Journal of Natural Medicines

, Volume 71, Issue 4, pp 617–631 | Cite as

Fermented Citrus reticulata (ponkan) fruit squeezed draff that contains a large amount of 4′-demethylnobiletin prevents MK801-induced memory impairment

  • Ichiro Kawahata
  • Tatsuya Suzuki
  • Evelyn Gutiérrez Rico
  • Shuichi Kusano
  • Hiroshi Tamura
  • Yoshihiro Mimaki
  • Tohru Yamakuni
Original Paper


A previous study reported biotransformation of a citrus peel polymethoxyflavone, nobiletin, by Aspergillus enabling production of 4′-demethylnobiletin, and the product’s antimutagenic activity. However, the effects of fermented citrus peel on the basal forebrain–hippocampal system remain unidentified. Citrus reticulata (ponkan) fruit squeezed draffs are generated as mass waste in beverage factories. In this study using PC12D cells and cultured central nervous system neurons, we therefore examined whether Aspergillus kawachii-fermented citrus fruit squeezed draff could affect cAMP response element (CRE)- and choline acetyltransferase gene (ChAT) promoter region-mediated transcriptional activities relevant to memory formation and cholinergic function. Our current fermentation yielded approximately 80% nobiletin bioconversion, and a sample of hot-water extract of the fermented fruit squeezed draff was stronger than that of the unfermented one in facilitating CRE-mediated transcription in cultured hippocampal neurons as well as in PC12D cells. A sample of 0–80% ethanol-eluted fraction of Diaion HP-20 column-adsorbed components of the preparation obtained by the fermentation concentration-dependently and more strongly facilitated CRE-mediated transcription than did the fraction of the unfermented one in both cell culture systems. In a separate study, this polymethoxyflavone-rich fraction of the fermented fruit squeezed draff showed a potent ability to facilitate CRE-mediated and ChAT transcription in a co-culture of hippocampal neurons and basal forebrain neurons. Repeated oral gavage of mice with the fermented fraction sample prevented MK801-impaired memory formation in mice. These findings suggest that the 4′-demethylnobiletin-rich fraction prepared from the Aspergillus-fermented ponkan squeezed draff has a potential anti-dementia effect.


Aspergillus kawachii-fermented Citrus reticulata fruit squeezed draff 4′-demethylnobiletin CRE-mediated transcription ChAT transcription CNS neurons Dementia 



We thank Dr. Brygida Berse of Boston University, School of Medicine, for providing the plasmid pGL3-ChAT.

Compliance with ethical standards

Conflict of interest

Tohru Yamakuni has received a research Grant from Fuji Sangyo Co., Ltd.

Supplementary material

11418_2017_1091_MOESM1_ESM.pdf (970 kb)
Supplemental Fig. 1. Analysis of the polymethoxyflavone-rich fraction of extract from Aspergillus-unfermented, liquid-fermented and solid-state-fermented ponkan squeezed draffs by HPLC. (a) Typical chromatograms of dried hot-water extracts of Aspergillus-unfermented ponkan squeezed draff (upper), solid-state-fermented draff (middle), and dried unprocessed liquid-fermented draff (bottom). (b) HPLC chromatograms of respective polymethoxyflavone-rich fractions of the Aspergillus-unfermented sample (upper), the solid-state-fermented sample (middle), and the liquid-fermented sample (bottom). Note that the Aspergillus-liquid fermentation of ponkan squeezed draff enabled complete conversion of nobiletin to 4′-demethylnobiletin. The chromatogram of the polymethoxyflavone-rich fraction of extract from Aspergillus-solid-state-fermented ponkan squeezed draffs is also shown at the bottom. Column: Mightysil RP-18 GP Aqua 250-4.6 mm (Kanto Chemical Co., INC.), Eluent: linear gradient: 0 min to 20 min, 7 to 75% acetonitrile in water, isocratic: 20 to 30 min: 75% acetonitrile in water, flow rate: 1.0 ml/min, detector: UV (254 nm), column temperature: 30 °C. Asterisk represents the second highest peak seen in the fraction of each sample that has a retention time identical to that of the 4′-demethyltangeretin-containing peak. Supplemental Fig. 2. Effects of polymethoxyflavone-rich fractions of Aspergillus-liquid-fermented, solid-state-fermented, or unfermented ponkan squeezed draff on the cell viability of PC12D cells. Cells were treated for 24 h with each sample at the concentrations indicated in the figure. Values represent the mean ± SEM (n = 4). One-way ANOVA with a post-hoc Tukey test for all pairs. * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. vehicle-treated cells. Supplemental Fig. 3. Effects of 4′-demethylnobiletin and nobiletin on CRE-mediated transcription in PC12D cells. Cells were treated with each of these natural compounds at 1-300 μM. Values represent the mean ± SEM (n = 8). One-way ANOVA with post-hoc Tukey test for all pairs. ** p < 0.01, *** p < 0.001 vs. CNT; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. nobiletin. 4′-DMNob: 4′-demethylnobiletin. Supplemental Fig. 4. Effects of nobiletin or 4′-demethylnobiletin on the cell viability of PC12D cells. Cells were treated for 24 h with nobiletin or 4′-demethylnobiletin at the concentrations shown in the figure. Values represent the mean ± SEM (n = 4). One-way ANOVA with a post-hoc Tukey test for all pairs. No significance was observed. Supplemental Fig. 5. Effects of 4′-demethylnobiletin and nobiletin on CRE-mediated transcription in PC12D cells cultured in the presence of 10 ng/ml NGF. Values represent the mean ± SEM (n = 8). One-way ANOVA with post-hoc Tukey test for all pairs. *** p < 0.001 vs. CNT; ### p < 0.001 vs. Nob. Nob: nobiletin; 4′-DMNob: 4′-demethylnobiletin; CNT: vehicle control. Supplemental Fig. 6. Effects of 4′-demethylnobiletin and nobiletin on the number of ChAT-positive neurons and ChAT immunoreactivity in basal forebrain neurons in primary culture. Cells were treated with 4′-demethylnobiletin or nobiletin at a concentration of 30 µM for each or with 0.1% DMSO as vehicle (control) for 2 days. Images were acquired by fluorescence microscopy (Olympus, Japan). For quantitative immunocytochemical analysis of cellular ChAT-immunoreactivity, 30–50 cells were counted for each group. Values represent the mean ± SEM. ** p < 0.01, *** p < 0.001 vs. control. ## p < 0.01 vs. nobletin. Scale: 20 μm. Supplemental Fig. 7. The effects of 4′-demethylnobiletin and nobiletin on CRE- mediated transcription (a), the effects of nobiletin, 4′-demetylnobiletin, and tangeretin on ChAT transcription in the co-culture of primary basal forebrain and hippocampal neurons (b), and the preventive effects of H-89 and U0126 against 4′-demetylnobiletin-induced increases in the levels for phosphorylated PKA substrates (c) and phosphorylated ERK (d) in the co-culture of the primary CNS neurons. (a) Cells were treated with the compounds at 30 μM. For pharmacological inhibition, the cells were pretreated with a 10 μM concentration of each inhibitor for 30 min prior to treatment with the both compounds. Values represent the mean ± SEM (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CNT. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. nobletin without H89 or U0126. §§§ p < 0.001vs. 4′-DMNob without H-89 or U0126. Two-way ANOVA with post-hoc Tukey test for all pairs. (b) Cells were treated with each natural compound at 30 µM. **** p < 0.0001 vs. CNT. #### p < 0.0001, $$$$ p < 0.0001. Tan: tangeretin. (c and d) Cells were pretreated with vehicle, 10 µM H-89 or U0126, and thereafter treated with 30 µM 4′-demetylnobiletin for 24 h in the presence or absence of the respective protein kinase inhibitor. Cell lysates (10 μg of protein/lane) were loaded on SDS-PAGE gel followed by Western blot analysis with anti-phospho-(Ser/Thr) PKA substrate, anti-phospho-p44/p42 MAPK (Erk1/2) (Thr202/Tyr204) or p44/p42 MAPK (Erk1/2) antibodies.Supplemental Fig. 8. Effects of an adenosine A2A antagonist on the polymethoxyflavone-rich fractions of Aspergillus-liquid-fermented, solid-state fermented, unfermented ponkan squeezed draff (a), or these fractionated samples-induced facilitation of CRE-mediated transcriptions (b) in PC12D cells. a Cells were treated for 24 h with fermented or unfermented samples at the indicated concentrations with or without ZM241385, an inhibitor of adenosine A2A receptor. Values represent the mean ± SEM (n = 4). Two-way ANOVA with a post-hoc Tukey test for all pairs. * p < 0.05, **** p < 0.0001 vs. control cells. #### p < 0.0001 vs. vehicle controls in each sample. A and B: hot-water extracts of unfermented ponkan squeezed draff and solid-state-fermented draff,respectively; C: unprocessed sample of the liquid-fermented fruit squeezed draff. (b) Cells were treated for 24 h with the ethanol-eluted fraction of solid-state fermented sample or the solid-state fermented sample with or without ZM241385. Values represent the mean ± SEM (n = 4). Two-way ANOVA with a post-hoc Tukey test for all pairs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control cells. #### p < 0.0001 vs. vehicle controls in each sample. S-ferm: solid-state fermented sample (PDF 970 kb)


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Copyright information

© The Japanese Society of Pharmacognosy and Springer Japan 2017

Authors and Affiliations

  • Ichiro Kawahata
    • 1
  • Tatsuya Suzuki
    • 1
  • Evelyn Gutiérrez Rico
    • 1
  • Shuichi Kusano
    • 2
  • Hiroshi Tamura
    • 2
  • Yoshihiro Mimaki
    • 3
  • Tohru Yamakuni
    • 1
  1. 1.Department of Pharmacology, Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan
  2. 2.Fuji Sangyo Co., Ltd. Research and Development CenterMarugameJapan
  3. 3.Laboratory of Medicinal Plant Science, School of PharmacyTokyo University of Pharmacy and Life ScienceHachiojiJapan

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