Anti-diabetic Activities of Ethanol Extracts from Persimmon Leaves

Abstract

Diospyros kaki L. (persimmon), cultivated in Korea for several centuries, is thought to have originated in southern China. The persimmon leaves are approved as “Traditional Chinese Medicine” in China for the treatment of stroke and apoplexy syndrome [Fan et al., 2006], and are utilized as a hypotensive drug in Japan [Funayama et al., 1979]. Furthermore, in Korea the leaves have long been used as herb tea and were traditionally employed for the treatment of hypertension in patients with type 2 diabetes mellitus [Sa et al., 2005]. The condensed tannins and flavonoids from the persimmon leaves were found to be mainly responsible for the antihypertensive, antimutagenic, anticarcinogenic, and antioxidative actions [Choo et al., 2000]. However, little studies have been performed on the anti-diabetic properties of the persimmon leaves. Type 2 diabetes mellitus is a heterogeneous metabolic disorder characterized by the impaired insulin secretion from the pancreatic beta cells and the insulin resistance in the peripheral tissues such as liver, adipose tissue, and skeletal muscle [Ward et al., 1984]. Skeletal muscles account for approximately 75% of the glucose absorption under the insulin-stimulated conditions, and glucose transport is the rate-limiting step in the primary glucose disposal and utilization, especially in the insulin-targeted skeletal muscle. Thus, the alteration of the glucose transport is one of the major defects in type 2 diabetes both in vitro and in vivo [Dohm et al., 1988; Ziel et al., 1988; DeFronzo et al., 1992]. For these reasons, some targeted anti-diabetic drugs have been developed; for example, metformin (dimethylbiguanide), a commonly used glucose-lowering reagent, is thought to increase the glucose uptake in the skeletal muscle. [Musi et al., 2002]. Rosiglitazone and pioglitazone both act by reducing the glucose production in the liver and increasing the insulindependent glucose uptake in the muscle cells [Yonemitsu et al., 2001]. Therefore, we investigated the anti-diabetic therapeutic potential of the ethanol extracts of the persimmon leaves to maximize the glucose transport in L6 myotubes, which have been widely used to study the mechanism of the insulin-stimulated glucose transport. Persimmon leaves (0.5 kg) were purchased from the herbal medicine co-operative association in Daejeon, Korea. The dried leaves were extracted with ethanol (1 L ×2), filtered,and evaporated. The ethanol extracts were then suspended in DMSO for further experiments. L6 rat myoblasts were obtained from the American Type Culture Collection, cultured in DMEM containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin, and incubated at 37oC under 5% CO2 humidified atmosphere. For the differentiation of myoblasts into myotubes, the medium was switched to DMEM with 2% horse serum for 6 days. To assess the anti-diabetic effects of the ethanol extracts of persimmon leaves, we conducted glucose transport assays at various concentrations (20, 40, 60, and 80 μg/ mL) (Fig. 1); incubation of the myotubes with the ethanol extracts of persimmon leaves for 1 h significantly enhanced the insulin-stimulated glucose transport in a dose-dependent manner by 114.3, 123, 160.7, and 183.7%, respectively, as compared to the negative control with the insulin treatment. These results suggest that persimmon leaves affect the insulin-stimulated muscular glucose transport, and, therefore, could be used as insulin sensitizers for the treatment of diabetes. To investigate the role of the ethanol extracts of persimmon leaves on the insulin receptor-regulated glucose transport activity, we assessed the tyrosine phosphatase1B in vitro. As a critical negative regulator of the insulin signal transduction cascade, tyrosine phosphatase1B acts to reverse the tyrosine kinase action and is a key phosphatase for the insulin receptor and the insulin receptor substrate-1, major mediators of the glucose transport pathways [Moller et al., 2001]. Tyrosine phosphatase1B has also been implicated in the insulin-dependent pathways, as well as in the insulin insensitivity that is the most common pathology of type 2 diabetes and obesity [Na et al., 2006]. Therefore, the *Corresponding author Phone: +82-42-821-6834; Fax: +82-42-821-8887 E-mail: cbsohn@cnu.ac.kr