In Vitro and In Vivo Antidiabetic, α-Glucosidase Inhibition and Antibacterial Activities of Three Brown Algae, Polycladia myrica, Padina antillarum, and Sargassum boveanum, and a Red Alga, Palisada perforata from the Persian Gulf

Background In recent decades, algae have attracted worldwide attention for their great biological activities, such as antidiabetic and antibacterial properties. Objectives We measured antibacterial and α-glucosidase inhibition potential of methanol and 80% methanol extracts of three brown algae species, Polycladia myrica, Padina antillarum, and Sargassum boveanum, and a red alga, Palisada perforata, from the Persian Gulf coasts. Methods Antibacterial activity of the algal extracts was assessed by broth dilution method against three gram-negative and -positive bacteria, including Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa; Staphylococcus epidermidis, Staphylococcus aureus, and Bacillus subtilis, respectively. Furthermore, the yeast’s α-glucosidase inhibition of the algal extracts was measured via colorimetric assay. In addition, we investigated the beneficial effect of 80% MeOH extract of S. boveanum on the blood glucose levels in streptozotocin-induced diabetic rats. Results The MeOH extract of S. boveanum was the best antibacterial extract with MIC = 2.5 mg/mL against all bacterial strains except for E. coli. The MeOH and 80% MeOH extracts of P. myrica and P. antillarum inhibited α-glucosidase at most with IC50 values of 12.70 ± 1.88 µg/mL and 13.06 ± 4.44 µg/mL, respectively. The oral gavage of S. boveanum extract in streptozotocin- (STZ-) induced diabetic rats resulted in decreasing their postprandial blood glucose levels. The algae and acarbose decreased blood glucose levels after sucrose administration in 60 minutes, compared to the non-drug-treated animals, with p values of 0.03 and 0.007, respectively. Conclusions Overall, due to the in vitro and in vivo antidiabetic potential of S. boveanum, we suggest the alga as a new source for the isolation and identification of potential antidiabetic and antibacterial compounds.

solvents (Appendix 2).For bioassays, the dried extracts of each sample was prepared by dissolving in the extracting solvents to achieve desired concentrations.

Thin layer chromatography (TLC) analytical conditions
We analysed the chemical constituents of the extracts using pre-coated TLC plates (silica gel 60 F254, 0.25 mm film thickness, Merck).To achieve ideal resolution and retention factor (R f ) various mobile phase composition were evaluated.The silica gel TLC analysis was performed using ethyl acetate: MeOH: formic acid (7:3.5:1) as the mobile phase followed by the spray with 0.5% thymol-sulfuric acid solution in 95 mL EtOH and heating at 100 ᵒ C which resulted in detection of pink-coloured which indicated the presence of glycosides (Appendix 3) (1).
Serial dilutions of the algal extracts or chloramphenicol as the positive control were dissolved in dimethyl sulfoxide (DMSO) in desired concentrations.Subsequently, 95 µL of the nutrient broth and 5 µL of the samples were added into a 96-well microplate and then mixed with 100 µL of bacterial suspension (OD = 0.1 at 600 nm) and incubated for 24 hours at 37 ºC.Then 0.5% INT solution in water was prepared and 10 µL of the solution was added to each well in microplate.Afterwards, the microplates were incubated for further 30 min, at the abovementioned condition.At the end, MICs of the extracts or antibacterial standard were defined as the lowest concentration that discoloured the purple solution of INT.The discolouration indicated the inhibition effects of the algal extracts on the tested bacterial strain.

α-Glucosidase inhibition assay
α-glucosidase enzyme (from Saccharomyces cerevisiae) inhibition activity was measured, using previously described method with minor modifications (3).Briefly, 5 μL of each MeOH and 80% MeOH extracts of the four seaweeds, were incubated in 96-well microplates followed by diluting with 90 μL of 0.1 mM potassium phosphate buffer (pH 6.8).After adding 20 μL of αglucosidase enzyme (0.25 U/mL) in phosphate buffer solution, the plates were incubated in the dark condition for 10 min at 37 ºC.Afterwards, 15 μL of 2.5 mM substrate (p-nitrophenyl-α-Dglucopyranoside) in buffer was added into the mixture and subsequently were stored at 37 ºC for further 30 min.Finally, 80 μl (0.2 M) Na 2 CO 3 was added into each well to quench the reaction and the absorbance reading (A) was measured at 405 nm using a microplate reader (Bio-Rad Model 680).The results were compared to a control solution, which involved 5μL buffer instead of the extract.In addition, a solution of acarbose in deionized water was used as the standard inhibitor.The percentage of α-glucosidase inhibitory activity was calculated as follows: Where A sample is the absorbance of the extract or positive standard and A control is the absorbance of the negative control.The IC 50 , was defined as the concentration (µg/mL) of the sample that inhibited 50% of the enzyme activity, which were evaluated using a serial dilution of the samples, from 8 to 120 µg/mL in MeOH and 80% MeOH, using Curve Expert 1.4 software (three replicates).

Kinetic of inhibition patterns on α-glucosidase
The kinetic of the enzyme was carried out using the extracts of S. boveanum against αglucosidase inhibition.Appendix 4 shows the Lineweaver-Burk plot of α-glucosidase inhibitory activity of the 80% MeOH extract of S. boveanum at 0, 1 and 2 mg/mL stock solution, with various substrate concentrations of PNPG (1-10 mM) (4,5).Statistical analysis results using one-way analysis of variance (ANOVA) followed by Tukey post-hoc test, showed that 1/V (∆OD/min) -1 for 1 and 2 mg/mL stock solution of the alga in 1/S = 1, 0.5 and 0.33 mM -1 , were significantly different (p value < 0.05) from that of the control group (no inhibitor).Data analysis revealed that, the increase in the extract's concentration has not affected the V max and remained at about 0.09 mM/min, while K m was increased significantly by 3.0, 5.4 and 24.9 mM, respectively (Appendix 4).These results indicated that the extract inhibits ⍺-glucosidase by competitive manner.

Experimental animals
We purchased about thirty healthy adult male Sprague-Dawley rats (180-220 g) from the Center of Comparative and Experimental Medicine, Shiraz University of Medical Sciences (Shiraz, Iran).The rats were fed on a standard laboratory diet and housed at room temperature for about 12 h light and dark cycles.The local Ethics Committee of Shiraz University of Medical Sciences approved the study protocols (No.IR.SUMS.REC.1399.279).

Induction of diabetes
One week after adaptation, diabetes was induced by a single intraperitoneal injection of 50 mg/Kg of streptozotocin (STZ; Sigma-Aldrich, UK).The rats were fasted for 10 h prior to the drug administration.STZ was dissolved in sodium citrate buffer solution (pH 4.5) and instantly injected to prevent degradation.A blood sample was collected from the tail vein, five days after STZ injection.Rats with blood glucose over 300 mg/dL were considered as diabetics (6).The Accu-Chek blood glucose test meter (Roche Diagnostic, Germany) was used for plasma glucose concentration checking.

Sucrose tolerance test
After an overnight fasting, diabetic animals were divided into three groups: 1. Diabetic controls, which received deionized water.2. Positive controls, diabetic rats which were treated with acarbose (30 mg/Kg) as a standard antidiabetic drug (7).3. Diabetic rats treated with 80% MeOH extract of S. boveanum (30 mg/Kg, the same dosage as acarbose, for the better comparison).Half an hour after the above-mentioned oral treatments, sucrose solution (2 g/Kg) was administrated orally.Plasma glucose levels were taken from the tail vein at 0, 30, 60, 90, 120 min after sucrose administration and compared among groups.

Statistical analysis
The data were displayed as mean ± standard error (SE).The statistical analysis was carried out using Excel 2016 software and inhibitory concentration (IC 50 ) values were calculated using CurveExpert 1.4 software.SPSS software (Ver.16) was another software which was used to evaluate the results by one-way analysis of variance (ANOVA) followed by post-hoc Dunnett's multiple range test.P-values less than 0.05 is considered significant.

Appendix 1 .
Species, voucher numbers, and collection details of all the investigated algae from the Persian Gulf