The leaves of Stevia rebaudiana BERTONI (Compositae), known colloquially as Caa-ehe, have been used for centuries by the Paraguayan Indians as a sweetening agent with maté tea.1) The steviol (ent-kaurene type diterpene) glycosides, stevioside, rebaudiosides A, C, and F, and dulcoside A have been isolated from S. rebaudiana and are widely used as sweeteners.2,3) In a previous paper we reported the inhibitory effect of the methanol extract of S. rebaudiana and its active component, lupeol palmitate, on inflammatory ear edema caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin.4) Steviol inhibited Epstein-Barr virus early antigen (EBV-EA) induced by TPA in Raji cells, although stevioside did not inhibit the induction of EBV-EA by TPA.5) In this paper we examine the antiinflammatory effect of steviol glycosides, stevioside, rebaudiosides A and C and dulcoside A against TPA-induced inflammation and found these compounds to possess a marked inhibitory effect. The 50% inhibitory dose (ID 50 ) of these compounds for TPA-induced inflammation was 54.1-291.6 mg/ear. Furthermore, the stevioside mixture (the steviol glycosides from S. rebaudiana) was found to suppress markedly the tumor-promoting effect of TPA on skin tumor formation initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in mouse skin.
MATERIALS AND METHODSChemicals Stevioside, rabaudiosides A and C, dulcoside A, and stevioside mixture was obtained from Tokiwa Phytochemical (Chiba, Japan). These compounds were identified by chromatographic and spectroscopic comparison with authentic samples.6) The stevioside mixture contained stevioside (48.9%), rebaudioside A (24.4%), rebaudioside C (9.8%), dulcoside A (5.6%), and unidentified components (11.3%). DMBA, indomethacin, hydrocortisone, quercetin, and dimethylsulfoxide were purchased from Sigma Chemical (St. Louis, MO, U.S.A.). TPA was obtained from Chemicals for Cancer Research (Minneapolis, MN, U.S.A.).Animals Female ICR mice (7 weeks old) were obtained from the Japan SLC (Shizuoka, Japan). The animals were housed in an air-conditioned specific pathogen-free room (22-23°C), lit from 08:00 to 20:00. Food and water were available ad libitum.Assay of TPA-Induced Inflammation TPA (1 mg/ear) dissolved in acetone (20 ml) was applied to the right ear only of ICR mice using a micropipette. A volume of 10 ml was delivered to both the inner and outer surfaces of the ear. The sample, or its vehicle (20 ml), chloroform-methanol-water (1 : 2 : 1) or methanol-water (1 : 1), as a control, was applied topically about 30 min before each TPA treatment. For thickness determinations, a pocket thickness gauge (Mitsutoyo, Tokyo, Japan) with a range of 0-9 mm, graduated at 0.01-mm intervals and modified so that the contact surface area was increased to reduce the tension, was applied to the tip of the ear.The ear thickness was measured before treatment (a). Edema was measured 6 h after TPA treatment (b: TPA alone; bЈ: TPA plus sample). The following values were then calculated: Pharmacy, Nihon University; 7-7-1 Narash...
