Phytomedicine 18 (2011) Contents lists available at ScienceDirect. Phytomedicine. journal homepage:

Transcription

1 Phytomedicine 18 (2011) Contents lists available at ScienceDirect Phytomedicine journal homepage: In vitro protective effects of colon-available extract of Camellia sinensis (tea) against hydrogen peroxide and beta-amyloid (A (1 42) ) induced cytotoxicity in differentiated PC12 cells E.J. Okello a,, G.J. McDougall b, S. Kumar a,c, C.J. Seal a a Medicinal Plant Research Group, School of Agriculture, Food and Rural Development, Newcastle University, Newcastle upon Tyne, NE1 7RU, United Kingdom b Scottish Crop Research Institute (SCRI), Plant Products and Food Quality Programme, Mylnefield, Invergowrie, Dundee DD2 5DA, Scotland, United Kingdom c University School of Biotechnology, GGS Indraprastha University, Kashmere Gate, Delhi 11403, India article Keywords: Camellia sinensis Polyphenols Neuroprotection Hydrogen peroxide -Amyloid Cytotoxicity info abstract There is mounting evidence that the deposition and aggregation of -amyloid peptides (A ) in the brain play a significant role in the development and pathogenesis of Alzheimer s disease. There is further evidence that free radical species such as hydrogen peroxide (H 2 O 2 ) mediate A induced toxicity. Previous studies have demonstrated that green tea polyphenols possess neuroprotective properties through their ability to ameliorate oxidative stress induced by free radical species. Green tea polyphenols have also been shown to enhance cognition in various animal models of induced cognitive impairment. Upon ingestion, green tea polyphenols are metabolised and undergo bio-transformation which affects their bioavailability and therefore efficacy. In this study, a green tea extract was subjected to a simulated gastrointestinal digestion and a colon-available extract (CAGTE) prepared and assessed for its potential protective effects against H 2 O 2 and A (1 42) induced cytotoxicity using differentiated PC12 cells (dpc12) as a model for neuronal cells. CAGTE represents green tea phytochemicals potentially available after upper gastrointestinal digestion. CAGTE which was depleted in flavan-3-ols, as shown by LC MS analysis, protected dpc12 cells at concentration ranges of g/ml and g/ml for H 2 O 2 and A (1 42), induced cytotoxicity, respectively. At high concentrations, CAGTE exhibited direct anti-proliferative effects, in line with the reputed anti-cancer properties of green tea polyphenols. These results demonstrate that potentially bioavailable green tea metabolites are able to ameliorate both H 2 O 2 and A (1 42) induced cytotoxicity Published by Elsevier GmbH. Introduction Alzheimer s disease (AD), the most common form of dementia, is a progressive and irreversible neurodegenerative disorder associated with cognitive dysfunction. There is mounting evidence that the deposition and aggregation of -amyloid peptides (A ) inthe brain plays a significant role in the development and pathogenesis of AD (Murphy and Levine 2010). There is further evidence that free radical species such as hydrogen peroxide (H 2 O 2 ) mediate A induced toxicity (Behl et al. 1994). Tea (Camellia sinensis, family Theaceae) is the most commonly consumed beverage after water. Epidemiological and laboratory studies, albeit sometimes conflicting, suggest that both black and green teas possess many pharmacologically protective properties mainly attributed to their polyphenol content. Green tea contains more flavan-3-ols whereas in black tea these components undergo enzymatic oxidation into more complex forms called theaflavins and thearubigins (Del Rio et al. 2004). The flavan-3-ols, are said to be efficient scavengers of the highly reactive free radical species, and exhibit anti-carcinogenic (Yang et al. 2009); hypocholesterolaemic (Imai and Nakachi 1985) and neuroprotective (Lee et al. 2000) properties. However, although the parent compounds of these flavan-3-ols have been reported to possess properties beneficial to health, they are known to undergo significant metabolism and conjugation in the gastrointestinal tract (Spencer 2003). It is unknown how such metabolism and conjugation may influence the putative properties of these polyphenols, hence the focus of our study on a digested green tea extract. The aim of this study was to investigate potential neuroprotective effects of a green tea extract after simulated gastrointestinal digestion against H 2 O 2 and A (1 42) induced cytotoxicity in differentiated PC12 cells as a model of neuronal cells. Materials and methods Green tea extract Corresponding author. Tel.: ; fax: address: e.j.okello@ncl.ac.uk (E.J. Okello). Extract preparation: An extract of green tea (GTE) (Temple of Heaven, China Green Tea, special gun powder, Shangai Tea Import /$ see front matter 2010 Published by Elsevier GmbH. doi: /j.phymed

2 692 E.J. Okello et al. / Phytomedicine 18 (2011) & Export Company, purchased from a local food store) was made using freshly boiled water (1:25, w/v) for 45 min. The infusions were left to cool to room temperature and centrifuged (12,000 rpm, 15 min, supernatant re-centrifuged). 1 ml aliquots of the supernatant were freeze dried in order to determine their dry weight equivalents. Freeze dried aliquots were reconstituted in de-ionised water prior to assay. In vitro digestion Triplicate samples of the freeze dried GTE were subjected to an in vitro procedure that simulates the digestive process as described previously (Coates et al. 2007). The method consists of two sequential steps; an initial pepsin/hcl digestion to simulate gastric conditions followed by a digestion with bile salts/pancreatin to simulate small intestine conditions. The GTE was used at a concentration of soluble phenols similar to that of the original green tea extract. Briefly, the GTE (final volume 20 ml) was acidified to ph 1.7 with 5 N HCl, pepsin (Sigma Chem. Co. Ltd.) was added at 315 units/ml and incubated at 37 C in a heated water bath for 2 h with shaking at 120 rpm. 2 ml aliquots of the post-gastric digestion were removed for analysis. The remainder was placed in a 2 ml glass beaker and 4.5 ml of 4 mg/ml pancreatin, 25 mg/ml bile salts mixture added. A segment of cellulose dialysis tubing (molecular mass cut-off 12 kda) containing sufficient NaHCO 3 to neutralise the sample s titratable acidity was added and the beaker sealed with parafilm. After 2 h incubation at 37 C, the solution outside the dialysis tubing was taken as the sample representing material that would reach the colon. The post gastric and CAGTE samples were centrifuged at 15,000 g in a microfuge and the supernatants assayed for phenol content using a modified Folin-Ciocalteau method (Singleton 1965). The colon-available green tea extract (CAGTE) was purified from bile salts using solid phase extraction as described previously. Samples were dried in a speed-vac (Thermo-Scientific Ltd., High Wycombe, UK) to suitable phenol concentrations for LCMS analysis. Cell line and cell culture Rat pheochromocytoma (PC12) cell line was a generous gift from the Medical School, Newcastle University. RPMI-1640 medium, penicillin streptomycin, foetal calf serum, glutamine and nerve growth factor (NGF) were purchased from Invitrogen (UK). A (1 42) and trypan blue were purchased from Sigma (UK). Cells were maintained in RPMI media supplemented with 10% heat-inactivated foetal bovine serum, 2 mm l-glutamine, IU/ml penicillin streptomycin in humidified 5% CO 2 and 95% air at 37 C. All cells were cultured in culture flasks pre-coated with poly-dlysine (0.1 mg/ml). Cells were differentiated for 2 3 days using ng/ml NGF. Prior to experimental treatments, cells were microscopically examined to assess differentiation. Eighty percent or more of cells with neurite outgrowth extensions over 2 3-fold cell body size were considered to be differentiated PC12 cells (dpc12). The medium was changed every second day. Just prior to confluency, cells were dislodged by mechanical scraping and split in 1:3 ratio. Before each experiment, cells were checked for viability using a trypan blue (0.5%) dye exclusion method (Freshney 2000). Cells were counted using a haemocytometer and the density was adjusted to cells/ml prior to plating in 96-well plates; cells in exponential growth phase were used. Assay of H 2 O 2 induced toxicity The viability of the dpc12 cells exposed to H 2 O 2 was assessed by incubating the cells with different concentrations of H 2 O 2 ranging from 12.5 M to400 M for 24 h. Before treatment, cells were plated at an appropriate density ( cells/ l) in a 96 well plate and incubated for 24 h at 37 C, so as to acclimatise the cells to the new environment. Cells were pre-incubated with CAGTE (at concentrations ranging from 0.3 to 10 g/ml), prior to exposure to H 2 O 2. Cell viability was determined by the MTT assay described below. Aˇ(1 42) fibrils preparation A (1 42) peptide was stored at 20 C until use. After warming to room temperature, the lyophilized peptide was dissolved in de-ionised water at a concentration of 0.1 mg/ml, with thorough mixing over a period of 2 min in order to clarify the solution. The solution was then incubated overnight at 37 C with constant oscillation to form the fibrils used to induce toxicity in dpc12 cells. Assay of ˇ-amyloid (1 42) cytotoxicity In vitro toxicity of A (1 42) to dpc12 cells was measured after incubating the cells overnight with increasing concentrations of aggregated A (1 42) peptide. The A concentrations (ranging from 0.03 to 2 g/) were added to the dpc12 cells in a 96 well plate. The plates were incubated at 37 C for 24 h, after which the cell viability was assessed by measuring cellular redox activity with the MTT assay described below. MTT assay PC12 cell viability was determined by the MTT reduction assay. In brief, MTT, a tetrazolium salt, is cleaved to formazan by succinate dehydrogenase (an enzyme of the mitochondrial respiratory chain), only by live cells. After pre-incubation of the dpc12 cells with the CAGTE extracts and toxicity inducers (H 2 O 2 or A ) for 24 h, cells were incubated with MTT (0.5 mg/ml) for 3 h. The dark blue formazan crystals formed in live cells were solubilised with dimethyl sulphoxide and ethanol (1:1) and the absorbance was measured at 570 nm using a spectrophotometer (Molecular Device Spectramax Plus 384, equipped with Softmax Pro V5 software) (Mossman 1983). Liquid chromatography mass spectroscopy (LC MS n ) Samples (containing 40 g gallic acid equivalents (GAE) by Folin assay) were analyzed on a LCQ-DECA system, comprising Surveyor autosampler, pump and photo diode array detector (PDAD) and a Thermo-Finnigan mass spectrometer iontrap. Three discrete channels were scanned at 280 nm, 365 nm and 520 nm. Samples were eluted over a gradient of 5% acetonitrile (0.1% formic acid) to 40% acetonitrile (0.1% formic acid) on a C18 column (Synergi Hydro C18 with polar end capping, 4.6 mm 1 mm, Phenomenex Ltd., Macclesfield, UK) over 60 min at a rate of 400 l/min. The LCQ- DECA LC MS was fitted with an electrospray ionisation interface and analyzed the samples in positive and negative ion mode. There were 2 scan events; full scan analysis followed by data dependent MS/MS of most intense ions. The data dependent MS/MS used collision energies (source voltage) of 45% in wideband activation mode. Representative chromatographs are presented. The % recoveries of each component were calculated by averaging the peak areas for each compound from triplicate injections (using the relevant m/z and the Xcalibur software). Although this method is not strictly a measure of content, in the absence of suitable standards for each component, it is a valid measurement of recovery as the MS detection is comparable for each component. The peak areas as measured by m/z were similar to those measured by PDA peak area for ( )-epigallocatechin-3-gallate (EGCG) and caffeine (results not shown).

3 E.J. Okello et al. / Phytomedicine 18 (2011) Table 1 Recoveries of major polyphenol components of green tea after simulated gastrointestinal digestion. Peak RT Compound PDA [M H] and main MS 2 ions Extract % Recovery Post gastric Colon available Gallic acid , 125 ± ± ± Galloyl quinic acid , 191, 169 ± ± Theobromine , 181 ± ± ± ( )Gallocatechin , 261, 221, 179 ± ± Epigallocatechin , 261, 221, 179 ± ± Unknown compound , 533, 339 ± ± Catechin , 245, 205 ± ± Caffeine , multiple ± ± ± Epicatechin , 245, 205 ± ± p-coumaroyl quinic acid , 173 ± ± ± Epigallocatechin gallate , 331, 305, 165 ± ± Unknown compound ND ± ± ± Myricetin hexose , 319 ± ± ± Myricetin hexose , 319 ± ± ± Quercetin glucosyl rutinoside , 301, 609, 301 ± ± ± Quercetin glucosyl rutinoside , 301, 609, 301 ± ± ± Kaempferol glucosyl rutinoside , 285, 609, 285 ± ± ± Kaempferol glucosyl rutinoside , 285, 609, 285 ± ± ± Quercetin rutinoside , 465, 301 ± ± ± Kaempferol glucosyl rutinoside , 285, 609, 285 ± ± ± epicatechin gallate , 289, 169 ± ± Kaempferol rutinoside , 447, 285 ± ± ± Kaempferol hexose , 285 ± ± Kaempferol hexose , 285 ± ± ± Quercetin rhamnose 3 447, 301 ± ± Epicatechin methyl gallate , 289, 183 ± ± Quercetin derivative , multiple ± ± ± Quercetin derivative , multiple ± ± ± Kaempferol derivative , multiple ± ± ± Quercetin derivative , 771, 753, 591, 301 ± ± ± Quercetin derivative , 741, 723, 301 ± ± Kaempferol derivative Multiple ± ± ± 1.2 G G , 109 ± ± G G , 331 ± ± ± 2.3 G3.46 Myricetin , * 0 G Quercetin , 273, 257, 179 ± ± C C , * C C , * C C , multiple ± ± 5.2 C C , 473, 353 ± ± ± 5.4 C C , 551, 359, 337 ± ± ± 7.5 C C , 3, 473, * * C C , 473, 353 ± ± ± 2.3 C C , 413, 293 ± ± ± 2.3 C C , 413, 293 ± ± ± 2.6 C C ND 0 * * Compounds 1 30 were identified in the original extract and are annotated in Fig. S1; compounds G1 G4 were more apparent in the post-gastric digest and compounds C1 10 were more apparent in the post-pancreatic, colon-available sample. + denoted detection in +ve mode MS only. Recoveries marked with * indicate that this component was not detected in the original GTE. Statistical analysis The results are expressed as the mean ± SEM. Student s t-test was used to compare differences between test groups and the negative control (H 2 O 2 or A only) using Graphpad statistical software; p < 0.05 were considered significant. Results In vitro digestion of green tea extract Gastric digestion did not greatly influence the total phenol content of the GTE (recovery = 90.7 ± 3.1% [mean ± SE]) but pancreatic conditions reduced this value to 35.2 ± 2.1%. However, the small drop in total phenol content did not reveal drastic changes in the phenolic composition of the gastric sample. The GTE had a polyphenol composition similar to previous reports (e.g. Del Rio et al. 2004) with a characteristic mixture of flavan-3-ols (including ( )-epigallocatechin (EGC), EGCG and epicatechin methyl gallate), various flavonol derivatives, caffeine, theobromine but an absence of theaflavins (Table 1 and Fig. 1). The content of flavan-3-ol derivatives dropped greatly after the gastric digestion (Table 1) and the CAGTE was effectively depleted in these major components. Gastric digestion also reduced the levels of certain flavonol glycosides with corresponding increases in the aglycones, myricetin, quercetin, and kaempferol, suggesting deglycosylation. Nevertheless, it was clear that CAGTE was relatively enriched in certain flavonols, hydroxycinnamates, caffeine, threobromine and a range of, as yet, unidentified phenolic components. Previous work also found that flavan-3-ols were particularly susceptible to the slightly alkaline conditions of pancreatic digestion (Zhu et al. 1997; Record and Lane 2001; Green et al. 2007; Neilson et al. 2007). However, some of these studies used an artificial mixture of flavan-3-ols reformulated at concentrations found in green tea or excluded oxygen from the incubation, which could markedly influence stability (Neilson et al. 2007). We also did not find evidence for the formation of theasinensin-type dimers from

4 694 E.J. Okello et al. / Phytomedicine 18 (2011) NL: 7.00E5 A Cell viability (%) B 1 8 G G G3 G4 NL: 7.00E Concentration of H 2 O 2 [um] Fig. 2. Concentration-dependent inhibition of cell viability by H 2O 2 in differentiated PC12 cells after treatment for 24. Values are expressed as percentage of control values (untreated). Data were obtained from means of three separate experiments performed in triplicates. 400 C 8 C6 C9 C7 17 C8 18 NL: 7.00E5 C5 3 C2 C3 C4 10 C C Time (min) Fig. 1. Liquid chromatography mass spectrometric (LC MS) analysis of green tea extracts. (A) A representative trace at 280 nm for the original green tea extract. (B) A representative trace at 280 nm for green tea after gastric digestion and (C) a representative trace at 280 nm for green tea after gastric and pancreatic digestion = colon-available extract. The full scale deflection is shown in the upper right corner. Peaks are labelled as described in Table 1 with components appearing in the gastric digest or the colon-available extract labelled, e.g. G1 and C1, etc., respectively. EGCG, ( )-epichatechin gallate (ECG) or EGC (Sang et al. 2005; Neilson et al. 2007) but these may themselves have broken down during the 2 h pancreatic digestion (Sang et al. 2005). Effect of H 2 O 2 on cell viability in dpc12 cell after treatment for 24 The viability of the dpc12 cells exposed to H 2 O 2 was assessed by the MTT test. When dpc12 cells were incubated with different concentrations of H 2 O 2 ranging from 12.5 M to 400 M for 24 h, a concentration-dependent cytotoxicity was observed (Fig. 2). The cell viability was decreased to approximately % at 200 M concentration over 24 h incubation (p < 0.001). Therefore, this concentration was selected to induce toxicity in further experiments with CAGTE. Effect of CAGTE extract against H 2 O 2 induced cytotoxicity CAGTE significantly protected dpc12 cells from the toxic effect of H 2 O 2 (Fig. 3) when the cells were pre-incubated with CAGTE extract for 24 h prior to H 2 O 2 exposure. All concentrations (ranging from g/mlto10 g/ml) caused significant increases in viability ( 80%) as compared with negative control (H 2 O 2 alone). Interestingly, the lowest concentrations seemed to afford the highest protection. Fig. 3. Protective effect of CAGTE against H 2O 2 induced cytotoxicity in differentiated PC12 cells. NC: negative control (H 2O 2 alone); PC: positive control (No H 2O 2 = % viability). Data are presented as mean ± SEM of three separate experiments performed in triplicates. *p < 0.05, **p < 0.01 and ***p < 0.001, compared with negative control. Effect of Aˇ(1 42) peptide aggregates in differentiated PC12 cells The viability of dpc12 cells exposed to A peptide was assessed by the MTT test. A concentration-dependent decrease of cell survival was observed after exposure to A (1 42) (Fig. 4). Cell viability was decreased to approximately % at 0.5 g/ml, with no further significant decrease noted at increased A concentrations. Cell viability (%) Concentration of beta-amyloid (1-42) ug/ml Fig. 4. Dose-dependent neurotoxicity of A (1 42) aggregates in differentiated PC12 cells measured by MTT assay. Values are expressed as percentage of control values (untreated). Data were obtained from as means of three separate experiments performed in triplicates. 2.0

5 E.J. Okello et al. / Phytomedicine 18 (2011) Fig. 5. Protective effect of CAGTE extracts against A (1 42) induced toxicity in differentiated PC12 cells. NC: negative control (A only); PC: positive control (No A ). Values are expressed as percentage of control values. Data are presented as mean ± SEM of three separate experiments performed in triplicates. ***p < 0.001, compared with negative control. This concentration of A was selected to induce toxicity in further experiments to test the protective effect of CAGTE. Effect of CAGTE on viability of dpc12 cells against Aˇ(1 42) induced toxicity Pre-incubation with CAGTE extract ( g/ml) for 24 h prior to A addition attenuated A -induced toxicity in PC12 cells at the lowest concentration (Fig. 5). However, at concentrations higher than 0.25 g/ml cell viability was significantly lower than negative control, perhaps due to direct cytotoxic or anti-proliferative activity widely reported for tea components. Discussion All forecasts indicate that the incidence of AD is likely to increase in an ageing population (Brookmeyer et al. 2007) with a huge associated societal and economic cost. AD is an irreversible, progressive brain disease with a complex pathophysiology with symptoms including cognitive dysfunction, primarily; memory loss, language deficit, depression, agitation, anxiety, and in some cases psychosis. Existing treatments are based on cholinesterase inhibitors (e.g. Tacrine, Revastigmine, Donepezil, Galanthamine) and various neuroleptics (e.g. Risperidone and Olanzapine) and treat the symptoms without providing a cure. Like many synthetic drugs, the benefits of AD treatments are accompanied by some unwanted side effects such as liver toxicity, anorexia, diarrhoea, nausea and vertigo, with a number of queries on efficacy, costs effectiveness and safety. The search for novel alternative and/or complementary approaches has been underway for a number of decades. Current developments in this search include products that seek to slow down the progression of the disease or indeed prevent its onset by targeting biochemical pathways associated with the disease mechanisms. These include, inter alia, products with cholinergic, receptor agonist/antagonist, antioxidant, anti-amyloid, anti-inflammatory, cholesterol-lowering and estrogenic activities. Many natural plant products possess these properties and their uses are increasingly guided by standardised preparations, mechanistic studies and clinical trials. A key target for AD treatment is H 2 O 2 - and A -induced toxicities which are said to play a major role in AD pathogenesis. The potential neuroprotective and cognition enhancing properties of both green and black tea have been explored by a number of researchers and attributed mainly to their polyphenolic content. Hasegawa et al. (2005) showed in a two-year follow up observation that increased consumption of green tea protected against cognitive decline in an elderly Japanese cohort. Tea polyphenols have also been shown to significantly reverse scopolamine-induced cognitive impairment in a mouse model (Kim et al. 2004), protect against hippocampal neural damage after transient global ischemia in gerbils (Lee et al. 2000), inhibit acetyl- and butyryl-cholinesterase (Kim et al. 2004; Okello et al. 2004), inhibit -secretase (Jeon et al. 2003; Okello et al. 2004), modulate amyloid precursor cleavage and reduce cerebral amylodosis in AD transgenic mice (Rezai-Zadeh et al. 2005) and to inhibit -amyloid induced cognitive dysfunction in a mouse model (Lee et al. 2009). There are known differences in chemical composition between non-digested green tea polyphenols and digested green tea polyphenols (metabolites). The focus of our study was on CAGTE (for cell viability assays) in experimental data shown in Figs. 3 and 5 as there is already published data on the cytoprotective effects of non-digested green tea polyphenols. For example, GTE (undigested) is reported to have a significantly strong cytoprotective effect against H 2 O 2 -induced cell death in Mel-Ab melanocytes (Jeong et al. 2005), to attenuated A -amyloid induced cell death in a PC12 cell model (Lee et al. 2005) and to reduce oxidative damage due to Fe 2+ treatment in a Jurkata T-Cell line model (Erba et al. 1999). There are no reports, to our knowledge, of similar effects exhibited by digested CAGTE, thus far. This study suggests that CAGTE, which effectively lacked flavan- 3-ols, had a protective effect on A (1 42) induced toxicity in PC12 cells in vitro at g/ml. Although the extracts were not quantified in the same manner, this figure is much lower than the 20 g/ml reported for effective protection in whole green tea extracts (Lee et al. 2005). However, the instability of flavan-3-ols in the CAGTE under in vitro digestion conditions raises the question of the effectiveness of circulating flavan-3-ols (especially at their low plasma concentrations) compared with other green tea components or their metabolites. Flavan-3-ols undergo extensive biotransformation and pharmacokinetic evidence suggests that the parent polyphenols only attain sub-to-low-micromolar peak plasma levels in human or animal subjects following oral administration (Chow et al. 2001; Henning et al. 2005; Yang et al. 2008). In tracer studies with stomach-administered 3 H EGCG in mice, the bulk of the radioactivity was excreted in faeces and urine but small amounts were also found in central tissues such as brain, kidney, lung and pancreas (Suganuma et al. 1998). However, the nature of these radioactive components was not explored. Nevertheless, substantial amounts of green tea flavanols enter the large intestine and are subject to intensive metabolism by the colonic microflora (Stalmach et al. 2009) and colonic metabolites are re-absorbed into the bloodstream before excretion in urine (Roowi et al. 2010). This re-absorption of breakdown products may explain the disparity between the low bioavailability of flavanols and the epidemiological studies suggest that polyphenolic compounds present in tea reduce the risk of a variety of diseases (Fujiki 2005; Kurahashi et al. 2008). Intake of green tea has been shown to be effective against cancers in experimental animal models (e.g. Wang et al. 1992) and epidemiological studies suggest a reduction in risk for prostate cancer in men (Kurahashi et al. 2008; Johnson et al. 2010) and other cancers (Fujiki 2005). Green tea extracts are known to have anti-cancer effects on cancer cells grown in vitro (e.g. Way et al. 2009). Therefore, the greater effectiveness of CAGTE against H 2 O 2 - or A (1 42) -induced toxicity at lower concentrations may be due to the overlaying of a protective effect against induced cytotoxicity onto a direct anti-proliferative effect of the CAGTE on the dpc12 cancer cells at higher concentration. Previous work has shown that green tea extracts or flavanol components of green tea, such as EGCG, can modulate nitric oxide-induced apoptosis (Jung et al. 2007) and prevent -amyloid-induced cell death in

6 696 E.J. Okello et al. / Phytomedicine 18 (2011) PC12 cells (Lee et al. 2005). Assuming that the average molecular weight of CAGTE is around 0 Da, then effective protection against A (1 42) induced toxicity at g/ml is the equivalent of M which is close to the physiological levels noted above. This study begins to address the limitations in the literature to date that do not take in to account the very low bioavailability of polyphenols, with peak plasma concentration reported to be in the sub-micromolar range. It is still not clear whether the putative effects are conferred at these sub-micromolar concentrations, or could it be that the parent molecules are producing more active metabolites post digestion or that the multiple compounds and/or metabolites exhibit polyvalent/synergistic effects (Berenbaum 1989; Williamson 2001; Savelev et al. 2003). These results provide a basis for further study of flavanol metabolites and their potential role in amelioration of some of the insults associated with AD pathophysiology. Acknowledgement This research was supported in part by a small grant from the Mental Health Foundation (Newcastle Branch), UK. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: /j.phymed References Behl, C., Davis, J.B., Lesley, R., Schubert, D., Hydrogen peroxide mediates amyloid protein toxicity. Cell 77, Berenbaum, M.C., What is synergy? Pharmacol. Rev. 41, Brookmeyer, R., Johnson, E., Ziegler-Graham, K., Arrighi, H.M., Forecasting the global burden of Alzheimer s disease. Alzheimers Dementia 3, Chow, H.H., Cai, Y., Alberts, D.S., Hakim, I., Dorr, R., Shahi, F., Crowell, J.A., Yang, C.S., Hara, Y., Phase 1 pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and polyphenon E. Cancer Epidemiol. Biomarkers Prev. 10 (1), Coates, E.M., Popa, G., Gill, C.I., McCann, M.J., McDougall, G.J., Stewart, D., Rowland, I., Colon-available raspberry polyphenols exhibit anti-cancer effects on in vitro models of colon cancer. J. Carcinog. 6 (4), Del Rio, D., Stewart, A.J., Mullen, W., Burns, J., Lean, M.E.J., Brighenti, F., Crozier, A., HPLC MS n analysis of phenolic compounds and purine alkaloids in green and black tea. J. Agric. Food Chem. 52, Erba, D., Riso, P., Colombo, A., Testolin, G., Supplementation of Jurkat T cells with green tea extract decreases oxidative damage due to iron treatment. J. Nutr. 129 (12), Freshney, R.I., Culture of Animal Cells: A Manual of Basic Techniques. Wiley Liss Inc. Fujiki, H., Green tea: health benefits as cancer preventive for humans. Chem. Rec. 5 (3), Green, R.J., Murphy, A.S., Schulz, B., Watkins, B.A., Ferruzzi, M.G., Common tea formulations modulate in vitro digestive recovery of green tea catechins. Mol. Nutr. Food Res. 51 (9), Hasegawa, T., Okello, E., Yamada, T., Protective effect of Japanese green tea against cognitive impairment in the elderly, a two-year follow-up observation. Alzheimers Dementia 1 (1), S. Henning, S.M., Niu, Y., Liu, Y., Lee, N.H., Hara, Y., Thames, G.D., Minutti, R.R., Carpenter, C.L., Wang, H., Heber, D., Bioavailability and antioxidant effect of epigallocatechin gallate administered in purified form versus as green tea extract in healthy individuals. J. Nutr. Biochem. 16 (10), Imai, K., Nakachi, K., Cross sectional study of effects of drinking green tea on cardiovascular and liver damage. Biochem. Med. J. 310, Jeon, S.Y., Bae, K., Seong, Y.H., Song, K.S., Green tea catechins as BACE1 ( secretase) inhibitor. Bioorg. Med. Chem. Lett. 13 (22), Jeong, Y.M., Choi, Y.G., Kim, D.S., Park, S.H., Yoon, Y.A., Kwon, S.B., Park, E.S., Park, K.C., Cytoprotective effect of green tea extract and quercetin against hydrogen peroxide-induced oxidative stress. Arch. Pharm. Res. 28 (11), Johnson, J.J., Bailey, H.H., Mukhtar, H., Green tea polyphenols for prostate cancer chemoprevention: a translational perspective. Phytomedicine 17 (1), Jung, J.Y., Han, C.R., Jeong, Y.J., Kim, H.J., Lim, H.S., Lee, K.H., Park, H.O., Oh, W.M., Kim, S.H., Kim, W.J., Epigallocatechin gallate inhibits nitric oxide-induced apoptosis in rat PC12 cells. Neurosci. Lett. 411 (3), Kim, H.K., Kim, M., Kim, S., Kim, M., Chung, J.H., Effects of green tea polyphenols on cognitive and acetylcholinesterase activities. Biosci. Biotechnol. Biochem. 68 (9), Kurahashi, N., Sasazuki, S., Iwasaki, M., Inoue, M., Tsugane, S., Green tea consumption and prostate cancer risk in Japanese men: a prospective study. Am. J. Epidemiol. 167 (1), Lee, S.Y., Lee, J.W., Lee, H., Yoo, H.S., Yun, Y.P., Oh, K.W., Ha, T.Y., Hong, J.T., Inhibitory effect of green tea extract on -amyloid-induced PC12 cell death by inhibition of the activation of NF-nB and ERK/p38 MAP kinase pathway through antioxidant mechanisms. Mol. Brain Res. 140, Lee, S.R., Suh, S.I., Kim, S.P., Protective effects of the green tea polyphenol ( )-epigallocatechin gallate against hippocampal neural damage after transient ischemia in gerbils. Neurosci. Lett. 287, Lee, J.W., Lee, Y.K., Ban, J.O., Ha, T.Y., Yun, Y.P., Han, S.B., Oh, K.W., Hong, J.T., Green tea ( )epigallocatachin-3-gallate inhibits -amyloid-induced cognitive dysfunction through modifications of secretase activity via inhibition of ERK and NF-kB pathways in mice. J. Nutr. 139 (10), Mossman, T., Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immuol. Methods 65, Murphy, M.P., Levine 3rd., H., Alzheimer s disease and the amyloid-beta peptide. J. Alzheimers Dis. 19 (1), Neilson, A.P., Hopf, A.S., Cooper, B.R., Pereira, M.A., Bomser, J.A., Ferruzzi, M.G., Catechin degradation with concurrent formation of homo- and heterocatechin dimers during in vitro digestion. J. Agric. Food Chem. 55 (22), Okello, E.J., Savelev, S.U., Perry, E.K., In vitro anti-beta-secretase and dual anticholinesterase activities of Camellia sinensis L. (tea) relevant to treatment of dementia. Phytother. Res. 18 (8), Record, I.R., Lane, J.M., Simulated intestinal digestion of green and black teas. Food Chem. 73 (4), Rezai-Zadeh, K., Shytle, D., Sun, N., Mori, T., Hou, H., Jeanniton, D., Ehrhart, J., Townsend, K., Zeng, J., Morgan, D., Hardy, J., Town, T., Tan, J., Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloidosis in Alzheimer s transgenic mice. J. Neurosci. 25 (38), Roowi, S., Stalmach, A., Mullen, W., Lean, M.E., Edwards, C.A., Crozier, A., Green tea flavan-3-ols: colonic degradation and urinary excretion of catabolites by humans. J. Agric. Food Chem. 58 (2), Sang, S., Lee, M.-J., Hou, Z., Ho, C.-T., Yang, C.S., Stability of tea polyphenol ( ) Epigallocatechin-3-gallate and formation of dimers and epimers under experimental conditions. J. Agric. Food Chem. 53 (24), Savelev, S., Okello, E., Perry, N.S.L., Wilkins, R.M., Perry, E.K., Synergistic and antagonistic interactions of anticholinesterase terpenoids in Salvia lavandulaefolia essential oil. Pharmacol. Biochem. Behav. 75 (3), Singleton, V.R.J., Colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. Am. J. Enol. Viticult. 16, Spencer, J.P.E., Metabolism of tea flavonoids in the gastrointestinal tract. Proceedings of the Third International Scientific Symposium on Tea and Human Health: Role of Flavonoids in the Diet, American Society for Nutritional Sciences. The Journal of Nutrition, 3255S 3261S. Stalmach, A., Troufflard, S., Serafini, M., Crozier, A., Absorption, metabolism and excretion of Choladi green tea flavan-3-ols by humans. Mol. Nutr. Food Res. 53 (Suppl. 1), S Suganuma, M., Okabe, S., Oniyama, S., Tada, Y., Ito, H., Fujiki, H., Wide distribution of 3 H EGCG, a cancer preventive tea polyphenol, in mouse tissue. Carcinogenesis 19, Wang, Z.Y., Huang, M.T., Ferraro, T., Wong, C.Q., Lou, Y.R., Reuhl, K., Iatropoulos, M., Yang, C.S., Conney, A.H., Inhibitory effect of green tea in the drinking water on tumorigenesis by ultraviolet light and 12-O-tetradecanoylphorbol-13- acetate in the skin of SKH-1 mice. Cancer Res. 52 (5), Williamson, E.M., Synergy and other interactions in phytomedicines. Phytomedicine 8, Yang, C., Lambert, J., Sang, S., Antioxidative and anti-carcinogenic activities of tea polyphenols. Arch. Toxicol. 83, Yang, C.S., Sang, S., Lambert, J.D., Lee, M.-J., Bioavailability issues in studying the health effects of plant polyphenolic compounds. Mol. Nutr. Food Res. 52, S139 S151. Way, T.D., Lin, H.Y., Kuo, D.H., Tsai, S.J., Shieh, J.C., Wu, J.C., Lee, M.R., Lin, J.K., Puerh tea attenuates hyperlipogenesis and induces hepatoma cell growth arrest through activating AMP-activates protein kinase (AMPK) in human HepG2 cells. J. Agric. Food Chem. 57 (12), Zhu, Q.Y., Zhang, A., Tsang, D., Huang, Y., Chen, Z.-Y., Stability of green tea catechins. J. Agric. Food Chem. 45 (12),