Review pubs.acs.org/JAFC
In Vitro and in Vivo Models of Colorectal Cancer: Antigenotoxic Activity of Berries Emma M. Brown,† Cheryl Latimer,† Philip Allsopp,† Nigel G. Ternan,† Geoffery McMullan,† Gordon J. McDougall,§ Derek Stewart,§,# Alan Crozier,⊥ Ian Rowland,⊗ and Chris I. R. Gill*,† †
Northern Ireland Centre for Food and Health, Centre for Molecular Biosciences, University of Ulster, Cromore Road, Coleraine, Northern Ireland BT52 1SA, U.K. § Environmental and Biochemical Sciences Group, Enhancing Crop Productivity and Utilization Theme, The James Hutton Institute, Mylnefield, Invergowrie, Dundee, Scotland DD2 5DA, U.K. # Bioforsk Nord Holt, Postboks 2284, Tromsø, Norway ⊥ Plant Products and Human Nutrition Group, Joseph Black Building, School of Medicine, University of Glasgow, Glasgow, Scotland G12 8QQ, U.K. ⊗ Hugh Sinclair Unit of Human Nutrition, Department of Food and Nutritional Sciences, University of Reading, P.O. Box 226, Whiteknights, Reading, England RC6 6AP, U.K. ABSTRACT: The etiology of colorectal cancer (CRC), a common cause of cancer-related mortality globally, has strong associations with diet. There is considerable epidemiological evidence that fruits and vegetables are associated with reduced risk of CRC. This paper reviews the extensive evidence, both from in vitro studies and animal models, that components of berry fruits can modulate biomarkers of DNA damage and that these effects may be potentially chemoprotective, given the likely role that oxidative damage plays in mutation rate and cancer risk. Human intervention trials with berries are generally consistent in indicating a capacity to significantly decrease oxidative damage to DNA, but represent limited evidence for anticarcinogenicity, relying as they do on surrogate risk markers. To understand the effects of berry consumption on colorectal cancer risk, future studies will need to be well controlled, with defined berry extracts, using suitable and clinically relevant end points and considering the importance of the gut microbiota. KEYWORDS: colon cancer, fruits and vegetables, berries, DNA damage, biomarker, (poly)phenols
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INTRODUCTION Epidemiological evidence suggests that diets rich in fruits and vegetables may contribute to a reduced risk of colorectal cancer (CRC).1,2 A recent meta-analysis reported a decreased risk associated with fruit consumption (RR = 0.85, 95% CI = 0.75− 0.96 for 3 servings/day) and highlighted that low fruit and vegetable consumption was associated with a moderately increased risk of colorectal cancer.3 Commonly consumed fruits include a variety of berries, such as strawberries (Fragaria), blueberries (Vaccinium), raspberries (Rubus), blackberries (Rubus), and cranberries (Vaccinium) as well as black currants (Ribes), bilberries (Vaccinium), lingonberries (Vaccinium), and cloudberryies (Rubus), which are also ingested in reasonable amounts in either fresh or processed forms (i.e., jams, yogurts, and juices). Berries accumulate high levels of (poly)phenols; for example, total phenol contents of 100−300 mg/100 g fresh weight are commonly reported for black currants, raspberries, and strawberries.4 The (poly)phenol composition of berry fruits is strongly influenced by genetic and environmental factors5 such as species and variety4,6 cultivation methods, weather, ripeness at and time of harvesting,7 and duration and conditions of storage8,9 and as a consequence may influence bioactivity. Berries contain simple phenolic acids such as benzoic (C6) and hydroxycinnamate (C6:C3) derivatives, which are usually conjugated to other components. Flavonoid derivatives with a © 2014 American Chemical Society
C6:C3:C6 structure are also common. For example, variations in anthocyanin composition are responsible for the red-to-purple coloration of berries; the red-orange color of strawberries is due to the presence of mainly pelargonidin anthocyanins, whereas the deep purple-black of black currants is caused by the accumulation of delphinidin and cyanidin anthocyanins in the skin. Flavonols are common in berries and also show speciesspecific variation. Certain berry species accumulate different tannin components (such as proanthocyanidins and/or ellagitannins), and these large and complex (poly)phenols can influence flavor. (Poly)phenolic compounds present in berries reported to have potential anticancer activity include proanthocyanidins, hydrolyzable tannins, anthocyanins, hydroxycinnamates, stilbenes, lignans, and phenolic acids10,11 (Poly)phenols are secondary plant metabolites commonly described as non-nutrient bioactives12 and are extensively metabolized and rapidly excreted postconsumption.13,14 Detailed (poly)phenol composition for many plant-based foods, including berries, is limited. This is confounded further Special Issue: 2013 Berry Health Benefits Symposium Received: Revised: Accepted: Published: 3852
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Review
mechanisms may be a feasible explanation for the protective effects of berry (poly)phenols against initiation of CRC. Unrepaired DNA damage can become incorporated into the genome as a permanent sequence change if the cell divides before the damage is repaired. For example, CRC is characterized by a multistep pathway involving sequential mutations and deletions of key oncogenes and tumor suppressor genes such as APC, K-ras, and p53 in epithelial cells.26 These genetic changes result in evasion of cell signaling pathways and cell cycle control points, leading to hyperproliferation, avoidance of apoptosis, and eventual invasion and metastasis. Under normal cellular conditions, reactive oxygen species (ROS) such as superoxide anions (•O2−) and hydroxyl radicals (•OH−) are produced by endogenous metabolism and used by the cell to modulate biological processes; for example, the generation of ROS can lead to activation of receptors including G protein-coupled receptors, tyrosine, and serine/ threonine kinases.27 Downstream signaling components are stimulated as a response, and these include MAPK and protein kinase C, which are tightly regulated and are involved in proliferation, differentiation, cell survival, and apoptosis.28 However, ROS production has also been implicated in sequential tumor development by a number of routes.27 Reaction of ROS with DNA results in the formation of oxidized DNA bases, apurinic/apyrimidinic sites (AP sites), or DNA strand breaks. In particular, 8-oxo-7,8-dihydro-2′deoxyguanosine (8-OHdG) is one of the most easily formed oxidative DNA lesions. Moreover, 8-OHdG can react with compounds such as peroxynitrate to produce further mutagenic lesions.29 ROS also react with phospholipids in the cell membrane and form lipid peroxidation products such as 4-hydroxynonenal, which in turn can react with DNA and to form cyclic-DNA adducts.30 Other abundant lesions of oxidative DNA damage, which are also highly mutagenic, result from GC to TA transversions.31 ROS assault on cells in areas prone to DNA damage increases the probability that changes will go unrepaired and lead to conformational changes of the structure of DNA, rendering replication more difficult and error prone.32,33 Furthermore, ROS may contribute toward carcinogenesis through differential regulation of cell signaling pathways. For example, modulation of MAPK, Jak/STAT, heat shock response, and PI3K/Akt pathways will affect gene expression, and, if activated, can in certain circumstances stimulate growth.34,35 Given that STATs are generally associated with transcriptional activation, STAT-dependent transcriptional repression has also been reported.36 Also, whereas activation of JNK signaling in response to a range of stress signals is generally thought to promote cell death or tumor suppression, there is evidence to suggest that JNK signaling may also be involved in the promotion of growth and the formation of tumors under certain circumstances.37 DNA damage from exogenous chemicals or radiation can have genotoxic activity and result in DNA modifications such as covalent bridging between bases as well as inducing single- and double-strand breaks. Therefore, cellular systems monitor the genome and repair DNA using different repair enzymes by removing damaged or incorrect sections of DNA and replacing them with the correct sequence. There are multiple different methods of DNA damage repair within the cell, including excision repair (for single-strand breaks), nonhomologous end joining and homologous recombination (for double-strand
by differences attributable to environmental conditions, ripeness at harvesting, and storage conditions. Additionally, the wide variety of structures and conjugates of (poly)phenols makes estimation in food substances extremely difficult, and therefore data on dietary intake are scarce. However, the recently developed and updated Phenol-Explorer database provides much useful information.15 In a recent study by PérezJiménez and co-workers,16 daily consumption of total (poly)phenols in a French population was estimated as 820 ± 335 mg/day (aglycone equivalents), which was similar to that previously observed in a Finnish cohort (863 ± 415 mg/day).17 In both studies phenolic acids were the main (poly)phenols consumed, with intake in the Finnish cohort being twice that of the French cohort. The anthocyanin intake was reported to be marginally higher in the Finnish cohort than in the French at 47 ± 79 and 35 ± 29 mg/day (aglycone equivalents), respectively. The large variations evident in consumption likely reflect individual preferences, but it was not surprising that berries were a more prominent source of (poly)phenols in the Finnish diet than in the French. Ileostomy feeding studies have demonstrated that substantial amounts of (poly)phenolic compounds in berries are not absorbed into circulation from the small intestine but rather pass into the large intestine where,18,19 it is reasonable to infer, they come into direct contact with the colonic epithelium.20,21 Moreover, the microbiota within the colon plays a key role in the fate of phytochemicals that are not absorbed in the small intestine. Once in the colon, berry (poly)phenolics are subject to the fermentative action of the microbiota, with catabolism giving rise to a diversity of phenolic acids.19,22 Moreover, fermentable carbohydrates (fiber) present in whole foods such as berries might act in synergy with the mixture of (poly)phenols present in berries and contribute to their overall antigenotoxic activities, as these fermentable dietary fibers can beneficially modulate both the composition and metabolic output of the human gut microbiota.23 Several theories on the anticancer activity of (poly)phenols are supported by an expanse of data demonstrating the potential mechanisms involved. The most studied effects are those that modify proliferation and apoptosis, and these have been extensively reviewed elsewhere.24 Other mechanisms responsible for the putative anticancer activity are still not fully understood but may include scavenging free radicals, induction of enzymes involved in metabolism of xenobiotics,25 regulation of gene expression, and modulation of cellular signaling pathways including those involved in DNA damage detection and repair. This review aims to assess the in vitro and in vivo evidence for anticancer activities for whole berries and berry extracts focusing particularly on the genoprotective element of these compounds.
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DNA DAMAGE AND CANCER DNA damage has a key role in cancer development. The initiating step in CRC development involves exposure to, or uptake of, carcinogens, resulting in permanent DNA damage. Considering the potentially toxic nature of the colon contents, it is not unsurprising that frequent mutations occur in colonic cells. Therefore, efficient DNA damage detection and repair mechanisms are present to prevent damage becoming a permanent sequence change. However, errors in detection and repair mechanisms may occasionally occur. Thus, reducing the frequency of DNA damage or enhancing the repair 3853
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3854
comet assay, endogenous and H2O2-induced DNA damage
modulation of 4E2/CuCl2induced oxidative DNA adducts intracellular oxidation
oxidative cytotoxicity (CellTiter-Glo assay)
1 μM or 100 μM
0−300 μM
3.15−50 μg/mL
0.8−25 μg/mL
ellagic acid
anthocyanin fraction from blackberry (Rubus f ruticosus)
phenolics isolated from strawberries (Fragaaria × ananassa)
quercetin
Caco-2
Caco-2
Caco-2
antioxidative ability, TEAC assay
comet assay, endogenous and H2O2-induced DNA damage base excision repair assay (BER) using enzyme 8oxoguanine DNA glycosylase 1 (OGG1)
5 μM or 10 μM
ursolic acid (UA)
HT29
10 mg/mL
antigenotoxic activity, comet assay intracellular ROS levels, CAA assay
10−500 μg/mL
Caco-2
anthocyanin-rich fraction from bilberry (Vaccinium myrtilus)
mutation frequency assay
fermented = raspberry, 15.5 μg/mL; strawberry, 13.9 μg/mL; black currant, 12.4 μg/mL
HT29
in vitro digested and in vitro fermented raspberry (Rubus idaeus), strawberry (Fragaria × ananassa) and black currant (Ribes nigrum) extracts antigenotoxic activity, comet assay
end point
IVD = 3.125−50 μg/mL GAE
dose intracellular ROS levels, CAA assay
Caco-2
anthocyanin-rich fraction from Vaccinium corymbosum and Vaccinium myrtilus
0.5−50 μg/L
berry or berry constituent
colonocyte cell line
three of the phenolics showed significant antioxidant capacity: cyanidin-3-glucoside (7156 μM Trolox/mg)
dose-dependent ↓ in intracellular oxidation no ↓ in cell viability ↓ cytotoxicity from 1.6 to 25 μg/mL
>60% ↓ of both unidentified oxidative adducts and 8-OHdG
↓ in strand breaks by 42% at 1 μM and 57% at 100 μM ↑ mRNA expression of human 8oxoguanine DNA glycosylase (hOGG1) at 0 and 4 h after H2O2 treatment
both concentrations yielded ∼40% ↓ in strand breaks following 2 h of preincubation 17% ↓ in strand breaks after 5 μM for 24 h 23.5% ↑ in BER activity, for Caco-2 cells pretreated (24 h) with UA
↓ in strand breaks by ∼50% after in Caco-2 after 50 μg/mL and 24 h ↓ in intracellular ROS by ∼30−40% in Caco-2 and HT29 after 250 μg/mL and 24 h
↓ in strand breaks by ∼40% in all IVD berry extracts from 6.25 to 50 μg/mL after 24 h ↓ in strand breaks by ∼30% in all fermented extracts after 24 h ↓in RMF >50% in all IVD berry extracts after 24 h ↓in RMF ∼40% in all fermented extracts after 24 h
↓ in intracellular ROS by ∼40% after 0.5 μg/L and 1 h
observation
Table 1. Effects of Berry Extracts or Berry Constituents on Models of DNA Damage and Scavenging Activitya
+b
+b
+b
+b
+b
+b
+b
+b
+b
+b
+b
+b
+b
protective/ adverse effect comment
isolation of the specific phenolics within strawberry extract has given a greater understanding on which compounds possess antioxidative capacity
suggests anthocyanins protect by scavenging AAPH-induced peroxyl radicals generated within cells
dose-dependent decrease in induced oxidative adducts after ellagic acid treatment
quercetin shows antigenotoxic activity at low concentrations; enhanced DNA repair by modulation of DNA repair enzyme expression
greater reduction in DNA damage after a short incubation period with UA; increase in BER activity
dose-dependent decrease in DNA damage and scavenging activity after anthocyanin treatment
fermented, phenolic acid rich extracts cause a significant decrease in DNA damage and mutation frequency
reduction of intracellular ROS at low concentrations of anthocyanin treatment
52
53
61
62
63
54
64
55
ref
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0−1 mM
quercetin
3855
observation
quercetin ↓ strand breaks by ∼50 and 65% after 0.5 and 18 h myricetin ↓ strand breaks at 1 mM kaempferol and rutin, no effect
′O2 ′O2 ′O2 ′O2 ′O2
+b −
+b
+b +b +b +b +b
> > > > >
OH• > H2O2 H2O2 > O2•− OH• > H2O2 OH• > H2O2 OH• > H2O2 O2•− OH• O2•− O2•− O2•−
+b +b +b
↓ strand breaks ∼39% ↓ strand breaks ∼35% ↓ strand breaks ∼30% > > > > >
+b
↓ in H2O2 induced strand breaks by ∼50% from 3.125 to 50 μg/mL
pelargonidin (4922 μM Trolox/mg) pelargonidin-3-rutinoside (5514 μM Trolox/mg)
protective/ adverse effect
specific antigenotoxic activity against H2O2induced strand breaks; no effect on oxidative base damage
scavenging activity varied with berry type and species of reactive oxygen
all flavonoids significantly reduced oxidantinduced strand breakage
dose-dependent decrease in DNA damage after berry treatment
comment
58
51
59
60
ref
a
GAE, gallic acid equivalents; ROS, reactive oxygen species; AAPH, 2,2′-azobis(2-methylpropanimidamide) dihydrochloride; NE, no effect; ↓, decrease; ↑, increase; 8-OHdG, 8-hydroxy-2′deoxyguanosine; CAA, cellular antioxidant activity; IVD, in vitro digested; RMF, relative mutation frequency; TEAC, Trolox equivalent antioxidant capacity; UA, ursolic acid. bSignificant result. +, protective effect; −, adverse effect.
myricetin kaempferol rutin
Caco-2
comet assay, endogenous and H2O2-induced DNA damage
inhibition of ROS formation through direct scavenging
100 μL of juice extract
human colon epithelial cells (HCEC)
cyanidin cyanidin-3-glucoside quercetin
blackberry (Rubus f ruticosus) blueberry (Vaccinium corymbosum) cranberry (Vaccinium oxycoccos) raspberry (Rubus idaeus) strawberry (Fragaria × ananassa)
HT29
raspberry (Rubus idaeus) in vitro digested extract
comet assay, endogenous and H2O2-induced DNA damage
end point
50 μM
dose
comet assay, endogenous and H2O2-induced DNA damage
colonocyte cell line
0−50 μg/mL GAE
berry or berry constituent
Table 1. continued
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breaks), and mismatch repair (for mismatched bases from DNA replication). However, if DNA damage goes undetected and unrepaired, this will become a mutation when the cell replicates.38
Review
IN VITRO STUDIES
Multiple in vitro studies have assessed the anticancer activities of berry components, using cell models in attempts to represent the initiation stage of CRC. These studies have mainly assessed the efficacy of whole berry extracts, fractionated berry extracts, or purified/commercial berry components in a range of human colon cell lines. These studies are summarized in Table 1. Berry (poly)phenols may exert potential anticancer effects by chelating redox active metals (such as iron) or by directly scavenging ROS [including hydrogen peroxide (H2O2), hydroxyl radicals (•OH−), superoxide radicals (•O2−), and singlet oxygen (1O2)] by being effective electron donors to the ROS. ROS scavenging activity51,52 and antioxidant capacity53−55 in vitro vary with type of berry extract or isolated components of berries (Table 1). The scavenging activity of berry (poly)phenols in simple, cell-free antioxidant systems through the scavenging of free radicals is remarkably high compared to those of other standard antioxidants such as ascorbic acid and α-tocopherol.51 However, although useful in assessing antioxidant potential, these chemical assays provide little in terms of mechanisms relating to biological activity. Modifications have included the use of simulated gastrointestinal digestion of fruits prior to testing: for example, cranberries mostly retained their antioxidant activity following digestion using cell-free (DPPH) and Caco-2-based assays, whereas blueberries lost 43% of their free radical scavenging activity.56 The measurement of intracellular ROS levels is a simple method to assess efficacy in a cellular model, which has greater biological relevance with uptake, retention, and metabolism of antioxidant (poly)phenols under physiologically relevant conditions. Several authors have utilized this technique to demonstrate a reduction in intracellular chemically induced ROS upon addition of the peroxyl radical (AAPH) to induce oxidative damage and associated cytotoxicity in various colon cancer cells.53−55 Although direct intracellular scavenging has been suggested as the mechanism for this decrease,53,54 this may be an oversimplification of the mechanisms involved. It may be that berry (poly)phenols interact with cell receptors or enzymes resulting in altered cell redox status, which triggers redox-dependent reactions.57 For example, Bornsek and colleagues55 hypothesized that the level of antioxidant activity invoked was not consistent with the low concentration of berry (poly)phenols (0.5 μg/mL) and that a signaling cascade may be triggered, possibly through their binding to an extracellular receptor or acting as an intercellular secondary messenger, resulting in activation of endogenous antioxidant systems. This mechanism would result in the amplification of the signal, so that low levels of (poly)phenols would have greater efficacy. Several in vitro studies have investigated the effects of berry (poly)phenols on H2O2-induced DNA strand breaks as a measure of genotoxicity and, overall, have shown a reduction in genotoxicity in response to berry extracts and isolated flavonoids 58−64 (Table 1). The suggested mechanisms responsible for this antigenotoxic activity have been described as increased antioxidant activity, enhanced DNA damage repair, or a combination of both. For example, this has been demonstrated with the flavonoid quercetin 62 and the pentacyclic triterpenoid acid ursolic acid,63 which is found in some berry types. Both of these compounds, at physiologically relevant (colonic) concentrations (100 and 10 μM, respectively), have demonstrated potent antioxidant effects in Caco-2 cells through reducing levels of H2O2-induced DNA damage
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MEASUREMENT OF DNA DAMAGE DNA damage can be determined in tissues and organs either by taking biopsies or by isolating exfoliated epithelial cells such as buccal, bladder, or intestinal cells. Systemic DNA damage is usually measured in peripheral blood mononuclear cells (PBMC) as they can be easily isolated from blood samples. In addition, they are believed to reflect DNA damage in other body tissues. Studies of DNA damage can be performed in vivo and ex vivo as well as in vitro. A recent study reported no genotoxic impact in human lymphocytes following exposure to radiofrequency electromagnetic field emission using four genotoxicity end points,39 including the alkaline single-cell gel electrophoresis (comet) assay,40 which is the most frequently applied assay to detect strand breaks and related events. The comet assay is applicable for a wide range of cell types and for in vivo and in vitro studies. It involves embedding a suspension of the target cells in agarose on a microscope slide, lysing the cells to liberate the DNA, and then treating under alkaline conditions, causing the DNA to unwind from sites of strand breakage. The slides are then subjected to electrophoresis, and DNA fragments induced by genotoxic agents migrate to the anode to form a comet “tail”. The DNA is stained with a fluorescent dye and the proportion of damaged DNA in the tail assessed by image analysis. A modification to the procedure, in which enzymes that convert oxidized bases to strand breaks are added, allows oxidative DNA damage to be quantified. For example, the enzyme endonuclease III (EndoIII), which specifically nicks DNA at sites of oxidized pyrimidines, or alternatively formamidopyrimidine DNA glycosylase (FPG), which recognizes 8-oxoGua and other oxidized purines, can be added after lysis.41 Conversely, the combined use of fluorescence in situ hybridization (FISH)/comet assay allows for the detection of DNA damage/repair to specific genes. By combining these two well-established methods, the hybrid assay allows fragmented DNA to be separated from nonfragmented DNA, whereas the FISH element allows detection of specifically labeled DNA sequences of interest such as APC, Tp53, or KRAS.42,43 The comet assay is widely regarded as a valuable tool for genotoxicity testing and biomonitoring of oxidative stress in humans,44 and its applicability to cancer risk studies has been well discussed;45,46 however, interlaboratory reproducibility remains an area for improvement.47 Systemic oxidative DNA damage can also be assessed by measurement of the formation of DNA adducts such as 8hydroxy-2′-deoxyguanosine (8-OHdG, one of the predominant forms of free radical-induced oxidative lesions48) or by detection of lipid peroxidation products. 8-OHdG is commonly detected by HPLC with electrochemical detection or tandem MS or by GC-MS. Lipid peroxidation products in plasma or urine include hydroxyeicosatetraenoic acids (HETE), which are generally measured using HPLC with MS, chemiluminescence, or fluorescence detection.49,50 It is interesting to note that there is evidence to suggestthat the enzymatic comet assay variant appears to be more accurate in the estimation of low background levels of oxidative damage than are the chromatographic methods.44 3856
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and specific oxidized adducts (8-oxoG) as detected using the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1) to specifically cleave DNA at these sites. Further analysis has revealed that pretreatment with ursolic acid prior to, but not following, exposure to oxidative insult enhanced the rate of repair of DNA strand breaks via base excision repair (BER),63 possibly through up-regulation of the repair enzyme OGG1 as evidenced by Min and colleagues with quercetin.62 However, earlier work did not detect changes in oxidative base damage with quercetin (50 μM), and Duthie et al.58 concluded that quercetin may in fact act by stabilizing the DNA directly. Although the exact mechanism responsible is still unknown, it may be affected via augmentation of p53 in response to antioxidant molecules, leading to up-regulation of the DNA excision repair pathway.65 Although studies have shown the genoprotective effects of isolated components from berries,54,58,59,62 few investigations have considered the effect of intestinal digestion and bacterial fermentation on (poly)phenols from whole berries, as would occur following human consumption. Post ingestion, the phytochemicals contained within berries undergo alterations in their structure, and possibly their function, because of the impact of the human digestive tract. We know, for instance, that substantial amounts of (poly)phenolic compounds in raspberries are not absorbed into circulation in the small intestine but pass into the large intestine66,67 where, it is reasonable to infer, they come into direct contact with the colonic epithelium and exert beneficial effects. For example, Coates et al. 60 demonstrated a genoprotective effect in HT 29 cells for a raspberry extract that had undergone in vitro digestion. However, it is also clear that berry (poly)phenols are extensively metabolized to simpler phenolics by the colonic microbiota during passage through the large intestine,67−69 and thus it seems reasonable to assume that the levels of intact berry (poly)phenols will decrease as they pass further through the colon, with a corresponding increase in microbiota-derived catabolites. Therefore, different parts of the proximal to distal colon will be exposed to different proportions of the original (poly)phenol components and their catabolites. Only a few studies have established that colonic catabolites of (poly)phenols have bioactivities (at least in vitro) relevant to colon cancer.64,70,71 We have previously demonstrated that in vitro digested and fermented raspberry, strawberry, and black currant extracts at physiological concentrations (
