Inhibition of Ocular Aldose Reductase by Constituents From Wrightia tinctoria Seeds

Abstract

Purpose: : In diabetes, the polyol pathway which includes aldose reductase and sorbitol dehydrogenase, accelerates the formation of sorbitol from glucose in insulin-insensitive tissues leading to the development of cataracts in the lens. The aim of the present study is to isolate and identify the aldose reductase inhibitors present in the seeds of the Ayurvedic herb, Wrightia tinctoria R. Br.

Methods: : Powdered plant material was extracted with 70% ethanol in a soxhlet apparatus for 9-12 hours. The dry extract was suspended in distilled water and fractionated in a separatory funnel by sequential extraction with chloroform, ethyl acetate and n-butanol. The ethyl acetate fraction was further purified on a silica gel column to yield active fractions 1 and 2. Fractions 1 and 2 showed strong aldose reductase activity and were selected for further purification. Fraction 1 was purified using a mixture of chloroform: methanol (90:10) on a silica gel column and fraction 2 was purified using water as eluent on a sephadex LH-20 column. The active compounds in each case were identified using NMR and LC-MS/MS and by comparison to authentic samples. IC₅₀ and enzyme kinetic assays were carried out on both active compounds. Aldose reductase was prepared as the 5000g supernatant from a rat lens 10% homogenate in 0.1M phosphate buffer pH 7.4.

Results: : All active fractions showed concentration dependent inhibition of aldose reductase. Fraction 1 had an IC₅₀ value of 2.2 µg/ml and fraction 2 an IC₅₀ of 2.0 µg/ml. Fraction 2 was identified as chlorogenic acid and showed competitive inhibition in the enzyme kinetic assay. The mass spectrum of Fraction 1 had m/z 283 and 265 representing [MH]⁺ and [MH-18]⁺ respectively. Further characterization of this compound is in progress.

Conclusions: : The Ayurvedic herb Wrightia tinctoria is a source for novel aldose reductase inhibitors which can be used as lead compounds for the possible treatment and/or prevention of diabetic cataracts.

Keywords: cataract • enzymes/enzyme inhibitors • diabetes