Mitigation of Hepatic Impairment with Polysaccharides from Red Alga Albidum corallinum Supplementation through Promoting the Lipid Profile and Liver Homeostasis in Tebuconazole-Exposed Rats

1. Introduction

Increasing interest in healthy human food worldwide introduces a rising consumption of natural compounds [1], which are known for their various biological activities and have been applied for a long time in the pharmaceutical area. They are often characterized as multi-component and multi-functional to treat diseases, which might be more effective due to possible synergistic actions [2].

Marine algae have recently been considered a new source of bioactive compounds [3]. Polysaccharides are the most common macromolecules produced by algae [4,5]. Like polynucleotides and proteins, polysaccharides play key roles in life activities such as fertilization, signal recognition, cell communication and adhesion, blood clotting, pathogenesis prevention, and system development [6,7,8]. Recently, pharmacological reports have shown that polysaccharides isolated from algae have various pharmacological activities, including immunomodulation, antitumor, anti-inflammatory, and antioxidant capacities [7,8]. The biological abilities of polysaccharides are probably related to the presence and positions of sulfate groups. The macromolecules have different levels of spatial organization, including primary, secondary, tertiary, and quaternary structures. Their primary forms can affect the various properties of polysaccharides, like their water-solubility, gel viscosity, and biological functions [9]. Functional polysaccharides could prevent the oxidative stress provoked by reactive oxygen species (ROS).

Generally, the endogenous antioxidant system can scavenge ROS amounts generated by cellular metabolism [10]. However, excessive environmental stresses, like ultraviolet irradiation and toxic chemicals, such as pesticides, can cause abnormal ROS production, which leads to oxidative stress.

Tebuconazole (TEB) is an effective fungicide used for the control of mildew and rust on wheat, barley, rice, fruits, and vegetables [11]. In the long term, it was one kind of toxicant for marine organisms, as it might induce an adverse effect on the aquatic environment [12]. Animals were sensitive to the influence of TEB because they could uptake and retain xenobiotics from circumstances via active or passive processes. TEB provokes adrenal gland hypertrophy in chronic dog studies and mouse teratogenic effects [13,14]. Sancho et al. [15] showed that short-term exposure to TEB induced physiological impairment and endocrine reproduction perturbation in male zebrafish. TEB is also deemed to cause hepatic and reproductive damage by inducing lipid peroxidation, decreasing antioxidant enzyme activities, and releasing free radicals [12,15]. To date, no investigation has been carried out on the biological activities of polysaccharides obtained from Alsidium corallinum (A. corallinum). Only a few studies have been realized on phenolic compounds extracted from A. corallinum, a red alga from the Mediterranean Sea, demonstrating their interesting biological activities, including antioxidant and hepatoprotective properties [16].

The present study aims to investigate the protective effect of ACPs against TEB-induced hepatotoxicity in adult rats. Identification of ACPs in vitro is necessary to understand their structural and functional properties and antioxidant potential. Furthermore, the binding affinities and molecular interactions of ACPs with TyrRS from S. aureus (1JIJ), human peroxiredoxin (1HD2), and Acyl-CoA: cholesterol acyltransferase (ACAT, 1WL4) were assessed using computational modeling.

2. Results and Discussion

2.1. Extraction Yield and Physicochemical Analysis of ACPs

The yield and chemical analysis of polysaccharides isolated from the alga Alsidium corallinum are reported in .

The yield percent of ACPs (20.93%) is better than the yields previously reported for polysaccharides from several other algae, including Gelidium crinale (2.6%) [17], Sargassum swartzii (11%) [18], and Chaetomorpha linum (16%) [19]. The yield is similar to polysaccharides isolated from fenugreek [20].

Polysaccharides are polar macromolecules that are easy to dissolve in water because they can replace water–water interactions with water–solute interactions. Polysaccharide extraction yield is influenced by algal species, period of collection, and extraction parameters. Moreover, the pH presented a 6.2 ± 03. Ktari et al. [21] reported that the pH of fenugreek polysaccharides solution at 37 °C is 6.4. ACPs have relatively low levels of moisture (2.96 ± 0.09%) and ash (3.00 ± 0.08%).

The quantitative estimation of ACPs showed a significant contribution of carbohydrates and less uronic acid and proteins. Proteins are part of cell wall structure, and are associated closely with polysaccharides. It has been considered a potential contaminant of polysaccharides [22]. After depigmentation and extraction of ACPs, we tried to denature proteins and eliminate the majority of lipids, and that is why we have acquired relatively low levels of proteins (3%) compared to other work done on the other polysaccharides [8], which suggests the efficiency of the extraction method. Amounts of proteins depend mainly on the way of extraction and deproteination processes. Fleury and Lahaye [23] indicated that precipitation of proteins during extraction at 100 °C contributed probably to their indigestibility. The homogeneity of the polysaccharide was confirmed by elemental microanalyses, which showed a protein content of 3% of protein residues. The ACPs protein contents were similar to those from the endodermis of Shaddock [24]. Our results indicate that extracting and isolating ACPs was suitable for yielding a compound free of undesired molecules that could interfere in the subsequent experiments.

Moreover, the results presented in revealed that ACPs had relatively high carbohydrate levels (66.06%).

On the other hand, the uronic acid content of ACPs (11.03%) was similar to those obtained by polysaccharides extracted from Sargassum vulgare (brown alga) [25]. The marine origin, seasonal periods, conditions, and extraction method are determining factors for the variations in all these contents.

2.2. Spectroscopic Analysis of ACPs

2.3. In Vitro Biological Activities of ACPs

Antioxidant activities of ACPs have been determined by various methods such as DPPH radical scavenging, ABTS radical scavenging, ferric-reducing antioxidant power, and nitric oxide scavenging assays.

2.4. Phytotoxicity Essay of ACPs

As shown in Figure 6, ACPs phytotoxicity was determined in different concentrations (1, 0.5, 0.25, 0.125, 0.0625 mg/mL) against cress seeds. Our results have shown that seed germination percent ≥80% with the different concentrations of ACPs, which confirms that polysaccharides from Alsidium corallinum are not phytotoxic [42].

Figure 6. Effects of ACPs on the seed germination (%).

2.5. In Vivo Biological Activities of ACPs

3. Materials and Methods

3.1. Source of Alga-Derived Polysaccharides

The marine red algae Alsidium corallinum was collected in the month of March from the coastal area of Sidi Mansour, Sfax, Tunisia. The voucher specimen of this species was deposited and identified in the herbarium of the Biology Laboratory at the Faculty of Sciences of Sfax. Alga was rinsed with tap water, followed by deionized water. Next, it was dried and ground before starting the extraction.

3.2. Extraction of Sulfated Polysaccharides (ACPs)

ACPs were extracted according to Chen’s method [65]. Briefly, A. corallinum powder was pre-extracted with ethanol (90%) to remove pigments. The dry residue was extracted twice with deionized water at 70 °C and stirred over six hours. The extract was combined and filtered, and the filtrate evaporated under a vacuum. The concentrated liquid was precipitated with ethanol for 24 h at 4 °C and then centrifuged for 15 min. The obtained residue was re-dissolved in double distilled water. The water phase was dialyzed at 4 °C against distilled water for 48 h. The dialysate was concentrated through rotary evaporation to obtain ACPs. The latter were stored at −20 °C for additional use.

3.3. Extraction Yield and Physicochemical Analysis of ACPs

ACPs extraction yield was measured based on the wet weight of A. corallinum powder. The yield of ACPs was expressed as a percentage (%) of the mass (g) of polysaccharides against the mass (g) of A. corallinum powder.

pH of ACPs (1% solution at 25 °C) was measured using a pH meter with complete immerging of the glass electrode into the solution.

The moisture and ash contents were determined at 105 and 550 ◦C, according to the AOAC standard methods 930.15 and 942.05, respectively [66].

The protein content was estimated by Bradford’s method [67]. Absorbance was measured by a spectrophotometer (595 nm). Protein levels were determined from a standard curve plotted using bovine serum albumin as a reference protein. All tests were carried out in triplicate.

Uronic acid amounts were determined by Blumenkrantz and Asbœ-Hansen’s method [68]. The absorbance was determined with a spectrophotometer (520 nm). The uronic acid amount was determined using a standard curve of glucuronic acid.

Carbohydrate amounts were determined by the method of Masuko [69]. Carbohydrate contents were measured by spectrophotometer at 490 nm. The result was estimated using glucose as a reference sugar from a standard curve.

3.4. Spectroscopic Analysis

3.5. In Vitro Biological Activities of ACPs

3.6. ACPs Phytotoxicity Analysis

The phytotoxicity of the polysaccharides obtained from A. corallinum was evaluated by methods of Zucconi and Monaco [81] using the seed germination technique. Throughout the assay, control included only distilled water sterile. The length of seedling growths was measured as mm. The phytotoxicity of our polysaccharides was evaluated by percentage of germination index (GI %).

3.7. In Vivo Antioxidant Activities of ACPs

3.8. Computational Analysis and Interactions Assay

The four monosaccharides in Alsidium corallinum, which had been identified, were used in the computational study to decipher their molecular interactions and confirm their potential antioxidant, antimicrobial, and hypolipidemic effects. The chemical structure of these monosaccharides was collected from the Pubchem website. The 3D crystal structure of TyrRS from Staphylococcus aureus (1JIJ), human peroxiredoxin (1HD2), and Acyl-CoA: cholesterol acyltransferase (ACAT, 1WL4) were obtained from the RCSB PDB. The studied monosaccharides and three targeted receptors were prepared, processed for depreciation, and then saved in pdbqt format [61,63]. As previously reported, they have been subjected to a CHARMm force field after targeting the grid box by selecting some key residues within the pocket region [57,58,59,60]. The reasons behind choosing these receptors are the massive responsibility of hospital-acquired infections by S. aureus, the key role of reducing hydrogen peroxide and alkyl hydroperoxides using equivalents, and the key involvement in fatty acid metabolism.

3.9. Statistical Analyses

All experiences and statistical analyses were made in triplicate. All results are expressed as the mean standard deviation. Statistical analysis was performed with the SPSS 17.0 statistical package for Windows (SPSS, Inc., Chicago, IL, USA). A two-way ANOVA followed by Tukey’s post hoc test was performed to compare treatment and control groups. Statistical significance was set at p = 0.05.

4. Conclusions

The polysaccharides from A. corallinum exhibited protective effects against TEB-induced toxicity by reducing ROS production. Both finding scores and molecular interactions of A. corallinum polysaccharides may explain the experimental in vitro and in vivo findings, which may result in the antioxidant, antimicrobial, and hypolipidemic activities. Further studies are required to clarify the mechanism of action of algae polysaccharides on liver cells and to probe the clinical availability of these compounds in the form of algal foods, food supplements, and regulated therapeutics.

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