In vitro α-amylase inhibitory assay
One therapeutic approach for treating Type 2 diabetes is to decrease postprandial hyperglycemia . The ability of a drug or diet to retard the production or absorption of glucose by inhibiting carbohydrate hydrolyzing enzymes such as α-amylase is one of the therapeutic approaches for decreasing postprandial hyperglycemia.
The bioactivity of HSSP was tested in vitro using α-amylase inhibitory assay. As shown in Table 1,HSSP showed strong α-amylase inhibitory activity under in vitro conditions. A concentration-dependent inhibitory activity against α-amylase was observed for the HSSP used at doses of 50, 100 and 200 mg/ml with IC50 value of 47.58 mg/ml as compared to glucor with IC50=17.26 mg/ml.
Postprandial hyperglycemia plays an important role in the development of type 2 diabetes mellitus and complications associated with the disease such as micro- and macro-vascular diseases has been proposed as independent risk factor . Therefore, control of postprandial hyperglycemia is suggested to be important in the treatment of diabetes and prevention of cardiovascular complications . Inhibiting glucose uptake in the intestines may help diabetic patients to control the blood glucose level in the postprandial state hence substances that inhibit amylase have been studied and some have been developed as drugs to treat diabetes mellitus .
Therefore, our extract can be considered a new natural source possessing properties for the fight against type 2 diabetes. To show equal preference for α-amylase enzyme it is always necessary to do the corresponding in vivo activity. Thus proof of concept needs to be demonstrated in preclinical animal studies and it was essential to confirm the in vivo experiments action following oral administration to live animals.
Effect of HSSP on pancreas β-cells architecture and α-amylase activity in serum, pancreas, intestine and blood glucose level
The pancreas is an important organ for blood sugar regulation. Research has shown that insulin resistance is closely associated with an impaired pancreatic β-cells function . As reported in Fig. 1, administration of alloxan to rats provoked a massive damage and death of pancreas β cells. Consequently, a significant increase in the α-amylase activity by 57.26%, 114.15%, and 132.1% in the serum, mucosal small intestine and the pancreas, were observed respectively in the untreated diabetic rats, which leads to an increase in the glucose rate by 240.17% as compared to untreated diabetic rats (Fig. 2). However, the administration of HSSP corrects partially the damage in the β cells, which leads to a decrease in α-amylase activity by 38.79%, 45.43% and 58.07% in the serum, small intestine and pancreas, respectively. Consequently reduction of blood glucose rate by 67.97%.
Alloxan produces free radicals in the body in which it causes pancreatic damage by preventing insulin secretion which induces a significant increase in serum glucose concentration of rats. This hyperglycemia is due to disorders in the use of glucose by tissues or to the increase of gluconeogenesis.
These results performed by OGTT in conscious fasted rats after HSSP administration. In fact, these results clearly showed that acute oral administration of HSSP to surviving diabetic rats reduced significantly the peak of blood glucose concentration 60 min after glucose administration as compared to untreated diabetic rats (Fig. 3).
This which clearly shows a very active use of glucose by peripheral tissues, explained by an increase in glucose tolerance in these tissues when animals are treated with some marine algae.
Effect of HSSP on liver function and antioxidant capacity on diabetic rats
This study showed that hyperglycemia provoked liver toxicity and stress; evidenced by a significant decrease in the SOD, CAT and GPX activities in liver tissues of diabetic rats (Fig. 4). Moreover, the liver toxicity and dysfunction were showed by the increase in blood liver indices such as AST, ALT and LDH in diabetic rats and confirmed by the apparition of fatty cysts in the hepatic tissues (Fig. 5).
In diabetic rats treated by HSSP, a significant protective action was showed. In fact, the administration of HSSP increases the antioxidant capacity in the liver tissues (increase in SOD, CAT and GPX activities by 240.25%, 299.42% and 120%, respectively and decrease in TBARS rate by 46.39% as compared to untreated diabetic rats). This increase in the antioxidant capacity prevents liver toxicity showed the decrease in the activity of AST, ALT and LDH in the blood and was confirmed by histological observation (Fig. 5).
This antihyperglycemic action is due to sulfated polysaccharides isolated from marine algae that are known to be important antioxidants for the prevention of pancreatic oxidative damage, which is an important contributor in diabetes mellitus .
It is believed that oxidative stress plays an important role in chronic complications of diabetes. Therefore, alleviation of oxidative stress is essential for preventing or reversing diabetic complications  and by a compound with antioxidant activity combined with inhibitory activities against α-glucosidase and α-amylase should be a more effective anti-diabetic agent.
In this study, indeed to the hypoglycemic action, HSSP stimulates the activity of CAT, SOD, and GPx, reduces the lipid peroxidation and suggests a compensatory response to oxidative stress as it reduces the endogenous H2O2 production thus diminishing the toxic effects due to this radical or other free radicals derived from secondary reactions [4-21]. This antioxidant action of HSSP on diabetic rats can prevents liver toxicity and damage showed by the decrease in the liver cells indices toxicity such as AST, ALT and LDH and also prevents the accumulation of lipid in liver tissues, evidenced by histological analysis.
Effect of HSSP on kidney function on diabetic rats
Results of this study revealed that hyperglycemia induced kidney toxicity, evidenced by the increase in albumin, creatinine and urea in blood by 19.94%, 35.36% and 111.57%, respectively as compared to untreated diabetic rats (Table 2). This can be explained by the accelerated degradation of hepatic and plasma proteins  or the degradation of somebody protein compounds due to the administration of alloxan or dietary compounds that can be degraded into amino acids and then into urea.
Our study showed that chronic hyperglycemia induces renal toxicity. The concentration of albumin, urea and creatinine is often considered as a clinical parameter to detect the toxic effects related to the treatment of some compounds on the kidneys in experimental animals . Also hyperglycemia induced stress oxidant and kidney cells damage showed by a decrease in the SOD, CAT and GPX activities by 43.65%, 44.51% and 90.62%, respectively and an increase in TBARS rate by 108.54% (Fig. 6). This toxic effect of diabetes on kidney was confirmed by histological analysis (a capsular space shrinkage and glomerular hypertrophy). However, the administration of HSSP normalized all these perturbations (Fig. 7).
Effect of HSSP on lipid profile on diabetic rats
Results of this study revealed that hyperglycemia associated with increase in the blood TC, TG, LDL-C and decreased in HDL-C concentration was in accordance with previous study . However, treatment with HSSP normalized all the lipid profile parameters.
The data in the table 2 showed that the supplement of HSSP normalizes lipid profile. In fact, the administration to HSSP to surviving diabetic rats decreases LDL-C and TG by 33% and 26%, respectively and increases HDL-C by 45% as compared to untreated diabetic rats.
The HSSP leads to a significant improvement in the lipid profile similar to that observed in diabetic rats treated with glucor. The increase in the level of HDL-C known as good cholesterol  is due mainly to their beneficial effect on cardiovascular complications, mainly atherosclerosis . This antihyperlipidemic activity attributed to sulfated polysaccharides, major constituent of extract as sulfated polysaccharides enhance the negative charges of cell surface so as to affect the aggradation of cholesterol in blood, thus decreasing the cholesterol in serum .
FT-IR spectrometric analysis
The characteristic absorption of HSSP was identified by the FT-IR spectra (Fig. 8). FTIR spectrum elucidates the structural information of polymers and determines their major functional groups. The infrared spectrum of HSSP displayed a broad stretching intense characteristic peak at 3315 cm-1 for the hydroxyl group . A weak band at 2967 cm-1 was attributed to the C-H stretching and bending vibration. The absorption band centered at 1725- 1641 cm-1 were caused by C=O asymmetric stretching vibration. The absorption at 1446 cm−1 was assigned to C–OH deformation vibration. The well defined peak at 1278 cm−1 attributed to the presence of sulfate ester groups (S=O)  and the sharp band at 753 cm−1 (C-S-O) suggested that the majority of sulphate groups occupy position 2 and/ or 3 (equatorial position) . Each particular polysaccharide has a specific band in the 1193- 1137 cm−1 region; this region was dominated by ring vibrations overlapped with stretching vibration of pyranosyl ring (C-O). A band at 1085 cm−1 of HSSP was found and corresponded to C-O-H deformation vibration. The relatively strong absorption peak at 924 cm−1 of HSSP reflected the absorption of the furan ring.
The X-ray diffraction determination
The X-ray diffraction was used to determine the structure and degree of crystallinity of the
HSSP. The diffracted intensities were recorded from 10° to 80° at 2 theta angles (Fig. 9). The results of X-ray diffractograms of HSSP suggest that HSSP was a semi-crystalline polymer with major crystalline reflection at 31.3°. Similar results were obtained by Maud Lemoine and William Helber  which reported that the sulfated polysaccharide was extracted from red algal cell wall as a dense network of semi-crystalline fibers. This structural arrangement is known to directly affect various properties, including tensile strength, flexibility, solubility, swelling or opaqueness of the bulk polymer. In fact, physical properties are dependent on the degree of order within the material .
Monosaccharide composition of HSSP
Sugar composition of HSSP has been carried out preliminarily by TLC analysis (Fig. 10). Indeed, after acid hydrolysis of HSSP, the retention time of acid-hydrolyzed HSSP sample was exactly the same as the monosaccharides such as glucose, arabinose, xylose, and galactose in the TLC analysis. A total hydrolysis of HSSP liberated glucose, arabinose, xylose, and galactose as a final hydrolysis product since the Rf value of acid-hydrolyzed HSSP was identical to that of glucose, arabinose, xylose, and galactose under our solvent ascending condition. To confirm the results obtained by CCM we applied a more efficient analytical technique to the study of the chemical composition, GC-MS. As a result, the chromatogram obtained makes it possible to determine the monosaccharide composition of the HSSP by reductive hydrolysis method (GC-MS) (Fig. 11).
The GC–MS analysis of the crude polysaccharide fractions revealed the presence of different carbohydrate moieties in varied proportions (Table 3). The acid hydrolysis of HSSP showed that the ribose was the most abundant sugar (14.76%), followed by mannose, lyxose, glucose, xylose, talose, galactopyranose and arabinose (11.12%, 8.88%, 8.64%, 7.68%, 6.84%, 6.08% and 5.91% respectively). It has been reported that glucose, xylose, arabinose, galactose and mannose were associated with antioxidant activities . In this study, HSSP was assumed to be responsible for the antioxidant activity; hence it was used for further experiments. In addition, the monosaccharides composition of sulfated polysaccharide isolated from brown seaweed Lobophora variegata showed that it is a polysaccharide composed of galactose, fructose, and xylose shows 36.8%, 29.2% and 0.1%, respectively . Another study had shown a sulfated galactofucan with high-level galactose (22%) and fructose of brown seaweed Adenocys tisutricularis was obtained . However, galactofucans often contain xylose in the monosaccharide composition from minor to significant. Cole et al.  describe that sulfate groups of polysaccharides from brown seaweed have action on several signaling events because its S-domains can interact with a number of growth factors, chemokines and their receptors.
These results confirm the biological activities of sulfated polysaccharide that would be related to the characterization of monosaccharides such as mannose, lyxose, glucose, xylose, talose, galactopyranose and arabinose contents in our HSSP sample.