Gastroprotective effect of mucilage fraction from Solenostemma argel via cytoprotection and attenuation of oxidative stress, inflammation and apoptosis

Introduction Plant mucilage as a chemical class of polysaccharides has obtained abundant interest nowadays due to their numerous applications in pharmacy (1), as well in pharmaceutical and food industries (2). They could be used for treatment of musculoskeletal, urinary, respiratory, gastrointestinal, immunomodulation, and skin disorders through oral or topical forms (3). Mucilage compounds are also used in pharmaceutical industries as disintegrating, binding, gelling, and suspending agents or emulsion stabilizers (4). Solenostemma argel (Delile) Hayne (Asclepiadaceae) is an Egyptian wild perennial dessert shrub, used in traditional medicine as an antispasmodic, antiinflammatory, and anti-rheumatic herb. It showed also hypoglycemic, hypolipidemic and weight loss activities in http://www.herbmedpharmacol.com doi: 10.34172/jhp.2021.26


Introduction
Plant mucilage as a chemical class of polysaccharides has obtained abundant interest nowadays due to their numerous applications in pharmacy (1), as well in pharmaceutical and food industries (2). They could be used for treatment of musculoskeletal, urinary, respiratory, gastrointestinal, immunomodulation, and skin disorders through oral or topical forms (3). Mucilage compounds are also used in pharmaceutical industries as disintegrating, binding, gelling, and suspending agents or emulsion stabilizers (4).
Solenostemma argel (Delile) Hayne (Asclepiadaceae) is an Egyptian wild perennial dessert shrub, used in traditional medicine as an antispasmodic, antiinflammatory, and anti-rheumatic herb. It showed also hypoglycemic, hypolipidemic and weight loss activities in experimental animals (5).
Gastric ulcer is a very common public health condition worldwide (6,7). It is provoked by an imbalance between destructive and defensive factors in the stomach (8).
The Helicobacter pylori infection, psychological stress, unhealthy dietary habits, ethanol consumption and the prolonged use of non-steroidal anti-inflammatory drugs are some factors, which can prompt the progress of gastric ulcers (9,10). Natural products are becoming nowadays more attractive targets than the available synthetic drugs for ulcer treatment due to their availability, perceived effectiveness and lower cost with fewer side effects (11). In this study, we examined the gastroprotective effects of mucilage fraction from Argel (MFA) with the elucidation of the mechanism of action involved in these effects, to find more safe and effective therapy instead of synthetic ones.

Plant material
The aerial parts of S. argel (Delile) Hayne were collected from Aswan, Egypt in 2019 and dried in shade at room temperature. The plant was authenticated by Mrs Therese Labib, the Botanical specialist and consultant at Orman and Qubba Botanical Gardens. A voucher specimen (No. 30.11.2019) was placed in the Herbarium of the Department of Pharmacognosy, College of Pharmacy, Cairo University, Egypt.
Preparation of MFA The powdered plant material (500 g) was mixed with 3 L of hot distilled water using ultrasonic (20 minutes) and left overnight. Then, the mucilage was prepared using the method as described by Radwan et al (12) and kept in vacuum desiccator over anhydrous calcium chloride to give 10 g of dried MFA.
Chemical analysis of MFA Acid hydrolysis of MFA MFA was hydrolysed according to a method described by Njintang et al (13). Briefly, 10 mg of dried MFA was hydrolysed in 0.5 mL 2 M trifluoroacetic acid for 2 hours at 80°C. The acid hydrolysate was analysed by HPLC. Monosaccharides were expressed as g.100 g −1 carbohydrates.
HPLC condition for analysis of monosaccharaides Agilent1260 infinity HPLC Series (Agilent, USA), equipped with Quaternary pump, Phenomenex® Rezex RCM-Monosaccharide column (300 mm × 7.8 mm, 5 µm) run at 80°C and RI detector run at 40°C. The separation was achieved using isocratic elution by HPLC grade water with 0.6 mL/min as flow rate and 20 μL injected volume.
HPLC condition for analysis of uronic acids HPLC (Knauer, Germany) condition was as follows: UV detector set at 214 nm, Rezex @ column (300 mm × 7.8 mm,5 µm), 0.6 mL/min flow rate, and oven temperature kept constant at 65°C. The mobile phase was 0.005M H 2 S0 4 data integration by ClarityChrom software.
Biological assay Animals and chemicals Sprague-Dawley male rats of 140-150 g were provided by the Animal House of the National Research Centre (Cairo, Egypt), grouped and housed under temperature and lightcontrolled conditions. All adopted procedures were in accordance with the guidelines of the EU Directive 1986; 2010/63/EU for animal experiments and were approved by the Institutional Research Ethics Committee at the Faculty of Pharmacy, Cairo University, Egypt (MP-2575).
Gastric ulcer induction Animals were randomly assorted into five groups with each group containing eight rats. Group I (control group): normal rats which received saline vehicle (5 mL/kg). Group II: (ethanol group): received a single intragastric dose of absolute ethanol (5 mL/kg) to induce gastric ulcer (14). Group III: received a single intragastric dose of ethanol+ Antodine (20 mg/kg) (15). Group IV: received a single intragastric dose of ethanol+ MFA (100 mg/kg). Group V: received a single intragastric dose of ethanol+ MFA (200 mg/kg). The rats were given treatments and Antodine 1 hour before ulcer induction by oral gavage. The animals were euthanized under deep anesthesia. Following immediate laparotomy, stomachs were excised, opened along the greater curvature and rinsed with normal saline.
Gastric ulcer index (UI) and protection percentage An observer examined the gastric mucosal lesions expressed as ulcer index (UI) as previously described by Salama et al (16). The percentage of protection was calculated by the following formula: [(UI ethanol control-UI treated)/UI ethanol control] × 100.
Preparation of tissue homogenate The stomach was excised, washed with saline and placed in ice-cold phosphate buffer (pH 7.4) to prepare the 20% homogenate using a tissue homogenizer (MPW−120, Bit-Lab Medical instruments, Poland). The supernatant was collected and stored at −80°C and then used for estimation of biochemical parameters.
Histopathological examination For histopathological study, the stomachs were dissected out, washed with cold water and kept in 10% neutral formalin saline. The sections (5 µm thickness) were cut into glass slides and stained with hematoxylin and eosin (H&E) (17). The glandular stomach in each animal was then assessed microscopically as previously described by Li et al (18).

Immunohistochemistry
For immunostaining, 5 µm sections were mounted on adhesive slides, deparaffinized and rehydrated. The sections were then subjected to heat induced epitope retrieval step in microwave for 15-20 minutes. Afterward, tissue sections were incubated with 1: 200 diluted primary antibodies (Mouse monoclonal Anti-Bax and Anti-BCl2, Santa Cruz) for 12 hours at 4 º C in humid chamber. After washing, the sections were then incubated with HRP labeled goat anti-mouse secondary antibody (Abcam, Cambridge, UK) for 2 hours followed by detection step using DAB Substrate kit (Thermo Scientific, IL, USA). Control negative slides were obtained by deletion of the primary antibody. Positive expression was quantified as area percent using Cell Sens dimensions (Olympus software).

Statistical analysis
One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test using GraphPad Prism software, version 5 (Inc., San Diego, USA) was used at < 0.05 for significance of difference. Data were expressed as mean ± standard error (SE).

Yield of the mucilage and extraction
The yield was 2% w/w. Monosaccharide composition is presented in Table 1. Glucose, fucose and N-acetyl glucosamine were the main monosaccharides, where galactomannan, glucomannan, or glucosamine polysaccharides were present in high amounts. With relatively lower amounts of glucuronic and galacturonic acids ( Table 1).
Effects of MFA on ethanol-induced ulcer Oral administration of absolute ethanol produced multiple mucosal lesions in the rat stomach. Where, the pre-treatment with Antodine, MFA, 100 and 200 mg/kg decreased the ethanol-induced gastric mucosal injuries (Figure 1) evidenced by a decrease in the ulcer index in rats (3.1 ± 0.12; 3.83 ± 0.13 and 3.5 ± 0.19, respectively), compared to that of the ethanol control group (6.27 ± 0.34) at P < 0.05. Moreover, they also produced a significant increase in the percentage of the ulcer protection by 50.1%, 38.58% and 43.73%, respectively, for Antodine, MFA 100 and MFA 200 mg/kg treated groups, compared to ethanol control ( Table 2).
Effects of MFA on oxidative stress biomarkers and inflammatory biomarkers A reduction in stomach content of GSH and an elevation in stomach content of MDA were observed in ethanol group by 38% and 40%, respectively, compared to normal data. Although, treatment with MFA 100 and 200 mg/ kg increased stomach content of GSH by 13% and 16%, respectively and decreased stomach content of MDA by 16% and 19%, respectively, as compared to ulcer group ( Figure 2).
Administration of ethanol produced an elevation in stomach contents of TNF-α, IL-6 and MPO by 1.2 fold, 27 fold and 2.7 fold, respectively, as compared to normal control values, where administration of MFA at 100 and 200 mg/kg reduced stomach contents of TNF-α by 41% and 39%; IL-6 by 22% and 33% and MPO by 24% and 29%, respectively, as compared to ulcer group ( Figure 3).
Effect of MFA on cytoprotective and cell proliferative markers Ethanol injection led to an inhibition of PGE2 secretion, while it reversed by MFA, 200 mg/kg to the normal levels. It elevated PGE2 by 29% as compared to reference drug Antodine ( Figure 4).
Ethanol administration produced a reduction in stomach contents of HSP-70 and VEGF by 87% and 71%, respectively, as compared to normal group. Treatment with MFA, 200 mg/kg increased the stomach contents of HSP-70 by 4 folds, and VEGF by 2.2 folds compared to ethanol group. In addition, MFA had a higher potency than Antodine in elevating HSP-70 by 31% (Figure 4).
Histopathological examination Microscopic examination of the glandular stomach of rats from normal group revealed the normal structure of glandular mucosa with its normal simple columnar epithelial cover and gastric glands containing chief and parietal cells (Figure 5a and 5b). Exposure to ethanol   All the values are presented as means ± standard error of the means (SE) and n=8. GSH; glutathione, MDA; malondialdehyde, MFA, mucilage fraction of Argel. Statistical analysis was carried out by one-way ANOVA followed by HSD Tukey's multiple comparisons test. a Significantly different from normal control at P < 0.05. b Significantly different from ethanol at P < 0.05. c Significantly different from Antodine at P < 0.05. mucus cover (Figure 5l). The only remarkable finding was the congestion at the deep mucosa with mild perivascular oedema at the submucosa (Figure 5m). Histological scoring of the lesions encountered in the glandular stomach is summarized in Figure 6; generally, the group received ethanol exhibited significant increase ( < 0.05) in all of the estimated parameters compared to the other groups. Administration of MFA, (200 mg/ kg) resulted in the highest significant decrease ( < 0.05) in the total histological score compared with the ethanol  group. The effect of MFA, (100 mg/kg) showed significant ( < 0.05) protective action which was similar to Antodine.

Immunohistochemistry of Bax and BCl2
The immunostaining of Bax and quantification as area  percentage is illustrated in Figure 7. Compared to the normal control group, ethanol administration caused significant triggering ( < 0.05) in Bax expression within the glandular stomach. No significant difference ( < 0.05) was detected between group III and IV, while the administration of MFA (200 mg/kg), showed the highest reduction ( < 0.05) in Bax expression compared to the ethanol group. Figure 8 shows the immune expression of BCl2 with the highest value recorded in the normal control group comparing to the other groups. Ethanol caused a significant reduction ( < 0.05) of BCl2 positive cells within the glandular stomach compared to the normal control group. Among the tested agents, both doses of MFA succeeded to keep significantly higher ( < 0.05) values of BCl2 within the stomach.

Discussion
Mucilage as a chemical class of polysaccharide could be used for several therapeutic purposes through oral or topical forms (3). The mucilage fraction isolated from S. argel was investigated for its ulcer healing potential as hope to replace the synthetic drugs having a lot of side effects and poor healing properties.
A lot of studies have reported that alcohol causes severe gastric mucosal injury and it is commonly used model to assess the gastroprotective effect of new medications and herbal products (19). The results of our study demonstrated that the intragastric administration of absolute ethanol caused multiple gastric mucosal lesions by gross examination along with a significant increase (P<0.05) in gastric UI in ethanol group II compared to normal rats (group I). The administration of MFA successively ameliorated these lesions and increased the ulcer protection percentages by 38.5% and 43.7%, respectively, at doses of 100 and 200 mg/kg. Likewise, these data were in agreement with the histopathological study of gastric mucosa, as ethanol-administered group showed several mucosal histological derangements including submucosal edema, congested blood vessels, necrosis and loss of epithelial cells and marked infiltration with inflammatory cells. While MFA pretreatment showed gastric mucosa as normal rats where all lesions were healed.
Oxidative stress has been well related to the pathological progression of gastric ulcer, leading to gastric mucosal injury with cell apoptosis in late stages (20,21). Our results showed also that ethanol as an aggressive factor, caused a significant oxidative stress-related damage leading to increase ( < 0.05) in oxidative stress biomarkers and inflammatory cytokines compared to the normal control group. The ulcer induced group showed also a significant increase in MDA level in stomach tissue and decrease in reduced GSH level. Where, the pretreatment with MFA (100 mg/kg) decreased significantly the level of MDA only, while MFA (200 mg/kg) elevated the levels of GSH and returned MDA to its normal value. From these results we can conclude that the gastroprotective effect of MFA was, in part, due to its potent anti-oxidant activity.
There is a strong relationship between gastric ulcer damage and inflammation induced by ethanol (22). The inflammatory response after ethanol leads to initiation of macrophages to release pro-inflammatory mediators such as TNF-α, IL-6, and MPO with the accumulation of neutrophils, resulting in destruction of the mucosal barrier (23). These cytokines in sequence cause the release of ROS (oxygen-derived free radicals), in that way assisting the occurrence of gastric ulcers. This, in turn, resulted in high levels of TNF-α, IL-6, and MPO in the ethanol control group. In contrast, MFA pre-treatment reserved the elevation of these levels.
Prostaglandin E2 (PGE2) is a vital mediator in maintaining the integrity of gastric mucosal defence and gastric ulcer healing. It has an important role as a gastric mucus secretion regulator. PGE2 increases blood flow, mucus secretion and bicarbonate. It also sustains the cellular integrity in the mucosa (24,25). Therefore, the decreased PGs levels are important events in the etiology of mucosal ulceration. Our results revealed that the gastroprotective effect of MFA might be partially associated with the stimulations of gastric PGs releases.
HSP-70 is another factor, which can protect stomach mucosa present in mammalian cells to protect the mitochondria from the different injurious factors and inhibit the stress-induced apoptosis process (26). Therefore, the overexpression of HSP-70 protects against the gastric mucosal damage and also repairs the damaged cells helping in ulcer healing of gastric mucosa (27). Ethanol-induced gastric ulcers inhibited the HSP70 release, where MFA (200 mg/kg) and Antodine significantly elevated its levels and further proved the cytoprotection of them.
VEGF is another factor with an important role in gastric  ulcer healing through its effect on angiogenesis (28,29). Furthermore, excess acid secretion reduces the process of ulcer healing via altering angiogenesis and decreasing VEGF in ethanol-induced gastric lesions (30). Our data indicated that MFA protected gastric mucosa against ethanol-induced gastric lesion via pro-angiogenic actions owing to upregulation of VEGF expression in gastric fibroblasts leading to ulcer repair. We can conclude that the reduction of gastric ulcer may be contributed to the ability of MFA to enhance the PGE2 generation at ulcer margin which leads to stimulation of VEGF that activates migration and proliferation of cells at the edge of the ulcer and promotes the angiogenesis, formation of granulation tissue, and remodelling of connective tissues during the process of ulcer repair.
A lot of studies have reported that Bax and BCl2 are associated with apoptosis throughout mitochondrial impairment and have an important role in interruption of stomach mucosal integrity later to ethanol administration (30). Free radicals lead to activation of intrinsic pathway of apoptosis (Bax) in areas of ulcer resulting in cell apoptosis. Our study showed that MFA pre-treatment downregulated the immunohistochemical of Bax expression in the ulcer-induced group, and successfully inhibited the cell death of gastric mucosal cells, consequently provided an anti-apoptosis approach against gastric ulcer destruction. Finally, we can conclude that polysaccharides are very interesting therapy for treatment of a lot of diseases such as gastric ulcer.

Conclusion
The potential usage of MFA for ulcer healing was assessed in ethanol induced gastric ulcer, where MFA showed a promising activity through anti-oxidant, anti-inflammatory, proangiogenic, cytoprotective and antiapoptotic role. Thus, the usage of MFA is highly recommended therapy for gastric ulcer treatment.