Rapid separation of cyanidin-3-glucoside and cyanidin-3-rutinoside from crude mulberry extract using high-performance countercurrent chromatography and establishment of a volumetric scale-up process.

This study describes the rapid separation of mulberry anthocyanins; namely, cyanidin-3-glucoside and cyanidin-3-rutinoside, using high-performance countercurrent chromatography, and the establishment of a volumetric scale-up process from semi-preparative to preparative-scale. To optimize the separation parameters, biphasic solvent systems composed of tert-butyl methyl ether/n-butanol/acetonitrile/0.01% trifluoroacetic acid, flow rate, sample amount and rotational speed were evaluated for the semi-preparative-scale high-performance countercurrent chromatography. The optimized semi-preparative-scale high-performance countercurrent chromatography parameters (tert-butyl methyl ether/n-butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 4.0 mL/min; sample amount, 200-1000 mg; rotational speed, 1600 rpm) were transferred directly to a preparative-scale (tert-butyl methyl ether/n-butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 28 mL/min; sample amount, 5.0-10.0 g; rotational speed, 1400 rpm) to achieve separation results identical to cyanidin-3-glucoside and cyanidin-3-rutinoside. The separation of mulberry anthocyanins using semi-preparative high-performance countercurrent chromatography and its volumetric scale-up to preparative-scale was addressed for the first time in this report.

Seoritae extract reduces prostate weight and suppresses prostate cell proliferation in a rat model of benign prostate hyperplasia.

Seoritae is a type of black soybean that is known to have health-promoting effects due to its high isoflavone and anthocyanin contents. We evaluated whether Seoritae extract (SE) had beneficial effects on the reduction of prostate weight in a rat model of benign prostatic hyperplasia (BPH). BPH was induced by intramuscular injections of testosterone enanthate once a week for 5 weeks in Sprague-Dawley rats, and rats were treated with or without daily oral doses of SE during BPH induction. After 5 weeks, the oxidative stress (superoxide dismutase and 8-hydroxy-2-deoxyguanosine), apoptosis (caspase-3), and activity of 5-alpha reductase were evaluated in the serum and prostate. The SE treatment group showed a significant decrease in prostate weight, oxidative stress, apoptosis, and 5-alpha reductase activity compared to the nontreated BPH group. These results show that SE is effective in decreasing the weight and proliferation of the prostate, and suggest that SE may be an effective treatment for BPH.

Inhibitory effects of acorn extract on glutamate-induced calcium signaling in cultured rat hippocampal neurons.

Various effects of acorn extract have been reported including antioxidant activity, cytotoxicity against cancer cells, and the levels of acetylcholine and its related enzyme activities in the dementia mouse models. However, it is unclear whether acorn extract inhibits glutamate-induced calcium signaling in hippocampal neurons. This study was an investigation into the effect of acorn extract on intracellular free Ca concentrations ([Ca]) in cultured rat hippocampal neurons using fura-2-based digital calcium imaging and photometry. Hippocampal neurons were used between 10 and 14 d in culture from embryonic day-18 rats. Treatment with acorn extract (1 µg/mL to 1 mg/mL) for 30 min inhibited glutamate (100 µM)-induced [Ca] increases in a dose-dependent manner (IC=46.9 µg/mL). After depletion of intracellular Ca stores by treatment with the inhibitor endoplasmic reticulum Ca-ATPase, thapsigargin (1 µM), treatment with acorn extract (50 µg/mL) for 30 min decreased the subsequent glutamate-induced [Ca] increases. Acorn extract significantly inhibited (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (30 µM)-induced [Ca] increases. In addition, acorn extract inhibited the AMPA-induced [Ca] responses in the presence of 1 µM nimodipine. Acorn extract also significantly inhibited N-methyl-D-aspartate (100 µM)-induced [Ca] increases. Acorn extract significantly inhibited 50 mM KCl -induced [Ca] increases. Acorn extract significantly inhibited (S)-3,5-dihydroxyphenylglycine-induced [Ca] responses. Moreover, acorn extract almost completely blocked synaptically mediated [Ca] spikes induced by decreasing extracellular Mg concentration to 0.1 mM. These results suggest that acorn extract inhibits synaptically induced frequent [Ca] spikes through multiple pathways such as ionotropic glutamate receptors, voltage-gated Ca channels and metabotropic glutamate receptors in cultured rat hippocampal neurons.

Grape seed proanthocyanidin extract inhibits glutamate-induced cell death through inhibition of calcium signals and nitric oxide formation in cultured rat hippocampal neurons.

BACKGROUND: Proanthocyanidin is a polyphenolic bioflavonoid with known antioxidant activity. Some flavonoids have a modulatory effect on [Ca²âº]i. Although proanthocyanidin extract from blueberries reportedly affects Ca²âº buffering capacity, there are no reports on the effects of proanthocyanidin on glutamate-induced [Ca²âº]i or cell death. In the present study, the effects of grape seed proanthocyanidin extract (GSPE) on glutamate-induced excitotoxicity was investigated through calcium signals and nitric oxide (NO) in cultured rat hippocampal neurons. RESULTS: Pretreatment with GSPE (0.3-10 µg/ml) for 5 min inhibited the [Ca²âº]i increase normally induced by treatment with glutamate (100 µM) for 1 min, in a concentration-dependent manner. Pretreatment with GSPE (6 µg/ml) for 5 min significantly decreased the [Ca²âº]i increase normally induced by two ionotropic glutamate receptor agonists, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). GSPE further decreased AMPA-induced response in the presence of 1 µM nimodipine. However, GSPE did not affect the 50 mM K+-induced increase in [Ca²âº]i. GSPE significantly decreased the metabotropic glutamate receptor agonist (RS)-3,5-Dihydroxyphenylglycine-induced increase in [Ca²âº]i, but it did not affect caffeine-induced response. GSPE (0.3-6 µg/ml) significantly inhibited synaptically induced [Ca²âº]i spikes by 0.1 mM [Mg²âº]o. In addition, pretreatment with GSPE (6 µg/ml) for 5 min inhibited 0.1 mM [Mg²âº]o- and glutamate-induced formation of NO. Treatment with GSPE (6 µg/ml) significantly inhibited 0.1 mM [Mg²âº]o- and oxygen glucose deprivation-induced neuronal cell death. CONCLUSIONS: All these data suggest that GSPE inhibits 0.1 mM [Mg²âº]o- and oxygen glucose deprivation-induced neurotoxicity through inhibition of calcium signals and NO formation in cultured rat hippocampal neurons.

Phospholipase C, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and redox state are involved in epigallocatechin gallate-induced phospholipase D activation in human astroglioma cells.

We show that epigallocatechin-3 gallate (EGCG), a major component of green tea, stimulates phospholipase D (PLD) activity in U87 human astroglioma cells. EGCG-induced PLD activation was abolished by the phospholipase C (PLC) inhibitor and a lipase inactive PLC-gamma1 mutant, which is dependent on intracellular or extracellular Ca(2+), with the possible involvement of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). EGCG induced translocation of PLC-gamma1 from the cytosol to the membrane and PLC-gamma1 interaction with PLD1. EGCG regulates the activity of PLD by modulating the redox state of the cells, and antioxidants reverse this effect. Moreover, EGCG-induced PLD activation was reduced by PKC inhibitors or down-regulation of PKC. Taken together, these results show that, in human astroglioma cells, EGCG regulates PLD activity via a signaling pathway involving changes in the redox state that stimulates a PLC-gamma1 [Ins(1,4,5)P(3)-Ca(2+)]-CaM kinase II-PLD pathway and a PLC-gamma1 (diacylglycerol)-PKC-PLD pathway.

Epigallocatechin-3-gallate increases intracellular [Ca2+] in U87 cells mainly by influx of extracellular Ca2+ and partly by release of intracellular stores.

Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [3H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca2+] ([Ca2+]i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca2+]i. The EGCG-induced [Ca2+]i increases were reduced to 20.9% of control by removal of extracellular Ca2+. The increases were also inhibited markedly by treatment with the non-specific Ca2+ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca2+ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca2+-ATPase thapsigargin (1 micro M) also significantly inhibited the EGCG-induced [Ca2+]i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 micro M) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 micro M) had no effect. EGCG increased [3H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 micro M) and flufenamic acid (100 micro M), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca2+]i increase in non-treated and thapsigargin-treated cells but indomethacin (100 micro M) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca2+]i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca2+ and partly by mobilizing intracellular Ca2+ stores by PLC activation. The EGCG-induced [Ca2+]i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.

Protective effects of epicatechin against the toxic effects of streptozotocin on rat pancreatic islets: in vivo and in vitro.

INTRODUCTION: Green tea catechins have diverse pharmacological effects such as anticarcinogenic and antioxidant activities. AIM: To study the protective effects of green tea (-)-epicatechin (EC) against the toxic effects of streptozotocin (STZ), a selective beta cell toxin, on pancreatic islets in vivo and in vitro. METHODOLOGY: Rats were randomly divided into four groups: control, EC (30 mg/kg)-treated, STZ (60 mg/kg)-treated, and EC plus STZ (same doses; EC+STZ)-treated rats. EC was administered twice a day for 6 days, and a single injection of STZ was used. In EC+STZ-treated rats, EC was administered 6 hours prior to STZ since posttreatment with EC had no beneficial effects on fully developed diabetes in our unpublished study. Insulin and insulin mRNA were detected by immunohistochemical analysis and in situ hybridization, respectively, and physiologic parameters including blood glucose concentration were measured daily. Following isolation of the islets, insulin release, nitrite levels, and islet morphology were observed in the four groups: control, EC (0.8 mM)-treated, STZ (5 mM)-treated, and EC+STZ (same doses)-treated islets. RESULTS: In EC+STZ-treated rats, hyperglycemia and weight loss were not observed and islet morphology was well preserved compared with STZ-treated rats. Compared with STZ treatment alone, insulin release was increased and nitrite production was decreased in EC+STZ-treated islets. CONCLUSION: EC appears to be helpful in protecting pancreatic islets against exposure to STZ in both in vivo and in vitro systems.

alpha-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate-induced phospholipase D activation in human embryonic kidney-293 cells.

alpha-Synuclein has been implicated in the pathogenesis of many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Although the function of alpha-synuclein remains largely unknown, recent studies have demonstrated that this protein can interact with phospholipids. To address the role of alpha-synuclein in neurodegenerative disease, we have investigated whether it binds phospholipase D (PLD) and affects PLD activity in human embryonic kidney (HEK)-293 cells overexpressing wild type alpha-synuclein or the mutant forms of alpha-synuclein (A53T, A30P) associated with Parkinson's disease. Tyrosine phosphorylation of alpha-synuclein appears to play a modulatory role in the inhibition of PLD, because mutation of Tyr(125) to Phe slightly increases inhibitory effect of alpha-synuclein on PLD activity. Treatment with pervanadate or phorbol myristate acetate inhibits PLD more in HEK 293 cells overexpressing alpha-synuclein than in control cells. Binding of alpha-synuclein to PLD requires phox and pleckstrin homology domain of PLD and the amphipathic repeat region and non-Abeta component of alpha-synuclein. Although biologically important, co-transfection studies indicate that the interaction of alpha-synuclein with PLD does not influence the tendency of alpha-synuclein to form pathological inclusions. These results suggest that the association of alpha-synuclein with PLD, and modulation of PLD activity, is biologically important, but PLD does not appear to play an essential role in the pathophysiology of alpha-synuclein.