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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

Erik.Larsen@agrsci.dk 6

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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Smith WL, Dewitt DL, Garavito RM. 2000. Cyclooxygenases: Structural, cellular, and 189

molecular biology. Annual Review of Biochemistry 69: 145-182 190

Claria J. 2003. Cyclooxygenase-2 biology. Current Pharmaceutical Design 9: 2177-2190 191

Coussens LM, Werb Z. 2002. Inflammation and cancer. Nature 420: 860-867 192

Lu HT, Ouyang WM, Huang CS. 2006. Inflammation, a key event in cancer development. 193

Molecular Cancer Research 4: 221-233 194

Wang H, Nair MG, Strasburg GM, Booren AM, Gray JI. 1999a. Antioxidant polyphenols 195

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He YH, Zhou J, Wang YS, Xiao C, Tong Y, Tang JCO, Chan ASC, Lu AP. 2006. Anti-205

inflammatory and anti-oxidative effects of cherries on Freund's adjuvant-induced 206

arthritis in rats. Scandinavian Journal of Rheumatology 35: 356-358 207

Christensen JV. 1990. A review of an evaluation of 95 cultivars of sour cherry. Tidsskrift for 208

Planteavl 94: 51-63 209

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Christensen JV. 1997. Evaluation of 12 sour cherry varieties. Erwerbsobstbau 39: 38-41 214

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induced production of nitric oxide and prostaglandin E2 in macrophage RAW264.7 221

cells. Planta Medica 68: 101-105 222

Mulabagal V, Lang GA, DeWitt DL, Dalavoy SS, Nair MG. 2009. Anthocyanin content, 223

lipid peroxidation and cyclooxygenase enzyme inhibitory activities of sweet and 224

sour Cherries. Journal of Agricultural and Food Chemistry 57: 1239-1246 225

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cyanidin, from tart cherries. Journal of Natural Products 62: 294-296 231

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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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