Synergistic Action of Flavonoids, Baicalein, and Daidzein in Estrogenic and Neuroprotective Effects: A Development of Potential Health Products and Therapeutic Drugs against Alzheimer's Disease.

Despite the classical hormonal effect, estrogen has been reported to mediate neuroprotection in the brain, which leads to the searching of estrogen-like substances for treating neurodegenerative diseases. Flavonoids, a group of natural compounds, are well known to possess estrogenic effects and used to substitute estrogen, that is, phytoestrogen. Flavonoid serves as one of the potential targets for the development of natural supplements and therapeutic drugs against different diseases. The neuroprotection activity of flavonoids was chosen for a possible development of anti-Alzheimer's drugs or food supplements. The estrogenic activity of two flavonoids, baicalein and daidzein, were demonstrated by their strong abilities in stimulating estrogen receptor phosphorylation and transcriptional activation of estrogen responsive element in MCF-7 breast cells. The neuroprotection effects of flavonoids against ß -amyloid (A ß ) were revealed by their inhibition effects on in vitro A ß aggregation and A ß -induced cytotoxicity in PC12 neuronal cells. More importantly, the estrogenic and neuroprotective activities of individual flavonoid could be further enhanced by the cotreatment in the cultures. Taken together, this synergistic effect of baicalein and daidzein might serve as a method to improve the therapeutic efficacy of different flavonoids against A ß , which might be crucial in developing those flavonoidsin treating Alzheimer's disease in the future.

Chemical fingerprinting and quantitative analysis of two common Gleditsia sinensis fruits using HPLC-DAD.

Gleditsiae Fructus Abnormalis and Gleditsiae Sinensis Fructus are obtained from different developmental stages of fruits from Gleditsia sinensis Lam. (Leguminosae). The possible interchangeable usage of the two fruits, however, has long been very controversial. Here, high performance liquid chromatography coupled with diode array detection was developed to explore their chemical fingerprinting profiles. Besides, the amounts of aglycones of saponin compounds, echinocystic acid and oleanolic acid in both fruits were quantified. The results indicated that there was no significant difference in the content of aglycones from the two types of fruits. However, their chromatographic fingerprints showed distinct characteristics. Therefore, the interchangeable application of these fruits has to be taken with a specific precaution.

Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor.

Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, share a chemical resemblance to estrogen, and indeed some of which have been used as estrogen substitutes. In searching for possible functions of flavonoids, the neuroprotective effect in brain could lead to novel treatment, or prevention, for neurodegenerative diseases. Here, different subclasses of flavonoids were analyzed for its inductive role in neurite outgrowth of cultured PC12 cells. Amongst the tested flavonoids, a flavonol aglycone, isorhamnetin that was isolated mainly from the leaves of Ginkgo biloba L. showed robust induction in the expression of neurofilament, a protein marker for neurite outgrowth, of cultured PC12 cells. Although isorhamnetin by itself did not show significant inductive effect on neurite outgrowth of cultured PC12 cells, the application of isorhamnetin potentiated the nerve growth factor- (NGF-)induced neurite outgrowth. In parallel, the expression of neurofilaments was markedly increased in the cotreatment of NGF and isorhamnetin in the cultures. The identification of these neurite-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer's disease and depression.

Molecular Assembly and Biosynthesis of Acetylcholinesterase in Brain and Muscle: the Roles of t-peptide, FHB Domain, and N-linked Glycosylation.

Acetylcholinesterase (AChE) is responsible for the hydrolysis of the neurotransmitter, acetylcholine, in the nervous system. The functional localization and oligomerization of AChE T variant are depending primarily on the association of their anchoring partners, either collagen tail (ColQ) or proline-rich membrane anchor (PRiMA). Complexes with ColQ represent the asymmetric forms (A(12)) in muscle, while complexes with PRiMA represent tetrameric globular forms (G(4)) mainly found in brain and muscle. Apart from these traditional molecular forms, a ColQ-linked asymmetric form and a PRiMA-linked globular form of hybrid cholinesterases (ChEs), having both AChE and BChE catalytic subunits, were revealed in chicken brain and muscle. The similarity of various molecular forms of AChE and BChE raises interesting question regarding to their possible relationship in enzyme assembly and localization. The focus of this review is to provide current findings about the biosynthesis of different forms of ChEs together with their anchoring proteins.

The assembly of proline-rich membrane anchor (PRiMA)-linked acetylcholinesterase enzyme: glycosylation is required for enzymatic activity but not for oligomerization.

Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal "t-peptide" in AChE catalytic subunit (AChE(T)). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChE(T) plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChE(T) mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher K(m) value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.

Baicalin, a flavone, induces the differentiation of cultured osteoblasts: an action via the Wnt/beta-catenin signaling pathway.

Flavonoids, a group of natural compounds found in a variety of vegetables and herbal medicines, have been intensively reported on regarding their estrogen-like activities and particularly their ability to affect bone metabolism. Here, different subclasses of flavonoids were screened for their osteogenic properties by measuring alkaline phosphatase activity in cultured rat osteoblasts. The flavone baicalin derived mainly from the roots of Scutellaria baicalensis showed the strongest induction of alkaline phosphatase activity. In cultured osteoblasts, application of baicalin increased significantly the osteoblastic mineralization and the levels of mRNAs encoding the bone differentiation markers, including osteonectin, osteocalcin, and collagen type 1α1. Interestingly, the osteogenic effect of baicalin was not mediated by its estrogenic activity. In contrast, baicalin promoted osteoblastic differentiation via the activation of the Wnt/ß-catenin signaling pathway; the activation resulted in the phosphorylation of glycogen synthase kinase 3ß and, subsequently, induced the nuclear accumulation of the ß-catenin, leading to the transcription activation of Wnt-targeted genes for osteogenesis. The baicalin-induced osteogenic effects were fully abolished by DKK-1, a blocker of Wnt/ß-catenin receptor. Moreover, baicalin also enhanced the mRNA expression of osteoprotegerin, which could regulate indirectly the activation of osteoclasts. Taken together, our results suggested that baicalin could act via Wnt/ß-catenin signaling to promote osteoblastic differentiation. The osteogenic flavonoids could be very useful in finding potential drugs, or food supplements, for treating post-menopausal osteoporosis.

Flavonoids from Radix Astragali induce the expression of erythropoietin in cultured cells: a signaling mediated via the accumulation of hypoxia-inducible factor-1α.

Radix Astragali (RA) is commonly used as a health food supplement to reinforce the body vital energy. Flavonoids, including formononetin, ononin, calycosin, and calycosin-7-O-ß-d-glucoside, are considered to be the major active ingredients within RA. Here, we provided different lines of evidence that the RA flavonoids stimulated the expression of erythropoietin (EPO), the central regulator of red blood cell mass, in cultured human embryonic kidney fibroblasts (HEK293T). A plasmid containing hypoxia response element (HRE), a critical regulator for EPO transcription, was tagged upstream of a firefly luciferase gene, namely, pHRE-Luc, which was being transfected into fibroblasts. The application of RA flavonoids onto the transfected cells induced the transcriptional activity of HRE. To account for the transcriptional activation after the treatment of flavonoids, the expression of hypoxia-inducible factor-1α (HIF-1α) was markedly increased: The increase was in both mRNA and protein levels. In addition, the degradation of HIF-1α was reduced under the effect of flavonoids. The regulation of HIF-1α therefore could account for the activation of EPO expression mediated by the RA flavonoids. The current results therefore reveal the function of this herb in enhancing hematopoietic functions.

The extract of Rhodiolae Crenulatae Radix et Rhizoma induces the accumulation of HIF-1α via blocking the degradation pathway in cultured kidney fibroblasts.

Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body resistance against hypoxia in mountain sickness. The mechanism of this adaptogenic property deriving from Rhodiola, however, has not been revealed. Erythropoietin (EPO) is an erythrocyte-specific hematopoietic hormone that increases the production of red blood cells: this hormone is a crucial factor in regulating the body balance in responding to hypoxia. In cultured kidney fibroblasts (HEK293T), application of water extract deriving from Rhodiola induced the expression of EPO both in mRNA and protein levels. The activation of the Hypoxia Response Element (HRE) located on the promoter region of the EPO gene is one of the mechanisms accounting for transcriptional activation. In addition, the Rhodiola-induced EPO expression was triggered by an increase of hypoxia-inducible factor-1 α (HIF-1 α) protein, via the reduction of HIF-1 α degradation but not the induction of HIF-1 α mRNA. Moreover, the same EPO induction effect by Rhodiola was also observed in cultured liver cells since liver is another vital organ to provide EPO regulation apart from the kidney. These results therefore elucidate one of the molecular mechanisms of this herb in mediating the anti-hypoxia function.

The expression of erythropoietin triggered by danggui buxue tang, a Chinese herbal decoction prepared from radix Astragali and radix Angelicae Sinensis, is mediated by the hypoxia-inducible factor in cultured HEK293T cells.

ETHNOPHARMACOLOGICAL EVIDENCE: Danggui buxue tang (DBT), a Chinese medicinal decoction that is being commonly used as hematopoietic medicine to treating woman menopausal irregularity, contains two herbs: radix Astragali and radix Angelicae Sinensis. Pharmacological results indicate that DBT can stimulate the production of erythropoietin (EPO), a specific hematopoietic growth factor, in cultured cells. AIM OF THE STUDY: In order to reveal the mechanism of DBT's hematopoietic function, this study investigated the activity of the DBT-induced EPO expression and the upstream regulatory cascade of EPO via hypoxia-induced signaling in cultured kidney fibroblasts (HEK293T). MATERIALS AND METHODS: DBT-induced mRNA expressions were revealed by real-time PCR, while the change of protein expressions were analyzed by Western blotting. For the analysis of hypoxia-dependent signaling, a luciferase reporter was used to report the transcriptional activity of hypoxia response element (HRE). RESULTS: The plasmid containing HRE, being transfected into HEK293T, was highly responsive to the challenge of DBT application. To account for the transcriptional activation of HRE, DBT treatment was shown to increase the mRNA and protein expressions of hypoxia-inducible factor-1α (HIF-1α). In addition, the activation of Raf/MEK/ERK signaling pathway by DBT could also enhance the translation of HIF-1α, suggesting the dual actions of DBT in stimulating the EPO expression in kidney cells. CONCLUSION: Our study indicates that HIF pathway plays an essential role in directing DBT-induced EPO expression in kidney. These results provide one of the molecular mechanisms of this ancient herbal decoction for its hematopoietic function.

Galangin, a flavonol derived from Rhizoma Alpiniae Officinarum, inhibits acetylcholinesterase activity in vitro.

Acetylcholinesterase (AChE) inhibitors are widely used for the treatment of Alzheimer's disease (AD). Several AChE inhibitors, e.g. rivastigmine, galantamine and huperzine are originating from plants, suggesting that herbs could potentially serve as sources for novel AChE inhibitors. Here, we searched potential AChE inhibitors from flavonoids, a group of naturally occurring compounds in plants or traditional Chinese medicines (TCM). Twenty-one flavonoids, covered different subclasses, were tested for their potential function in inhibiting AChE activity from the brain in vitro. Among all the tested flavonoids, galangin, a flavonol isolated from Rhizoma Alpiniae Officinarum, the rhizomes of Alpiniae officinarum (Hance.) showed an inhibitory effect on AChE activity with the highest inhibition by over 55% and an IC(50) of 120 microM and an enzyme-flavonoid inhibition constant (K(i)) of 74 microM. The results suggest that flavonoids could be potential candidates for further development of new drugs against AD.

PRiMA directs a restricted localization of tetrameric AChE at synapses.

Acetylcholinesterase (AChE), a highly polymorphic enzyme with various splicing variants and molecular isoforms, plays an essential role in the cholinergic neurotransmission by hydrolyzing acetylcholine into choline and acetate. The AChE(T) variant is expressed in the brain and muscle: this subunit forms non-amphiphilic tetramers with a collagen tail (ColQ) as asymmetric AChE (A(12) AChE) in muscle, and amphiphilic tetramers with a proline-rich membrane anchor (PRiMA) as globular AChE (G(4) AChE) in the brain and muscle. During the brain development, the expression of amphiphilic G(4) AChE is up regulated and becomes the predominant form of AChE there. This up-regulation of G(4) AChE can be attributed to the increased expressions of both AChE(T) and PRiMA. A significant portion of this membrane-bound G(4) AChE is localized at the membrane rafts of the cell membranes derived from the brain. This raft association could be directed by PRiMA via its CRAC (cholesterol recognition/interaction amino acid consensus) motif and C-terminus. In cultured cortical neurons and muscles, the PRiMA-linked AChE was clustered and partially co-localized with synaptic proteins. The restricted localizations suggest that the raft association of PRiMA-linked AChE could account for its synaptic localization and function.

Expression and Localization of PRiMA-linked globular form acetylcholinesterase in vertebrate neuromuscular junctions.

Acetylcholinesterase (AChE) is well known to process different molecular forms via the distinct interacting partners. Proline-rich membrane anchor (PRiMA)-linked tetrameric globular AChE (G4 AChE) is mainly found in the vertebrate brain; however, recent studies from our laboratory have suggested its existence at neuromuscular junctions (nmjs). Both muscle and motor neuron express AChE at the nmjs. In muscle, the expression of PRiMA-linked AChE is down-regulated during myogenic differentiation and by motor neuron innervation. As compared with muscle, spinal cord possessed higher total AChE activity and contained PRiMA-linked AChE forms. The spinal cord expression of this form increased during development. More importantly, PRiMA-linked G4 AChE identified as aggregates localized at nmjs. These findings suggest that the restricted localization of PRiMA-linked G4 AChE at the nmjs could be contributed by the pre-synaptic motor neuron and/or the post-synaptic muscle fiber.

A new variant of proline-rich membrane anchor (PRiMA) of acetylcholinesterase in chicken: expression in different muscle fiber types.

Proline-rich membrane anchor (PRiMA) is a molecule to organize acetylcholinesterase (AChE) into tetrameric globular form (G(4)) that anchors onto the plasma membrane in brain and muscle. In mammal, PRiMA is encoded by a single gene with two splicing variants, PRiMA I and PRiMA II: PRiMA II is different to PRiMA I by its absence of a C-terminal cytoplasmic domain. The existence of these isoforms has not been revealed in avian specie. By using RT-PCR and bioinformatic analyses, two splicing variants of PRiMA were identified in chicken cerebrum. One variant contains very similar domains as compared to mammalian PRiMA I. The other variant, named as PRiMA II, has a very distinct cytoplasmic C-terminus of having 26 amino acids. Both forms of chicken PRiMA were able to organize the formation of G(4) AChE when that was over expressed together with AChE(T) subunit in cultured cells. The level of PRiMA mRNA, mainly PRiMA I, was higher in slow-twitch muscle than that of in fast-twitch muscle of chicken. This finding suggests that the muscle fiber type-specific expression of G(4) AChE in chicken could be a result of the different expression pattern of PRiMA in fast- and slow-twitch muscles.

Hibifolin, a flavonol glycoside, prevents beta-amyloid-induced neurotoxicity in cultured cortical neurons.

The toxicity of aggregated beta-amyloid (A beta) has been implicated as a critical cause in the development of Alzheimer's disease (AD). Hibifolin, a flavonol glycoside derived from herbal plants, possessed a strong protective activity against cell death induced by aggregated A beta. Application of hibifolin in primary cortical neurons prevented the A beta-induced cell death in a dose-dependent manner. In cultured cortical neurons, the pre-treatment of hibifolin abolished A beta-induced Ca(2+) mobilization, and also reduced A beta-induced caspase-3 and caspase-7 activation. Moreover, DNA fragmentation induced by A beta could be suppressed by hibifolin. In addition to such protection mechanisms, hibifolin was able to induce Akt phosphorylation in cortical neurons, which could be another explanation for the neuroprotection activity. These results therefore provided the first evidence that hibifolin protected neurons against A beta-induced apoptosis and stimulated Akt activation, which would be useful in developing potential drugs or food supplements for treating AD.

Quality evaluation of Rhizoma Belamcandae (Belamcanda chinensis (L.) DC.) by using high-performance liquid chromatography coupled with diode array detector and mass spectrometry.

A high-performance liquid chromatography coupled with diode array detector and mass spectrometry (HPLC-DAD-MS) method was developed to evaluate the quality of Rhizoma Belamcandae (Belamcanda chinensis (L.) DC.) through establishing chromatographic fingerprint and simultaneous determination of seven phenolic compounds. The analysis was achieved on an Alltima C(18) analytical column (250 mm x 4.6 mm i.d. 5 microm) using linear gradient elution of acetonitrile-0.1% trifluoroacetic acid. The correlation coefficients of similarity were determined from the HPLC fingerprints, and they shared a close similarity. By using an online APCI-MS/MS, twenty phenols were identified. In addition, seven of these phenols including mangiferin, 7-O-methylmangiferin, tectoridin, resveratrol, tectorigenin, irigenin and irisflorentin were quantified by the validated HPLC-DAD method. These phenols are considered to be major constituents in Rhizoma Belamcandae, and are generally regarded as the index for quality assessment of this herb. This developed method by having a combination of chromatographic fingerprint and quantification analysis could be applied to the quality control of Rhizoma Belamcandae.