Nobiletin improves brain ischemia-induced learning and memory deficits through stimulation of CaMKII and CREB phosphorylation.

Decreased cerebral blood flow causes cognitive impairments and neuronal injury in the progressive age-related neurodegenerative disorders such as Alzheimer's disease (AD) and vascular dementia. In the present study, we for the first time found that nobiletin, a novel leading compound for AD therapy, improved cerebral ischemia-induced memory deficits in vivo. Treatment with 50 mg/kg of nobiletin (i.p.) for the consecutive 7 days before and after brain ischemia significantly inhibited delayed neuronal death in the hippocampal CA1 neurons in a 20-min bilateral common carotid arteries occlusion (BCCAO) ischemia. However, the contextual memory assessed by passive avoidance task was not improved. On the other hand, a 5-min BCCAO-induced contextual memory deficit was significantly improved by the nobiletin treatment. In the 5-min BCCAO mice, Western blot analysis evidently showed that the levels of synaptic proteins, including calcium/calmodulin-dependent protein kinase II (CaMKII), microtubule-associated protein 2 (MAP2) and glutamate receptor 1 (GluR1), significantly decreased in the hippocampal CA1 region. The nobiletin treatment prevented the reduction in CaMKII, MAP2 and GluR1 protein levels in the hippocampal CA1 region, accompanied by restoration of both ERK and CREB phosphorylation and CaMKII autophosphorylation. Consistent with the restored CaMKII and ERK phosphorylation, an electrophysiological study showed that the impaired hippocampal long-term potentiation (LTP) observed in the 5-min ischemic mice was significantly improved by the nobiletin treatment. These findings suggest that the activation of CaMKII and ERK signaling in part mediates improvement of ischemia-induced learning and memory deficits by nobiletin.

Identification of nobiletin, a polymethoxyflavonoid, as an enhancer of adiponectin secretion.

Adiponectin, an adipocyte-derived protein with insulin-sensitizing, anti-diabetic and anti-atherogenic activities, is known to be induced during adipocyte differentiation. Nobiletin, a citrus polymethoxy flavonoid, was found to induce the differentiation of ST-13 preadipocytes into mature adipocytes and enhance the production of adiponectin protein at a concentration of 10 microM.

Nobiletin, a citrus flavonoid, improves memory impairment and Abeta pathology in a transgenic mouse model of Alzheimer's disease.

Increasing evidence suggests that the elevation of beta-amyloid (Abeta) peptides in the brain is central to the pathogenesis of Alzheimer's disease (AD). Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from citrus peels, enhances cAMP/protein kinase A/extracellular signal-regulated kinase/cAMP response element-binding protein signaling in cultured hippocampal neurons and ameliorates Abeta-induced memory impairment in AD model rats. For the first time, we report that this natural compound improves memory deficits in amyloid precursor protein (APP) transgenic mice that overexpress human APP695 harboring the double Swedish and London mutations [APP-SL 7-5 transgenic (Tg) mice]. Our enzyme-linked immunosorbent assay (ELISA) also showed that administration of nobiletin to the transgenic mice for 4 months markedly reduced quantity of guanidine-soluble Abeta(1-40) and Abeta(1-42) in the brain. Furthermore, consistent with the results of ELISA, by immunohistochemistry with anti-Abeta antibody, it was evidently shown that the administration of nobiletin decreased the Abeta burden and plaques in the hippocampus of APP-SL 7-5 Tg mice. These findings suggest that this natural compound has potential to become a novel drug for fundamental treatment of AD.

Nobiletin, a citrus flavonoid with neurotrophic action, augments protein kinase A-mediated phosphorylation of the AMPA receptor subunit, GluR1, and the postsynaptic receptor response to glutamate in murine hippocampus.

Nobiletin isolated from citrus peels prevents bulbectomy- and amyloid-beta protein-induced memory impairment in rodents. In the present study, using combined methods of biochemistry and electrophysiology, we examined the effects of nobiletin on phosphorylation of GluR1 receptor, the subunit of alpha-amino-3-hydroxy-5-methyl-D-aspartate (AMPA) receptors, and the receptor-mediated synaptic transmission in the hippocampus, a region implicated in memory formation, in culture and/or in slices. Western blot analysis showed that nobiletin-stimulated phosphorylation of multiple protein kinase A (PKA) substrates at 10 min following the treatment in cultured hippocampal neurons. In the cultured neurons, this natural compound also increased not only PKA activity, but also phosphorylation of GluR1 receptor at a PKA phosphorylation site, Ser 845, which has been demonstrated to be critical for synaptic plasticity, including enhancement of postsynaptic glutamate response, and important for spatial memory in vivo. The increased phosphorylation of GluR1 receptor at Ser 845 was abolished by H89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride), the PKA inhibitor, but not U0126 (1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene), the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, in the cultured neurons. An increment of the phosphorylation of GluR1 receptor at Ser 845 was induced by nobiletin in the hippocampal slices as well. Furthermore, our electrophysiological analysis showed that nobiletin potentiated the AMPA receptor-mediated synaptic transmission at Schaffer collateral-CA1 pyramidal cell synapses in the hippocampal slices. This potentiation induced by the natural compound was not accompanied by the changes in paired-pulse ratio, and partially occluded the long-term potentiation, indicating the possible involvement of the postsynaptic mechanism. These findings suggest that nobiletin probably up-regulates synaptic transmission via the postsynaptic AMPA receptors at least partially by stimulation of PKA-mediated phosphorylation of GluR1 receptor in the hippocampus.

Nobiletin, a citrus flavonoid that improves memory impairment, rescues bulbectomy-induced cholinergic neurodegeneration in mice.

We have recently reported that nobiletin, a citrus flavonoid, improves impaired memory in olfactory-bulbectomized (OBX) mice, which have been widely utilized as a useful paradigm that shares some major clinical features of Alzheimer's disease. Here, we examined the effects of nobiletin on OBX-induced cholinergic neurodegeneration in mice. OBX mice showed reduced acetylcholinesterase (AChE) staining and choline acetyltransferase (ChAT) expression in the hippocampus. An 11-day administration of nobiletin rescued OBX-induced decrease in the density of AChE-staining and ChAT expression in the hippocampus. These results suggest that nobiletin rescues OBX-induced cholinergic neurodegeneration, accompanied by improvement of impaired memory in OBX mice.

Nobiletin, a citrus flavonoid, reverses learning impairment associated with N-methyl-D-aspartate receptor antagonism by activation of extracellular signal-regulated kinase signaling.

Recent studies have indicated that learning-induced activation of extracellular signal-regulated kinase (ERK) signaling via N-methyl-D-aspartate (NMDA) receptors is required for consolidation of the resultant learning. These findings raise an idea that control of ERK signaling may be a potential target for treatment of cognitive dysfunction. Our recent studies have demonstrated that nobiletin, a polymethoxylated flavone from Citrus depressa, enhances cAMP/protein kinase A/ERK signaling in cultured rat hippocampal neurons and PC12D cells. Here, we, for the first time, present the evidence that this natural compound reverses learning impairment associated with NMDA receptor antagonism by activation of ERK in the hippocampus. Treatment with 50 mg/kg nobiletin reversed the NMDA receptor antagonist MK-801 (dizocilpine maleate)-induced learning impairment in mice. Western blot analysis also showed that nobiletin reversed MK-801-induced inhibition of learning-associated ERK activation in the hippocampus of the animals. Furthermore, consistent with these results, in cultured rat hippocampal neurons, nobiletin restored MK-801-induced impairment of NMDA-stimulated phosphorylation of ERK in a concentration-dependent manner. Taken together, the present study suggests that compounds that activate ERK signaling improve cognitive deficits associated with NMDA receptor hypofunction and that nobiletin may give us a new insight into therapeutic drug development for neurological disorders exhibiting cognitive impairment accompanied by a hypofunction of NMDA receptor-ERK signaling.

Neocimicigenosides A and B, cycloartane glycosides from the rhizomes of Cimicifuga racemosa and their effects on CRF-stimulated ACTH secretion from AtT-20 cells.

Two new cycloartane glycosides, named neocimicigenosides A (1) and B (2), were isolated from the rhizomes of Cimicifuga racemosa. The structures of 1 and 2 were determined on the basis of extensive spectroscopic analysis and enzymatic hydrolysis followed by chromatographic and spectroscopic analyses to be (16S,23R,24S)-24-acetoxy-16,23:16,25-diepoxy-15alpha-hydroxycycloartan-3beta-yl alpha-L-arabinopyranoside (1) and (16S,23R,24S)-24-acetoxy-16,23:16,25-diepoxy-15alpha-hydroxycycloartan-3beta-yl beta-D-xylopyranoside (2), respectively. Neocimicigenosides A and B enhanced CRF-stimulated ACTH secretion from AtT-20 cells.

Nobiletin restoring beta-amyloid-impaired CREB phosphorylation rescues memory deterioration in Alzheimer's disease model rats.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive and memory deterioration. Production and accumulation of beta-amyloid peptide (Abeta) is central to the pathogenesis of AD. Recent studies have demonstrated that PKA/CREB-dependent signaling pathway and long-term potentiation are inhibited by sublethal concentrations of Abeta(1-42) in cultured hippocampus neurons. Here, we examined the effects of nobiletin on the Abeta-induced inhibition of CREB phosphorylation in cultured rat hippocampus neurons. A sublethal concentration of Abeta(1-42) or Abeta(1-40) decreased glutamate-induced CREB phosphorylation, whereas pretreatment with nobiletin reversed the Abeta-induced decrease in CREB phosphorylation. The effects of nobiletin on impairment of learning ability were also examined in chronically Abeta(1-40) infused AD model rats using the eight-arm radial maze. In the AD model rats, nobiletin showed protective effects on Abeta(1-40)-induced impairment of learning ability. These results suggest that nobiletin has the potential for becoming a novel lead compound for drug development for AD.

Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities.

cAMP response element (CRE) transcription is dysregulated in neurodegenerative disorders in the central nervous system (CNS), including polyglutamine diseases. As the first step to find natural compounds with protective action against neurodegeneration in the CNS, we here examined whether six citrus flavonoids, namely nobiletin, 5-demethylnobiletin, tangeretin, sinensetin, 6-demethoxytangeretin, and 6-demethoxynobiletin, stimulated CRE-dependent transcription and induced neurite outgrowth in PC12D cells. Among the compounds, nobiletin most potently enhanced CRE-dependent transcription and neurite outgrowth by activating ERK/MAP kinase-dependent signalling to increase CREB phosphorylation. The transcription and neurite outgrowth were stimulated by nobiletin in a concentration-dependent manner, with a strong correlation between them. Furthermore, a 11-day oral administration of nobiletin rescued impaired memory in olfactory-bulbectomized mice documented to be accompanied by a cholinergic neurodegeneration. These results suggest that nobiletin with the activity to improve impaired memory may become a potential leading compound for drug development for neurodegenerative disorders exhibiting the dysregulated CRE-dependent transcription.

Mechanism of neurotrophic action of nobiletin in PC12D cells.

Nobiletin is a nonpeptide compound with a low molecular weight from a citrus fruit and has the activity to rescue bulbectomy-induced memory impairment. Here we describe that nobiletin itself induces neurite outgrowth in PC12D cells, a rat pheochromocytoma cell line, like NGF, and the molecular mechanism of its neurotrophic action. As cultured in the presence of nobiletin or NGF for 48 h and then assayed using a scanning electron microscope, PC12D cells treated with nobiletin showed morphology with flatter and larger cell bodies than the cells cultured with NGF. Nobiletin-induced neurite outgrowth was inhibited by PD98059 and U0126 but not K252a. Consistently, nobiletin caused a concentration-dependent enhancement of Erk/MAP kinase phosphorylation and a sustained increment of phosphorylation of MEK and Erk/MAP kinase, resulting in a stimulation of CREB phosphorylation and CRE-mediated transcription. This compound also increased intracellular cAMP and CRE-mediated transcription in the presence of forskolin and enhanced PKA activity to stimulate phosphorylation of multiple PKA substrates in PC12D cells. Furthermore, nobiletin preferentially inhibited Ca2+/CaM-dependent phosphodiesterase in vitro. This compound failed to stimulate phosphorylation of Erk5, which is known to be induced by NGF/TrkA signaling. These results suggest that nobiletin induces neurite outgrowth by activating a cAMP/PKA/MEK/Erk/MAP kinase-dependent but not TrkA-dependent signaling pathway coupling with CRE-mediated gene transcription and may thus become a novel type of biochemical probe for elucidation of the molecular mechanism of neuronal differentiation.

New glycosides of the campesterol derivative from the rhizomes of Tacca chantrieri.

Seven new glycosides of the campesterol derivative (24R,25S)-ergost-5-ene-3beta,26-diol (1-7) were isolated from the rhizomes of Tacca chantrieri (Taccaceae). Their structures were determined by extensive spectroscopic analysis, including 2D NMR data, and a few chemical transformations.

Effects of inhaling the vapor of Lavandula burnatii super-derived essential oil and linalool on plasma adrenocorticotropic hormone (ACTH), catecholamine and gonadotropin levels in experimental menopausal female rats.

The effects of inhaling the vapor of Lavandula burnatii super-derived essential oil and one of the main components of lavender oil, linalool on plasma adrenocorticotropic hormone (ACTH), catecholamine and gonadotropin levels in menopausal model rats under ether-inhalation were studied. The increased plasma ACTH levels induced by ether-inhalation tended to decrease by pre-inhalation of Lavandula burnetii super and linalool vapor was induced the decrease of ACTH level. The decrease in adrenaline, noradrenaline and dopamine levels induced by ether-inhalation tended to recover, especially, the dopamine level significantly recovered to the normal level by the inhalation of Lavandula burnetii super and linalool vapor. However, the increased plasma gonadotropin levels in ovariectomized retired female rats (menopausal model rats) was significantly decreased by the inhalation of linalool. These results suggest that lavender oil or one of the main components, linalool may contribute to relieving tension and may be applicable to the treatment of menopausal disorders in human beings.

Curcuminoids and sesquiterpenoids in turmeric (Curcuma longa L.) suppress an increase in blood glucose level in type 2 diabetic KK-Ay mice.

Turmeric, the rhizome of Curcuma longa L., has a wide range of effects on human health. The chemistry includes curcuminoids and sesquiterpenoids as components, which are known to have antioxidative, anticarcinogenic, and antiinflammatory activities. In this study, we investigated the effects of three turmeric extracts on blood glucose levels in type 2 diabetic KK-A(y) mice (6 weeks old, n = 5/group). These turmeric extracts were obtained by ethanol extraction (E-ext) to yield both curcuminoids and sesquiterpenoids, hexane extraction (H-ext) to yield sesquiterpenoids, and ethanol extraction from hexane-extraction residue (HE-ext) to yield curcuminoids. The control group was fed a basal diet, while the other groups were fed a diet containing 0.1 or 0.5 g of H-ext or HE-ext/100 g of diet or 0.2 or 1.0 g of E-ext/100 g of diet for 4 weeks. Although blood glucose levels in the control group significantly increased (P < 0.01) after 4 weeks, feeding of 0.2 or 1.0 g of E-ext, 0.5 g of H-ext, and 0.5 g of HE-ext/100 g of diet suppressed the significant increase in blood glucose levels. Furthermore, E-ext stimulated human adipocyte differentiation, and these turmeric extracts had human peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand-binding activity in a GAL4-PPAR-gamma chimera assay. Also, curcumin, demethoxycurcumin, bisdemethoxycurcumin, and ar-turmerone had PPAR-gamma ligand-binding activity. These results indicate that both curcuminoids and sesquiterpenoids in turmeric exhibit hypoglycemic effects via PPAR-gamma activation as one of the mechanisms, and suggest that E-ext including curcuminoids and sesquiterpenoids has the additive or synergistic effects of both components.

27-norlanostane glycosides from the bulbs of Muscari paradoxum.

Eight 27-norlanostane glycosides (1-8), including five new compounds (3 and 5-8), were isolated from the MeOH extract of the bulbs of Muscari paradoxum. The structures of the new compounds were determined on the basis of extensive spectroscopic analysis, including 2D NMR data, and the results of hydrolytic cleavage. The cytotoxic activity of 1-8 against HSC-2 human oral squamous cell carcinoma cells is also reported.

Taccasterosides A-C, novel C28-sterol oligoglucosides from the rhizomes of Tacca chantrieri.

Three novel C(28)-sterol oligoglucosides, named taccasterosides A-C (1-3), were isolated from the rhizomes of Tacca chantrieri (Taccaceae). Their structures were determined by detailed spectroscopic analysis, including 2D NMR data, and a few chemical transformations.

Steroidal glycosides from the bulbs of Ornithogalum thyrsoides.

Phytochemical analyses have been carried out on the fresh bulbs of Ornithogalum thyrsoides with particular attention to the steroidal glycoside constituents, resulting in the isolation of four new spirostanol saponins and seven new cholestane glycosides, together with three known steroidal compounds. The structures of the new glycosides were determined on the basis of their spectroscopic data, including 2D NMR spectroscopy, and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activities against HL-60 human promyelocytic leukemia cells and HSC-2 human oral squamous cell carcinoma cells.

Ultraviolet derivatization of steroidal saponin in garlic and commercial garlic products as p-nitrobenzoate for liquid chromatographic determination.

A method is described for determination of the steroidal saponin, eruboside B, originating in garlic and garlic products as the p-nitrobenzoyl chloride (PNBC) derivative by reversed-phase liquid chromatography (with ultraviolet detection at 260 nm. Proto-eruboside B was extracted from garlic (Allium sativum L.); subjected to solid-phase extraction (SPE) with a C18 cartridge, Florisil column chromatography, and silica gel column chromatography; and then enzymatically converted to eruboside B, which was applied as an external standard. Steroidal saponins in garlic and commercial garlic products were extracted with methanol and purified by SPE cartridges, followed by enzymatic treatment. A frostanol saponin such as proto-eruboside B is enzymatically transformed to a spirostanol saponin, eruboside B. After the derivatization with PNBC, the saponin derivative was chromatographed on a C8 column with a gradient elution of (A) 80% aqueous acetonitrile and (B) 100% acetonitrile. The detection limit of the developed method was 1 microg/g for the samples. The method was applied to the analysis of garlic and garlic health food products available in Japan.

Triterpene saponins from the roots of Clematis chinensis.

A saponin-enriched fraction prepared from the MeOH extract of the roots of Clematis chinensis showed cytotoxic activity against HL-60 promyelocytic leukemia cells, from which five new triterpene saponins based on oleanolic acid, along with three known saponins, were isolated. The structures of the new saponins were determined on the basis of spectroscopic analysis, including extensive 1D and 2D NMR data and hydrolysis followed by chromatographic and spectroscopic analysis. Among the isolated saponins, monodesmosidic saponins exhibited cytotoxic activities against cultured tumor cells.

The structure of a new crystalline cevane alkaloid: its identity with persicanidine B and harepermine.

The structure of a new crystalline base (melting point (mp) 167-169 degrees C) obtained from Fritillaria imperialis was elucidated as (20R, 25R)-5alpha,17beta-cevanine-3beta,6beta-diol, X-ray diffraction analysis of the mono-hydrate. The base was found to be identical with persicanidine B and also with harepermine.