!Prevention and treatment of Alzheimerʼs disease are urgent problems for elderly people in developed countries. To seek useful compounds that treat, delay the onset, or prevent the development of Alzheimerʼs disease, we screened natural resources using the cyclic AMP response element-dependent transcription in the PC12 cell line. We previously found that honey bee royal jelly stimulates the cyclic AMP response element-dependent transcription activity in PC12 cells. Here, we report that R-limonene, a monoterpene, enhanced the royal jelly-induced cyclic AMP response element-dependent transcription activity, whereas R-limonene showed no effect in absence of royal jelly. The enhancement was inhibited by ZM241385, an antagonist for adenosine A 2 A receptor. R-limonene also enhanced the cyclic AMP response element-dependent transcription activity activated by adenosine and AMP. Thus, R-limonene represents a novel type of modulator that enhances the CRE-dependent transcription via adenosine A 2 A receptor pathway. To find a substance that potentiates the antidementia activity of RJ, we screened extracts derived from herbal plants. Preliminary screening identified limonene as a candidate for the activator of the RJ-induced stimulation of CRE-dependent transcription in PC12 cells. To confirm this finding, we tested commercially available limonenes for the CRE-dependent transcription induced by RJ in PC12 cells. As shown in l " Fig. 1 A, R-limonene dose-dependently enhanced the CRE-dependent transcription activity induced by 10 µg/mL RJ. Interestingly, R-limonene had no effect on the CRE-dependent transcription activity in the absence of RJ. Rlimonene was effective on both low and high concentrations of RJ (l " Fig. 1 B). We next examined the effect of R-limonene on RJ along the timecourse of its stimulation of CRE-dependent transcription (l " Fig. 2). RJ at 10 µg/mL significantly increased the CRE-dependent transcription activity 1-5 h after administration. The activating effect of RJ diminished at 24 and 48 h after administration. The addition of 100 µM R-limonene to RJ significantly enhanced the CRE-dependent transcription activity induced by RJ from 1 to 24 h. Again, R-limonene alone had no effect on the CRE-dependent transcription activity at all time points examined. We next tested the effect of several analogues of R-limonene on the CRE-dependent transcription induced by RJ (l " Fig. 3). (S)-(−)-limonene and (4R)-limonene oxide enhanced the RJ-induced CRE-dependent transcription comparable to the level of R-limonene. (4 S)-limonene oxide significantly increased the CRE-dependent transcription induced by RJ, but the potency of the limonene oxide was weaker than that of R-limonene. Interestingly, all these limonene analogues had no effect on the CRE-dependent * These authors contributed equally to this work. Letter THIEME
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