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Effects of anthocyanins and other phenolics of boysenberry and blackcurrant as inhibitors of oxidative stress and damage to cellular dna in sh-sy5y and hl-60 cells

Journal of the Science of Food and Agriculture Effects of anthocyanins and other phenolics of
boysenberry and blackcurrant as inhibitors of
oxidative stress and damage to cellular DNA in
SH-SY5Y and HL-60 cells
Dilip Ghosh,1∗ Tony K McGhie,2 Jingli Zhang,1 Aselle Adaim1 and Margot Skinner1
1Food Sector, The Horticulture and Food Research Institute of New Zealand Ltd, Auckland, New Zealand
2Future Horticulture Sector, The Horticulture and Food Research Institute of New Zealand Ltd, Palmerston North, New Zealand
Abstract: There is growing interest both from consumers and researchers in the role that berries play in
human health. The objective of this study was to investigate whether anthocyanins and other phenolics
present in boysenberries and blackcurrants are effective in protecting cells against the oxidative damage
induced by hydrogen peroxide (H2O2). The concentrations of polyphenols used were within the human
physiological range. The data showed that SH-SY5Y human neuroblastoma cells were protected against
H2O2-induced toxicity by the anthocyanins and phenolic fractions. The concurrent addition of either
fractions of these berries with H2O2 significantly inhibited the increase in intracellular reactive oxygen
species (ROS) production. Pre-incubation of cells with the same concentrations had no effect on the ROS
level—a result that may be due to the metabolic conversion to inactive compounds. Anthocyanins and
phenolic fractions of blackcurrant were better at protecting DNA of HL-60 human promyelocytic cells
from damage than similar fractions from boysenberry. The phenolic extract of blackcurrant demonstrated
the highest protective effect against H2O2-induced neurotoxicity, oxidative stress and DNA damage and
may be a good candidate for inclusion into a processed functional food.
2006 Society of Chemical Industry
Keywords: antioxidants; berries; DNA damage; human health; oxidative stress; polyphenols
anti-carcinogenic and protection from cardiovascu- Polyphenolic anthocyanins are a subclass of flavonoids lar damage and allergy.1,2,9 – 11 Although anthocyanins and are present in high concentrations in highly appear to have limited bioabsorption,12,13 the com- coloured fruits and vegetables such as berries, red ponents and metabolites resulting from anthocyanin grapes, and cabbages.1 Based on experimental and consumption have not been fully determined. Once epidemiological evidence it has been proposed that absorbed, the systemic antioxidative effects of circulat- anthocyanins, along with other polyphenols, may ing anthocyanins (and metabolites) might be expected exert therapeutic activities on human diseases such to reduce oxidative stress and ultimately the risk of as coronary heart disease, cancer and neurodegenera- developing certain chronic and degenerative diseases.
tive diseases associated with oxidative stress.2,3 Many The objective of this study was to investigate of the biological effects of anthocyanins and other phe- whether anthocyanins and other phenolics present in nolics have been related to their antioxidant properties.
boysenberry and blackcurrant are capable of providing These properties include the ability to scavenge free protection to human cells (SH-SY5Y, HL-60) when radicals,4 to chelate metal ions,5 to inhibit lipoprotein H2O2-mediated oxidative stress is imposed.
oxidation6 and to form complexes with DNA.7 Thereis also some evidence on the protection afforded byanthocyanins against oxidative damage.8 During the MATERIALS AND METHODS
past two decades the results of an increasing num- Chemicals
ber of studies suggest that polyphenolics present in All cell culture media and reagents were purchased fruits and vegetables have diverse effects on bio- from Gibco-Invitrogen Corporation (Auckland, New logical systems. These effects include antioxidant, Zealand). Other chemicals used in these studies were anti-allergic, anti-diabetic, anti-inflammatory, antivi- obtained from Sigma Chemical Co. (St Louis, MO, ral, anti-proliferative, anti-mutagenic, antimicrobial, ∗ Correspondence to: Dilip Ghosh, Food Sector, The Horticulture and Food Research Institute of New Zealand Ltd, Auckland, New ZealandE-mail: 25 April 2005; accepted 17 October 2005)  2006 Society of Chemical Industry. J Sci Food Agric 0022–5142/2006/$30.00 Extract preparation and HPLC analysis
For assessment of their metabolic integrity, SH-SY5Y Boysenberry (Rubus loganbaccus × baileyanus Britt cv.
cells were incubated with extracts made up in culture Riwaka Choice) and blackcurrant (Ribes nigrum L. cv.
medium for 24 h prior to addition of H2O2. For Ben Ard) fruit were used in this project for extract measurement of oxidative stress, anthocyanin and preparation and were supplied by Berryfruit Export phenolic fractions were added simultaneously with Ltd (Richmond, New Zealand) and Blackcurrants H2O2 to the SH-SY5Y cell suspension. HL-60 cells New Zealand Ltd (Christchurch, New Zealand).
were used to assess oxidative damage to DNA using the To extract non-anthocyanin polyphenols, portions Comet assay. The cells were incubated with extracts (50 g) of both berries were first homogenised in a for 24 h prior to exposure to H2O2 for 30 min on ice to Waring blender with ethyl acetate and anhydrous induce DNA damage and minimise the possibility of cellular DNA repair. The concentration ranges removed by filtration and the solid residue was (0.5 – 0.065 µg mL−1) used in these experiments are further homogenised with methanol to extract the within the human physiological range.16,17 Moreover, anthocyanins. The methanol extract was separated all fractions at 1 mg mL−1 and above were found to be from the solid residue by filtration and the methanol toxic to cells in a preliminary study.
was evaporated by rotary evaporation. The resultingaqueous anthocyanin extract was loaded onto an Cell toxicity assay
XAD column previously conditioned with 1.3 mol L−1 Cell toxicity was determined by assessing effects formic acid. The column was eluted with 1.3 mol L−1 on metabolic activity (mitochondrial succinate dehy- formic acid to remove sugars and other water- soluble compounds not bound to the column. The 5-diphenyl-tetrazoliumbromide (MTT) colorimetric anthocyanins were then eluted with 1.3 mol L−1 assay.18 Briefly, SH-SY5Y cells were pre-incubated in formic acid in methanol. This methanolic extract 96-well plates for 24 h with different concentrations of was concentrated and dried under vacuum to yield extract (from 0.25 to 0.075 µg mL−1) in triplicate and an anthocyanin-rich fraction of boysenberry (ByAcy) then treated with H2O2 at 100 µmol L−1 for 30 min.
and blackcurrant (BcAcy). The ethyl acetate extract At the end of treatment, 0.4 µg mL−1 of MTT dis- containing the non-anthocyanin polyphenols was solved in PBS was added to the medium in each well, filtered twice and concentrated on a rotary evaporator.
and incubated for 2 h at 37 ◦C. The medium was then Residual aqueous-soluble compounds were removed removed, and the blue formazan formed quantified at by washing the ethyl acetate extract twice with 570 nm using a microtitre plate reader (Spectra Max 0.1 mol L−1 HCl followed by drying with anhydrous Gemini, Molecular Devices). Cell toxicity was calcu- sodium sulfate. It was then evaporated to dryness.
lated by measuring the difference in optical density The residue was redissolved in 1.3 mol L−1 formic of treated samples with respect to control cells (with- acid/methanol and washed three times with hexane out H2O2 treatment) and expressed as percentage of to remove lipids and other fat-soluble compounds such as carotenoids. Finally the dark-brown methanolextract was evaporated to dryness and freeze dried to Oxidative stress assay
yield a polyphenolic-enriched fraction of boysenberry Oxidative stress was measured using the DCF assay.19 (ByPhen) or blackcurrant (BcPhen). Anthocyanins14 This assay measures the generation of intracel- and non-anthocyanin polyphenol15 concentrations in lular reactive oxygen species (ROS) through the the extracts were determined by high-performance peroxide-dependent oxidation of intracellular 2 7 - liquid chromatography (HPLC) following solid phase dichlorofluorescein (DCFH) to the fluorescent com- pound 2 7 -dichlorofluorescein (DCF). Fluorescencewas measured using a fluorescence plate reader (Spec- Cell culture and treatment
tra Max Gemini, Molecular Devices). SH-SY5Y cells Human neuroblastoma (SH-SY5Y) and promyelo- were seeded at a density of 1.5 × 105 cells per well cytic (HL-60) cells were obtained from ATCC in non-fluorescent 96-well plates (NUNC, Rosklide, (Rockville, MD, USA). SH-SY5Y cells were grown in Denmark) one day before the experiments. For the DMEM/F-12 nutrient mixture medium supplemented pre-incubation studies, SH-SY5Y cells were treated at with 10% fetal bovine serum (FBS), 100 IU mL−1 37 ◦C for 24 h with different concentrations of berry penicillin and 100 µg mL−1 streptomycin. HL-60 fruit extracts ranging from 0.5 to 0.065 µg mL−1 for cells were grown in RPMI-1640 medium supple- anthocyanins and 0.25 µg mL−1 for other phenolics.
mented with 10% FBS, 100 IU mL−1 penicillin and After incubation, the cells were washed three times 100 µg mL−1 streptomycin. Both cell lines were sub- with Kreb’s Ringer solution (KRS) and then incubated cultured every 3 – 4 days and kept at 37 ◦C in a 5% in 1% FBS-KRS containing 100 µmol L−1 DCFH- DA for an additional 60 min at 37 ◦C. The cells were Preliminary dose – response experiments carried out washed and maintained in 1% FBS-KRS (100 µL).
with H2O2 revealed that 100 µmol L−1 H2O2 induced After adding H2O2 at 100 µmol L−1, the fluorescence a moderate amount of oxidative damage to cells and was monitored for up to 60 min at excitation and emis- was therefore used at this concentration throughout.
sion wavelengths of 485 nm and 538 nm, respectively.
Effects of boysenberry and blackcurrant polyphenics on oxidative stress In some experiments the berryfruit extract was added each, followed by chilled absolute ethanol for 10 min at the same time as the H2O2, 60 min after the addition and left to dry overnight. The slides were then of the DCFH-DA. Values are expressed as the percent- stained by placing 500 µL ethidium bromide solution age increase in DCF fluorescence intensity compared (20 µg mL−1) on each slide for 10 min and destained for another 10 min in deionised water. They wereviewed under an epifluorescence microscope (Leitz DNA damage assay (comet assay)
Fluovert F8, Germany) with an attached CCD camera The alkaline comet assay was performed as described and computer. Images of 100 individual cells and their by Singh et al.20 with minor modifications. Quarter- associated comets were acquired digitally, saved as frosted microscopic slides were dipped into hot 1.0% electronic files and quantitatively analysed using Scion normal melting point agarose (Sigma) to one-half of image-processing software with add-on macros. The the frosted area, drained of excess agarose and the final measure of DNA damage was expressed as the underside of the slide wiped to remove agarose. All ‘tail moment’ calculated from the length of the comet pre-coated slides were dried in a 37 ◦C oven overnight and the ratio of DNA in the comet and the remaining and stored at room temperature. An 80 µL drop of cell nucleus based on the definition by Olive and 0.5% low melting point agarose (LMPA, Sigma) at 37 ◦C was mixed with a 10 µL suspension of 10 000HL-60 cells and the mixture was poured onto a pre- Statistical analysis
coated slide. It was levelled by placing a cover slip Results are expressed as mean ± standard deviation.
over the agarose – cell mixture. After the agarose had All data were evaluated for statistical significance using set, the cover slip was removed and a third layer of one-way ANOVA. The confidence level for statistical agarose (80 µL) was added. A cover slip was reapplied significance was set at a probability value of 0.05.
and removed after the agarose layer hardened. Theslides were immersed in lysis buffer (2.5 mol L−1NaCl, 100 mmol L−1 Na2EDTA, 10 mmol L−1 Tris,NaOH to pH 10.00, 1% Triton X-100 and 10% DMSO) at 4 ◦C for at least 2 h to remove cell Identification and characterisation of
protein. The slides were then soaked in a Coplin compounds
jar containing electrophoresis solution (300 mmol L−1 The boysenberry (cv. Riwaka Choice) and black- NaOH, 1 mmol L−1 Na2EDTA, HCl to pH 13), to currant (cv. Ben Ard) extracts were analysed by unwind DNA, for 40 min and electrophoresed at reverse-phase HPLC. HPLC analysis confirmed the a constant current of 300 mA for 30 min. After presence of the four major anthocyanins — cyanidin electrophoresis, the slides were neutralised with Tris- glucoside, cyanidin rutinoside, cyanidin sophoroside HCl buffer at pH 7.5 by three washes for 5 min and cyanidin glucorutinoside — in the boysenberry Figure 1. HPLC chromatograms of extracts: (A) blackcurrant anthocyanins; (B) blackcurrant polyphenolics; (C) boysenberry anthocyanins;
(D) boysenberry polyphenolics.
% Cytotoxicity
% Increase in fluorescence
Figure 3. Protective effects of boysenberry and blackcurrant
anthocyanins and other phenolic compounds against H2O2-induced
Figure 2. Protective effects of blackcurrant and boysenberry
oxidative stress (+). SH-SY5Y neuroblastoma cells were treated with anthocyanins and other phenolic compounds against H2O2-induced boysenberry or blackcurrant extracts and H2O2 at the same time and cytotoxicity (+). SH-SY5Y neuroblastoma cells were incubated with oxidative stress was measured by the DCF assay method. The mean blackcurrant or boysenberry extracts before exposure to H2O2 and of six determinants is shown with standard deviations. All sample cell viability was assessed by the MMT assay. The mean of six treatments are statistically significant from treatment-matched control determinants is shown with standard deviations. All sample (H2O2 alone) (P < 0.001).
treatments are significantly different from treatment-matched control(H2O2 alone) (P < 0.001).
both berries significantly inhibited the increase inintracellular ROS production at all concentrations anthocyanin extract (Fig. 1C). The Ben Ard black- used (P < 0.001) (Fig. 3). Among all the extracts currant anthocyanin extract showed the presence of tested, the phenolic extract of blackcurrant (BcPhen) cyanidin glucoside, cyanidin rutinoside, delphinidin showed the highest degree of protection and this was glucoside and delphinidin rutinoside (Fig. 1A). Minor significantly higher than the same dose of the phenolic components were also found in both extracts and con- extract of boysenberry (ByPhen, P < 0.001). Although firmed by liquid chromatography – mass spectrometry the anthocyanin extracts of both blackcurrant and (LC-MS). These were derivatives of anthocyanins and boysenberry gave similar levels of protection at doses probably produced during the extraction and purifica- ranging from 0.5 to 0.125 µg mL−1, there was a tion procedure. The chromatograms of both Ben Ard significantly higher effect again with the blackcurrant blackcurrant and Riwaka Choice boysenberry pheno- extract when a dose of 0.065 µg mL−1 was used lic extracts demonstrated a very complex mixture of (P < 0.001). Pretreatment of the cells with the berry extracts for 24 h prior to adding the H2O2 insult hadno protective or detrimental effect on the cells (data Neuroprotection against H2O2-induced toxicity
Neuroprotective activities of anthocyanins and otherphenolics from boysenberry and blackcurrant were Effect of extracts on H2O2-induced DNA damage
evaluated by assessing the viability of human neurob- In the comet assay cellular DNA damage is detected by lastoma cells injured with H2O2. The results, shown the size of the ‘comet’ DNA. The nucleoid from each in Fig. 2, demonstrated that all anthocyanins and phe- cell typically appears as either an intact spherical mass nolic extracts gave a high degree of protection at con- (i.e., no DNA damage) or a ‘comet’ (i.e., DNA dam- centrations ranging from 0.075 to 0.25 µg mL−1 (P < age) upon staining with ethidium bromide. The overall 0.001). None of the compounds alone at concentra- span of the tail region of the comet is a general indica- tions ranging from 0.075 to 0.25 µg mL−1 significantly tion of the extent of DNA single-strand breakage. An affected cell viability compared to control (data not experiment was carried out to determine whether boy- senberry or blackcurrant anthocyanins or phenolicscould reduce the comet size after exposure to H2O2 Protective effect of extracts against oxidative
and thus protect cells from DNA damage. Groups of cells were incubated with berry anthocyanins or To test for the protective effect of anthocyanins phenolic fractions at 0.25 and 0.125 µg mL−1 for 24 h and other phenolic fractions against oxidative stress, and exposed to H2O2. Included were control groups both pre-incubation and concurrent incubation of of untreated cells, cells treated with berry extract fractions with H2O2 were carried out. The intracellular alone or cells treated with H2O2 alone. Represen- concentration of ROS in SH-SY5Y cells, as assayed tative slides of treated and untreated cells are shown by DCFH oxidation, was increased nine-fold after in Fig. 4. The untreated cells were relatively intact exposure to H2O2. The concurrent addition of either (Fig. 4A), large ‘comets’ were observed in cells treated the anthocyanin extracts or the phenolic extracts from with H2O2 (Fig. 4D) and incubation of cells with Effects of boysenberry and blackcurrant polyphenics on oxidative stress Figure 4. Single-strand breakage of DNA in HL-60 cells treated with anthocyanins and other phenolic compounds from blackcurrant and
boysenberry, as visualised by the comet assay: (A) untreated control cells; (B) blackcurrant anthocyanin extract at 0.25 µg mL−1 plus H2O2;
(C) boysenberry anthocyanin extract at 0.25 µg mL−1 plus H2O2; (D) H2O2 alone.
berry extracts prior to exposure with H2O2 reduced to the oxidative challenge by hydrogen peroxide. Both the size of the ‘comet’ (Fig. 4B, C). In order to the anthocyanin and non-anthocyanin fractions of quantify these effects and examine whether antho- boysenberry and blackcurrant were able to protect cells cyanins and phenolic fractions of boysenberry and against H2O2-induced cell toxicity effects and DNA blackcurrant themselves induced any DNA damage, damage, with the blackcurrant extracts exhibiting the the mean tail moments of 100 individual cells from highest protection against cell toxicity.
each experimental and control group were calculated.
The boysenberry (cv. Riwaka Choice) antho- When cells were exposed to H2O2, the tail moment cyanin extract contained four main anthocyanins exceeded 400 and that value was significantly dif- ferent from that of untreated control (P < 0.0001).
cyanidin-3-O-glucoside, cyanidin-3-O-2G glucosyl- The results are shown in Fig. 5. Decreased damage rutinose and cyanidin-3-O-rutinose, based on our is associated with a low tail moment and indicates a previous report.14 The main anthocyanins present protective effect of flavonoid pre-treatment. Results in the blackcurrant (cv. Ben Ard) anthocyanin demonstrated that both boysenberry anthocyanins extract were cyanidin-3-O-glucoside, cyanidin-3-O- (ByAcy) and phenolic (ByPhen) fractions were sig- rutinose, delphinidin-3-O-glucoside, and delphinidin- nificantly protective at 0.25 µg mL−1 (P < 0.05), but 3-O-rutinose as identified by Slimestad and Solheim15 not at 0.125 µg mL−1 concentration. Similarly, pre- treatment with blackcurrant anthocyanins (BcAcy) at In addition to the anthocyanins, both boysenberry 0.25 µg mL−1 was significantly protective (P < 0.001) and blackcurrant contain non-anthocyanin polyphe- but phenolic blackcurrant fractions (BcPhen) signifi- nolics. Chromatogram traces of these are shown cantly decreased the tail moment at both concentra- in Fig. 1(B, C) for blackcurrant and boysenberry, tions tested (P < 0.001). Interestingly, the phenolic respectively. The chromatograms show that these extract of blackcurrant (BcPhen) demonstrated again non-anthocyanin fractions contain a complex mix- the highest protective effect on DNA damage (Fig. 5).
ture of different compounds. Previous experimental None of the fractions, when used alone, induced less results22,23 have also shown that the inhibitory effects DNA damage than was present in untreated control of fruit extracts against oxidative stress is significantly cells that were not exposed to H2O2.
correlated with the content of individual categories ofphenolic compounds and that the total phenolics andanthocyanins may also be associated with oxidative DISCUSSION
stress inhibition. It is possible that the different phe- This study has demonstrated the protective effects nolics present in a particular berry extract synergise of boysenberry and blackcurrant extracts on human with each other to give an enhanced effect. (Zhang J neuroblastoma and promyelocyte cells exposed in vitro Protective
Effects of boysenberry and blackcurrant polyphenics on oxidative stress Experiments have shown that dietary supplemen- substances against DNA damage. Epidemiological tation with berries rich in anthocyanins are effec- and some in vivo and in vitro experimental studies tive in reducing stress-induced disease manifestation suggest that diets rich in fruit and vegetables may (McGhie T, unpublished).17,24 Our results confirm exert protective effects against various stages of cancer the in vivo effects reported using an in vitro cell- and cardiovascular diseases.32,33 Protection against based system, and the concentration range used in oxidant challenge may decrease the rate of mutation this present study was within the human physiolog- and hence help prevent ageing and age-related ical range. Some recent human and animal feeding diseases including cancer.8,34,35 Oxidant challenge trial experiments showed that the plasma/serum con- can induce potentially mutagenic DNA damage by, centration of anthocyanins was in the 12 — 100 µg L−1 for example, direct action of reactive oxygen species range.16,17 The extracts of boysenberry and black- (ROS) on DNA, or indirectly via aldehydic lipid currant containing anthocyanins and phenolic com- peroxidation degradation products.36,37 There are pounds displayed significant inhibition against the various intracellular antioxidant mechanisms of DNA oxidative challenge of H2O2 at concentrations rang- protection,38 which include scavenging of damaging ing from 0.065 to 0.5 µg mL−1. These extracts had no ROS, enzymatic inactivation of ROS and binding effect when added to cells in tissue culture medium, of iron. DNA damage can be assessed by levels suggesting that there was no production of perox- of oxidised bases, for example 7,8-dihydro-8-oxo- ide as reported for other polyphenolic compounds.25 deoxyguanosine (8oxodG), and this has been used for Additional experiments (data not shown) indicated studies by measuring levels in plasma and/or urine.39 that these results were not complicated by effects on Alternatively, the single-cell gel electrophoresis test, cellular viability, as measured by the MTT assay, known as the comet assay, can be used, and as the extracts were not cytotoxic in this dose range.
this was the technique employed in the current Human neuroblastoma cells were used to demonstrate study. It is a well-validated and relatively simple that the two types of extracts from both boysenberries technique for detecting DNA strand breaks.20,40 The and blackcurrants protected them from H2O2-induced ‘standard’ comet assay has been used extensively to cell toxicity. A neuronally derived cell line was used determine DNA damage in whole cells before and for these studies as the neuroprotective effect of after incubation with potentially genotoxic agents, berries is of great interest. Recently, Rice-Evans and and to investigate the putative protective effect of co-workers demonstrated, in an in vitro experiment, feeding dietary antioxidants.41 – 44 Our findings with that flavonoids, including dietary anthocyanins and anthocyanins and other phenolic compounds in DNA some metabolites, are able to traverse the blood – brain damage protection are consistent with those of other barrier, and that potential for permeability (Papp) is investigations.45,46 In the current study, no DNA consistent with compound lipophilicity.26 Intriguingly, damaging effect was seen at doses up to 0.25 µg mL−1, pre-incubation of the neuroblastoma cells for 2 h with the highest concentration tested. Contrary to our the extracts prior to H2O2 insult had no effect on results, Glei et al.47 showed that anthocyanin-rich the H2O2-induced ROS level, whereas the concurrent black carrot extract did not protect cells from H2O2- addition of fractions with H2O2 significantly inhib- induced DNA damage despite containing the aglycon ited the increase of intracellular ROS production. It cyanidin, which was shown to be protective. In has been postulated that dietary flavonoids can exert recent in vivo and in vitro experiments, Duthie et al.48 differential protective effects on ROS-induced intra- demonstrated that cyanidin-3-glucoside did not alter cellular oxidative stress following their metabolism lipid peroxidation or DNA damage in rats. However, it during absorption and circulation.27 It is possible was chemoprotective against DNA damage in human that pre-incubation of the cells with the berry antho- colonocytes. Commercially prepared grape, bilberry cyanins and other phenolic compounds resulted in and chokeberry anthocyanin-rich extracts have been metabolism to compounds that were ineffective at shown to differentially inhibit the growth of human inhibiting the increase in intracellular ROS produc- colon cancer cells.49 Interestingly, all these extracts tion. The metabolic conversion of catechin has been have no inhibitory effect on the growth of non- shown to have no effect on free radical scavenging tumorigenic colon cells at lower concentration (25 or activities, but exhibits significant negative effects on 50 µg mL−1), illustrating greater growth inhibition of ROS regulation.28 Another example of the metabolic colon cancer, as compared to non-tumorigenic colon conversion of this class of compounds is found in the work of Boulton et al.,29 where quercetin aglycon The diverse protective effects that boysenberry and was subject to oxidative degradation when incubated blackcurrant anthocyanins and phenolic compounds with HepG2 cells, with the resulting formation of the appear to elicit in vitro and in vivo have contributed O-methylated metabolite, isorhamnetin.
toward the growing interest in the role that the The results of this study also show that all four berries play in human health. It is unrealistic to claim berry fruit extracts provide protection to HL-60 cells any specific health benefits based on in vitro results; and prevent DNA damage. Although anthocyanins are however, it is important to understand the protective well known to have antioxidant activity,30 – 33 there is effects of fruit extracts and the mechanisms involved, so far limited evidence for the protective role of these as well as the purified phenolic components that they contain. There may also be synergies between 15 Slimestad R and Solheim H, Anthocyanins from black currants the different components of berries and the in vitro (Ribes nigrum L.). J Agric Food Chem 50:3228–3231 (2002).
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Bidden or Unbidden God is present. My sisters and brothers, God is always workingin our lives whether we name him or not. What is most conspicuous about the Book ofEsther is the absence of God being named. Esther is the only book of the Bible that doesnot mention the word God. But God is present, God acts in the world, God’s work amongus is apparent in the story of Esther and in our daily li

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