Isolation of Vacuoles from the Leaves of the Medicinal Plant Catharanthus roseus.

The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H+-ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H+-ATPase and the V-H+-pyrophosphatase, also producing further information on vacuole purity.

Antioxidant potentiality of three herbal teas consumed in Bandundu rural areas of Congo.

The aim of this study was to evaluate and compare the cellular antioxidant activities of Lantana montevidensis, Lippia multiflora, and Ocimum gratissimum leaves often consumed as herbal teas in a rural area of Bandundu severely affected by konzo, which is related to oxidative damage. Consequently, dietary supplements with proven antioxidant potentialities could be of real interest to promote in this area. Phytochemical screening by TLC and HPLC-DAD of extracts revealed the presence of verbascoside as a major phenolic compound. Verbascoside in L. montevidensis and O. gratissimum is reported here for the first time. All extracts displayed high ABTS and DPPH radical-scavenging activities at the concentration range of 1-40 µg mL-1 according to order: L. multiflora > O. gratissimum > L. montevidensis. L. multiflora showed the best cellular antioxidant activity using DCFH-DA on HL-60 monocytes assay at 1-20 µg mL-1. These herbal teas may be used as nutraceuticals for their potent antioxidant activity.

Perihematomal Cellular Injury Is Reduced by Trans-sodium Crocetinate in a Model of Intracerebral Hemorrhage.

The carotenoid compound trans-sodium crocetinate (TSC) has been shown to increase oxygenation in various tissues, including the brain. Notably, TSC can enhance oxygenation under conditions of reduced blood flow, thus attenuating the depth of an ischemic challenge. This study examined the impact of TSC on neuronal loss in an animal model of intracerebral hemorrhage (ICH). Utilizing a rat model of collagenase injection, TSC was shown to reduce perihematomal cellular loss after ICH, as assessed by Fluoro-Jade B staining in tissue sections. This is the first evidence demonstrating that TSC is capable of limiting hemorrhagic injury to neurons in the brain. The finding supports the concept that TSC may represent a candidate therapeutic for early intervention regardless of whether a stroke is hemorrhagic or ischemic in nature.

Screening of epidermal growth factor receptor inhibitors in natural products by capillary electrophoresis combined with high performance liquid chromatography-tandem mass spectrometry.

A method for screening of inhibitors to epidermal growth factor receptor (EGFR) in natural product extracts with capillary electrophoresis (CE) in conjunction with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is reported. The method was established by employing 5-carboxyfluorescein labeled substrate peptide, two commercially available EGFR inhibitors OSI-744 and ZD1839, and a small chemical library consisted of 39 natural product extracts derived from the Traditional Chinese Medicines. Biochemical assay of crude natural product extracts was carried out by using CE equipped with a laser induced fluorescence detector. The CE separation allowed an accurately quantitative measurement of the phosphorylated product, hence the measurement of the enzymatic activity as well as the inhibition kinetics. The hits are identified if the peak area of the phosphorylated product is reduced in comparison with the negative control. The active constituents in the natural product extract were then identified by an assay-guided isolation with HPLC-MS/MS system. With the method, the flavonoids component of the Lycopus lucidus extract, namely quercetin and rutin were identified to be the active ingredients. Their IC50 values were determined as 0.88 µM and 10.1 µM, respectively. This result demonstrated a significant merit of our method in the identification of the bioactive compounds in natural products.

Desferrioxamine-caffeine (DFCAF) as a cell permeant moderator of the oxidative stress caused by iron overload.

Desferrioxamine (DFO) is a potent iron chelator used in the treatment of iron overload (IO) disorders. However, due to its low cell permeability and fast clearance, DFO administration is usually prolonged and of limited use for the treatment of IO in tissues such as the brain. Caffeine is a safe, rapidly absorbable molecule that can be linked to other compounds to improve their cell permeability. In this work, we successfully prepared and described DFO-caffeine, a conjugate with iron scavenging ability, antioxidant properties and enhanced permeation in the HeLa cell model.

Flavonostilbenes from Sophora alopecuroides L. as multidrug resistance associated protein 1 (MRP1) inhibitors.

Flavonoids have always attracted much attention due to their reversal activity on multidrug resistance (MDR). Eight flavonoids isolated from traditional Chinese medicine Sophora alopecuroides L. were applied to test their effect on MDR associated protein 1 (MRP1) through the established predicting assay. Three flavonostilbenes (alopecurone A, B and D) were first found exhibiting potent inhibitory activity on MRP1. All of them dramatically increased 6-carboxyfluorescein diacetate and doxorubicin accumulation in MRP1-transfected U-2 OS cells. The compounds significantly increased the cytotoxicity and decreased the IC50 value of doxorubicin on the MDR cells (12-, 5- and 8-fold, respectively) at a non-toxic concentration (20 µM). Besides, Q-PCR analysis reveals that the MRP1 mRNA level in U-2 OS/MRP1 was also markedly decreased by the three compounds. These findings indicate a new therapeutic role of the herb. The three flavonostilbenes may have the possibility for further development as novel therapeutic reversal agents against MDR.

The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside.

The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine=12.240 mg/g, theobromine=6.733 mg/g and total catechins=4.336 mg/g. Cells were exposed to 10 µM SNP during a 6 h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20 mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5 mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels.

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.

Resveratrol attenuates L-DOPA-induced hydrogen peroxide toxicity in neuronal cells.

A variety of polyphenol antioxidant compounds derived from natural products have demonstrated neuroprotective activity against neuronal cell death. The objective of this study was to investigate the effect of resveratrol (RESV) and bioflavonoids in attenuating hydrogen peroxide (H(2)O(2))-induced oxidative stress in neuronal cells. H2O2 levels were increased by the addition of L-3,4-dihydroxyphenylalanine (L-DOPA) to cultured dopaminergic SKNSH cells. H(2)O(2) was monitored by peroxyfluor-1, a selective H(2)O(2) optical probe. To examine the neuroprotective effects of RESV and bioflavonoids against L-DOPA, we cotreated RESV, quercetin, or (-) epigallocatechin gallate with L-DOPA and monitored for H(2)O(2) levels. The combination of RESV and L-DOPA was 50% more effective at reducing H(2)O(2) levels than the combination of quercetin or epigallocatechin gallate with L-DOPA. However, the combination of each antioxidant with L-DOPA was effective at preserving cell viability.

Low-power 808-nm laser irradiation inhibits cell proliferation of a human-derived glioblastoma cell line in vitro.

It has been reported that low-power laser irradiation (LLI) can modulate various biological processes including cell proliferation. Some reports suggest that LLI interferes with the cell cycle and inhibits cell proliferation, while others suggest that LLI has a stimulatory effect. Mechanisms underlying the effects of LLI remain unclear. Since the effects of LLI on cancer cells are not well understood, with the aim of developing an LLI therapy for malignant glioblastoma, we investigated the effects of LLI on the cell proliferation of the human-derived glioblastoma cell line A-172. Glioblastoma cell cultures were irradiated with a diode laser at a wavelength of 808 nm and the effects on cell viability and proliferation were examined. Cell counting at 24 and 48 h after irradiation showed that LLI (at 18, 36 and 54 J/cm(2)) suppressed proliferation of A-172 cells in a fluence-dependent manner (irradiation for 20, 40 and 60 min). A reduction in the number of viable cells was also demonstrated by a fluorescent marker for viable cells, calcein acetoxymethylester (calcein-AM). The reduction in cell viability was not associated with morphological changes in the cells or with necrotic cell death as demonstrated by propidium iodide staining. LLI also had little effect on cell proliferation as shown by 5-bromo-2'-deoxyuridine staining. We discuss possible mechanisms underlying the suppressive effect of 808-nm LLI on the viability of human-derived glioblastoma A-172 cells.

A high throughput in vitro mrp2 assay to predict in vivo biliary excretion.

Prediction of biliary excretion is a challenge for drug discovery scientists due to the lack of in vitro assays. This study explores the possibility of establishing a simple assay to predict in vivo biliary excretion via the mrp2 transport system. In vitro mrp2 activity was determined by measuring the ATP-dependent uptake of 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) in canalicular plasma membrane vesicles (cLPM) from rat livers. The CDCF uptake was time- and concentration-dependent (K(m) of 2.2 ± 0.3 µM and V(max) of 115 ± 26 pmol/mg/min) and strongly inhibited by the mrp2 inhibitors, benzbromarone, MK-571, and cyclosporine A, with IC(50) values ≤ 1.1 µM. Low inhibition of CDCF uptake by taurocholate (BSEP inhibitor; 57 µM) and digoxin (P-gp inhibitor; 101 µM) demonstrated assay specificity towards mrp2. A highly significant correlation (r(2) = 0.959) between the in vitro IC(50) values from the described mrp2 assay and in vivo biliary excretion in rats was observed using 10 literature compounds. This study demonstrated, for the first time, that a high throughput assay could be established with the capability of predicting biliary excretion in the rat using CDCF as a substrate.

Rhinacanthus nasutus protects cultured neuronal cells against hypoxia induced cell death.

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) is an herb native to Thailand and Southeast Asia, known for its antioxidant properties. Hypoxia leads to an increase in reactive oxygen species in cells and is a leading cause of neuronal damage. Cell death caused by hypoxia has been linked with a number of neurodegenerative diseases including some forms of dementia and stroke, as well as the build up of reactive oxygen species which can lead to diseases such as Huntington's disease, Parkinson's disease and Alzeheimer's disease. In this study we used an airtight culture container and the Mitsubishi Gas Company anaeropack along with the MTT assay, LDH assay and the trypan blue exlusion assay to show that 1 and 10 µg mL⁻¹ root extract of R. nasutus is able to significantly prevent the death of HT-22 cells subjected to hypoxic conditions, and 0.1 to 10 µg mL⁻¹ had no toxic effect on HT-22 under normal conditions, whereas 100 µg mL⁻¹ reduced HT-22 cell proliferation. We also used H2DCFDA staining to show R. nasutus can reduce reactive oxygen species production in HT-22 cells.

Symplastic connection is required for bud outgrowth following dormancy in potato (Solanum tuberosum L.) tubers.

To gain greater insight into the mechanism of dormancy release in the potato tuber, an investigation into physiological and biochemical changes in tuber and bud tissues during the transition from bud dormancy (immediately after harvest) to active bud growth was undertaken. Within the tuber, a rapid shift from storage metabolism (starch synthesis) to reserve mobilization within days of detachment from the mother plant suggested transition from sink to source. Over the same period, a shift in the pattern of [U-(14)C]sucrose uptake by tuber discs from diffuse to punctate accumulation was consistent with a transition from phloem unloading to phloem loading within the tuber parenchyma. There were no gross differences in metabolic capacity between resting and actively growing tuber buds as determined by [U-(14)C]glucose labelling. However, marked differences in metabolite pools were observed with large increases in starch and sucrose, and the accumulation of several organic acids in growing buds. Carboxyfluorescein labelling of tubers clearly demonstrated strong symplastic connection in actively growing buds and symplastic isolation in resting buds. It is proposed that potato tubers rapidly undergo metabolic transitions consistent with bud outgrowth; however, growth is initially prevented by substrate limitation mediated via symplastic isolation.

Five essential oils from aromatic plants of Cameroon: their antibacterial activity and ability to permeabilize the cytoplasmic membrane of Listeria innocua examined by flow cytometry.

AIMS: To investigate the antibacterial effect of five essential oils (EO) extracted from aromatic plants (Cymbopogon citratus, Ocimumbasilicum, Ocimum gratissimum, Thymus vulgaris and Zingiber officinale) of Cameroon against strains of Listeria monocytogenes, L. innocua and Staphylococcus aureus. The ability of selected EO to permeabilize the cytoplasmic membrane of L. innocua was also examined. METHODS AND RESULTS: The antibacterial activity of the EO determined by the agar diffusion method showed that T. vulgaris had the highest activity followed by O. gratissimum and C. citratus. Lowest activity was recorded from Z. officinale and O. basilicum. Significant differences in sensitivity between strains of Listeria and S. aureus were observed. Flow cytometry of L. innocua stained with carboxy-fluorescein diacetate showed that the fluorescence intensity of cells exposed to EO decreased faster than nonexposed cells, indicating that EO permeabilized the cytoplasmic membrane with the leakage of carboxy-fluorescein. CONCLUSIONS: Almost all the EO tested showed antibacterial activity to a different extent. The antibacterial effect was due to permeabilization of the cytoplasmic membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has identified the preservative potential of the EO examined. The use of sensitive method, such as flow cytometry, is advantageous for quick generation of data on the antibacterial effect of EO.

Intestinal transmission of macromolecules in newborn dairy calves of different ages at first feeding.

Four groups of eight newborn calves were used to study the intestinal transmission of colostral immunoglobulin from the intestinal lumen to the blood circulation. The first feed was given one, eight, 16 or 24 hours after birth. Thereafter, three feeds were given with eight hour intervals. All feeds were from the same pool of colostrum and the amount fed each time corresponded to 3 per cent of the calves birthweight. To estimate the transmission of macromolecules in each feed four different macromolecules were used as markers. For the first feed, the marker was bovine IgG, in the second FITC-dextran, in the third ovalbumin and in the fourth human serum albumin. Blood samples were taken eight hours after each feed and at one week old. There were no differences in transmission for the first feed although the calves varied in age between one and 24 hours, but in the second, third and fourth feeds the calves that received a first feed at one hour old, transmitted significantly more of the marker molecules than did the other three groups. The substantial transmission of macromolecules at the first feed in all four groups indicates that a base level of transmission capacity is maintained during the first 24 hours or longer and that, under certain conditions, acceptable passive immunisation is possible in calves given their first colostrum as late as 24 hours after birth.