Inhibitory effects of a sulfated polysaccharide isolated from edible red alga Bangia fusco-purpurea on α-amylase and α-glucosidase

Figures & data

Figure 1. Purification and characterization of BFP.

(a) Elution profile of polysaccharide fraction extracted from B. fusco-purpurea on a Sephadex G75 column. (b) TLC pattern of BFP developed with n-butanol/glacial acetic acid/water, 2: 2: 1, v/v/v. The first line (a line) is the solvent front, and the second line (b line) is the origin. (c) Profile of BFP in HPLC on a TSK-gel G4000 PW_XL column (7.8 mm I.D. × 300 mm) eluted with ultra-pure water (pH = 7.0) at a flow rate of 1.0 mL/min. (d) FT-IR spectrum of BFP.

Table 1. Chemical compositions and molecular mass of polysaccharide isolated from B. fusco-purpurea.

	Neutral monosaccharides (%)^a(mol of D-glucose was considered as 1.00)
Sample	Gal	Man	Glc	Fuc	Xyl	Ara	3,6-AnGal (%)^b(Molar ratio)	UA (%)^c(Molar ratio)	SO4^2- (%)^d(Molar ratio)	MM (kDa)^e
BFP	66.55 (11.23)	6.04 (1.02)	5.92 (1.00)	1.82 (0.34)	1.71 (0.35)	1.27 (0.18)	2.81 (0.43)	7.27 (1.14)	10.34 (0.93)	133.18

^aDetermined by HPLC analysis after hydrolysis with 2 M of trifluoroacetic acid.

^bDetermined by the resorcinol method.

^cDetermined by carbazole-sulfuric acid assay as glucuronic acid equivalent (UA, uronic acids).

^dDetermined by turbidity assay using K₂SO₄ as a standard after hydrolysis with 2 M HCl at 105°C for 4 h.

^eDetermined by a HPLC system equipped with a TSK-gel G4000 PW_XL column (MM, molecular mass).

Figure 2. Inhibitory effects of BFP on α-amylase and α-glucosidase.

(a) Inhibitory effects of BFP (0–1.0 mg/mL) or acarbose (0.01 mg/mL) on α-amylase. (b) Inhibitory effects of BFP (0–1.0 mg/mL) or acarbose (0.50 mg/mL) on α-glucosidase. All experiments were performed in triplicate, and values are the means ± SD. Asterisks indicate significant differences between with and without polysaccharides (or acarbose). *p <0.05.

Figure 3. The inhibitory kinetics of BFP on α-amylase and α-glucosidase.

(a) Linear regression of reaction rates versus different concentrations of α-amylase in the presence (closed circles) and absence (open circles) of BFP. The final concentration of starch was 5.0 mg/mL, and the reaction time was 3 min. (b) Linear regression curves of reaction rates versus different concentrations of α-glucosidase in the presence (closed circles) and absence (open circles) of BFP. The final concentration of pNPG was 2.5 mM, and the reaction time was 3 min. (c) Lineweaver–Burk plot of the reactions of α-amylase in the presence (closed circles) and absence (open circles) of BFP. (d) Lineweaver–Burk plot of the reactions of α-amylase in the presence (closed circles) and absence (open circles) of BFP. All experiments were performed in triplicate, and values were the means ± SD.

Table 2. Effects of BFP on the secondary structures of α-amylase and α-glucosidase.

Enzyme	BFP (1000 µg/mL)	α-Helix (%)	β-Sheet (%)	β-Turn (%)	Random (%)
α-amylase	–	15.8	21.3	28.1	37.9
	+	29.0	8.1	31.3	31.8
α-glucosidase	–	12.7	32.9	19.6	35.0
	+	15.6	26.6	21.2	35.0

Figure 4. Effects of BFP on the CD spectrum of α-amylase or α-glucosidase.

CD spectra of α-amylase (a) or α-glucosidase at 5.0 mg/mL (b) in the presence (dotted line) or absence (solid line) of BFP (1000 µg/mL) were measured. Measurements were carried out after 15 min incubation of the mixture of enzyme and BFP at 25°C.

Figure 5. Effects of BFP on the fluorescence spectrum of α-amylase or α-glucosidase.

Fluorescence emission spectra of α-amylase (a) or α-glucosidase (b) at 1.0 mg/mL in the presence (dotted line) or absence (solid line) of BFP (1000 µg/mL) were measured. Measurements were carried out after 15 min incubation of the mixture of enzyme and BFP at 25°C. Fluorescence emission spectrum of BFP alone (c) at 1000 µg/mL.