Vasodilatory Effect of Alpinia officinarum Extract in Rat Mesenteric Arteries.

BACKGROUND: Alpinia officinarum (A. officinarum) is known to exhibit a beneficial effect for anti-inflammatory, anti-oxidant, and anti-hyperlipidemic effects. However, no sufficient research data are available on the cardiovascular effect of A. officinarum. Thus, in this study, we investigate whether A. officinarum extract has direct effects on vascular reactivity. METHODS: To examine whether A. officinarum extract affects vascular functionality, we measured isometric tension in rat mesenteric resistance arteries using a wire myograph. After arteries were pre-contracted with high-K+ (70 mM), phenylephrine (5 µM), or U46619 (1 µM), A. officinarum extract was treated. RESULTS: A. officinarum extract induced vasodilation in a concentration-dependent manner, and this effect was endothelium independent. To further investigate the mechanism, we incubated arteries in a Ca2+-free and high-K+ solution, followed by the cumulative addition of CaCl2 (0.01-2.5 mM) with or without A. officinarum extract (30 µg/mL). Pre-treatment of A. officinarum extract reduced the contractile responses induced by cumulative administration of Ca2+, which suggests that extracellular Ca2+ influx was inhibited by the treatment of A. officinarum extract. These results were associated with a reduction in phosphorylated MLC20 in VSMCs treated with A. officinarum extract. Furthermore, eucalyptol, an active compound of A. officinarum extract, had a similar effect as A. officinarum extract, which causes vasodilation in mesenteric resistance arteries. CONCLUSION: A. officinarum extract and its active compound eucalyptol induce concentration-dependent vasodilation in mesenteric resistance arteries. These results suggest that administration of A. officinarum extract could exert beneficial effects to treat high blood pressure.

Vasodilatory Effect of Phellinus linteus Extract in Rat Mesenteric Arteries.

Phellinus linteus is a well-known medicinal mushroom that is widely used in Asian countries. In several experimental models, Phellinus linteus extracts were reported to have various biological effects, including anti-inflammatory, anti-cancer, hepatoprotective, anti-diabetic, neuroprotective, and anti-angiogenic activity. In the present study, several bioactive compounds, including palmitic acid ethyl ester and linoleic acid, were identified in Phellinus linteus. The intermediate-conductance calcium-activated potassium channel (IKCa) plays an important role in the regulation of the vascular smooth muscle cells' (VSMCs) contraction and relaxation. The activation of the IKCa channel causes the hyperpolarization and relaxation of VSMCs. To examine whether Phellinus linteus extract causes vasodilation in the mesenteric arteries of rats, we measured the isometric tension using a wire myograph. After the arteries were pre-contracted with U46619 (a thromboxane analogue, 1 µM), Phellinus linteus extract was administered. The Phellinus linteus extract induced vasodilation in a dose-dependent manner, which was independent of the endothelium. To further investigate the mechanism, we used the non-selective K+ channel blocker tetraethylammonium (TEA). TEA significantly abolished Phellinus linteus extract-induced vasodilation. Thus, we tested three different types of K+ channel blockers: iberiotoxin (BKca channel blocker), apamin (SKca channel blocker), and charybdotoxin (IKca channel blocker). Charybdotoxin significantly inhibited Phellinus linteus extract-induced relaxation, while there was no effect from apamin and iberiotoxin. Membrane potential was measured using the voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)) in the primary isolated vascular smooth muscle cells (VSMCs). We found that the Phellinus linteus extract induced hyperpolarization of VSMCs, which is associated with a reduced phosphorylation level of 20 KDa myosin light chain (MLC20).

Protective Effects of Radish Extract Against Neurotoxicity in Mice and PC12 Cells.

A variety of natural compounds have been used to reduce the oxidative stress associated with Alzheimer's disease, and many of these defend cells from oxidative stress-induced neuronal toxicity. In this study, the protective effect of radish (Raphanus raphanistrum) extract was investigated in mice and PC12 cells. In vivo behavioral tests were completed to examine the protective effects of the extract on amyloid beta (Aß)-peptide1-42-induced learning and memory impairment in a mouse model. The extract increased spontaneous alternation behaviors and step-through latency in mice. We discovered that administration of the extract reduced lipid peroxidation and Aß aggregation in a biochemical study of mice brain tissues. Treatment with the extract also increased acetylcholine and catalase activity in the brain. Furthermore, the radish extract attenuated H2O2-induced oxidative stress in cells. Through sequential fractionation of the radish extract, the active compound was identified as oleamide. These results suggest that the radish extract could have a protective role against oxidative stress-induced neuronal toxicity, possibly owing to the antioxidative activity of oleamide.

Separation of nine novel triterpene saponins from Camellia japonica seeds using high-performance countercurrent chromatography and reversed-phase high-performance liquid chromatography.

INTRODUCTION: Camellia japonica L. (Theaceae) is an evergreen shrub, which is cultivated as a popular ornamental tree in Korea, China, and Japan and its seeds have been used as a source of cooking oil, in cosmetics and as a traditional medicine. Intensive phytochemical works have revealed that oleanane-type saponins are the characteristic compounds of the seeds of C. japonica. OBJECTIVE: The purpose of the present study is to isolate and determine oleanane-type saponins from C. japonica using high-performance countercurrent chromatography (HPCCC) coupled with reversed-phase high-performance liquid chromatography (RP-HPLC) and spectroscopic evidences, respectively. METHODOLOGY: HPLC electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) was applied to profile the saponin composition of an enriched saponin extract of C. japonica seeds. The enriched saponin extract was separated by HPCCC using a dichloromethane/methanol/isopropanol/water (9:6:1:4, v/v/v/v) system and RP-HPLC. The structures of the isolates were determined utilising ESI-Q-TOF-MS, one-dimensional and two-dimensional NMR and optical rotation. RESULTS: HPCCC on enriched saponin extract of C. japonica yielded four saponin fractions in the order of the number of sugars attached to the triterpene aglycone, and preparative RP-HPLC on each saponin fraction led to the isolation of nine novel saponins, namely camoreoside A-I, along with six known ones. CONCLUSIONS: This study indicates that combination of HPLC-ESI-Q-TOF-MS analysis and HPCCC coupled with RP-HPLC are excellent tools for discovering saponins from natural sources.

Synergistic effect of Korean red ginseng and Pueraria montana var. lobata against trimethyltin-induced cognitive impairment.

Many edible plant extracts exhibit biological activities. For example, the ethanol extract of Pueraria montana var. lobata (P. montana) inhibits acetylcholinesterase (AChE), and red ginseng is well known for promoting health. In this study the authors investigated the synergistic effect of P. montana and red ginseng extracts on AChE activity in vitro and in mouse brain tissues and trimethyltin (TMT)-induced cognitive impairment in a mouse model of TMT-induced neurodegeneration. A diet containing a mixture of P. montana and red ginseng extracts reversed learning and memory impairments in Y-maze and passive avoidance behavioral tests. In addition, the mixture inhibited AChE activity and lipid peroxidation synergistically.

Erucamide from Radish Leaves Has an Inhibitory Effect Against Acetylcholinesterase and Prevents Memory Deficit Induced by Trimethyltin.

In this study, we investigated a potent acetylcholinesterase inhibitor that was isolated from radish leaf (Raphanus sativus L.) extracts. Through sequential fractionation of radish leaf extract, the active constituent was identified as cis-13-docosenamide (erucamide). To validate the potency, erucamide derived from radish leaves was supplemented in diets and then fed to trimethyltin (TMT)-exposed mice. Specifically, mice had free access to a control diet or diets containing different concentrations of erucamide for 3 weeks, followed by an injection of TMT (2.5 mg/kg body weight). Our results showed that pretreatment of mice with erucamide (20 and 40 mg/kg body weight per day) significantly attenuated the TMT-induced learning and memory deficits that were assessed by Y-maze and passive avoidance tests. These findings suggest that radish leaves, and possibly its isolated erucamide, may have preventive effects against memory deficits related to Alzheimer's disease by modulation of cholinergic functions.

Cinnamomum loureirii Extract Inhibits Acetylcholinesterase Activity and Ameliorates Trimethyltin-Induced Cognitive Dysfunction in Mice.

The pathogenesis of Alzheimer's disease (AD) has been linked to the deficiency of neurotransmitter acetylcholine (ACh) in the brain, and the main treatment strategy for improving AD symptoms is the inhibition of acetylcholinesterase (AChE) activity. In the present study, we aimed to identify potent AChE inhibitors from Cinnamomum loureirii extract via bioassay-guided fractionation. We demonstrated that the most potent AChE inhibitor present in the C. loureirii extract was 2,4-bis(1,1-dimethylethyl)phenol. To confirm the antiamnesic effects of the ethanol extract of C. loureirii, mice were intraperitoneally injected with the neurotoxin trimethyltin (2.5 mg/kg) to induce cognitive dysfunction, and performance in the Y-maze and passive avoidance tests was assessed. Treatment with C. loureirii extract significantly improved performance in both behavioral tests, suggesting that this extract may be neuroprotective and therefore beneficial in preventing or ameliorating the degenerative processes of AD, potentially by restoring cholinergic function.

Perilla frutescens Extract Ameliorates Acetylcholinesterase and Trimethyltin Chloride-Induced Neurotoxicity.

One of the critical features of Alzheimer's disease is cognitive dysfunction, which is, in part, due to decreases in acetylcholine (ACh). The ethanol extract of Perilla frutescens was selected for isolating the acetylcholinesterase (AChE) inhibitor based on preliminary screening. In vivo behavioral tests were performed to examine the effects of the P. frutescens extract on trimethyltin chloride-induced impairment of learning and memory in mice. A diet containing P. frutescens extract effectively reversed learning and memory impairment on the Y-maze and passive avoidance tests. To isolate the active compound from the P. frutescens extract, solvent partitioning, silica gel open column chromatography, thin-layer chromatography, and high-performance liquid chromatography were used. The AChE inhibitor was identified as rosmarinic acid.

Effect of Chaenomeles sinensis Extract on Choline Acetyltransferase Activity and Trimethyltin-Induced Learning and Memory Impairment in Mice.

The aim of this study was to search for a novel choline acetyltransferase (ChAT) activator from plants traditionally grown in Korea. An ethanol extract from Chaenomeles sinensis Koehne showed the highest ChAT-activating effect in vitro in an assay that used human neuroblastoma cells and [(14)C]acetyl-CoA. The active compound was speculated to be stearic acid methyl ester (SAME). In an in vivo experiment, C. sinensis extract and SAME improved trimethyltin (TMT)-induced deficits in learning and memory in mice as assessed by a Y-maze behavioral test and a passive avoidance test. The C. sinensis extract might attenuate the TMT-induced brain disorder. This study suggests that SAME from C. sinensis might be useful in the treatment of Alzheimer's disease.

Separation of polyphenols and caffeine from the acetone extract of fermented tea leaves (Camellia sinensis) using high-performance countercurrent chromatography.

Leaves from Camellia sienensis are a popular natural source of various beverage worldwide, and contain caffeine and polyphenols derived from catechin analogues. In the current study, caffeine (CAF, 1) and three tea polyphenols including (-)-epigallocatechin 3-O-gallate (EGCg, 2), (-)-gallocatechin 3-O-gallate (GCg, 3), and (-)-epicatechin 3-O-gallate (ECg, 4) were isolated and purified by flow-rate gradient high-performance countercurrent chromatography (HPCCC) using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:9:1:9, v/v). Two hundred milligrams of acetone-soluble extract from fermented C. sinensis leaves was separated by HPCCC to give 1 (25.4 mg), 2 (16.3 mg), 3 (11.1 mg) and 4 (4.4 mg) with purities over 98%. The structures of 1-4 were elucidated by QTOF-MS, as well as 1H- and 13C-NMR, and the obtained data were compared to the previously reported values.

Two new phenolic glucosides from Lagerstroemia speciosa.

Two new phenolic glucosides, 1-O-benzyl-6-O-E-caffeoyl-ß-d-glucopyranoside and 1-O-(7S,8R)-guaiacylglycerol-(6-O-E-caffeoyl)-ß-d-glucopyranoside, were isolated from the aerial parts of Lagerstroemia speciosa, along with ten known compounds. The structures of the isolated compounds were determined based on 1D- and 2D-NMR, Q-TOF MS and optical rotation spectroscopic data. All of the compounds showed moderate inhibitory activities against nitric oxide production in lipopolysaccharide-treated RAW264.7 cells, with IC50 values of 69.5-83.3 µM.

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.

Application of high-performance countercurrent chromatography for the isolation of steroidal saponins from Liriope plathyphylla.

High-performance countercurrent chromatography (HPCCC) with electrospray light-scattering detection was applied for the first time to isolate a spirostanol and a novel furostanol saponin from Liriope platyphylla. Due to the large differences in KD values between the two compounds, a two-step HPCCC method was applied in this study. The primary HPCCC employed methylene chloride/methanol/isopropanol/water (9:6:1:4 v/v, 4 mL/min, normal-phase mode) conditions to yield a spirostanol saponin (1). After the primary HPCCC run, the solute retained in the stationary phase (SP extract) in HPCCC column was recovered and subjected to the second HPCCC on the n-hexane/n-butanol/water system (1:9:10 v/v, 5 mL/min, reversed-phase mode) to yield a novel furostanol saponin (2). The isolated spirostanol saponin was determined to be 25(S)-ruscogenin 1-O-ß-D-glucopyranosyl (1→2)-[ß-D-xylopyranosyl (1→3)]-ß-D-fucopyranoside (spicatoside A), and the novel furostanol saponin was elucidated to be 26-O-ß-D-glucopyranosyl-25(S)-furost-5(6)-ene-1ß-3ß-22α-26-tetraol-1-O-ß-D-glucopyranosyl (1→2)-[ß-D-xylopyranosyl-(1→3)]-ß-D-fucopyranoside (spicatoside D).

Ligularia fischeri extract protects against oxidative-stress-induced neurotoxicity in mice and PC12 cells.

Alzheimer's disease (AD) is pathologically characterized by the presence of amyloid plaques in brain and the overproduction of amyloid beta (Aß), leading to learning and memory impairment and intense oxidative stress. In this study, the protective effect of Ligularia fischeri extract was investigated using PC12 cells. L. fischeri extract attenuated hydrogen-peroxide-induced DNA fragmentation in cells. In vivo behavioral tests were performed to examine the effects of the extract on amyloid-ß peptide1-42-induced impairment of learning and memory in mice. A diet containing the extract increased alternation behaviors in the Y-maze test and step-through latency of passive avoidance task. Moreover, we found that consumption of the extract decreased lipid peroxidation in a biochemical study of brain tissue in mice. High-performance liquid chromatography was used to identify the active compounds in the extract. These results suggest that L. fischeri extract could be protective against Aß-induced neurotoxicity, possibly due to the antioxidative capacity of its constituent, 3-O-caffeoylquinic acid.

Effects of brussels sprouts and their phytochemical components on oxidative stress-induced neuronal damages in PC12 cells and ICR mice.

In this study, the protective effects of Brussels sprouts extract and its major constituents against oxidative stress-induced damages were investigated in rat pheochromocytoma cells and Institute of Cancer Research mice. The major constituents of Brussels sprouts (3,4',5,7-tetrahydroxyflavone (kaempferol), indole-3-carbinol, and phenethyl isothiocyanate) were selectively tested. Of these, the flavonoid compound, kaempferol exhibited the highest potency in radical scavenging activity (1,1-diphenyl-2-picryl hydrazyl assay and oxygen radical absorbance capacity assay) and was most protective against oxidative stress in neuronal cell assays (measurement of intracellular oxidative stress levels and cell viability). In mice, after 4 weeks of kaempferol administration, significant protection against amyloid beta (Aß) peptide-induced neurotoxicity was also observed, as assessed through the passive avoidance test. Taken together, the results suggest that Brussels sprouts could be protective against Aß-induced neurotoxicity, possibly due to the antioxidative capacity of its major constituent, kaempferol.

2,4-Di-tert-butylphenol from sweet potato protects against oxidative stress in PC12 cells and in mice.

In this study, the protective effect of sweet potato extract against hydrogen peroxide-induced oxidative stress and cytotoxicity on the pheochromocytoma cell line (PC12) was investigated. The active component of the sweet potato extract was purified and determined to be 2,4-di-tert-butylphenol. The antioxidant capacity of 2,4-di-tert-butylphenol was measured by using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical. To examine the effects of 2,4-di-tert-butylphenol on amyloid-beta peptide (Aß11₋42)-induced learning and memory impairment in mice, in vivo behavioral tests were performed. Administration of 2,4-di-tert-butylphenol increased alternation behavior in mice injected with Aß1₋42. These results suggest that sweet potato extract could be protective against Aß-induced neurotoxicity, possibly due to the antioxidative capacity of its constituent, 2,4-di-tert-butylphenol.

Rubus coreanus Miquel inhibits acetylcholinesterase activity and prevents cognitive impairment in a mouse model of dementia.

To find acetylcholinesterase (AChE) inhibitors for the prevention of neurological disorders, such as Alzheimer's disease, ethanol extracts of promising traditional edible Korean plants were tested. Among them, Rubus coreanus Miquel extract exhibited the most significant AChE inhibitory activity. The effect of R. coreanus extract on trimethyltin-induced memory impairment in mice was investigated using Y-maze and passive avoidance tests. Our results showed that administration of R. coreanus extract significantly improved alternation behavior and step-through latency. In addition, R. coreanus extract was sequentially fractionated, and the purified constituent was determined to be 3,4,5-trihydroxybenzoic acid.

Antiamnesic effects of ethyl acetate fraction from chestnut (Castanea crenata var. dulcis) inner skin on Aß(25-35)-induced cognitive deficits in mice.

To investigate neuronal cell protective effects of an ethyl acetate fraction from chestnut inner skin, in vitro assays, including 2',7'-dichlorofluorescein diacetate, 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), and lactate dehydrogenase (LDH), were performed. Intracellular accumulation of reactive oxygen species resulting from hydrogen peroxide (H(2)O(2)) treatment of PC12 cells was significantly reduced when ethyl acetate fractions were present in the medium compared to PC12 cells treated with H(2)O(2) only. In a cell viability assay using MTT, the ethyl acetate fraction protected against H(2)O(2)-induced neurotoxicity, and inhibited LDH release into the medium. In addition, the ethyl acetate fraction improved in vivo cognitive ability against amyloid ß-peptide (Aß)-induced neuronal deficit. High-performance liquid chromatography analyses showed that gallic acid, catechin, and epicatechin were predominant phenolics in the ethyl acetate fraction. Consequently, the results suggest that chestnut inner skin, including above phenolics, could ameliorate Aß-induced learning and memory deficiency, and be utilized as effective substances for neurodegenerative disorders, notably Alzheimer's disease.

Effects of methoxsalen from Poncirus trifoliata on acetylcholinesterase and trimethyltin-induced learning and memory impairment.

Previously, we identified methoxsalen (8-methoxy-2',3',6,7-furocoumarin) as the bioactive compound probably responsible for acetylcholinesterase (AchE) inhibition achieved by feeding crude extract of Poncirus trifoliate. To confirm the activity of methoxsalen, Institute of Cancer Research (ICR) mice were fed a control or a methoxsalen-supplemented diet for 4 weeks, and then learning and memory enhancing effects with respect to trimethyltin (TMT)-induced neurotoxicity were evaluated. The brain tissues of ICR mice were dissected after completion of the behavioral tests for biochemical analysis. Methoxsalen effectively reversed TMT-induced memory impairment on both Y-maze and passive avoidance tests. Brain AchE activity was inhibited by the oral consumption of all concentrations of methoxsalen. Moreover, the level of oxidative stress was significantly ameliorated in the groups on methodsalen containing diets. This is the first in vivo study conducted with methoxsalen in the field of AD research, and it indicates that further investigation of methoxsalen is warranted.

Punica granatum protects against oxidative stress in PC12 cells and oxidative stress-induced Alzheimer's symptoms in mice.

Alzheimer's disease (AD) is a progressive degenerative brain disorder that is characterized by neuronal loss, neurofibrillary tangles, and the abnormal deposition of senile plaque and amyloid ß peptide (Aß). The brains of AD patients are under intense oxidative stress. The overproduction of Aß leads to Aß-associated free radical oxidative stress. In this study, the antioxidative and neuronal protective effects of Punica granatum extract were investigated against oxidative stress-induced cytotoxicity in PC12 cells. The ethanol extracts of P. granatum protected PC12 cells from hydrogen peroxide (H2O2-induced oxidative stress. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assays revealed a significant increase in cell viability when oxidatively stressed PC12 cells were treated with the P. granatum extract. To examine the effects of P. granatum on Aß1₋42-induced learning and memory impairment in mice, in vivo behavioral tests were performed. Treatment with the extract of P. granatum increased step-through latency in mice injected with Aß1₋42. The results of this study suggest that the ethanol extract of P. granatum mitigated H2O2-induced oxidative stress in PC12 cells. In addition, the extract inhibited neuronal cell death caused by Aß-induced oxidative stress and Aß-induced learning and memory deficiency.