Aldose reductase (AR) is an enzyme to catalyze the reduction of glucose to sorbitol in the polyol pathway. AR is more active in diabetes than in normal condition. And then the polyol pathway in diabetes accelerates the formation of sorbitol in insulin-insensitive tissues such as nerve, lens, retina and kidney, so that diabetic complications, such as neuropathy, cataract, retinopathy and nephropathy are induced.1) In fact, sorbitol accumulation is observed in the crystalline lens of experimental diabetic rats, resulting in induction of osmotic stress followed by tissue damage.2) A number of AR inhibitors have been developed to treat for these complications, however, none of them has achieved worldwide use because of limited efficacy or undesirable side effects.
3)Ellagic acid (EA, Fig. 1 8) The inhibitory activity of EA against AR was ten times higher than that of quercetin.7) It is known that derivatives of EA such as 3,3Ј,4-tri-O-methylellagic acid 4Ј-sulfate potassium salt, 2-(2,3,6-trihydroxy-4-carboxyphenyl)ellagic acid (TEA, Fig. 1) and 4-(a-rhamnopyranosyl)ellagic acid showed about three times higher activities than EA. 5,7,8) Although EA and its derivatives have been reported to be AR inhibitors, their effects on sorbitol accumulation in vitro and in vivo have not been previously reported.In this report, the effects of EA and TEA on sorbitol accumulation in erythrocytes, lens and sciatic nerve incubated with glucose in vitro, and the effect of EA on sorbitol accumulation in erythrocytes, lens and sciatic nerve of diabetic rats in vivo are discussed. Animals Male Wistar rats were purchased from Japan SLC Inc. (Hamamatsu, Japan).
MATERIALS AND METHODS
MaterialsIn Vitro Experiments Under ether anesthesia, blood was collected into a heparinized tube from the abdominal aorta of the rats, and then lenses and sciatic nerve were quickly removed. Erythrocytes were obtained by centrifugation at 1500ϫg and washed with cold saline twice, the content of hemoglobin in erythrocytes was measured with a kit (Hemoglobin B-test Wako; Wako Chemical Co.), 1 ml of erythrocytes was added into 3 ml of Krebs-Ringer bicarbonate buffer containing 28 mM glucose, and each compound dissolved in 25 ml of dimethyl sulfoxide (DMSO) to prepare from 0 to 1.0ϫ10 Ϫ4 M, and was incubated under the circumstances equilibrated with 95% air and 5% CO 2 at 37°C for 3 h.The removed lens and sciatic nerve were separately put into 5 ml of Krebs-Ringer bicarbonate buffer containing 50 mM glucose, and each compound dissolved in 25 ml of DMSO to prepare from 0 to 4.0ϫ10 Ϫ4 M, and were incubated under the circumstances equilibrated with 95% air and 5% CO 2 at 37°C for 4 and 6 h, respectively.The sorbitol contents were measured by the method of
Effects of Ellagic Acid and 2-(2,3,6-Trihydroxy-4-carboxyphenyl)ellagic Acid on Sorbitol Accumulation in Vitro and in VivoHidenori UEDA, Kazuko KAWANISHI,* and Masataka MORIYASU Department of Natural Medicinal Chemistry, Kobe Pharmaceutical University; 4-19-1 Motoyamakitamachi, Higashinada-ku, Kobe 658-8558, Japa...