Gastric ulcer prevention, harmlessness and antioxidant activity of astaxanthin extracted from a new Algerian strain of Haematococcus pluvialis

Astaxanthin is a high-value carotenoid (3, 3’ dihydroxy-β carotene-4, 4’-dione) with multiple biological properties of interest. It is produced by a microalga, Heamatococcus pluvialis, in substantial amounts especially under stressful conditions such as nitrate starvation and high-light intensity. The present study investigated the gastric ulcer prevention, harmlessness, and antioxidant activity of dimethyl sulfoxide-extracted (DMSO) astaxanthin (DMSO-AE) of a newly isolated Haematococcus pluvialis Algerian strain. The experiment was carried out using the ethanol-induced gastric ulcer model in mice. Changes in behavior, physical appearance, convulsion, and death rate were regularly monitored during the first 3h and after the next 24h. Antioxidant activity of H. pluvialis DMSO-AE was evaluated with DPPH (2, 2’diphenylpicrylhydrazyl) method. Ethanol-induced gastric ulcer was significantly (P  0.05) reduced in mice treated with 250 and 500µg of H. pluvialis DMSO-AE/Kg BW, when compared to the negative and the positive control groups. Histopathological examination of stomach sagittal sections of H. pluvialis DMSO-AE pretreated mice did not show any modification of tissue architecture. There was no evidence of toxicity or changes in the behavior or the mortality rate of the mice at the administrated dose of 500 mg H. pluvialis DMSO-AE/Kg BW. The DPPH scavenging activity of H. pluvialis DMSO-AE used at a concentration of 200µg/mL, was about 89.97% with an IC50 value of 25.82µg/mL. These results highlighted the astaxanthin protective effects on ethanol-induced gastric ulcers and lipid peroxidation which open up the prospects for the use of this carotenoid in the food, pharmaceutical, and cosmetic industry.


Introduction
Gastric ulcer is a chronic disease affecting up to 10% of the world's population (Kuna et al., 2019).It is damage in the gastric or duodenal mucosa down to the submucosa (Karaoglan et al., 2018) and is characterized by different a stages of necrosis, neutrophil infiltration, blood flow reduction and inflammation (Da Silva et al., 2013).Its occurrence depends on exposure to several aggressive factors such as stress, non-steroidal anti-inflammatory drugs (NSAIDs) uptake, smoking, alcohol consumption, Helicobacter pylori infection, and high secretion of hydrochloric acid (Hudson and Christopher, 1993;De Souza et al., 2018;Kuna et al., 2019).This disease may be treated using several synthetic anti-ulcer drugs such as proton-pump inhibitors, H2-receptor antagonist, antacid, and prostaglandin (Khushtar et al., 2016).However, the wide use of these drugs is associated with a range of side effects namely bone fracture (Yang et al., 2018), stroke (Sherwood et al., 2015) and renal disorder, among others (Khushtar et al., 2016).Furthermore, over intake of these drugs may be related to the risk of developing ulcer cancer (Cheung et al., 2017).The above-mentioned limitations of use and side effects of conventional ulcer drugs have led to look for new alternative therapeutic substances from more effective and safer natural sources like plants and microorganisms (bacteria, fungi, microalgae) that can be used in ulcer gastric treatment (Lavinya et al., 2012).Astaxanthin, a xanthophyll carotenoid, is a very powerful antioxidant with a unique molecular structure with hydroxyl and keto groups on both ends (Guerin et al., 2003).Moreover, this carotenoid has several biological properties such as protection against cancer, inflammation, cardiovascular diseases, and diabetes (Yang et al., 2013;Ranga Rao et al., 2014).Likewise, epidemiological studies have shown that high consumption of astaxanthin has reduced gastric ulcer disease (Kamath et al., 2008).Synthetic astaxanthin is a petrochemical product that is much more widespread on the world market than natural astaxanthin which is extracted from salmon, trout, krill, yeast or even algae (Capelli et al., 2013;Ranga Rao et al., 2014).The green microalga H. pluvialis is believed to be the best source of natural astaxanthin, since it accumulates up to 1-5% of cell dry weight under unfavorable (stressful) growth conditions (He et al., 2007) such as salinity (Torres-Carvajal et al., 2017), high temperature (Giannelli et al., 2015), strong light and nutrient starvation like nitrogen or phosphorus (Nagaraj et al., 2012).Under these stressful conditions, H. pluvialis cells undergo a dormant stage and accumulate astaxanthin as a protective mechanism for their survival beyond 40 years without nitrogen or phosphorus (Capelli et al., 2013).Stronger antioxidant activity of natural astaxanthin compared to that of the petrochemical form has been previously reported by Capelli et al. (2013) who estimated it 50 and 20 times higher in singlet oxygen quenching and free radical elimination, respectively; while human benefits were not determined yet.The goal of this study is to investigate the in vitro antioxidant activity and in vivo gastroprotective effect of astaxanthin, produced under absolute nitrogen starvation and strong light by an Algerian H. pluvialis strain isolated from freshwater, on ethanol-induced gastric ulcer in mice.

Material and Methods
All reagents used in this study were obtained from Sigma-Aldrich (St Louis, Mo USA).

Microalga culture and astaxanthin determination
H. pluvialis was obtained from our laboratory collection of the previously isolated strain (Sadoud et al., 2019), and grown, first on Bold's Basal medium (BBM) containing 250 mg/L NaNO3 for 10 days under continuous illumination of 40 µmol photons m -2 S -1 at 25 ± 2°C and bicarbonate/carbonate mixture generated carbon dioxide atmosphere (1.5%: V/V) inside the flask.Cells were then transferred to fresh modified absolutely nitrogen-free BBM medium at final cell concentration of 10 5 cells/mL and exposed to continuous illumination of 200 µmol photons m -2 S -1 at 25 ± 2°C to induce astaxanthin accumulation as previously described by Sadoud et al. (2019).Extraction and determination of Astaxanthin were carried out using the slightly modified (Sadoud et al., 2019) method of Wan et al. (2014).Astaxanthin accumulated in the encysted cells was extracted using dimethyl sulfoxide (DMSO).To do so, 5 mL of culture of encysted cells were centrifuged (3000 g / 15 min).The issued pellet was treated with a 5% (w/v) methanolic hydroxide potassium solution at 65°C for 15 min, and then washed three times with 2 mL of distilled water.Carotenoids were extracted with 5 mL of preheated (55 °C) DMSO vigorously mixed for 10 min to break the cells and recover the maximum of cell pigments content.This procedure was repeated at least three times until the cell debris were almost colorless.Absorbance of the extract was measured at 490 nm, and astaxanthin concentration (AC) was calculated using the following formula (1) (Davies, 1976): AC (mg/mL) = 4.5 (A490).Va/Vb (1) Where: AC: Astaxanthin concentration, A490: Absorbance of the extract at 490 nm, Va: Volume of extract, Vb: Volume of the algal culture.

Experimental animals
Seven-week-old male Mus musculus mice weighing 25g to 30g were purchased from Pasteur Institute (Algiers, Algeria) and acclimatized for two weeks in plexiglass cages in an air-conditioned room at 23 ± 1°C and 55 ± 10% relative humidity and 12h light-dark cycle.Female mice were excluded from the experiment because of the possible interference of hormones and estrous cycle in data variability (Becker et al., 2005).They were fed ad libitum with a standard pellet diet (Animal Food, Bouzereah, Algiers, Algeria) and tap water.The experiment was conducted following the recommendations of the Algerian Ethics Committee for Research on Animals of Abdelhamid Ibn Badis University of Mostaganem, Algeria (AECRA/AIBUM).

Gastroprotective determination of H. pluvialis DMSO astaxanthin extract
Gastroprotective effect of the H. pluvialis DMSO astaxanthin extract (H.pluvialis DMSO-AE) was carried out as described by Kamath et al. (2008) with a slight modification.Animals were divided into 5 groups of 6 mice each and were fasted overnight (16h) before the experiment.The different groups of mice were all given the standard diet and tap water ad libitum.Group 1 (G1) received nothing else (sham control); whereas groups G2 and G3 orally received in addition either 1mL of 0.9% (P/V) saline solution (G2: negative control) or 30mg omeprazole/Kg BW (G3: positive control).The assay groups (G4 and G5) were orally given 250 and 500µg H. pluvialis DMSO-AE/Kg BW, respectively.Then 1 hour after, 1 mL of absolute ethanol (90%: V/V) was orally administered to all mice groups, except the sham control (G1).One hour after induction of gastric ulceration, all mice were anesthetized with chloroform and the abdomen was opened; the stomachs were rapidly removed, excised along the greater curvature, washed with saline solution (0.9% P/V), and stored at 4°C until analysis.

Macroscopic evaluation of stomach tissues
Cleaned stomachs were fixed on a glass slide and the mucosa was macroscopically examined for red coloration, spot ulcer, hemorrhagic lesions, deep ulcers, or perforation, and then stomach samples were photographed using a digital camera.The images were registered and the total ulcerative lesions in each stomach were measured using Image-J software (1.51 d).The ulcerated area rate in each stomach and the protection percentage against ulcer (I %) were calculated using the following formulas ( 2) and (3) (Salga et al., 2011) : Ulcerated Area Rate (%) = UAR % = (TLa / TSa).100 (2) Where: TLa : total lesions area, TSa : total stomach area Where: UARcontrol : Ulcerated Area Rate in a negative control group, UARassay : Ulcerated Area Rate in assay group

Microscopic evaluation of stomach tissues
Stomach tissues were fixed in formalin 10% (V/V) for 24h, embedded in paraffin, and sectioned into 5 µm thick slices that were stained with hematoxylin and eosin (H&E); then the gastric lesions were analyzed using an optical microscope (Chung et al., 2008).

Acute toxicity test
Acute toxicity test was carried out as described by OECD guidelines (Organization for Economic cooperation and Development no. 425) (2008) using 2 groups of five mice each: the assay group and the control group received a limit dose of 500 mg DMSO-AE of H. pluvialis/Kg BW and a saline solution, respectively.Changes in behavior (salivation and locomotor activity), physical appearance, convulsion, and death rate were regularly monitored during the first 3h and after the next 24h.

DPPH radical scavenging activity
The DPPH radical scavenging activity of H. pluvialis DMSO astaxanthin extract was determined as originally described by Blois (1958).1mL of different concentrations of the H. pluvialis 12.5,25,50,100,and 200

Results and Discussion
The results of the present study confirm the previously reported (Sadoud et al., 2019) accumulation of astaxanthin (about 67.25 ± 2.28 mg/L) in nitrogen-free medium and under strong light exposure.

Gastroprotective effect of H. pluvialis DMSO astaxanthin extract Macroscopic aspect of gastric mucosa
Figure 1 illustrates the gastric mucosa appearance of all the mice groups used in the present experiment.Gastric mucosa of ethanol given mice (G2: negative control), but not those treated with H. pluvialis DMSO-AE (G4 and G5) or opremazole (G3: positive control), was markedly damaged, and characterized by perforations, deep ulcerations, visible bleeding strips, and edema (Fig. 1B); while that of sham control (G1 mice group) did not show any damage (Fig. 1A).Omeprazole (30mg/Kg BW) (G3: positive control: Fig. 1C) or H. pluvialis DMSO-AE (250 and 500µg/Kg BW) (G4, Fig. 1D and G5, Fig. 1E, respectively) treatment of ethanol-induced gastric ulcer remarkably reduced damage of the gastric mucosa comparatively to the negative control group (G2).Sequelae of mild damage with slight interstitial hemorrhagic lesions of the gastric mucosa of these G4 and G5 mice groups were still observed after treatment (Fig. 1D and 1E).

Figure-1. Macroscopic aspects of mice gastric mucosa:
A: sham control (G1); B: untreated ethanol-induced gastric ulcer (G2: negative control); C: omeprazole (30mg/Kg BW) treated ethanol-induced gastric ulcer (G3: positive control); D and E: ethanol-induced gastric ulcer treatment with doses of 250 (assay group G4) and 500µg H. pluvialis DMSO-AE/Kg BW (assay group G5).This is in line with the results of Kamath et al. (2008) who reported this same dose-dependent effect of H. pluvialis extract on ethanol-induced gastric lesions in rat.These authors attributed this effect to an inhibition of proton-potassium (H+, K+-ATPase) pump, a regulation of mucin content, and an increase of antioxidant status.
H. pluvialis DMSO-AE effect is likely dose-dependent since its increase provides better protection against an ethanol-induced gastric ulcer in mice compared to the reference drug (omeprazole).

H. pluvialis DMSO-AE protection rate against gastric ulcer
The protection rate of H. pluvialis DMSO-AE against ethanol-induced gastric ulcer is about 27.58 ± 2.5 and 71.59 ± 2.19% when used at a concentration of 250 and 500µg/Kg BW, respectively; whereas that of omeprazole was 60.65 ± 4.15 (Fig. 2).

Histopathological examination
Gastric mucosa histopathological examination of sham control mice group (G1) showed intact wellorganized cell structure (figure 3 A and B); whereas that of negative control one (G2: untreated ethanolinduced gastric ulcer) was markedly damaged showing deep tissue changes with necrosis, hemorrhage, neutrophil infiltration in submucosa, inflammation, edema, and loss of the epithelial cells continuity (figure 3 C, D, and E).Several studies have shown involvement of ethanol in gastric mucosal damage characterized by gastric injuries via dehydration which disrupts mucosal cell barriers and cytotoxicity (Kim et al., 2005a).Therefore, ethanol causes depletion of the gastric wall mucosal barrier via Reactive Oxygen Species (ROS) and cytokine upward regulation, which promotes oxidative stress-induced damage in gastric tissue, inflammatory reactions, and epithelial cell apoptosis.Thus, ethanol is actually one of the most frequent causes of gastric ulceration (Chen et al., 2019;Liu et al., 2020).Ethanol induces lesions and injuries to the gastric mucosa and causes a reduction in bicarbonate secretion and inflow of gastric blood with depletion of gastric wall mucus and an increase in the release of histamine, the influx of calcium and sodium ions, generation of reactive oxygen species (ROS) and leukotrienes, resulting in severe gastric damage (Balan et al., 2015).Histopathological examination of stomach sections of mice given absolute ethanol and pretreated with 30 mg omeprazole/Kg BW (G3: positive control mice group) has shown an important reduction in gastric injury and erosion as compared with the negative control mice group (G2) (fig.4 A and B).These findings are in agreement with those of Raish et al. (2021) who observed that omeprazole pretreatment of ethanolinduced gastric ulcer in rat led to a reduction in lesions in the gastric wall mucosa, a decrease in the gastric volume and free acidity, and an increase in the gastric pH and mucosa levels.Thus, Omeprazole, a proton pump inhibitor, ensures good protection of gastric mucosa and is an effective drug widely used in the treatment of gastric disorders related to gastric acid secretion (Liu et al., 2020).A significant (P<0.05)reduction in ethanol-induced gastric lesions was observed in H. pluvialis DMSO-AE pretreated mice groups (G4 and G5 groups).Microscopic examination of the gastric mucosa of mice pretreated with 250µg of H. pluvialis DMSO-AE/kg BW (G4) has shown minor damage.Gastric epithelium surface is protected from ethanol erosion and necrotic lesions; the leukocytes infiltration is reduced compared to that of the negative control group (G2) and a moderate inflammation of the submucosa was observed in the G4 group (Fig. 5 A, B, and C).

Figure-5. Histopathological examination of sectioned stomachs of ethanol-induced gastric ulcer mice pretreated with 250 (A, B, C) (G4 group) or 500 µg H. pluvialis DMSO-AE/kg BW (D, E, F) (G5 group) (G10x100).
However, the histopathological aspect of the gastric mucosa of 500 µg H. pluvialis DMSO-AE /kg BW pretreated mice group (G5) has shown intact gastric mucosa and absence of gastric ulcer indicator signs such as leucocytes infiltration, inflammation, edema formation, erosion or epithelium destruction.The preservation of the continued formation of the gastric epithelium after pretreatment of mice with 500µg of H. pluvialis DMSO-AE/kg BW shows the efficiency of this dose to ensure excellent protection of the integrity of gastric mucosa against ethanol harmfulness (Fig. 5 D, E, and F).Similar observations have been reported by Murata et al. (2012) and Kim et al. (2005a, b) who have shown that treatment of mice and rats with astaxanthin significantly (P<0.05)reduced gastric lesions induced by ethanol, hydrochloric acid secretion or drug consumption.The ethanol-induced gastric ulcer can be promoted by free radicals production and lipid peroxidation which unbalances the ratio between cellular antioxidant and oxidant capacity, thereby increasing oxidative stress (Kim et al., 2005b).The preventive effect of astaxanthin on ulcer gastric damage is related to its high antioxidant potential in removing free radicals species involved in gastric lesions (Kamath et al., 2008).Kim et al. (2005a) have shown that the gastric mucosa of Xanthophylomyces dendrorhousis astaxanthin orally given rats was significantly protected from ethanol-induced damage and in turn from lipid peroxidation.
The histopathological examination has shown that in the present study, gastric lesions disappeared after H. pluvialis DMSO-AE treatment.Kamath et al. (2008) study on astaxanthin has shown that this carotenoid exhibits a dose-dependent protective effect on ethanol-induced gastric ulcer which might be due to an increase in the levels of the antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase in stomach homogenate.Furthermore, astaxanthin ensures a gastroprotective effect by regulating gastric acid secretion (Kim et al., 2005a, b;Kamath et al., 2008).Astaxanthin provides a protective effect against gastric lesions induced by non-steroidal antiinflammatory drugs such as indomethacin and naproxen, suggesting that this carotenoid may represent an alternative strategy for gastric ulcer treatment (Kim et al., 2005a).

Acute toxicity
The harmlessness of 500mg/Kg H. pluvialis DMSO-AE was evidenced by the absence of clinical toxicity signs during the whole experiment period for all mice.This is in agreement with observations made by Regnier et al. (2015) on H. pluvialis astaxanthin harmlessness.These authors have reported no changes either in the morphology or in the viability of human vascular cells when cultured in presence of up to 10 µM astaxanthin concentrations.

Antioxidant activity of the H. pluvialis DMSO astaxanthin extract DPPH assay
Radical scavenging activity of H. pluvialis DMSO-AE is dose-dependent in that it reaches respective values of 49.35±1.45,64.35±4.98,78.61±2.23%,and 89.07±2.23%for extract concentrations of 25, 50, 100 and 200µg/mL, respectively.The concentration of H. pluvialis DMSO-AE extract required to scavenge 50% of the initial DPPH radicals (IC50) was calculated in order to be compared to that of the controls (BHT and ascorbic acid).Obtained results have shown that H. pluvialis DMSO-AE IC50 (24.56 μg/mL) is significantly (P<0.01)lower than that of BHT (141.61μg/mL) and ascorbic acid (68.82 μg/mL), reflecting the high anti-free radical capacity of the algal extract (Fig. 6).2016) who reported the related high H. pluvialis extract DPPH scavenging capacity to the richness of this microalga in hydrogen donor molecules which can convert free radicals to stable products and terminate the radical chain reaction.Astaxanthin is the carotenoïd that represents such molecules (Shimada et al., 1992) and its production by H. pluvialis is increased by the applied unfavorable environmental conditions herein such as nitrate starvation and strong light exposure.Ranga Rao (2011) and Zhao et al. (2016) reported similar results that they attributed the same interpretation; while Karppi et al. (2007) also recorded the high scavenging capacity of astaxanthin that Dose et al. (2016) attribute to the presence of carbonyl and hydroxyl functional groups.Astaxanthin is also known as an antioxidant whose potency exceeds that of α-tocopherol and other carotenoids such as lutein, β-carotene or lycopene (Yang et al., 2013;Focsan et al., 2017).This suggests that the antioxidant potential of astaxanthin may be one of the mechanisms responsible of its gastro-protective activity, since ulcer gastric process is related to oxidative stress (Kim et al., 2005a).

Conclusion
This investigation evidenced that a new Algerian strain of Haematococcus pluvialis DMSO-astaxanthin extract is a powerful gastroprotective extract whose harmlessness at a dose of 500mg/Kg BW has been herein demonstrated in mice.By the way, it has been also shown that this algal extract has a dose-dependent antioxidant activity.These biological properties are likely related to the astaxanthin content of the algal extract which depends on the unfavorable growth (absolute nitrate starvation and strong light exposure) conditions of H. pluvialis.These findings suggest that H. pluvialis is an Algerian endemic alga that is easy to grow, and whose exploitation for the astaxanthin production could be of interest.

Figure- 6 .
Figure-6.IC50 values (µg/mL) of BHT, ascorbic acid and H. pluvialis DMSO astaxanthin extract Values are the mean ± SD (n = 3).*** (P<0.001)difference H. pluvialis extract Vs BHT, # (P<0.05)difference H. pluvialis extract Vs ascorbic acid This is in line with the results of Zhao et al. (2016) who reported the related high H. pluvialis extract DPPH scavenging capacity to the richness of this microalga in hydrogen donor molecules which can convert free radicals to stable products and terminate the radical chain reaction.Astaxanthin is the carotenoïd that represents such molecules(Shimada et al., 1992) and its production by H. pluvialis is increased by the applied unfavorable environmental conditions herein such as nitrate starvation and strong light exposure.RangaRao (2011) andZhao et al. (2016) reported similar results that they attributed the same interpretation; whileKarppi et al. (2007) also