration of sulfated polysaccharides has been shown to offer protection against such inflammation, as evidenced by a reduction in pro‐inflammatory cytokines such as interleukin (IL)‐6, IL‐1β, and tumor necrosis factor (TNF)‐α, along with an increase in the anti‐inflammatory cytokine IL‐10 [59]. This modulation of the immune response contributes to the preservation of gastric mucosal integrity and a significant reduction in ulcer severity. Moreover, the study by Dutra et al. [60] demonstrated that sulfated polysaccharides extracted from the seaweed Gracilaria caudata protect against dexamethasone‐induced ulcerative colitis, primarily through the suppression of inflammation and oxidative stress. Another study by Qin et al. [61] reported that sulfated polysaccharides derived from Ishige okamurae effectively protect against DSS‐induced ulcerative colitis by downregulating inflammation through the reduction of pro‐inflammatory cytokines such as TNF‐α and IL‐6, as well as by modulating gut microbiota composition.
Effects of PSUT and PSUM on H2O2 and TBARS Levels in Gastric Juice of Ulcerated Rats
2.7.2
Our study demonstrates that lymphocyte infiltration and gastric inflammation are closely associated with elevated levels of reactive oxygen species (ROS), particularly hydrogen peroxide (H_2_O_2_), which increased by 149% in the gastric juice of ulcerated rats. This oxidative stress was joined by severe cellular damage, as evidenced by a 303% rise in thiobarbituric acid reactive substances (TBARS) levels compared to healthy controls. These findings are illustrated in Figure 8A–B.
*Effects of PSUT and PSUM on hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances (TBARS) levels in gastric juice and gastric mucosa of ethanol‐induced ulcerated rats. (A) Gastric juice H2O2 level. (B) mucosa juiceTBARS rate. Abbreviations: Con = control; GU = gastric ulcer; U‐PSUT200 = ulcer + PSUT (200 mg/kg); U‐PSUM200 = ulcer + PSUM (200 mg/kg); U‐Omz = ulcer + omeprazole. Each value represents the mean ± SEM for each group (n = 6). Differences were considered statistically significant at p < 0.05. Statistical significance is indicated as follows: p < 0.05 vs normal rats; @p < 0.05 vs untreated gastric ulcer (GU) rats; ≠p < 0.05 vs GU rats treated with PSUT; &p < 0.05 vs GU rats treated with PSUM.
This study demonstrates that PSUT and PSUM effectively reduce oxidative stress markers in gastric juice by significantly decreasing hydrogen peroxide (H_2_O_2_) levels and lipid peroxidation, as indicated by a marked reduction in TBARS levels. These findings highlight the antioxidant potential of both polysaccharides in protecting gastric tissues against ethanol‐induced oxidative damage.
Oral administration of PSUT and PSUM significantly attenuated this oxidative imbalance by reducing H_2_O_2_ levels by 61 and 49%, respectively. Consequently, lipid peroxidation was markedly suppressed, with TBARS levels decreasing by 71 and 67% in PSUT‐ and PSUM‐treated rats, respectively, compared to untreated gastric ulcer rats. Our findings are congruent with previous studies revealing that the administration of polysaccharides in ulcerated rats enhances the antioxidant capacity of the gastric system. For instance, Lajili et al. [62] observed that oral administration of a sulfated polysaccharide extracted from the red alga Laurencia obtusa in rats with ethanol/HCl‐induced gastric ulcers significantly strengthened the antioxidant defense of the gastric mucosa, thereby preventing tissue damage and ulceration. Similarly, another study by Son et al. [63] reported that treatment with sulfated rhamnoglucuronan isolated from the Korean seaweed Ulva pertusa in DSS‐induced ulcerative colitis rats effectively suppressed the levels of inflammatory cytokines and downregulated MAPK‐ and NF‐κB‐related signaling pathways in both serum and colon tissues. Moreover, the study by Li et al. [64] demonstrated that administration of Chaenomeles sinensis polysaccharide provides protective effects against ulcerative colitis in rats. This protection was mediated by a reduction in the expression of myeloperoxidase (MPO), inflammatory cytokines [tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), and IL‐6], as well as oxidative stress markers including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and nitric oxide (NO).
Effect of PSUT and PSUM on the Activity of Key Gastric Acidity Enzymes: H+/K+‐ATPase and Pepsin
2.7.3
This study shows that absolute ethanol ingestion leads to a significant increase in the activity of key gastric acidity enzymes, namely H^+^/K^+^‐ATPase and pepsin, which contributes to heightened gastric acidity and subsequent mucosal ulceration. These findings are illustrated in Figure 9A–B.
*Effect of PSUT and PSUM on the activity of key gastric acidity enzymes (H+/K+‐ATPase and Pepsin) in relation to ethanol‐induced ulceration. (A) Gastric juice H+/K+ATPase activity. (B) Gastric juice pepsin activity. Abbreviations: Con = control; GU = gastric ulcer; U‐PSUT200 = ulcer + PSUT (200 mg/kg); U‐PSUM200 = ulcer + PSUM (200 mg/kg); U‐Omz = ulcer + omeprazole. Each value represents the mean ± SEM for each group (n = 6). Differences were considered statistically significant at p < 0.05. Statistical significance is indicated as follows: p < 0.05 vs normal rats; @p < 0.05 vs untreated gastric ulcer (GU) rats; ≠p < 0.05 vs GU rats treated with PSUT; &p < 0.05 vs GU rats treated with PSUM.
PSUT and PSUM exhibited a strong protective effect against ethanol‐induced overactivation of H^+^/K^+^‐ATPase and pepsin, contributing to their anti‐ulcer potential. PSUM demonstrated a more pronounced inhibitory effect, supporting its superior efficacy in preserving gastric mucosal integrity.
Supplementation with PSUT and PSUM provided notable gastroprotective effects by suppressing the activity of these enzymes. Specifically, PSUT reduced H^+^/K^+^‐ATPase and pepsin activities by 33 and 32%, respectively, while PSUM achieved a more pronounced inhibition of 47 and 55%, compared to untreated ulcerated rats. These findings suggest that PSUT and especially PSUM mitigate ethanol‐induced gastric damage through the downregulation of acid‐secreting enzymes. To date, no previous studies have directly linked the effects of sulfated polysaccharides to the activity of the key gastric acidity enzyme H^+^/K^+^‐ATPase or its involvement in ulcer progression. However, numerous studies have demonstrated the critical role of this enzyme in the pathogenesis of gastric ulcers, indicating that its inhibition could help preserve gastric function. Elevated H^+^/K^+^‐ATPase activity leads to excessive secretion of gastric acid, resulting in a drop in intragastric pH [65]. This hyperacidity compromises the gastric mucosal barrier, promotes mucosal erosion, and facilitates the activation of pepsin, ultimately leading to tissue damage, inflammation, and ulcer formation. Such an environment also impairs the healing process and intensifies pain and discomfort, contributing to chronic gastric dysfunction. This protective effect of PSUT and PSUM on the gastric system could be attributed to multiple mechanisms. Firstly, these sulfated polysaccharides inhibit the inflammatory response, reducing the recruitment and activation of immune cells that contribute to mucosal damage. Secondly, they suppress oxidative stress by scavenging reactive oxygen species and enhancing the antioxidant defense system. By decreasing both inflammation and oxidative stress, PSUT and PSUM help preserve the structural integrity of the gastric mucosa. This maintenance of mucosal integrity prevents further damage caused by excessive gastric acidity. Consequently, the proper functioning of the gastric system is supported, reducing ulcer formation and promoting healing.
Effect of PSUT and PSUM on Mucin and Gastric Mucus Levels
2.7.4
The results of this study demonstrate that both polysaccharides, PSUT and PSUM, enhance the gastric defense system against ethanol‐induced ulcerative effects. This protective action is evidenced by a significant increase in mucin levels by 62 and 100%, and in adherent gastric mucus content by 32 and 71%, respectively, following the administration of PSUT and PSUM compared to untreated rats with gastric ulcers. These findings are illustrated in Figure 10A–B.
*Effect of PSUT and PSUM on the levels of protective gastric components: mucin and adherent mucus content. (A) Gastric juice mucin level. (B) Adherent gastric mucus weight. Abbreviations: Con = control; GU = gastric ulcer; U‐PSUT200 = ulcer + PSUT (200 mg/kg); U‐PSUM200 = ulcer + PSUM (200 mg/kg); U‐Omz = ulcer + omeprazole. Each value represents the mean ± SEM for each group (n = 6). Differences were considered statistically significant at p < 0.05. Statistical significance is indicated as follows: p < 0.05 vs normal rats; @p < 0.05 vs untreated gastric ulcer (GU) rats; ≠p < 0.05 vs GU rats treated with PSUT; &p < 0.05 vs GU rats treated with PSUM.
PSUT and PSUM significantly restored the ethanol‐induced depletion of mucin and adherent mucus, two key protective factors of the gastric mucosa. This recovery supports their mucosal defense‐enhancing and anti‐ulcer properties.
In fact, mucin and mucus play a crucial role in protecting the gastric mucosa from damage that can lead to ulcers. The increased mucus production induced by PSUT and PSUM in rats with gastric ulcers forms a thick, viscous layer covering the stomach lining. This physical barrier prevents direct contact between the epithelium and harmful agents like hydrochloric acid and digestive enzymes such as pepsin, thus protecting against gastric acidity [58]. Mucin, the main component of mucus, is a glycoprotein that gives mucus its viscoelastic and adhesive properties, ensuring its stability and constant renewal. Furthermore, the mucus traps bicarbonate ions that neutralize acid at the mucosal surface, creating an alkaline microenvironment favorable for cell survival. This defense system also restricts pathogen invasion and supports tissue repair. When mucin or mucus production is reduced or impaired, the mucosa becomes vulnerable, increasing the risk of ulceration and chronic inflammation.
Effect of PSUT and PSUM on Gastric Ulcer Area and Gastric Juice Volume
2.7.5
This study shows that PSUT and PSUM exhibit effective anti‐gastric ulcer activity, attributed not only to their potential anti‐inflammatory and antioxidant effects in the gastric environment, but also to their ability to reduce both the ulcerated gastric area and the volume of gastric juice. Oral administration of PSUT and PSUM to rats with gastric ulcers resulted in a reduction of gastric juice volume by 39 and 48%, and a decrease in gastric ulcer area by 39% and 65%, respectively. These effects led to a curative index of 61% and 68% compared to untreated ulcerated rats. These findings are illustrated in Figure 11A–C.
*Effect of PSUT and PSUM on gastric ulcer area, ulcer index, and curative index (CI). (A) Gastric juice volume. (B) Gastric ulcer area. (C) Curative index (CI). Abbreviations: Con = control; GU = gastric ulcer; U‐PSUT200 = ulcer + PSUT (200 mg/kg); U‐PSUM200 = ulcer + PSUM (200 mg/kg); U‐Omz = ulcer + omeprazole. Each value represents the mean ± SEM for each group (n = 6). Differences were considered statistically significant at p < 0.05. Statistical significance is indicated as follows: p < 0.05 vs normal rats; @p < 0.05 vs untreated gastric ulcer (GU) rats; ≠p < 0.05 vs GU rats treated with PSUT; &p < 0.05 vs GU rats treated with PSUM.
Oral administration of PSUT and PSUM markedly reduced the ulcerated gastric area and ulcer index, while significantly increasing the curative index. These findings highlight the effective gastroprotective potential of both polysaccharides, with PSUM showing greater efficacy in promoting mucosal healing.
The results of this study show that oral administration of the sulfated polysaccharides PSUT and PSUM in rats with gastric ulcers significantly reduced both the volume of gastric juice (by 39% and 48%, respectively) and the ulcerated gastric area (by 39% and 65%). These reductions reflect a substantial protective and curative effect of these compounds, further supported by high healing indices of 61% for PSUT and 68% for PSUM. Reducing gastric juice volume is critical in preventing and treating gastric ulcers, as excessive secretion of hydrochloric acid and digestive enzymes like pepsin exacerbates mucosal damage and promotes ulcer formation and expansion. The ability of PSUT and PSUM to decrease gastric juice volume suggests they may modulate gastric acid secretion, possibly through inhibition of the H^+^/K^+^‐ATPase proton pump, a key enzyme responsible for acid secretion. This inhibition limits chemical injury to the gastric mucosa and promotes ulcer healing. Moreover, the decreased ulcerated area directly indicates the effectiveness of these polysaccharides in limiting mucosal damage. This protective effect likely involves not only reduced gastric acidity but also their anti‐inflammatory and antioxidant properties, which help maintain mucosal integrity and accelerate tissue repair. A smaller ulcerated area corresponds to improved healing and faster restoration of damaged tissue. The healing index, which integrates multiple clinical and morphological parameters, confirms the overall efficacy of the treatments. The high values observed for PSUT and PSUM demonstrate a strong capacity to protect the mucosa from further injury while promoting ulcer repair. This index is a key marker in evaluating new therapeutic agents, and these results suggest that sulfated polysaccharides could be promising candidates for natural management of gastric ulcers.
In conclusion, the reduction in gastric juice volume and ulcerated area, alongside improved healing indices, highlights the combined effectiveness of PSUT and PSUM in protecting and healing the gastric mucosa. These effects likely result from a multifactorial mechanism involving acid secretion regulation, oxidative stress protection, and inflammation modulation.
Conclusion
3
This study aimed to comparatively evaluate sulfated polysaccharides extracted from U. lactuca collected in Tunisia (PSUT) and Morocco (PSUM), focusing on their structural characteristics and biological activities. FTIR analysis confirmed the presence of sulfate groups in both extracts, while GC–MS revealed distinct monosaccharide profiles, with PSUT showing a more complex and diverse composition than PSUM. Both polysaccharides exhibited strong in vitro antioxidant activity, with PSUT displaying slightly higher reducing power. In contrast, PSUM demonstrated superior in vivo bioactivity, with significant anti‐inflammatory, antioxidant, and gastroprotective effects, including reductions in oxidative stress markers and protection of the gastric mucosa. These results indicate that structural differences between the polysaccharides govern their distinct biological effects, emphasizing the importance of chemical characterization in predicting functional properties. Overall, this study provides solid preliminary evidence of the therapeutic potential of U. lactuca polysaccharides, particularly PSUM, and lays the foundation for future mechanistic studies, translational research, and sustainable exploitation of this marine resource for pharmacological applications.
Materials and methods
4
Collection and Identification of Macroalgal Samples
4.1
U. lactuca samples from Tunisia were collected from Sidi Mansour (Sfax, Tunisia) in December 2021. The species was authenticated by Professor Asma HAMZA. The voucher specimen (N° LCSN160) was deposited at the herbarium of the Laboratory of Organic Chemistry, Natural Substances team, Faculty of Sciences of Sfax. The Moroccan samples were collected from Sidi Rahal (Morocco) in December 2021 and authenticated by Dr Abderrahmane AMAIRI. The voucher specimen (LSEB150) was deposited at the herbarium of the Health and Environment Laboratory, Department of Biology, University Hassan II, Faculty of Sciences, Ain Choc, Casablanca, Morocco.
Fresh plants have been carefully removed from their epiphytes, washed with saltwater on the spot, and then packed in plastic bags. When they arrived at the laboratory, the seaweed samples were cleaned again with distilled water and dried in the dark. They were then milled into a fine, homogenous powder and kept at room temperature (25°C).
Extraction of Sulfated Polysaccharides
4.2
The sulfated polysaccharides extracted from U. lactuca (PSUT, SPUM) were made as described by Ananthi et al. [66]. Overnight at room temperature, the dry powdered plant was continuously stirred with acetone to depigment it. Ethanol under reflux was used twice for the second depigmentation. After refluxing the depigmented combination three times with hot water at 90 to 95°C for three to four hours, it was filtered and concentrated to ¼ of its initial volume. The resulting cooled mixture was incubated with ethanol for precipitation for a whole night at 4°C. The resulting precipitate was centrifuged for 15 min at 8000 rpm. After that, ethanol was used to wash it three times. The residue was lyophilized and dehydrated to get the final crude polysaccharide.
Analytical Methods
4.3
The total phenols content (TPC) and total flavonoids content (TFC) were established colorimetrically, as referenced in [67, 68, 69]. The results were expressed as mg of standard compounds (gallic acid equivalent (GAE) for TPC and quercetin equivalent (QE) for TFC) per g of dried extract (DE). Samples were examined in triplicate.
Spectroscopic Analysis
4.4
The Fourier transform infrared spectrophotometer (Spectrum Two Perkin Elmer System, USA) was used to identify the functional groups in the structure of the sulfated polysaccharides. The wavenumber range used to examine the sulfated polysaccharides was 4000–370.cm^−1^.
DSC Measurements
4.5
The phase transition was characterized using differential scanning calorimetry (DSC). The thermal analysis of sulfated polysacchrides was carried out using a Perkin‐Elmer DSC4000 with mechanical cooling system. Each sulfated polysaccharide sample (6 mg) was deposited in aluminum pans [70] and scanned from ‐50°C to 300°C with an empty aluminum pan as a reference.
Hydrolysis of the Polysaccharide
4.6
Hydrolysis of sulfated polysacchrides was made by a partially modified version of the method described by Boual et al. [71]. The polysaccharide sample (50 mg) was dissolved in 2 mL of 2 M trifluoroacetic acid (TFA) in a 10‐ml bulb and incubated at 100°C for 5 h. The hydrolysis tube was returned to room temperature. The reaction mixture was then neutralized to pH 7 by adding NaOH (1 M), and the sample was thoroughly mixed by vortexing and filtering.
Determination of Sugar Constituents via Gas Chromatography‐mass Spectrometry
4.7
The neutral monosaccharide composition, including the type and mole ratio of monosaccharide, was ascertained using GC‐MS (gas chromatography‐mass spectrometry), as previously reported. Using 50 µL per mg of dried hydrolysate, the resulting hydrolysate was lyophilized and subsequently silylated using a pyridine‐hexamethyldisilazane‐trimethylchlorosilane (9:3:1, v/v/v) mixture. A Varian Saturn 2000 ITD spectrometer and a fused silica capillary column (30 m × 0.25 mm) coated with DB‐225MS (Durabond) were employed in the Varian 3800 chromatograph to analyze the derivatives of the obtained trimethylsilyl polysaccharides. At 1 mL min^−1^, helium was utilized as the carrier gas.
Antioxidant Activity
4.8
Radical scavenging on DPPH; 2,2‐diphenyl‐1‐picrylhydrazyl [72], FRAP; ferric reducing antioxidant power [73], and TAC; total antioxidant capacity [74] tests, were carried out. All samples and the extracts' antioxidant activity were tested in triplicate. The total antioxidant capacity (TAC) of the samples was determined using the 50% inhibitory concentration (IC_50_) of the radical scavenging tests and expressed as mg gallic acid equivalents mg GAE/g of DE. The concentration of extract/ pure compound was 1 mg/mL. Vitamin C and Trolox were used as standards. The total radical scavenging activity of PSUT and PSUM was evaluated using the ABTS•+ (2,2‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonate)) assay, following a modified protocol based on the method described by Re et al. [75]. The ABTS radical cation was generated by mixing 7 mM ABTS with 2.45 mM K_2_S_2_O_8_ and incubating the solution in the dark for 16 h. Following this, the solution's absorbance was measured at 734 nm and then corrected to 0.700 using ethanol. 150 µL of ABTS^•+^ solution was mixed with 150 microliters of the sulfated polysaccharides (83.33–33.33 µg/mL). The polysaccharide‐free ABTS radical solution was used in the control experiment. A microtiter plate reader was used to measure the absorbance at 734 nm after 15 min.
In Vivo Biological Activities and Biochemical Analyses
4.9
The study was conducted using thirty adult male Wistar rats, two months old, with an average body weight of 181.9 ± 7 g, with each group consisting of 6 rats. The animals were housed under controlled environmental conditions, with ad libitum access to a standard diet and water. The rats were randomly assigned to five experimental groups. The ethanol‐induced gastric ulcer model was selected because it represents one of the most reliable, reproducible, and widely used experimental models for evaluating the gastroprotective activity of natural bioactive molecules. Marine sulfated polysaccharides including those extracted from green, red, and brown algae are well known for their antioxidant, anti‐inflammatory, cytoprotective, and mucin‐protective properties, which are directly relevant to ulcer prevention and healing. However, very few studies have investigated these effects specifically in polysaccharides extracted from U. lactuca. Therefore, this model was considered the most appropriate to explore the potential anti‐ulcer activity of PSUT and PSUM and to allow meaningful comparison with existing studies on marine polysaccharides. The dose of 200 mg/kg for PSUT and PSUM was chosen based on previously published in vivo studies evaluating marine algal polysaccharides with comparable structural features, in which similar concentrations demonstrated both efficacy and safety. The reference anti‐ulcer drug, omeprazole, was administered at the widely established therapeutic dose of 20 mg/kg. All dose selections were based on validated protocols and supported by the scientific literature.
Group 1 served as the healthy control (C), while Group 2 included rats in which gastric ulcers were induced via oral administration of absolute ethanol at a dose of 5 mL/kg body weight (U). Groups 3 and 4 consisted of ulcerated rats treated daily with algae extract PSUT and PSUM at a dose of 200 mg/kg body weight, respectively. Group 5 comprised ulcerated rats treated with omeprazole at a therapeutic dose of 20 mg/kg/day. At the end of the treatment period, the rats were sacrificed three hours after ethanol administration. Stomachs were immediately excised, gently rinsed with ice‐cold physiological saline, and examined for ulcerative lesions. The ulcerated surface area was assessed using an inverted microscope connected to a digital camera. Quantification was performed with ImageJ software, using calibrated measurements in millimeters. Gastric mucosal damage was evaluated following the methodology of Ofusori et al. [58]. The ulcer index (UI) and the curative index (CI) were calculated based on protocols previously described in our earlier work [76]. All experimental procedures complied with the ethical standards outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85‐23, revised 1996) and were approved by institutional ethical committees (approval numbers: CEEA‐ENMV 23/20 and CER‐SVS 0013/20222020‐0205).
Immediately after sacrifice, gastric juice was collected for biochemical analyses., three hours after ulcer induction. Following abdominal opening, the stomach was isolated, ligated at both ends, removed, and opened along the greater curvature. The gastric contents were carefully aspirated using a sterile pipette and transferred into clean tubes. To remove debris, the fluid was centrifuged at 3000 rpm for 10 min at 4 °C. The clear supernatant, representing the gastric juice, was used immediately for biochemical analyses or stored at −80 °C. The pH was measured directly using a digital pH meter (Amagase et al., 2001), and the total volume was recorded after centrifugation. The activity of H^+^/K^+^‐ATPase was assessed from gastric mucosa homogenates incubated with ATP in the presence of Mg^2^ ^+^ and K^+^, and inorganic phosphate (Pi) release was colorimetrically measured using the technique of Nagaya et al. [77]. Pepsin activity was established by hydrolysis of bovine albumin at acidic pH, followed by absorbance measurement at 280 nm, as described by Anson [78]. Lipoxygenase (LOX) activity was evaluated by monitoring the oxidation of linoleic acid at 234 nm, according to the method of Axelrod et al. [79]. Mucin content was assessed following trichloroacetic acid (TCA) precipitation and Alcian blue staining, with spectrophotometric reading at 620 nm according to Hall et al. [80]. Adherent gastric mucus was quantified following Corne et al.’s [81] method, by scraping the gastric mucosa, drying the mucus, and weighing it gravimetrically. Gastric juice MPO activity was determined spectrophotometrically at 460 nm using o‐dianisidinedihydrochloride and 0.005% hydrogen peroxide. One unit of MPO activity was described as that degrading 1 µmol peroxide/min/25 °C [82]. The degree of lipid peroxidation in the pancreas of the control and treatment groups of animals was quantified by measuring TBARS using the technique developed by Buege and Aust [83]. Superoxide dismutase activity was determined using the spectrophotometric approach described by Marklund & Marklund [84]. GPX activity was measured using the approach developed by Pagila and Valentine [85]. Catalase (CAT) activity was measured colorimetrically at 240 nm and expressed as moles of H_2_O_2_ consumed per minute per milligram of protein, following Aebi description [86]. Protein content was calculated using the technique outlined by Lowry et al. [87]. The gastric H_2_O_2_ level was measured using the method of Dingeon et al. [88] where hydrogen peroxide combines with 4‐aminoantipyrine and p‐hydroxybenzoic acid to produce quinoneimine, which is detectable at 505 nm.
Statistical Analysis
5
All extraction, phytochemical, and antioxidant procedures were performed in triplicate. Results are expressed as means ± standard deviation. Statistical analysis was conducted using one‐way ANOVA at a significance level of p ≤ 0.05, followed by Tukey's multiple range test with GraphPad Prism version 9.1.0 (221). For the in vivo study, each group included eight animals.
ANOVA and Fisher's test were used to identify significant differences using StatView 5.0 software (SAS Institute Inc., Cary, NC, USA).
Data are expressed as mean ± standard error of the mean (SEM). Statistical differences between groups were assessed using one‐way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons. For comparisons between two groups, an unpaired t‐test was used. Differences were considered statistically significant at p < 0.05. Statistical significance are as follows: *p < 0.05 versus normal rats; @p < 0.05 versus untreated gastric ulcer (GU) rats; ≠p < 0.05 versus GU rats treated with PSUT; &p < 0.05 versus GU rats treated with PSUM. All statistical analyses were performed using StatView 5.0 software (SAS Institute Inc., Cary, NC, USA).
Author Contributions
Nourhene kharrat: conceptualization, methodology, formal analysis, software, writing – original draft, writing – review and editing, resources, visualization. Yasmine TOUHAMIA: formal analysis, resources, methodology. Rihab ben abdallah kolsi: formal analysis, methodology. Khaled Hamden: analysing the in vivo biological activity, methodology, writing, review and editing. Rafik Ben Said: sampling methodology, sites and species identification, visualization. Touria OULD BEL LAHCEN: Conceptualization, Methodology, Writing – review and edit, Supervision, Project administration, Validation. Hichem BEN SALAH: Conceptualization, Methodology, Writing – review and edit, Supervision, Project administration, Validation.Noureddine ALLOUCHE: Conceptualization, Methodology, Software, Writing – original draft, Writing – review and edit, Supervision, Validation, Visualization.
Conflicts of Interest
The authors declare no conflicts of interest.
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