Diarylheptanoids from Rhizomes of Alpinia officinarum Inhibit Aggregation of α‑Synuclein

Two new diarylheptanoids, alpinin A (<b>1</b>) and alpinin B (<b>2</b>), together with 18 known diarylheptanoids (<b>3</b>–<b>20</b>), were isolated from the rhizomes of Alpinia officinarum. Their structures were elucidated by comprehensive spectroscopic analysis, including high-resolution mass spectrometry, infrared spectroscopy, and one- and two-dimensional nuclear magnetic resonance spectroscopy. Structurally, alpinin A is a new member of the small family of oxa-bridged diarylheptanoids and contains the characteristic 2,6-<i>cis</i>-configured tetrahydropyran motif (C₁–C₅ oxa bridge). The absolute configuration of alpinin A was confirmed by asymmetric total synthesis of the enantiomer (<i>ent</i>-<b>1</b>), corroborating the assignment of the molecular structure. The absolute configuration of alpinin B was determined on the basis of the analysis of the circular dichroism exciton chirality spectrum. We evaluated the inhibitory activity of all isolated diarylheptanoids against α-synuclein aggregation at 10 μM. Alpinins A and B significantly inhibited α-synuclein aggregation by 66 and 67%, respectively

AA3 reduces the disease severity of EAE mice.

<p>(A) We orally administered AA3 or water (control) daily starting from the day of immunization. The time courses of symptom development (reflected by the EAE score) and the incidence of EAE scores at day 20 are shown (table below graph). (B) We orally administered AA3 or water (control) to EAE mice daily starting from day 8 post-immunization; the time courses of EAE scores are shown. Data are mean ± SEM (<i>n</i> = 24 per treatment group); *<i>p</i> < 0.05, Student’s <i>t</i>-test. (C) Histopathologic examination of spinal cord tissues by hematoxylin and eosin (top and middle panels) and Luxol Fast Blue staining (bottom panels). We obtained tissues from mice injected with complete Freund’s adjuvant (CFA), or EAE mice treated with water (Con/EAE) or AA3 (AA3/EAE). Representative pictures are shown. Scale bar, 100 μm.</p

Oral administration of AA3 reduces the inflammatory cytokine response of lymphocytes upon MOG_35–55 re-stimulation.

<p>We stimulated primary lymphocytes isolated from spleens of the control EAE mice (open bar) or the AA3-treated EAE mice (black bar) with MOG_35–55 peptide (exMOG) for 2 days. We quantified cytokine expression in the conditioned media by ELISA; the expression of (A) IFN-γ, (B) IL-4, (C) IL-17, and (D) IL-6 are shown. Data are mean ± SEM (<i>n</i> = 24 per treatment group); *<i>p</i> < 0.05, one-way ANOVA.</p

AA3 attenuates the production of pro-inflammatory cytokines in MOG-reactive lymphocytes.

<p>(A) We re-challenged primary lymphocytes from EAE mice with MOG_35–55 peptide in the presence of AA3. We quantified IFN-γ, IL-4, and IL-17 levels in the culture supernatants by ELISA. (B) Western blots of phosphorylated and total STAT4, STAT6, and STAT3 from whole-cell lysates collected from the cultures in (A). α-tubulin served as an equal loading control. Densitometric analysis of signals from western blots in (B). (C) qPCR analysis of <i>foxp3</i> and <i>il-17</i> in the MOG-reactive lymphocytes. (D) We examined the cell survival of the MOG-reactive lymphocytes by MTT assay. Data are mean ± SEM (<i>n</i> = 3 per treatment group); *<i>p</i> < 0.05, one-way ANOVA.</p

AA3 reduces Th17 cell differentiation.

<p>(A) We stimulated T helper cells isolated from naïve mice with TGF-β and IL-6, followed by IL-23 and PGE2, in the presence or absence of AA3 in order to differentiate them into Th17 cells. We analyzed IL-17–positive cells by flow cytometry after intracellular staining for IFN-γ and IL-17. Cells without AA3 treatment were used as a control. Representative cytograms are shown. (B) Histograms of the percentages of CD4+ IL-17⁺ IFN-γ⁻ and CD4⁺ IL-17⁻ IFN-γ⁺ cells obtained from the experiment conducted in (A). (C) We stimulated T helper cells with IL-23 to induce IL-17 cell expansion in the presence or absence of AA3. We quantified IL-17 levels in the culture media by ELISA and determined the numbers of viable and non-viable cells by Trypan Blue staining. Data are mean ± SEM (<i>n</i> = 3 per treatment group); *<i>p</i> < 0.05, one-way ANOVA.</p

AA3 inhibits PGE2–EP4 signaling.

<p>We treated THP-1 cells with PGE2 or PGE-1-OH (an EP4 agonist) in the presence of increasing concentrations of AA3 (A and B, respectively) or with increasing concentrations of PGE-1-OH together with AA3 or L-161982 (C). We measured cellular cAMP levels, and the concentration—response curves are shown. (D) We examined THP-1 cell survival after 2 days of AA3 treatment by MTT assay. Data are mean ± SEM (<i>n</i> = 3 per treatment group).</p