BIOACTIVE PLANT FOODS - AU
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Bioactivity of sour cherry cultivars grown in Denmark
Journal: Phytotherapy Research
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Complete List of Authors: Khoo, Gaik Ming; Aarhus University, Food Science Clausen, Morten; Aarhus University, Food Science Pedersen, Bjarne; Aarhus University, Horticulture Larsen, Erik; Aarhus University, Food Science
Keyword: Prunus cerasus, sour cherries, ORAC, cancer, PGE2
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Bioactivity of sour cherry cultivars grown in Denmark 1
Gaik Ming Khooa, Morten Rahr Clausena, Bjarne Hjelmsted Pedersenb, and Erik Larsena* 2
a Department of Food Science, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark. 3
b Department of Horticulture, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark. 4
*Corresponding Author: Tel: +45-89993367, Fax: +45-89993495, e-mail address: 5
Abstract 7
Thirty four varieties of sour cherries (Prunus cerasus) were investigated for their total 8
antioxidant activity, Caco-2 cancer cell proliferation inhibitory activity and effect on 9
prostaglandin E2 (PGE2) production. Total phenolic content, oxygen radical absorbance 10
capacity (ORAC) and cancer cell proliferation inhibitory activity of sour cherries were 11
closely correlated but not PGE2 production. The cultivars ‘Birgitte x Böttermö’, ‘Fanal’ and 12
‘Tiki’ were the three cultivars with the highest ORAC values (180, 147 and 133 µmol TE/g, 13
respectively) and inhibition against Caco-2 cancer cell proliferation (74, 79 and 73%, 14
respectively). ‘Stevnsbaer Birgitte’ (22%) and ‘Stevnsbaer Viki’ (22%) inhibited PGE2 15
production with a similar potency as the positive controls indomethacin and NS-398. 16
Significant differences between cultivars in all bioactivity experiments indicated that 17
selection of cultivars is important to obtain sour cherries with better potential health 18
promoting effects. 19
Keywords: Prunus cerasus, sour cherries, ORAC, cancer, PGE2 20
21
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1 Introduction 22
Cyclooxygenase-2 (COX-2) is the key enzyme in the process of inflammation responsible 23
for the biosynthesis of prostaglandin E2 (PGE2) an important mediator that initiates typical 24
symptoms of inflammation including fever, swelling and redness (Smith et al., 2000; Claria, 25
2003). Furthermore it has been shown that COX-2 and PGE2 are involved in cancer 26
development, since over-expression of COX-2 and high levels of PGE2 were detected in 27
different cancer cells (Coussens et al., 2002; Lu et al., 2006). 28
Sour cherries (Prunus cerasus) one of the important fruit crops in Denmark. Sour cherries 29
have traditionally been used for wine making and liquers. In addtion, there are some 30
finished products available commercially, such as jam, syrup, sweets and canned fruits. Sour 31
cherries contain a substantial amount of phenolics including anthocyanins which are 32
reported to have anti-inflammatory and antioxidant properties (Wang et al., 1999a; Wang et 33
al., 2000). Sour cherry anthocyanins inhibited tumor development in APCMin mice and 34
reduced the growth of human colon cancer cells (Kang et al., 2003). In addition, the 35
anthocyanins showed potential anti-inflammatory effects against arthritis in rats, by 36
decreasing the level of tumor necrosis factor-α and PGE2 (He et al., 2006). Most of the 37
research on sour cherries focused on ‘Montmorency’ and ‘Balaton’ cultivars because of 38
their high anthocyanin content. However, ‘Montmorency’ often gives a very low yield and 39
low anthocyanin content when grown in Denmark (Christensen, 1990). 40
Evaluation of sour cherry cultivars that are suitable for growing in Denmark has been 41
conducted earlier (Christensen, 1986; Christensen, 1988; Christensen, 1990; Christensen, 42
1997). A thorough investigation of sour cherry cultivars grown in Denmark with high 43
content of anthocyanins and better bioactivity is needed. This study aimed at determining 44
the bioactivity of selected sour cherry cultivars in Denmark, targeting for cultivars with 45
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potential health promoting effects. Thirty four sour cherry cultivars were evaluated using the 46
oxygen radical absorbance capacity (ORAC) assay, cancer cell proliferation inhibition 47
activity and PGE2 inhibition activity assays. 48
49
2 MATERIALS AND METHODS 50
2.1 Plant materials 51
The sour cherries from 34 different cultivars were harvested from fields at the Department 52
of Horticulture, Aarhus University, Aarslev, Denmark, between the period of July and 53
August 2009 depending on the ripening status. Sour cherries were stored at -24 ºC 54
immediately after harvest. For the experiments, frozen sour cherries were removed from the 55
freezer and pitted. The pitted sour cherries were then homogenized (2 g cherry per 1 mL 56
water) in a food blender. Homogenized samples were centrifuged for 30 min at 12000 rpm 57
at 4ºC. The supernatants were collected and filtered through a 0.45 µm filter. Filtered 58
samples were kept at -80 ºC prior to use. 59
2.2 Chemicals 60
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fluorescein, sodium 61
dihydrophosphate, acetonitrile, trifluoroacetic acid (TFA) and acetylsalicylic acid were 62
purchased from Sigma-Aldrich (Missouri, USA). Dulbecco's Modified Eagle Medium-63
GlutamaxTM (DMEM) was purchased from Invitrogen (Paisley, UK). Trypsin-64
ethylenediaminetetraacetic acid (Trypsin-EDTA) and phosphate buffer saline-65
ethylenediaminetetraacetic acid (PBS-EDTA) were purchased from Lonza (Braine, 66
Belgium). Penicillin G-potassium salt and streptomycin sulfate were purchased from Serva 67
(Heidelberg, Germany). Fetal calf serum (FCS) was purchased from PAA Laboratories 68
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(Pasching, Austria). WST-1 cell proliferation reagent was purchased from Roche 69
Diagnostics (Mannheim, Germany). Lipopolysaccharide (LPS) was purchased from 70
Calbiochem, Merck (Damstadt, Germany). Monoclonal PGE2 enzyme immunoassay (PGE2 71
EIA) kit and 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) were 72
purchased from Cayman Europe (Tallinn, Estonia). 73
2.3 Oxygen radical absorbance capacity (ORAC) assay 74
The ORAC assay was performed as described by Huang with minor modification (Huang et 75
al., 2002). The fluorescein solution was prepared (1.2 x 10-8 mM) in phosphate buffer (75 76
mM, pH 7.42) and AAPH was dissolved in phosphate buffer to a final concentration of 15 77
mM. The assay was performed in triplicate in a Nunc black 96-well plate (Thermo Fischer 78
Scientific, Leicestershire, UK) with two independent tests. Trolox was used for standard 79
curve calibration. A 25 µL subsample of standard or filtered samples was mixed with 150 80
µL of fluorescein solution and incubated at 37 ºC for 30 minutes. The assay was initiated by 81
adding 25 µL AAPH solution. Fluorescence was read every minute for 60 minutes with an 82
excitation wavelength of 485 nm and an emission wavelength of 515 nm using a BioTek 83
Synergy 2 multi-mode microplate reader. Mean values and standard deviations were 84
calculated. The results were expressed as trolox equivalent per gram (TE/g) sour cherry. 85
2.4 Caco-2 cancer cell proliferation assay 86
Caco-2 (European Collection of Cell Cultures, Salisbury, UK) were grown in DMEM 87
medium supplemented with 10% FCS, 100 IU/mL penicillin and 100 µg/mL streptomycin. 88
Medium was changed every second day and cells were passaged every fourth day. Trypsin-89
EDTA was used for detachment of cells from culture flask. Cells were incubated in 8000 90
WJ CO2 incubator (Thermo Scientific, UK) at 37 ºC with 5% humidified CO2. 91
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Cells were seeded into 96-well plate at a density of 1x104 cells per well and incubated for 24 92
hours. Filtered samples were added at a final concentration of 50 µL/mL and the cells were 93
incubated for another 72 hours. Proliferation assay was performed using WST-1, based on 94
the cleavage of tetrazolium salt to formazan by the mitochondrial dehydrogenase. After 3 95
hours of incubation with WST-1, the absorbance was detected at a wavelength of 450 nm 96
and with 630 nm for background correction using BioTek Synergy 2 multi-mode microplate 97
reader. All samples were measured in triplicate in two identical experiments. Wells without 98
sample were used as control and inhibition was calculated relative to the control for each 99
sample. 100
2.5 PGE2 assay 101
PGE2 assay was used to determine COX-2 inhibitory activity of sour cherries. In brief, 102
Caco-2 cells were grown in DMEM medium supplemented with 10% FCS, 100 IU/mL 103
penicillin and 100 µg/mL streptomycin. The medium was changed every second day and the 104
cells were passaged every fourth day. Trypsin-EDTA was used for detachment of cells from 105
culture flask and cells were cultivated at 37 ºC 5% CO2. 1x104 cells was seeded into each 106
well of transparent 96-well plate and incubated for 48 hours. Cells were then incubated with 107
500µM aspirin for 3 hours to inactivate the endogenous cyclooxygenase-1 (Bang et al., 108
2002). After that, the cells were washed twice with PBS-EDTA, and 200 ng/mL LPS was 109
added with or without filtered sample. After incubation for 4 hours, media were collected 110
and centrifuged. All media were tested according to the given protocol for the PGE2 111
content with Cayman PGE2 EIA kit set. 112
2.6 HPLC analysis 113
Filtered samples were diluted 10 times and separated on a column (Kinetex 2.6 µm C18 114
100Å, 100 x 4.6 mm, Phenomenex, California, USA) using Dionex Ultimate 3000 HPLC 115
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system (Dionex, California, USA) equipped with Chameleon software program. All samples 116
were measured twice using a linear gradient elution with a mobile phase consisting of 0.5% 117
TFA aqueous (solvent A) and acetonitrile (solvent B) at a flow rate of 1.0 ml/min. The 118
gradient was 5-25 % B (0-13 min), 25-5% B (13-14 min) and 5% B (14-15 min). 119
Measurement of phenolic compounds and anthocyanins were determined at 365 nm and 520 120
nm, respectively. Total phenolic content was quantified with quercitrin standard at the 121
wavelength of 365 nm and the results were expressed as quercitrin equivalent per 100 gram 122
(QE/100g) sour cherries. Total anthocyanin content was quantified with cyanidin chloride at 123
the wavelength of 520nm and the results were expressed as cyanidin equivalent per 100 124
gram (CE/100g) sour cherries. 125
2.7 Statistical analysis 126
Analysis of variance and Students T-test were performed using SAS 9.1 software package 127
(SAS Institute, North Carolina, USA) with a confidence level of 95 %. Linear regressions 128
between total phenolic content total anthocyanin content, ORAC value, PGE2 assay and 129
anticancer cell proliferation activities were investigated to determine the correlation 130
significance. 131
132
3 RESULTS AND DISCUSSION 133
Table 1 shows the total phenolic and total anthocyanin content, ORAC value, Caco-2 cancer 134
cell proliferation inhibition activity and the PGE2 production assay in the 34 different sour 135
cherry cultivars. Sour cherry cultivars with high total phenolics and total anthocyanins 136
content were ‘Tiki’, ‘Aarslev 2510’, ‘Fanal’ and ‘Aarslev 2403’. In contrast ‘Gerema’, 137
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‘Skyggemorel Hannover’, ‘Zagarvysne’, ‘Vytenu Star’, ‘Surefire’ and ‘Favorite’ contained 138
the lowest amount of total phenolics and anthocyanins. 139
ORAC values, Caco-2 cancer cell inhibitory effect and PGE2 assay of the 34 sour cherry 140
cultivars were significantly different between varieties (P < 0.001). ORAC values ranged 141
from 15 to 180 µmol TE/g; inhibitory effects against Caco-2 varied from no inhibition to a 142
strong inhibitory effect up to 79 %. As shown in table 2, total phenolics, total anthocyanin, 143
ORAC values and Caco-2 cancer cell inhibitory activity are significantly correlated (P< 144
0.001).‘Birgitte x Böttermö’, ‘Fanal’ and ‘Tiki’ displayed the strongest antioxidant activity 145
in the ORAC assay; these 3 cultivars also displayed the strongest inhibitory effect against 146
the growth of Caco-2 cancer cell line. In contrast, ‘Surefire’ and ‘Favorite’ had the lowest 147
ORAC value and also displayed very weak inhibitory effects against Caco-2 cancer cell 148
proliferation. 149
The effect of sour cherry cultivars against PGE2 production were compared against a blank 150
and two positive controls. The positive controls, indomethacin and NS-398, decreased the 151
PGE2 production to 24 and 33% respectively. The 34 sour cherry cultivars exhibited 152
different levels of inhibition against PGE2 production. ‘Stevnsbaer Birgitte’ and ‘Stevnsbaer 153
Viki’ displayed the strongest suppression against PGE2 production which was similar to the 154
positive control. However, ‘Nefris,’ ‘Sumadinka’, ‘Pernilla’ and ‘Surefire’ showed no 155
inhibitory effect against PGE2 production. However, PGE2 assay was independent and not 156
correlated with other results in present study (P > 0.05). In vitro anti-inflammatory activity 157
of sour cherry anthocyanins and their aglycons has been reported and assigned to inhibition 158
of COX-2 (Mulabagal et al., 2009; Reddy et al., 2005; Wang et al., 1999b). Other research 159
pointed out that sour cherry juice displaying strong anti-inflammatory effect by inhibiting 160
the COX-2 level Freund’s adjuvant mice (Šáric et al., 2009). 161
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[Table 1 about here] 162
The majority of the sour cherry cultivars with high total phenolics, high ORAC values and 163
better inhibitory effect against Caco-2 cancer cell proliferation were either Stevnsbaer-type, 164
Fanal-type or new cross breeding cultivars. Stevnbaer-type and Fanal-type sour cherries 165
shared very similar characteristics and produced high quality dark red sour cherries 166
(Christensen, 1986). ‘Tiki’ is a hybrid between ‘Stevnbaer’ and ‘Fanal’ (DLBR 167
Landbrugsinfo, 2008), ‘Birgitte × Böttermö’ is a cross breeding cultivar of ‘Stevnbaer 168
Birgitte’ and ‘Erdi Böttermö’. In the present study we observed that both ‘Tiki’ and ‘Birgitte 169
x Böttermö’ contained higher amount of phenolics as compared to the parental cultivars. 170
‘Aarslev 2510’, ‘Aarslev 2403’, ‘Aarslev 1803’ and ‘Aarslev 2504’ are new cross breeding 171
cultivars; ‘Fanal’ and ‘Nefris’ are sharing a similar characteristic and both belong to Fanal-172
type sour cherry cultivars. ‘Heimann Rubin 4’ is a cultivar origin from Germany with large 173
fruits and produce high quality juices, similar to the characteristic of Fanal-type sour 174
cherries (Christensen, 1990). 175
[Table 2 about here] 176
4 Conclusions 177
Sour cherry phenolics are strongly correlated with antioxidative and anticancer effects of 178
sour cherries. The cultivars that are rich in phenolics display stronger antioxidant activity 179
and anticancer activity. However, we suggest that there may be some other compounds in 180
sour cherries that are responsible for the COX-2 inhibitory activity beside anthocyanins. In 181
addition, more in vivo experiments and clinical research on sour cherries is needed to 182
confirm the bioavailability and mechanism. The significant difference of the bioactivity 183
results suggests that selection of cultivars is an important procedure for growers to obtain 184
high quality sour cherries with potential health promoting effects. 185
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186
Reference List 187
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induced production of nitric oxide and prostaglandin E2 in macrophage RAW264.7 221
cells. Planta Medica 68: 101-105 222
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Table 1 Results of sour cherry cultivars on their total phenolic (TP, QE = quercitrin 250
equivalent), total anthocyanin (TA, CE = cyanidin equivalent), oxygen radical absorbance 251
capacity (ORAC, TE = trolox equivalent), Caco-2 proliferation inhibitory activity, PGE2 252
assay and the least significant difference (LSD). Results for ORAC, Caco-2 inhibition and 253
PGE2 assay are expressed in a form of mean±SD. Values in the same column followed by 254
identical letters show no significant difference. Level of significance, ***P < 0.001. 255
Cultivars TP, mg QE/100g
TA, mg CE/100g
ORAC, µmol TE/g
Caco-2 inhibition, %
PGE2 production, %
Tiki 140.2 188.6 133 ± 18.6 bc 73 ± 2.1 ab 50 ± 9.2 fghijk Aarslev 2510 133.0 154.8 103 ± 23.7 defg 14 ± 5.1 jk 68 ± 2.6 bcdefghi Fanal 127.3 172.6 147 ± 16.9 b 79 ± 1.3 a 69 ± 24.8 bcdefghi Aarslev 2403 125.2 177.1 97 ± 21.9 defgh 9 ± 5.0 jk 58 ± 6.2 efghij Heimanns Rubin 4 106.2 129.1 115 ± 15.9 cd 72 ± 0.6 ab 85 ± 4.6 abcdef Birgitte x Böttermö 106.1 100.8 180 ± 14.5 a 74 ± 0.7 ab 73 ± 20.4 abcdefgh Bofa 97.2 126.7 112 ± 12.3 cde 65 ± 2.5 bc 76 ± 9.9 abcdefg Aarslev 1803 92.7 103.8 114 ± 10.6 cd 53 ± 3.2 def 58 ± 8.9 efghij Nefris 89.5 108.8 115 ± 15.7 cd 70 ± 1.5 bc 101 ± 6.2 abc Recta 85.7 105.0 91 ± 10.1 defghi 29 ± 2.0 i 42 ± 4.9 ghijk Safir 83.1 107.9 72 ± 11.1 hijklm 44 ± 5.1 fg 86 ± 6.6 abcdef Stevnsbaer,PH 82.8 80.6 103 ± 23.8 defg 44 ± 9.2 g 61 ± 7.9 efghi Aarslev 2504 72.9 93.1 66 ± 9.3 ijklm 15 ± 3.2 j 79 ± 8.4 abcdef Stevnsbaer Birgitte 70.4 85.3 108 ± 16.5 cdef 50 ± 1.2 efg 22 ± 2.3 k Nadwislanka 67.7 74.7 85 ± 27.8 efghij 55 ± 3.6 de 88 ± 11.4 abcde Stevnsbaer Viki 67.4 80.9 113 ± 18.8 cde 61 ± 0.9 cd 22 ± 3.0 k Sumadinka 66.7 72.2 61 ± 17.3 jklmn -5 ± 4.7 no 103 ± 23.5 ab Danax 1 66.0 88.7 81 ± 26.9 fghijk 14 ± 8.3 j 55 ± 12.4 fghijk K27/2 60.2 41.9 79 ± 14.6 ghijkl 68 ± 2.6 bc 64 ± 5.4 defghi Kelleris 16 60.2 45.5 56 ± 12.7 klmn 4 ± 2.1 klm 52 ± 1.2 fghijk M7 55.2 74.6 51 ± 10.5 lmnop 5 ± 2.7 kl 70 ± 5.8 bcdefgh Lutovka 54.9 55.8 65 ± 16.1 ijklmn 3 ± 12.3 klmn 68 ± 4.1 bcdefghi Pernilla 49.0 60.8 72 ± 19.2 hijklm -2 ± 3.6 lmn 106 ± 21.0 a Cigganymeggy 7 46.0 67.8 44 ± 10.0 mnopq 2 ± 1.4 klmn 74 ± 12.9 abcdefgh Erdi Böttermö 45.1 38.4 37 ± 6.7 nopqr 2 ± 3.1 klmn 91 ± 6.3 abcde Zigeunerkirschen 44.3 56.2 50 ± 16.0 mnop 10 ± 2.3 jk 58 ± 9.3 efghij Oblachinska Holo 43.1 60.8 49 ± 14.3 mnop 7 ± 9.8 jk 57 ± 7.4 efghijk Ungarische Traubige 41.5 34.4 22 ± 5.5 pqr 4 ± 1.6 klmn 65 ± 13.3 cdefghi Gerema 40.8 13.6 53 ± 14.6 klmno -4 ± 3.1 mno 79 ± 14.5 abcdef Skyggemorel Hannover 38.5 24.6 59 ± 7.5 jklmn -11 ± 3.4 o 56 ± 1.7 efghijk Zagarvysne 31.2 23.7 51 ± 14.0 lmno 3 ± 3.9 klmn 40 ± 5.8 hijk Vytenu Star 25.5 15.3 25 ± 8.0 opqr -5 ± 5.9 mno 72 ± 4.4 abcdefgh Surefire 22.4 11.0 15 ± 3.4 r 2 ± 1.9 klmn 101 ± 5.4 ab Favorite 16.0 18.2 16 ± 7.7 qr 7 ± 2.1 jk 58 ± 17.9 efghij
Blank - - - - 100 ± 0.0 abcd Indomethacin - - - - 24 ± 0.2 jk NS-398 - - - - 33 ± 0.0 ijk LSD (T=95%) - - 28.5*** 8.98*** 35.95***
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Table 2. Linear regression (R2) of total phenolic (TP), total anthocyanin (TA), oxygen 256
radical absorbance capacity (ORAC), Caco-2 proliferation inhibitory activity and PGE2 257
assay. Level of significance, ***P < 0.001. 258
TP TA ORAC Caco-2
TA 0.96*** - - -
ORAC 0.85*** 0.77*** - -
Caco-2 0.66*** 0.61*** 0.82*** -
PGE2 -0.06 -0.08 -0.17 -0.11
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