Isolation and characterization of a novel glutathione S-transferase-activating peptide from the oriental medicinal plant Phellodendron amurense

Abstract

The aim of this study was to elucidate the characteristics of glutathione S-transferase(GST)-activating compounds from medicinal plants. Among 265 kinds of medicinal plants, Phellodendron amurense showed the highest GST activity at 174.8%. The GST-activating compound of P. amurense was maximally extracted when treated with distilled water at 30 °C for 12 h. The compound was purified by ultrafiltration, Sephadex G-10 gel filtration chromatography, and reverse-phase HPLC. The purified GST-activating compound from P. amurense was a novel tetrapeptide with an amino acid sequence of Ala-Pro-Trp-Cys and its molecular weight was estimated to be 476 Da. It also displayed a clear detoxicative effect in 1-chloro-2,4-dinitrobenzene treated mice at a dosage of mg/kg body weight.

Introduction

Glutathione S-transferases (EC 2.5.1.18, GST) are phase II enzymes that catalyze the nucleophilic attract of glutathione (GSH) to a wide range of hydrophobic and electrophilic compounds. GST belongs to a family of enzymes that play a major role in the dextoxication of potential carcinogenic compounds, including electrophilic agents that are potentially harmful to DNA [3] and potential alkylating agents including pharmacologically active compounds [26]. They also play a role in the elimination of many cellular alkylating agents through glutathione conjugation and subsequent mercapturic acid formation [14].

Attempts have been made to classify such enzyme activities from rat liver on the basis of the carbon skeleton of the electrophilic molecule or the specific leaving group involved [4], hence the common use of the terms aryl [9], alkyl [15], alkene [6], and epoxidetransferase [5], [7]. Six transferases have been separated from rat liver and are designated E, D, C, B, A, and AA (9). Transferase B is ligandin [10]. The transferases show broad and overlapping substrate specificities and similar physical properties [26]. Only transferases A and C share a common antigenic determinant in the rat [10]; however, the five electrophoretically distinguishable forms of glutathione S-transferase in human liver all cross react antigenically [16]. The glutathione S-transferases have been calculated to represent some 10% of soluble rat liver proteins [13]. Transferse B, as measured by quantitative immunoprecipitation, comprises 4.5% of soluble protein in rat liver and 2% in the small intestine and kidney [8]. Ligandin has also been detected in rat ovary and testis [1].

Ketley et al. [18] have reported that rat liver transferases AA, A, and C, as well as ligandin, bind a broad range of nonsubstrate ligands including bilirubin, indocyanine green, and hematin, with varying affinities. These ligands markedly inhibited the glutathione S-transferase activities of these proteins with 1-chloro-2,4-dinitrobenzene as a substrate. Furthermore, Schultz et al. [23] and Bladeren et al. [2] reported on inhibitors of glutathione S-transferase as therapeutic agents. Meanwhile, Mukhatar and Bresnick [11] recently demonstrated that glutathione S-epoxide transferase activity is inducible in rat liver by injection with phenobarbital or 3-methylcholanthrene. Ligandin is markedly induced by phenobarbital [8], apparently by an increase in de novo synthesis [12]. Nemoto et al. [22] have reported that rat transferase A, B, C, E, and AA have significant glutathione S-transferase activity towards the aryl-epoxide benzo(α)pyrene-4,5-oxide but only transferase A and E show activity towards alkyl-epoxide substrates [24]. A similar number of glutathione S-transferases prepared in homogeneous form from human liver exhibit a similar broad spectrum of activity [17]. Recently, Willmore and Storey [27] reported on purification and properties of GST from the anoxia-tolerant turtle, Trachemys scripta elegans.

Recently, medicinal plants and herbs have attracted attention for their health-stimulating properties and medicinal effects. Few studies, however, have been conducted on bioactive compounds from medicinal plants as raw material for functional foods. This study describes the extraction and characterization of a novel glutathione S-transferase-activating peptide from Phellodendron amurense for potential use as a liver function-activating drug or in medicinal food.