Extract from the leaves of “Amacha” (Hydramgea macrophylla Seringe var. thunbergii Makino) has been traditionally used as a sweet seasoning in Japan. Recently, several Japanese investigators have become interested in the pharmacological properties of the constituents of “Amacha” extract (Nozawa et al. 1981; Yamahara et al. 1995). We initially examined the effects of “Amacha” extract on steroidogenesis in bovine adrenocortical cells. We found that a low dose of the extract potentiated cyclic AMP-induced steroidogenesis. The major constituent of “Amacha” extract is phyllodulcin, which gives it the sweet flavour. Similar to the action of “Amacha” extract, low concentrations of phyllodulcin enhanced cyclic AMP-induced steroidogenesis in bovine adrenocortical cells. Therefore, we investigated the mechanism of action of phyllodulcin on cyclic AMP-induced steroidogenesis. And we found that phyllodulcin acts as an inhibitor of adrenocortical phosphodiesterase.
Materials.[2,8-3H] cyclic AMP (specific activity 925 Gbq/ mmol) from New England Nuclear Co. (Boston, MA, USA); 2-bis-(2-hydroxylethyl) amino ethanesulfonic acid (BES) and phyllodulcin from Wako Pure Chemicals Co. (Tokyo, Japan); cyclic AMP, 5'-AMP, snake venom, theophylline and 3-isobutyl-1-methylxanthine (IBMX) from Sigma Chemical Co. (St. Louis, MO, USA); DEAE-Sephadex A-25 column from Nacalai Tesque. Inc. (Tokyo, Japan). All the other chemicals used were reagent grade. The structure of (+)-phyllodulcin is shown in fig. 1.
Measurement of steroidogenic activity. Bovine adrenocortical fasciculata cells were isolated aseptically from freshly obtained bovine adrenal cortex using Krebs-Ringer bicarbonate buffer (123.4 mM NaCl, 25.0 mM NaHCO3, 5.9 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO2, 1.2 mM CaCl2, 0.2% glucose and 0.3% bovine serum albumin; pH 7.4) containing 0.1% collagenase, 0.005% deoxyribonuclease I and gentamicin as described previously (Matsui 1991). The isolated cells were cultured in Ham's F-10 medium supplemented with 5% foetal calf serum, 10% newborn calf serum, 2.5% horse serum, penicillin and streptomycin in 24-well plate at 37° under 5% CO2 in the air as a gas phase. The 3-day primary cultured cells were incubated in Krebs-Ringer bicarbonate buffer for 1 hr at 37° in a CO2 (5%)-incubator as reported (Kawamura et al. 1984). Glucocorticoid content in the incubation medium was determined fluorometrically with cortisol as a standard (Salvinski et al. 1976). The results were expressed as picomoles cortisol production per 105 cells par 1 hr (pmol/ 105 cells/ hr).
Measurement of phosphodiesterase activity. The crude phosphodiesterase fraction in adrenal cortex was prepared as follows. Fresh bovine adrenal cortex was hashed and homogenized by polytoron with 5 volumes of 25 mM Tris buffer (pH 7.4) containing 0.1 mM phenylmethysulforyl fluoride. The homogenate was centrifuged at 700 × g for 10 min. at 4° and the supernatant was subsequently centrifuged at 10,000 × g for 10 min. at 4°. The resultant supernatant was preserved at −80° as the crude phosphodiesterase fraction. Protein concentration was determined by the method of Lowry et al. (1951).
Phosphodiesterase activity was determined by the method of Elks et al. (1983). Briefly, the crude phosphodiesterase fraction (40∼50 μM protein/tube) was incubated in 0.1 M BES buffer (8.3 mM MgCl2, 2 mM CaCl2, 0.2 mM EGTA, 0.5 μM [2,8-3H]cyclic AMP: pH 7.0) for 30 min. at 30° in the presence or absence of phyllodulcin. Total incubation medium was 0.3 ml. Phyllodulcin was dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in incubation medium was less than 1%. At this concentration dimethyl sulfoxide itself does not affect the phosphodiesterase activity. The reactions were stopped by the addition of a solution containing 0.25 M HCl, 5 mM cyclic AMP and 4 mM 5'-AMP, and neutralized by 0.25 M NaOH and 0.25 M Tris. Subsequently, the samples were further incubated by adding 0.1 M Tris buffer (pH 7.4) containing 0.18 mg snake venom for 20 min. at 30°. Then [3H] adenosine was separated by DEAE-sephadex A-25 column, and detected by a liquid scintillation counter. The results were expressed as picomoles adenosine production per mg protein per 30 min. (pmol/mg/30 min.).
Statistical analysis. Results are expressed as mean±S.E. Analysis of variance and Dunnet multiple comparison test were used for the statistical analysis of the data. P<0.05 was considered statistically significant.
We first examined the effect of phyllodulcin on steroidogenesis induced by 5 mM cyclic AMP in bovine adrenocortical cells. As shown in fig. 2, phyllodulcin potentiated cyclic AMP-induced steroidogenesis in a concentration-dependent manner (2.5∼40 μM). Because cyclic AMP is biotransformed to a biologically inactive compound (5'-AMP) by intracellular phosphodiesterase, one possible mechanism explaining our result could be that phyllodulcin exhibits a phosphodiesterase inhibitory effect. Therefore, we examined the effect of phyllodulcin on phosphodiesterase activity by use of the crude phosphodiesterase fraction of bovine adrenal cortex. Fig. 3 showed that phyllodulcin inhibited the phosphodiesterase activity in a concentration-dependent manner (50∼800 μM). Control value was 838.7±37.9 pmol/mg/30 min. (mean±S.E.). We were able to add phyllodulcin up to 800 μM only, due to precipitation of the agent at higher concentration. Apparent IC50 on phosphodiesterase activity is 100 μM. Phosphodiesterase are subdivided into 7 isozymes (phosphodiesterase 1∼7) (Beavo 1995). In bovine adrenal cortex, we could detect at least 4 isozymes of phosphodiesterase (i.e.; phosphodiesterase 2, 3, 4, 5 or 6) pharmacologically by use of the isozyme selective inhibitors (data not shown). Before the effect of phyllodulcin on each phosphodiesterase isozyme was investigated, we compared the inhibitory effects of IBMX and theophylline, non-selective phosphodiesterase inhibitors, on adrenocortical crude phospodiesterase fraction to the effect of phyllodulcin. As shown in fig. 4A and B, both IBMX (6.25∼200 μM) and theophylline (50∼800 μM) inhibited the phosphodiesterase activity in a concentration-dependent manner. The inhibitory profiles of these xanthines are similar to that of phyllodulcin. Apparent IC50 of IBMX and theophylline on phosphodiesterase activity are 6 μM and 100 μM, respectively. Therefore the potency order might be IBMX >> theophylline=phyllodulcin. Although there is no analogy of chemical structure between phyllodulcin and the xanthines, phyllodulcin seems to act as a non-selective phosphodiesterase inhibitor with similar potency to theophylline. The results also show that phyllodulcin could be the precusor of novel isozyme-selective phosphodiesterase inhibitors. Furthermore, consumption of “Amacha” extract as a crude drug might avert an attack of bronchial asthma and/or minor exacerbation of heart failure.