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Anthocyanin-Rich Black Currant Extract Suppresses the
Growth of Human Hepatocellular Carcinoma Cells
Anupam Bishayeea*, Erzsébet Háznagy-Radnaib, Thomas Mbimbaa, Péter Siposc,
Paolo Morazzonid, Altaf S. Darvesha, Deepak Bhatiaa and Judit Hohmannb
aCancer Therapeutics and Chemoprevention Group, Department of Pharmaceutical Sciences,
Northeastern Ohio Universities Colleges of Medicine and Pharmacy, 4209 State Route 44,
Rootstown, OH 44272, USA
bDepartment of Pharmacognosy, University of Szeged, Eötvös u. 6, 6720 Szeged, Hungary
cDepartment of Pharmaceutical Technology, University of Szeged, Eötvös u. 6, 6720 Szeged, Hungary
dIndena S.p.A., Milan, Italy
abishayee@neoucom.edu
Received: June 4th, 2010; Accepted: July 15th, 2010
Dietary antioxidants, such as anthocyanins, are helpful in the prevention and control of various diseases by counteracting the
imbalance of oxidative and antioxidative factors in the living systems. Black currant (Ribes nigrum L., Grossulariaceae) is
known to contain high amounts of anthocyanins (250 mg/100 g fresh fruit). Black currant fruits have been used in Asian and
European traditional medicine for the treatment of a variety of diseases. Black currant extract has recently been found to be the
second most effective amongst nine different berry extracts studied for their free radical scavenging activity. Constituents
present in black currant juice have been found to exert a number of health-promoting effects, including immunomodulatory,
antimicrobial and antiinflammatory actions, inhibition of low-density lipoprotein, and reduction of cardiovascular diseases.
Although antioxidant and antiinflammatory effects of black currant juice could be of value in preventing and treating oxidative
stress- and inflammation-driven cancers, no experimental evidence is available to now. The objective of the present study was
to evaluate the potential antiproliferative effects of black currant fruit skin extract against HepG2 human liver cancer cells. The
aqueous extract yielded an anthocyanin-rich fraction with cyanidin-3-O-rutinoside as one of the major anthocyanins. This
fraction exhibited a potent cytotoxic effect on HepG2 cells and this effect was more pronounced than that of delphinidin and
cyanidin, two major aglycones of anthocyanins present in black currant. Our results indicate, for the first time, that black
currant skin containing an anthocyanin-rich fraction inhibits the proliferation of liver cancer cells, possibly due to additive as
well as synergistic effects. This product could be useful in the prevention and treatment of human hepatocellular carcinoma.
Keywords: Black currant, Ribus nigrum, anthocyanin, delphinidin, cyanidin, cyanidin-3-O-rutinoside, cytotoxicity, HepG2,
hepatocellular carcinoma.
Epidemiological studies over the past few years have
suggested that regular intake of fruits and vegetables
may significantly reduce the risk of age-related chronic
illnesses in certain populations [1a]. Dietary
antioxidants, including anthocyanins, play a vital role in
the prevention and control of several major chronic
illnesses, such as arthritis, atherosclerosis, diabetes,
cardiovascular ailments, Alzheimer’s disease, and
Parkinson’s disease, as well as cancer by counteracting
the imbalance of oxidative and antioxidative factors
in the living systems [1b-1d]. Anthocyanins, a
predominant group of water-soluble pigments belonging
to the flavonoid class, are present in various plants,
contributing to most of the blue, red, violet and purple
colors [2a]. It has been estimated that the average daily
intake of anthocyanins in the United States population is
180−215 mg [2b]. Edible berry fruits such as blueberry,
strawberry, black and red raspberry, as well as black
and red currants represent an abundant source of
structurally diverse anthocyanins in quantitative as well
as qualitative terms [2c,2d]. Black currant (Ribes
nigrum L., Grossulariaceae) fruits are known to contain
high amounts of anthocyanins (250 mg/100 g fresh
fruit) [2e]. Black currant fruits and leaves have been
used in both Asian as well as European traditional
medicine for the treatment of a variety of diseases
including inflammatory disorders [3a,3b]. Black currant
extract has been found to be the second most effective
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1614 Natural Product Communications Vol. 5 (10) 2010 Bishayee et al.
antioxidant amongst nine different berry extracts
studied for their free radical scavenging activity [3c].
Black currants have been recently termed as
"superfruits” as they are believed to possess a number
of health benefits including alleviation of chronic
oxidative stress-related ailments [3d]. Constituents
present in black currant have been found to
exert a variety of health-promoting effects,
including immunomodulatory, antimicrobial and
antiinflammatory actions, inhibition of low-density
lipoprotein, as well as reduction of cardiovascular
diseases [4a-4e]. Black currant juice and extract have
also been found to suppress the proliferation of breast,
prostate, stomach, intestine and colon cancer cells in
vitro [5a-5c] and significantly inhibit the growth of
Ehrlich carcinoma in vivo [5d,5e]. In a recent clinical
study, it has been found that an anthocyanin-rich black
currant extract exhibited antioxidant, antiinflammatory
and immunostimulatory properties [5f].
Berries including black currants are used to prepare
juice, wines, jams, jellies, ice cream, and cake toppings,
as well as other food products. The solid residue that
remains following the extraction of black currant juice
results in the production of a byproduct, known as
pomace. The pomace of black currant can be regarded
as a good source of phenolic antioxidants, including
anthocyanins and flavonols, which predominantly
contribute to the high radical scavenging property of
this press residue [6a,6b]. The skin of black currant fruit
represents a considerable portion of the pomace.
Although this dark-colored fraction is likely to contain
anthocyanins and other constituents with important
biological activities, data on characterization of the
phytochemicals exclusively from the skin of black
currant are very limited. Likewise, the anticancer
potential of the constituents of black currant skin has
not been investigated until now. In view of this, the aim
of the current study was to prepare and characterize an
extract from black currant skin. As black currant
anthocyanins possess potent antioxidant and
antiinflammatory properties [2c,4e,5f,6c,7a], an
anthocyanin-rich extract from the skin of this berry fruit
has been tested for its efficacy in treating oxidative
stress- and inflammation-driven hepatocellular
carcinoma (HCC) using an in vitro cell culture model.
In the present study, the skin of R. nigrum fruit was
extracted with water. Following evaporation and spray-
drying, a dark pink-colored solid material was obtained.
This extract was used for in vitro experiments. The
chemical composition of the extract was characterized
by total anthocyanin content, which was determined by
spectrophotometry and high performance liquid
chromatograpy (HPLC). Moreover, the content of
cyanidin-3-O-rutinoside, as one of the major
anthocyanins of R. nigrum skin has also been
quantified. The reverse phase-HPLC determination
resulted in 1.15 ± 0.05% total anthocyanin and 0.29 ±
0.06% cyanidin-3-O-rutinoside. In accordance with
these data, the total anthocyanin content of the black
currant extract was measured by spectrophotometry as
1.10 ± 0.05%, expressed as cyanidin-3-O-rutinoside
chloride.
Our data are in accordance with previous studies that
have indicated that anthocyanins (anthocyanidin
glycosides) dominate in black currant fruits with the
concentrations ranging from 0.17 to 0.36% (fresh
weight) depending on specific cultivars [6c,7b]. The
four major anthocyanins previously found in black
currant fruit extracts are cyanidin-3-O-glucoside,
cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside and
delphinidin-3-O-rutinoside, which collectively represent
94-98% of the total anthocyanin content depending on
the cultivar [2c-2e]. Moreover, the aforementioned four
anthocyanins have also been detected by HPLC in
extracts prepared from black currant pomace and
residues, accounting for approximately 90% of total
anthocyanin content [6a]. Additionally, acidic
hydrolysis following extraction yielded two
anthocyanidins, namely delphinidin and cyanidin [6a].
In order to evaluate the antitumor effects of
anthocyanin-rich black currant skin extract on HCC, we
treated HepG2 human liver cancer cells with varying
concentrations of this extract. The cytotoxic potential of
the extract was investigated by 3-(4,5-dimethyl thiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.
As illustrated in Figure 1A, addition of the extract to the
cell culture medium exhibited a striking cell killing
effect in a concentration-dependent manner, with an
estimated IC50 value (half maximal inhibitory
concentration) of 1.35 mg/mL. A statistically significant
(P<0.05) result was obtained at an extract concentration
of 1.5 mg/mL or above. Delphinidin and cyanidin were
also found to suppress the growth of HepG2 cells in our
experimental conditions (Figure 1B). Although both
anthocyanidins exhibited cytotoxicity against HepG2
cells, delphinidin (IC50 = 240 μM) appears to be more
potent than cyanidin (IC50 = 360 μM).
Our results are in agreement with previous studies
showing cytotoxicity of anthocyanins on various cancer
cells, including those from hepatic origin. For example,
a procyanidin-rich fraction from grapes significantly
reduced the viability of HepG2 cells [7c]. In another
study, an aqueous extract from the Korean vine plant
meoru (Vitis coignetiae, Pulliat), containing
diglucosides of cyanidin and delphinidin as major
constituents, exhibited antiproliferative, antiinvasive
and apoptotic effects on human hepatoma Hep3B cells
Black currant and HCC Natural Product Communications Vol. 5 (10) 2010 1615
[7d]. Cyanidin and delphinidin, two anthocyanin
aglycones, have been found to exert strong growth
inhibitory effects against HepG2 and to a lesser extent
against Hep3B cells [7e]. Recently, Feng and coworkers
[7f,7g] have reported that cyanidin-3-O-rutinoside,
extracted and purified from black raspberry (Rubus
occidentalis L.), had selective cytotoxicity against HL-
60 human leukemic cells, but not on SMMC7721,
HCCLM3 and MHCC97L liver cancer cells. The same
group has also shown that cyanidin-3-O-rutinoside and
delphinidin (the latter extracted from bilberry) caused
growth retardation of HCC cells by macroautophagy
[7g]. Cyanidin-3-O-rutinoside (extracted from
mulberry, Morus alba L.) exerted a dose-dependent
inhibitory effect on the migration and invasion of highly
metastatic A549 human lung carcinoma cells [7h].
Cyanidin-3-O-rutinoside has also been identified as one
Figure 1: Effects of black currant skin extract (A), delphinidin and
cyanidin (B) on the proliferation of HepG2 cells. Cells were plated into
96-well dishes (4×103 cells/well) 24 h prior to the addition to the extract
of either delphinidin or cyanidin at various concentrations. Following 24
h incubation, the cell proliferation was determined by MTT assay. Results
are presented as mean±SE based on quadruplicate determinations in three
independent experiments. Statistical analysis performed by one-way
analysis of variance followed by Student-Newman-Keuls test. *P<0.05
and •P<0.05 as compared with corresponding control.
of the major bioactive constituents of black raspberry
[7i,8], with multiple anticarcinogenic effects, as
reviewed by Wang and Stoner [9a].
The underlying mechanisms of the antitumor effects of
black currant skin extract on HepG2 cells, as observed
in the current study, remain to be elucidated. We have
identified cyanidin-3-O-rutinoside as one of the major
anthocyanins in the skin fraction that supports previous
studies, as mentioned above. Delphinidin and cyanidin
have also been reported to be present in black currant
[2c]. Additionally, other bioactive phytochemicals, such
as phenolic acids, proanthocyanidins and other
flavonoids may also be present in black currant skin
extract. All these constituents are known to possess
“pleiotropic” biochemical and pharmacological
effects, including antioxidant, antiinflammatory,
immunostimulatory, anti-apoptotic, cell cycle arrest-
inducing, anti-invasive and anti-angiogenic properties
through modulation of multiple signal transduction
pathways [9b,9c]. All these could contribute to the
observed cytotoxicity of the extract tested in our study.
Our experimental results indicate that a complex
mixture of phytochemicals present in black currant skin
extract is more effective in inhibiting the growth of
HCC cells than the individual constituents evaluated.
This effect may be due to either the presence of other
active compounds or through additive or synergistic
effects. Our results are in agreement with a previous
study showing a better antiinflammatory effect of a total
flavonoid-rich black currant extract than its two major
components [10]. Accumulating evidence suggests that
several plant phytochemicals from diverse dietary
sources, including cranberry, raspberry, pomegranate
and green tea, may be more effective anticancer agents
when used in combination rather than in single pure
form [11-14]. It is also plausible that over enrichment or
purification may result in the loss of pharmacological
activities and hence therapeutic benefits of plant
extracts [13].
In conclusion, skin of black currant fruit should not be
considered as low-value waste as it contains an
anthocyanin-rich fraction with potent antitumor activity
against HepG2 human liver cancer cells. The cytotoxic
effects of black currant skin extract could be due to the
presence of diverse bioactive phytochemicals rather
than a single constituent. A better understanding of the
observed inhibitory effects of black currant skin extract
on the proliferatation of liver cancer cells would benefit
the development of this product for the prevention and
treatment of HCC.
Experimental
Plant material: Ripe black currant (Ribes nigrum L.
Grossulariaceae) fruits were gathered in July 2008 from
A
B
*
Concentration (mg/mL)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Cell Viability (% of control)
0
10
20
30
40
50
60
70
80
90
100
110
*
*
*
*
*
Cyanidin
Concentration (μM)
0 50 100 150 200 250 300 350 400 450 500
Cell Viability (% of control)
0
10
20
30
40
50
60
70
80
90
100
110 Delphinidin
*
*
*
*
*
*
*
*
•
•
•
•
•
•
•
•
•
•
1616 Natural Product Communications Vol. 5 (10) 2010 Bishayee et al.
cultivated plants in the region of Csíkkarcfalva
(Romania). A voucher specimen (No. 770) has been
deposited in the herbarium of the Department of Phar-
macognosy, University of Szeged, Szeged, Hungary.
Preparation of the extract: The collected fresh fruits
were crushed with a fruit-grinder and after 48 h
standing the juice was pressed. Because of the high
pectin content, the fruit mass was stirred several times
during the standing with the aim of facilitating effective
squeezing. The pressed material, containing seeds and
skin, was dried at room temperature, and the seed and
skin of the fruits were separated by sifting. The dried
skin was extracted with a 5-fold volume of water for 24
h at room temperature. After squeezing the plant
material, the extract was then filtered and evaporated
under reduced pressure to one-tenth of its volume.
Spray drying of the extract: The aqueous extract of
black currant skin was spray-dried, with continuous
mechanical stirring, using a Büchi B-191 Laboratory
Spray-dryer (Büchi Co., Flawil, Switzerland) with a
standard 0.7 mm nozzle. The particles were separated in
the cyclone, with a high separation and recovery rate.
Spray-drying was carried out under the following
conditions: 10 L/min air flow, 5 bar pressure, and
3 mL/min pump flow rate. The inlet temperature was set
to 110°C, and the outlet temperature varied in the range
80 ± 5°C. The final product was a dark pink colored
fine powder, which was stored under controlled
humidity at room temperature.
Reagents and solutions: Cyanidin-3-O-rutinoside
chloride (keracyanin chloride CDX-00011325-005) was
purchased from LGC Standard GmbH (Wesel,
Germany). Chromatographic grade acetonitrile was
purchased from Merck (Darmstadt, Germany) and water
plus for HPLC from Carlo Erba (Rodano, Italy). The
other chemicals were of analytical reagent grade and
obtained from local firms. The extraction and all
aqueous solutions were made up with deionized water.
Formic acid (98-100%) was purchased from Molar
Chemicals Kft (Budapest, Hungary).
Preparation of standard and sample solutions: Stock
solution of cyanidin-3-O-rutinoside chloride was
prepared by dissolving accurately weighted portions of
the standards in 10% aqueous formic acid. The stock
solution was diluted to make 1, 10, 20, 50, 70 and 100
μg/mL concentrations, and the calibration curve was
determined with these concentrations. The spray-dried
black currant extract was dissolved in 10% aqueous
formic acid to yield 1% concentration. The same
standard and test solutions were used for HPLC
determination and spectrophotometry.
HPLC conditions: The HPLC analyses were performed
on a Shimadzu system (Shimadzu, Kyoto, Japan)
equipped with a SPD 10A/10 solvent delivery unit,
SCL-10A system controller and SPD-10A UV-VIS
detector. The samples were separated on LiChrospher
10 RP-18 (4 x 250 mm, 10 μm particle size; Merck) and
the column temperature was maintained at 25°C. The
analysis was performed using a gradient solvent system,
consisting of solvent A: formic acid/H2O 1:9 (v/v), and
solvent B: acetonitrile (AcCN). Elution profile was: 0-
0.5 min 1% B (v/v); 0.5-1 min linear gradient from 1 to
7% B (v/v); 1-4 min, linear gradient from 7-14% B
(v/v); 4-9.5 min linear gradient from 14-20% B (v/v);
9.5-10.5 min linear gradient from 20-60% B (v/v); 10.5-
14 min, linear gradient from 60-90% B (v/v); 14-18 min
linear gradient from 90-100% B (v/v); 18-19 min
column wash with 100% B. Post time: 11 min with 1%
B (v/v). Detection was made at 520 nm. The flow rate
throughout the chromatographic analysis was 1.00
mL/min, and the total run time was 30 min. The
injection volume was 10 µL. The HPLC determination
was carried out using cyanidin-3-O-rutinoside chloride
as external standard.
Spectrophotometric determination: Total anthocyanin
content was determined on a HELIOS Beta single-beam
UV-VIS spectrophotometer (Thermo Spectronic) at 520
nm. Aqueous formic acid (10%) was used as the blank
solution. The calibration and sample analyses were
carried out in triplicate.
Cell culture: HepG2 cells were purchased from
American Tissue Culture Collection (Manassas, VA)
and maintained in Dulbecco’s modified Eagle’s medium
(Sigma-Aldrich, St. Louis, MO) supplemented with
10% fetal bovine serum, 100 units/mL penicillin, and
100 μg/mL streptomycin and grown at 37°C under a
humidified atmosphere containing 5% CO2 in a
humidified incubator. Cells were cultured at
preconfluent densities by the use of 0.25% tryptin-
EDTA solution (Millipore, Phillipsburg, NJ).
Cell proliferation assay: Cell proliferation was
determined by the ability of HepG2 cells to cleave
tetrazolium salt to formazan. In brief, HepG2 cells were
seeded onto 96-well plates at a density of 4x103
cells/well in 100 µL of the aforementioned medium.
After 24 h of culture, the medium was removed
carefully and replaced with 100 µL fresh medium, or
medium with various concentrations of either black
currant extract or pure compounds (delphinidin,
cyanidin) for an additional 24 h. At the end of this time-
point, 50 µL of MTT (5 mg/mL) was added to the wells
containing 100 µL of media and cells were incubated
for 4 h. Subsequently, the culture medium was removed
Black currant and HCC Natural Product Communications Vol. 5 (10) 2010 1617
and the formazan crystals (produced by metabolically
active cells) were dissolved by the addition of 100 µL of
DMSO and 15 µL of glycine. The absorbance was
measured at 570 nm with a SpectraMax 340 PC
microplate spectrophotometer (Molecular Devices,
Sunnyvale, CA). All experiments were performed at
least 3 times with 4 samples for each concentration.
Statistical analysis: Data are presented as an average
value ± the standard error (SE) of the mean. The one-
way analysis of variance (ANOVA), followed by
Student-Newman-Keuls test was used for statistical
analysis. A value of P less than 0.05 was considered as
statistically significant.
Acknowledgements - Financial support from a New
Faculty Startup Fund from the Northeastern Ohio
University College of Medicine and Pharmacy to A.B.
and the Hungarian Scientific Research Fund (grant
OTKA K72771) and Hungarian National Development
Agency (grant TÁMOP 4.2.2-08/1) to J.H. is gratefully
acknowledged. The authors sincerely thank Elemér
Zágoni, Ph.D. (Csíkkarcfalva, Romania) for the supply
of the plant material and Werner J. Geldenhuys, Ph.D.,
for technical assistance with the chemical structures.
Cyanidin and delphinidin are generous gifts from
Indena S.p.A., Milan, Italy.
References
[1] (a) World Cancer Research Fund/American Institute for Cancer Research. (2007) Food, Nutrition, Physical Activity, and the
Prevention of Cancer: A Global Perspective; AICR: Washington, DC, USA; (b) Bishayee A, Darvesh AS. (2010) Oxidative stress
in cancer and neurodegenerative diseases: prevention and treatment by dietary antioxidants. In Handbook of Free Radicals:
Formation, Types and Effects; Kozyrev D, Slutsky V. (Eds). Nova Science Publishers: Hauppauge, USA. 1-55; (c) Darvesh AS,
Carroll RT, Bishayee A, Geldenhuys WJ, Van der Schyf CJ. (2010) Oxidative stress and Alzheimer's disease: dietary polyphenols as
potential therapeutic agents. Expert Review of Neurotherapeutics, 10, 729-745; (d) Thomasset S, Teller N, Cai H, Marko D, Berry
DP, Steward WP, Gescher AJ. (2009) Do anthocyanins and anthocyanidins, cancer chemopreventive pigments in the diet, merit
development as potential drugs? Cancer Chemotherapy and Pharmacology, 64, 201-211.
[2] (a) Clifford MN. (2000) Anthocyanins – nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture, 80,
1063-1072; (b) Hertog MG, Hollman PC, Katan MB, Kromhout D. (1993) Intake of potentially anticarcinogenic flavonoids and
their determinants in adults in The Netherlands. Nutrition and Cancer, 20, 21-29; (c) Wu X, Gu L, Prior RL, McKay S. (2004)
Characterization of anthocyanins and proanthocyanins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant
capacity. Journal of Agricultural and Food Chemistry, 52, 7846-7856; (d) Scalzo J, Currie A, Stephens J, McGhie T, Alspach P,
The Horticulture and Food Research Institute of New Zealand Limited (HortResearch). (2008) The anthocyanin composition of
different Vacc i niu m , Ribes and Rubus genotypes. Biofactors, 34, 13-21; (e) Slimestad R, Solheim H. (2002) Anthocyanins from
black currants (Ribes nigrum L.). Journal of Agricultural and Food Chemistry, 50, 3228-3231.
[3] (a) Declume C. (1989) Anti-inflammatory evaluation of a hydroalcoholic extract of black currant leaves (Ribes nigrum). Journal of
Ethnopharmacology, 27, 91-98; (b) Suzutani T, Ogasawara M, Yoshida I, Azumura M, Knox YM. (2003) Anti-herpes virus activity
of an extract of Ribes nigrum L. Phytotherapy Research, 17, 609-613; (c) Amakura Y, Umino Y, Tsuji S, Tonogai Y. (2000)
Influence of jam processing on the radical scavenging activity and phenolic content in berries. Journal of Agricultural and Food
Chemistry, 48, 6292-6297; (d) Lister CE, Wilson PE, Sutton KH, Morrison SC. (2002) Understanding the health benefits of
blackcurrants. Proceeding of 8th International Rubus and Ribes Symposium, 1-2, 443-449.
[4] (a) Hertog MGL, Feskens EJM, Hollman PCH, Katan MB, Kromhout D. (1993) Dietary antioxidant flavonoids and risk of
coronary heart disease: the Zutphen elderly study. Lancet, 342, 1007-1011; (b) Heinonen IM, Meyer AS, Frankel EN. (1998)
Antioxidant activity of berry phenolics on human low-density lipoprotein and liposome oxidation. Journal of Agricultural and
Food Chemistry, 46, 4107-4112; (c) Puupponen-Pimia R, Nohynek L, Meier C, Kahkonen M, Heinonen M, Hopia A, Oskman-
Caldentey KM. (2001) Antimicrobial properties of phenolic compounds from berries. Journal of Applied Microbiology, 90, 494-
507; (d) Garbacki N, Tits M, Angenot L, Damas J. (2004) Inhibitory effects of proanthocyanidins from Ribes nigrum leaves on
carrageenin acute inflammatory reactions induced in rats. BMC Pharmacology, 4, 25; (e) Hurst SM, McGhie TK, Cooney JM,
Jensen DJ, Gould EM, Lyall KA, Hurst RD. (2010) Blackcurrant proanthocyanidins augment IFN-gamma-induced suppression of
IL-4 stimulated CCL26 secretion in alveolar epithelial cells. Molecular Nutrition and Food Research, 54, 1-12.
[5] (a) Olsson M, Gustavsson K-E, Andersson S, Nilsson Å, Duan R-D. (2004) Inhibition of cancer cell proliferation in vitro by fruit
and berry extracts and correlations with antioxidant levels. Journal of Agricultural and Food Chemistry, 52, 7264-7271; (b) Wu
QK, Koponen JM, Mykkänen HM, Törrönen AR. (2007) Berry phenolic extracts modulate the expression of p21(WAF1) and Bax
but not Bcl-2 in HT-29 colon cancer cells. Journal of Agricultural and Food Chemistry, 55, 1156-1163; (c) Boivin D, Blanchette
M, Barrette S, Moghrabi A, Béliveau R. (2007) Inhibition of cancer cell proliferation and suppression of TNF-induced activation of
NF-κB by edible berry juice. Anticancer Research, 27, 937-948; (d) Takata R, Yamamoto R, Yanai T, Konno T, Okubo T. (2005)
Immunostimulatory effects of a polysaccharide-rich substance with antitumor activity isolated from black currant (Ribes nigrum
L.). Bioscience, Biotechnology and Biochemistry, 69, 2042-2050; (e) Takata R, Yanai T, Yamamoto R, Konno T. (2007)
Improvement of the antitumor activity of black current polysaccharide by an enzymatic treatment. Bioscience, Biotechnology and
Biochemistry, 71, 1342-1344; (f) Lyall KA, Hurst SM, Cooney J, Jensen D, Lo K, Hurst RD, Stevenson LM. (2009) Short-term
blackcurrant extract consumption modulates exercise-induced oxidative stress and lipopolysaccharide-stimulated inflammatory
responses. American Journal of Physiology – Regulatory, Integrative and Comparative Physiology, 297, R70-R81.
1618 Natural Product Communications Vol. 5 (10) 2010 Bishayee et al.
[6] (a) Kapasakalidis PG, Rastall RA, Gordon MH. (2006) Extraction of polyphenols from processed black currant (Ribes nigrum L.)
residues. Journal of Agricultural and Food Chemistry, 54, 4016-4021; (b) Kapasakalidis PG, Rastall RA, Gordon MH. (2009)
Effect of cellulose treatment on extraction of antioxidant phenols from black currant (Ribes nigrum L.) pomace. Journal of
Agricultural and Food Chemistry, 57, 4342-4351; (c) Constantino L, Albasini A, Rastelli G, Benvenuti S. (1992) Activity of
polyphenolic crude extracts as scavengers of superoxide radicals and inhibitors of xanthine oxidase. Planta Medica, 58, 342-344.
[7] (a) Garbacki N, Angenot L, Bassleer C, Damas J, Tits M. (2002) Effects of prodelphinidins isolated from Ribes nigrum on
chondrocyte metabolism and COX activity. Naunyn-Schmeideberg’s Archives of Pharmacology, 365, 434-441; (b) Määttä K,
Kamal-Eldin A, Törrönen R. (2001) Phenolic compounds in berries of black, red, green, and currants (Ribus sp.). Antioxidants and
Redox Signaling, 3, 981-393; (c) Jo JY, de Mejia EG, Lila MA. (2006) Cytotoxicity of bioactive polymeric fractions from grape cell
culture on human hepatocellular carcinoma, murine leukemia and non-cancerous PK15 kidney cells. Food and Chemical
Toxicology, 44, 1758-1767; (d) Shin DY, Ryu CH, Lee WS, Kim DC, Kim SH, Hah YS, Lee SJ, Shin SC, Kang HS, Choi YH.
(2009) Induction of apoptosis and inhibition of invasion in human hepatoma cells by anthocyanidins from meoru. Annals of New
York Academy of Science, 1171, 137-148; (e) Yeh CT, Yen GC. (2005) Induction of apoptosis by anthocyanidins through regulation
of Bcl-2 gene and activation of c-jun N-terminal kinase cascade in hepatoma cells. Journal of Agricultural and Food Chemistry, 53,
1740-1749; (f) Feng R, Ni HM, Wang SY, Tourkova IL, Shurin MR, Harada H, Yin XM. (2007) Cyanidin-3-rutinoside, a natural
polyphenol antioxidant, selectively kills leukemic cells by induction of oxidative stress. Journal of Biological Chemistry, 282,
13468-13476; (g) Feng R, Wang SY, Shi YH, Fan J, Yin XM. (2010) Delphinidin induces necrosis in hepatocellular carcinoma cells
in the presence of 3-methyladenine, an autophagy inhibitor. Journal of Agricultural and Food Chemistry, 58, 3957-3964; (h) Chen
PN, Chu SC, Chiou HL, Kuo WH, Chiang CL, Hsieh YS. (2006) Mulberry anthocyanins, cyanidin 3-rutinoside and cyanidin 3-
glucoside, exhibited an inhibitory effect on the migration and invasion of a human lung cancer cell line. Cancer Letters, 235, 248-
259; (i) Hecht SS, Huang C, Stoner GD, Li J, Kenney PM, Sturla SJ, Carmella SG. (2006) Identification of cyanidin glycosides as
constituents of freeze-dried black raspberries which inhibit anti-benzo[a]pyrene-7,8-diol-9,10-epoxide induced NF-κB and AP-1
activity. Carcinogenesis, 27, 1617-1626.
[8] Tulio AZ Jr, Reese RN, Wyzgoski FJ, Rinaldi PL, Fu R, Scheerens JC, Miller AR. (2008) Cyanidin 3-rutinoside and cyanidin 3-
xylosylrutinoside as primary phenolic antioxidants in black raspberry. Journal of Agricultural and Food Chemistry, 56, 1880-1888.
[9] (a) Wang LS, Stoner GD. (2008) Anthocyanins and their role in cancer prevention. Cancer Letters, 269, 281-290; (b) Stoner GD,
Wang LS, Castro C. (2008) Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries. Carcinogenesis,
29, 1665-1674; (c) Moskaug JØ, Borge GI, Fagervoll AM, Paur I, Carlsen H, Blomhoff R. (2008) Dietary polyphenols identified as
intracellular protein kinase A inhibitors. European Journal of Nutrition, 47, 460-469.
[10] Chanh PH, Ifansyah N, Chahine R, Mounayar-Chalfoun A, Gleye J, Moulis C. (1986) Comparative effects of total flavonoids
extracted from Ribes nigrum leaves, rutin and isoquercitrin on biosynthesis and release of prostaglandins in the ex vivo rabbit heart.
Prostaglandins, Leukotrienes, and Medicine, 22, 295-300.
[11] Seeram NP, Adams LS, Hardy ML, Heber D. (2004) Total cranberry extract versus its phytochemical constituents: antiproliferative
and synergistic effects against human tumor cell lines. Journal of Agricultural and Food Chemistry, 52, 2512-2517.
[12] Lansky EP, Jiang W, Mo H, Bravo L, Froom P, Yu W, Harris NM, Neeman I, Campbell MJ. (2005) Possible synergistic prostate
cancer suppression by anatomically discrete pomegranate fractions. Investigational New Drugs, 23, 11-20.
[13] de Kok TM, van Breda SG, Manson MM. (2008) Mechanisms of combined action of different chemopreventive dietary
compounds. European Journal of Nutrition, 47, 51-59.
[14] Zikri NN, Riedl KM, Wang LS, Lechner J, Schwartz SJ, Stoner GD. (2009) Black raspberry components inhibit proliferation,
induce apoptosis, and modulate gene expression in rat esophageal epithelial cells. Nutrition and Cancer, 61, 816-826.
