Inhibition behaviours of some phenolic acids on rat kidney aldose reductase enzyme: an in vitro study

Figures & data

Figure 1. Mechanism of the polyol pathway.

Figure 2. The relationship between oxidative stress and diabetic complications of the polyol pathway: Accelerated flux polyol pathway plays a critical role in the development of diabetic complications. Cataract is one of the diabetic complications. It is known that sorbitol accumulates in tissues and causes an increase in the osmotic pressure. Thus, water enters into the cells and swelling takes place, which can cause a cataract. Also, AR competes with glutathione reductase (GR) for their co-factor NADPH. The activity of GR is decreased when the activity of AR increased and this leading to a decrease in GSH level. Increased NADH causes NADH oxidase (NOx) to produce ROS. Fructose-3-phosphate (F-3-P) and 3-deoxyglucosone (3-DG), metabolites of fructose, increase AGE and binding of advanced glycation end product (AGE) to receptor of AGE (RAGE) increase oxidative stress4.

Figure 3. The molecular structures of phenolic acids used in this study.

Figure 4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of purified rat kidney aldose reductase. Lane 1: Standard proteins (kDa). Lane 2 and lane 3: Purified rat kidney aldose reductase enzyme.

Table 1. Summary of purification of aldose reductase from rat kidney.

Purification steps	Activity (EU/ml)	Protein (mg/ml)	Total volume (ml)	Total activity (EU)	Total protein (mg)	Specific activity (EU/mg)	Purification fold	Yield%
Homogenate	0.14	3.080	45	6.3	138.6	0.0455	1	100
(NH4)2SO4 precipitation and dialyze	0.19	3.24	30	5.7	97.2	0.0586	1.288	90.47
DE-52 cellulose anion exchange chromatography	0.138	1.405	20	2.76	28.1	0.098	2.154	43.8
Gel filtration chromatography	0.053	0.25	15	0.795	3.75	0.212	4.659	12.61
2′5′ ADP-sepharose 4b affinity chromatography	0.022	0.025	10	0.22	0.25	0.88	19.34	3.49

Table 2. IC₅₀, K_i values and inhibition types for phenolic acids used in this study.

Compounds	IC50	Ki		Inhibition type
Tannic acid	0.5 μM	0.598 ± 0.148 μM		Noncompetitive
		Ki	Ki^'
Chlorogenic acid	5.47 μM	1.563 ± 0.436 μM	28.05 ± 10.210 μM	Mixed
Sinapic acid	0.033 mM	0.0434 mM		Noncompetitive
Protocatechuic acid	0.048 mM	0.0463 ± 0.0315 mM		Noncompetitive
4-Hydroxybenzoic acid	0.05 mM	0.0522 ± 0.0208 mM		Noncompetitive
p-Coumaric acid	0.057 mM	0.0531 ± 0.002 mM		Uncompetitive
Ferullic acid	0.069 mM	0.0725 ± 0.0299 mM		Noncompetitive
Vanillic acid	0.086 mM	0.103 ± 0.0563 mM		Noncompetitive
Syringic acid	0.095 mM	0.107 ± 0.0216 mM		Noncompetitive
α-Resorcylic acid	0.11 mM	0.127 ± 0.0220 mM		Noncompetitive
3-Hydroxybenzoic acid	0.15 mM	0.176 ± 0.0245 mM		Noncompetitive
Gallic acid	0.176 mM	0.219 ± 0.0563 mM		Noncompetitive

Figure 5. A: Lineweaver–Burk graph and inhibition mechanism of tannic acid using three different tannic acid concentrations for determination of K_i and inhibition type. B: Lineweaver–Burk graph and inhibition mechanism of chlorogenic acid using three different chlorogenic acid concentrations for determination of K_i and inhibition type. C: Lineweaver–Burk graph and inhibition mechanism of p-coumaric acid using three different p-coumaric acid concentrations for determination of K_i and inhibition type.

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