Bioactive Plant Foods

BIOACTIVE PLANT FOODS

PhD Dissertation

Gaik Ming Khoo, MSc

Faculty of Agricultural Sciences Aarhus University 2011 Preface

This thesis represents three years of work from May 2008 to May 2011, to fulfill the requirement for a PhD at The Department of Food Sciences (IFK), Faculty of Agricultural Sciences (DJF), Aarhus University, Denmark.

The project is funded by The Danish Agency for Science, Technology and Innovation, Hyben Vital A/S and DJF-SAFE school.

Main supervisor was Erik Larsen, Co-supervisor was Morten R. Clausen and project supervisor was Anette K. Thybo from Aarhus University.

I would never have managed to complete my work without the help and support from my associates. Firstly, I would like to thank my supervisors for their help on the guidance of work on my project experiments and writing up the papers. I would like to thank the skillful technician Nina Eggers, who helped me on experiment setups and trouble-shooting. Thanks to Hanne L. Pedersen and Eleftheria Stavridou from The Department of Horticulture, who assisted me on part of the statistical analyses.

A special thank to Sandie Møller, my office mate who always took time to listen to me, talk to me and gave me a lot of help. It is great to be in the same office with you!

Thanks to all my colleagues in Aarslev, who gave me a lot of support during this three years time. This will be part of my unforgettable experience for being in Europe for the first time working.

I would like to take this opportunity to thank my beloved family in Malaysia giving me full support throughout the years.

At last a special thank to my good friend Lisbeth B. Nielsen in Denmark, who has given me a lot of care and support, when I was experiencing the hardest time in Denmark.

Abstract

In inflammatory events, cyclooxygenase-2 (COX-2) is the key enzyme in catalyzing the formation of prostaglandin E2 (PGE2) from arachidonic acid. Persistent inflammation leads to release of reactive species, pro-inflammatory cytokines and other agents which cause the development of cancer. Recent research has shown that these processes could be inhibited by bioactive phytochemicals from plants. In addition, consumption of plant food is beneficial for prevention of cancer and cardiovascular diseases. Therefore, investigation of whole plant food is important for the society.

Sour cherries ( Prunus cerasus ), blackcurrants ( Ribes nigrum ) and rose hip ( Rosa canina ) are three common plant foods in Denmark, which are consumed in a processed form. All three of these plant foods are recognized as having a very high nutritional value, in addition rose hip have been recognized as a medicinal plant in Denmark since the old days. Blackcurrants and sour cherries are well known as being rich in bioactive phenolics, which are believed to be potential health promoting compounds.

Analysis of the phytochemical content, total antioxidant capacity and the effect PGE2 production and the anticancer cell proliferation activity was conducted in a variety of sour cherries, and blackcurrants grown with and without pesticide treatment. In addition high performance liquid chromatography (HPLC) was applied to collect the fingerprint of phenolic compounds, and nuclear magnetic resonance (NMR) was applied to collect the fingerprint of the whole juice extracts. All data was combined and multivariate data analysis was used as a tool to distinguish the difference between the varieties.

The phenolics and the biological activity of the sour cherries were significantly different among the cultivars. The cultivars which displayed a better antioxidant and anticancer activity contained higher amount of phenolics. However, the ability to inhibit the PGE2 production was independent from the phenolics, antioxidant and anticancer cell proliferation activity. Multivariate data analysis on NMR was able to distinguish sour cherry cultivars according to the bioactivity.

Applying pesticide treatment in the production of blackcurrants had a significant impact on the yield, vegetative growth of plants, leaf spot, ascorbic acid content and anticancer cell

ii proliferation activity. However, no significant difference was observed on berry sizes, total anthocyanin content and antioxidant capacity of blackcurrants. Nevertheless, the plant growth and physical conditions, total anthocyanin content and effect on PGE2 production were significantly different among cultivars. Furthermore, multivariate data analysis on both NMR and HPLC data were unable to distinguish the difference between cultivars. However, multivariate data analysis was able to partly distinguish the blackcurrants with and without pesticide treatment.

For the first time ever the in vitro anticancer cell proliferation activity was investigated in the rose hip extracts. However the crude extracts did not display strong inhibitory effect against cancer cell proliferation. Betulinic acid, oleanolic acid and ursolic acid were reported before as potential anticancer agents and combination effects of these three compounds were primarily investigated. Only the combination of oleanolic acid and ursolic acid showed additive effect against HT-29 cancer cells. In addition, these three triterpene acids showed no inhibition against COX-2 activity. Linoleic acid and α-linolenic acid showed inhibition against pure COX-2 enzyme, but neither of them inhibited the PGE2 production in cancer cells.

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Sammendrag

Ved inflammation spiller cyclooxygenase-2 (COX-2) en stor rolle som katalysator for dannelsen af prostaglandin E2 (PGE2) fra arachidonsyre. Vedvarende inflammation fører til frigivelse af reaktive materialer, pro-inflammatoriske cytokiner og andre komponenter, der forårsager udviklingen af kræft. Nyere forskning har vist, at disse processer kan hæmmes af bioaktive fytokemikalier fra planter. Desuden er forbruget af vegetabilske fødevarer til gavn for forebyggelsen af kræft og hjerte-kar sygdomme. Af disse årsager er undersøgelser af fødevareplanter vigtige for samfundet.

Surkirsebær (Prunus cerasus), solbær (Ribes nigrum) og hyben (Rosa canina) er tre almindelige fødevareplanter i Danmark, alle tre forbruges i forarbejdet form. Disse tre fødevareplanter er kendt for at have en meget høj næringsværdi, og hyben er siden gamle dage blevet anvendt som lægeplante. Solbær og surkirsebær er kendt for at være rige på bioaktive fenoler, som menes at være potentielle sundhedsfremmende stoffer.

Indholdet af fytokemikalier, samlet antioxidantkapacitet og deres effekt på PGE2 produktion og kræftcelleaktivitet blev undersøgt i en række surkirsebær og solbær, dyrket med eller uden pesticider. Yderligere, blev high performance liquid chromatography (HPLC) anvendt til at undersøge fenolforbindelserne, og nuclear magnetic resonance (NMR) blev anvendt til at indsamle oplysninger om andre komponenter, forekommende i solbær og surkirsebær saft. Alle data blev samlet og multivariat dataanalyse blev anvendt til at undersøge forskellene mellem sorterne.

Mængden og sammensætningen af fenoler samt biologisk aktivitet i forskellige surkirsebær sorter var signifikant forskellige. De sorter, der påviste en bedre antioxidant og anticancer aktivitet havde et højere indhold af fenoler. Men evnen til at hæmme PGE2 produktionen var uafhængig af fenolerne, antioxidant og aktiviteten af anticancer celledeling. Multivariat dataanalyse på NMR data var i stand til at skelne surkirsebærsorter efter bioaktivitet.

Anvendelse af pesticider i produktionen af solbær havde en betydelig indvirkning på udbyttet, væksten af planten. bladplet, adcorbinsyre indhold samt aktiviteten af anticancer celledeling. Anvendelsen af pesticider viste ingen signifikante forskelle i bærstørrelse, anthocyanin indhold og antioxidantkapacitet i solbær. Dog blev en signifikant effekt af sort på plantevækst

iv og fysiske forhold, anthocyaninindhold og påvirkning af PGE2 produktion påvist. Multivariat dataanalyse på både NMR og HPLC-data var ikke i stand til at skelne mellem sorter, men var til en hvis grad i stand til at skelne mellem solbær, dyrket med eller uden pesticider.

For første gang nogensinde er aktiviteten af in vitro anticancer celledeling blevet undersøgt i hybenekstrakter. De rå ekstrakter viste ikke stærk hæmmende effekt af kræftcelledeling. Betulinic syre, oleanolic syre og ursolic syre er før blevet rapporteret som potentielle anticancerkomponenter og kombinationseffekter af disse tre stoffer blev primært undersøgt. Kun kombinationen af oleanolic syre og ursolic syre viste additiv effekt mod HT-29 kræftceller. Desuden viste disse tre triterpene syrer ingen hæmning af COX-2 aktivitet. Linolsyre og α-linolensyre viste hæmning af ren COX-2 enzym, men ingen af dem hæmmede produktionen af PGE2 kræftceller.

Table of Contents

Preface……………………………………..……………………………………………. i Abstract…………………………………………………………………………………. ii Sammendrag……………………………………………………………………………. iv Table of Contents……………………………………………………………………...... vi List of Papers………………………………………………………………………....… vii Abbreviations………………………………………………………………………….... viii 1. Introduction………………………………………………………………………… 1 2. Inflammation and Cancer……………………..…………………………….……... 4 2.1 Cyclooxygenase family………………………………………………………….. 4 2.2 Inflammation, cancer and oxidative stress……..…………………………….….. 5 2.3 Anti-inflammatory drugs…………………………………………………..…….. 7 3. Plant, Agriculture and Metabolomics…………………………………….……….. 8 3.1 Plant secondary metabolites……………………………………………..………. 8 3.2 Plant evolution and agriculture……………………………………….………….. 9 3.3 Sustainable agriculture………………………………………………….……….. 10 3.4 Plant metabolomics………..……………………………………….……………. 11 4. Rose Hip……………….……………………………………………………………. 13 4.1 Folk remedy………………………………………………….………………….. 13 4.2 Phytochemical content and biological activities……………..………………….. 13 4.2.1 Ascorbic acid and carotenoids……………………….……………………. 13 4.2.2 Pharmacological properties……………………………………..…………. 14 4.2.3 Clinical trials………………………………………………..……………… 16 4.2.4 Cancer cell proliferation activity…………………………….…………….. 17 5. Bioactive Pure Compounds…………………………………………...……………. 20 5.1 Triterpene acids……………………………………………………….…………. 20 5.2 Linoleic acid and α-linolenic acid………………………………….……………. 22 6. Conclusions...………………………………………………………………………... 24 7. Perspectives…………………………………………………………………………. 25 Appendix………………………………………………………………………………… 26 References…………………………………………………………….………………… 29 Papers…………………………………………………………………………………… 47

List of Papers

Paper 1

Gaik Ming Khoo, Morten Rahr Clausen, Bjarne Hjelmsted Pedersen, Erik Larsen. Bioactivities and total phenolics of 34 sour cherry cultivars. Journal of Food Composition and Analysis , 2011, doi: 10.1016/j.jfca.2011.03.004

Paper 2

Gaik Ming Khoo, Morten Rahr Clausen, Hanne Lindhard Pedersen, Nina Eggers, Erik Larsen. Total content of anthocyanin, ascorbic acid and bioactivities of pesticide treated and pesticide free blackcurrant ( Ribes nigrum ) cultivars. Food Chemistry . (Accepted with revision)

Paper 3

Gaik Ming Khoo, Morten Rahr Clausen, Bjarne Hjelmsted Pedersen, Erik Larsen. Bioactivity of sour cherry cultivars grown in Denmark. Phytotheraphy Research (Submitted)

Paper 4

Gaik Ming Khoo, Morten Rahr Clausen, Erik Larsen. Investigation of three triterpene acids against cancer cell proliferation activity. Phytotheraphy Research (Submitted)

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Abbreviations

COX Cyclooxygenase COX-1 Cyclooxygenase-1 COX-2 Cyclooxygenase-2 COX-3 Cyclooxygenase-3 PGE2 Prostaglandin E2 PGH2 Prostaglandin H2 NSAIDs Non steroidal anti-inflammatory drugs COXIBs Cyclooxygenase-2 inhibitors ROS Reactive oxygen species RNS Reactive nitrogen species EIA Enzyme immunoassay LPS Lipopolysaccharide DMEM Dulbecco’s modified eagle’s medium FCS Fetal calf serum PBS-EDTA Phosphate buffered saline – ethylenediaminetetraacetic acid

CO 2 Carbon dioxide DMSO Dimethyl sulfoxide NMR Nuclear magnetic resonance MS Mass spectroscopy HPLC High performance liquid chromatography PCA Principal Component analysis PLS Partial least square Hex Hexane DCM Dichloromethane MeOH Methanol

H2O Water THF Tetrahydrofuran AGJ Artificial gastric acid juice

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1 1. Introduction

3 Plants produce a number of chemical compounds, defined as phytochemicals. Different 4 plants can synthesize various classes of phytochemicals. Apart from the primary metabolites 5 that are responsible for the essential growth of plants, there are a diverse classes of 6 phytochemicals produced by plants which are not involved in the primary metabolism [1]. 7 Since ancient times, plants have been utilized by human for nutritional and medicinal purpose. 8 In early days, humans were consuming the unmodified plant parts or boiling the plant 9 materials in hot water. Later in 19 th century, development in chemistry research had 10 successfully led to the isolation and purification of some bioactive substances from plants. 11 Nowadays, there are still up to 80 % of the population in developing countries fully 12 dependent on plants for the primary health care [2]. In addition, over 25 % of the prescription 13 drugs available on the market are derived directly or indirectly from plants, and it might reach 14 up to 50% when over-the-counter (OTC) market is taken into consideration [3].

15 Biological effects of phytochemicals in the human daily diet are not yet fully understood, 16 except for some primary metabolites like carbohydrate, protein and fat. Secondary 17 metabolites such as phenolics, terpenes and alkaloids which are considered as non-nutritive 18 compounds, are a class of compounds that was for once considered as having no health 19 impact on humans. However, rapid development in science enabled the isolation and 20 identification of these compounds, and numerous results have amplified the important values 21 of these neglected phytochemicals, by pointing out that these classes of compounds were 22 biologically active against different assays either in vitro or in vivo , which might potentially 23 benefit humans by preventing or combating different diseases, instead of providing basic 24 nutrition [4].

25 Advanced development of modern technology has improved the quality of human life over 26 the last few decades. At the same time the occurrence of some life-threatening diseases 27 including different kinds of cancers, cardiovascular diseases, obesity and diabetes has 28 increased due to the change in human diet. [5]. Consumer demands, increasing healthcare 29 costs and increased population ageing give a rise to a new general interest in plant food. This 30 increased interest emphasizes the scope of these bioactive phytochemicals and it is based on 31 epidemiological studies promoting the notion that an increased consumption of plant foods is

32 able to prevent or reduce the risk of diseases, including cardiovascular events and cancers [6- 33 8]. During the past two decades, there has been a dramatic increase in the use of 34 phytochemical-based products in many countries, representing one of the fastest growing 35 segments in food industry, with an estimated growth of 5 % per annum [9]. Plants are not 36 only used as a drug source, they are also promoted as functional foods, to improve health 37 conditions in humans by providing extra nutrients [10-12].

38 The crude fraction from a single part of a plant can easily contain up to more than 2000 39 phytochemical compounds [13]. Although a large number of phytochemical compounds have 40 been identified from plants, there are still many unknown phytochemicals waiting to be 41 discovered. A problem during the screening process is to judge whether the individual 42 compound will maintain the same effect as if it remained in the crude extract, because in the 43 crude extracts there might be a synergistic effect increasing or suppressing the biological 44 activity performances [14, 15]. In addition, pure compounds might have different effects on 45 different assays, and thus it is hard to judge if the compound is valuable, even if it is inactive 46 in one of the bioassays. Newer techniques, for example high throughput screening and 47 combinational chemistry have been developed in the attempt to speed up the screening 48 process and detect the bioactive compounds more effectively [16].

49 The aim of this thesis was to investigate the anti-inflammatory, in vitro anticancer and 50 antioxidant activity of plant food, and the correlation between the assays and phytochemical 51 content. The experiment was based on the theory that cyclooxygenase-2 being related to 52 cancer development [17, 18], and that consumption of fruits and vegetables which are rich in 53 antioxidants may prevent the development of cancer [19]. In addition, it is known that the 54 production of the phytochemicals in plants does not only depend on species, but also depend 55 on cultivars and environment [20]. Different sour cherry cultivars and blackcurrant cultivars 56 with and without pesticide treatment were evaluated using the assays. In addition, correlation 57 between the assay data and the NMR data was investigated using multivariate data analysis. 58 Furthermore, in vitro anticancer effect on rose hip extracts was investigated for the first time. 59 In vitro anticancer effects and anti-inflammatory activity was evaluated on selected pure 60 compounds from rose hip.

61 This thesis consists of three main parts reviewing different topics that make up this project. 62 The first part reviews the bioassays which have been applied in the project study, including 63 the occurrence of cyclooxygenase and the physiological function, the relationship of

64 cyclooxygenase with cancer and oxidative stress, and the current development in anti- 65 inflammatory drugs (Chapter 2). The second part consist of a review on the difference of 66 cultivars within plants and the recent development of plant metabolomics (Chapter 3), 67 referring to papers 1, 2 and 3. A literatures review on rose hip and part of the unpublished in 68 vitro anticancer experiment results and the discussion is included (Chapter 4). Further, anti- 69 inflammatory and anticancer effect of three triterpene acids and two fatty acids from rose hip 70 were reviewed (Chapter 5), partly referring to paper 4.

89 2. Inflammation and Cancer

91 2.1 Cyclooxygenase family

92 Cyclooxygenase (COX) is the enzyme that catalyzes the first two committed steps in the 93 biosynthesis pathway of prostaglandins from arachidonic acid, namely the cyclooxygenation 94 which forms prostaglandin G2 (PGG2) and the peroxidation which forms prostaglandin H2 95 (PGH2). PGH2 is transformed by specific synthases or isomerases into a range of primary 96 prostanoids including prostaglandins, prostacyclins and thromboxanes. These prostanoids act 97 as mediators giving signals for d ifferent physiological functions in the body [21] . Figure 1 98 shows the biosynthesis pathway of prostanoids.

99 Figure 1. Prostanoids biosynthesis pathway

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101 The cyclooxygenase family comprises three known members namely cyclooxygenase -1, -2 102 and -3 (COX-1, COX-2 and COX -3). COX-1 is considered as an enzyme which is 103 constitutively expressed in normal mammalian cells. The metabolites of arachidonic acid 104 derived from the COX-1 are responsible for maintaining the basic physiological conditio ns in 105 the body, including cytoprotection to gastric mucosa, integrity of platelet and renal perfusion 106 [22]. COX-3 is a splice variant of COX -1. It is abundant in the cerebral cortex and the heart.

4 107 The actual function of COX-3 is yet to be investigated [23]. COX-2, in contrast to COX-1 is 108 an inducible enzyme which is not found in most of the normal mammalian cells, but it can 109 rapidly be up-regulated in response to a variety of stimuli related to inflammatory response 110 such as growth factors and cytokines [22], thus COX-2 is engaged in the inflammatory 111 process.

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113 2.2 Inflammation, cancer and oxidative stress

114 Inflammation is a complex biological response of vascular tissues to injury, involving 115 migration of leukocytes and damage to local tissues [24]. It is a protective attempt by the 116 organism, to remove injurious stimuli as well as initiating a healing process of the tissues. 117 Inflammation can be classified to acute and chronic inflammation. Acute inflammation is an 118 initial response of the body to stimuli, achieved by the movement of plasma and leukocytes 119 from the blood into injured tissue [25]. Chronic inflammation is a prolonged inflammation 120 that leads to a progressive shift of leukocytes, lymphocytes and other inflammatory cells to 121 the inflammation site, contributing to the breakdown and healing of tissues in the 122 inflammatory process. But if a chronic inflammation is persistent, abnormal cell proliferation 123 might lead to neoplasia which contributes to formation of cancers [24-26].

124 When the inflammation system is activated by stimuli, arachidonic acid is released from the 125 cell membrane phospholipids by phospholipase A2. COX-2 is activated in response to 126 inflammation, converting arachidonic acid to PGE2, one of the major metabolites in 127 inflammation that acts as mediator, contributing to the initiation of fever, swelling, redness 128 and increase in of vascular permeability, which is a typical character of inflammation [27]. 129 Recent research pointed out that COX-2 and PGE2 are connected to cancer development, as 130 over expression of COX-2 and high levels of PGE2 were found in different cancer cells [28- 131 32]. Furthermore, COX-2 and PGE2 might be associated with neurodegenerative disease 132 development, for example Parkinson and Alzhemier, via activation of microglial in prion [33- 133 38].

134 Exposing the body towards a stressful environment such as pollution, radiation, ultraviolet 135 lights, or harmful chemicals resulted in oxidative stress and generation of reactive oxygen 136 species (ROS) and reactive nitrogen species (RNS), both known as chemically unstable 137 molecules that are able to modify and decompose the biological components [39]. ROS and

138 RNS provoke a large number of pro-inflammatory cytokines and inflammatory cytokines [40, 139 41]. Release of these cytokines creates complex pro-inflammatory microenvironment that is 140 associated with tissue injury and leads to aggressive inflammation, and a number of cancers 141 and neurodegenerative diseases [42, 43]. A review of evidences pointed out that ROS and 142 RNS are associated with inflammatory liver injury [44]. In addition, ROS was associated 143 with aggressive inflammatory breast cancer, up-regulating the COX-2 mRNA expression and 144 PGE2 production in cells [45]. Thus, antioxidants play an important role in suppressing 145 inflammation and cancer development. Figure 2 summarizes the mechanism for the 146 involvement of reactive species and inflammation in cancer development [25].

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148 149 Figure 2. Summary of mechanism for the involvement of inflammation in cancer development. 150 Inflammatory microenvironment promotes the development of tumors by activation of different 151 cytokines, chemokines and growth factors. 152

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6 155 2.3 Anti-inflammatory drugs

156 NSAIDs is one of the most commonly used drugs in the world with activity against 157 inflammatory symptoms without any glucocorticoid actions [46]. Traditional NSAIDs, for 158 example aspirin, naproxen and ibuprofen are known as non selective COX-1/COX-2 159 inhibitors. Use of these NSAIDs leads to a decreased level of prostaglandins; not only 160 prostaglandins synthesized by COX-2, but also prostaglandins synthesized by COX-1 that 161 mainly occurs in gastrointestinal mucosa and renal [47]. Thus use of NSAIDs is related to 162 some adverse effects such as gastrointestinal complications and renal dysfunction [48]. A 163 new class of NSAIDs named as COXIBs has been developed in recent years for example 164 celecoxibs, parecoxibs and etoricoxibs, focus on the inhibition of COX-2 and lack of the 165 mentioned side effects. However, use of COXIBs might elevate the level of thromboxane and 166 lead to an increased risk of thrombosis and heart attack [49]. Furthermore, long term use of 167 both classes of drugs contains potential risk of cardiovascular events, such as myocardial 168 infarction and stroke [50].

169 Plant secondary metabolites that display COX inhibitory effect are mainly phenolic 170 compounds [51]. Flavonoids are mainly found to exhibit inhibition against COX-1 activity, 171 but the selectivity towards COX-2 can be increased by structural modifications [52]. Some 172 compounds displaying significant COX-2 inhibitory activity include rutaecarpine [53], 173 eugenol [54], curcumin [55], linoleic acid [56] and α-linolenic acid [56]. Non-selective COX 174 inhibitors from natural products still deserve further investigation, and perhaps structural 175 modifications may increase the COX-2 selectivity. For example, the curcumin pyrazole 176 analogue showed increased selectivity compared to curcumin [57].

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184 3. Plant, Agriculture and Metabolomics

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186 3.1 Plant secondary metabolites

187 It is a simple fact that, unlike animals and other organisms, very few plants have the ability to 188 escape from their living environment. Therefore, plants produce diverse classes of 189 compounds through different pathways to survive in the nature. For example the Shikimic 190 acid pathway for production of nitrogen containing compounds, the phenylpropanoid 191 pathway to synthesize phenolic compounds, and the terpenoid pathway producing a number 192 of terpernoid metabolites [58]. These compounds act as poison to avoid the predators [59]; 193 some of them are volatile compounds or color compounds which attract animals for 194 pollination purposes [60-62]. Another necessary surviving competence is that plants produce 195 rigid compounds such as lignin and cellulose. These compounds support them to grow 196 straight, strengthen water transportation and make them resistant to herbivores attack [63]. 197 Figure 3 summarizes the biosynthesis pathway of major secondary metabolites in plants.

198 Figure 3. Biosynthesis pathway of major secondary metabolites in plants.

199 3.2 Plant evolution and agriculture

200 Adaptability to the ever-changing environment is the key to determine if a species can 201 survive in nature. Mutation and recombination allow organisms to modify their genetic 202 information during evolution to create new combinations that may or may not work in the 203 environment, and only the combinations that work successfully with the environment will 204 survive to the next generation [64]. In addition, genetic diversity is a critical factor for each 205 species to survive, and therefore plants have various methods of sexual reproduction to 206 promote out-crossing recombination when breeding [64, 65]. For example, plants breed 207 through pollination to share genetic information among individual species. In addition, some 208 plants have a self-incompatibility system to avoid self-fertilization or fertilization by similar 209 individuals [66].

210 Development of agriculture often involves domestication of crops from different origin to 211 fulfill the local requirements, but domestication crops might differ morphologically and 212 physiologically from the wild type due to the shift of environment [67, 68]. The introduction 213 of genomics studies boosts the knowledge in plant breeding by creating, selecting and fixing 214 plant phenotypes with the ambition to develop improved cultivars. The development is aimed 215 to improve yield, and nutritional quality and commercial value traits. The development of 216 plant breeding is considered to be successful, as many new cultivars are being developed and 217 benefits the global society with the purpose of meeting the needs for food and feed [69]. A 218 number of research including research on sour cherries (Paper 1 and 3) and blackcurrants 219 (Paper 2), have shown that phytochemicals and bioactivity of the same plant species differ 220 depending on individual cultivars [70-73], and these individual cultivars showed different 221 responses against environmental stress such as heat, drought and nutrient supply [74-76]; the 222 same plant cultivars showed different on phytochemical content and bioactivity when it was 223 grown in different geographical region or different agricultural condition [77, 78]. For 224 example, significant difference in ascorbic acid content and anticancer activity was observed 225 in same variety of blackcurrants but treated with and without pesticide (Paper 4).

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229 3.3 Sustainable agriculture

230 Agriculture was industrialized after World War II to increase crop production due to the 231 growth in world population. In addition, pesticides were applied to the conventional 232 industrialized farming to ensure crop productivity [79-81]. However, the continuous use of 233 pesticide has led to contamination of all basic necessities of life including water and food, 234 resulting in public health risk problems [82]. In addition, pesticide toxicity has caused 235 deleterious effects to the ecosystem [83]. According to an investigation of the United States 236 Department of Agriculture (USDA), pesticide contamination was found in most of the 237 vegetables and fruits in the United States, with a higher percent for imported foods [84].

238 The concept of sustainable agriculture has been introduced to the community recently, aiming 239 to develop a balance between the community and nature (Figure 4). Sustainable agricultural 240 production includes a variety of alternative agricultural methods using the principles of 241 ecology, which over a long term period is able to adapt the production to satisfy the human 242 food need, meanwhile enhancing environmental quality and improving the economic 243 condition of farmers and community as a whole [85]. Alternative agricultural methods 244 include for example organic farming, pesticide free farming, crop rotation farming, cover 245 crop farming, etc. Although food from alternative agriculture might still contain pesticides, 246 the amount is much lower than the food from conventional farming [86]. A number of 247 research point out that the nutritional quality of fruits and vegetables from alternative farming 248 is higher compared to the fruits and vegetables from conventional farming and may be more 249 health promoting [87].

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251 252 Figure 4. Balance system of sustainable agriculture is like a 3-legged stool, the balance will be destroyed if 253 one of the “legs” collapses. 254

10 255 3.4 Plant metabolomics

256 Metabolomics (or metabonomics) is the study of systems biology based on analysis of a 257 complete metabolite profile in a system under a given condition [88]. Metabolites are the end 258 products of genomic expression in response to the environment. In the 1970s, the 259 metabolomic study was first developed slowly from metabolite profiling and has recently 260 become a major research interest [88]. Metabolomics is considered as a new member of the “- 261 omics” family, joining genomics, transcriptomics and proteomics for studying and 262 understanding the global systems biology.

263 Nuclear magnetic resonance (NMR) and mass spectroscopy (MS) are the most commonly 264 used analytical tools for collecting the data of metabolomes [89]. NMR is a simple, 265 comprehensive and reproducible method, and the spectrum contains vast information 266 regarding the samples [86]. Nevertheless, the application has several limitations, such as low 267 sensitivity and overlapping of signals. However, these problems have been solved by the 268 recent developments in the equipment and two-dimensional NMR [90]. MS is a sensitive and 269 high resolution analytical tool that has a wide range of choice for fragmentation pattern 270 collections and different instrument combinations to obtain the specific fragment patterns for 271 metabolic profiling [91]. Data resulting from metabolomic work usually require multivariate 272 data analysis for interpretation. Principal component analysis (PCA) and partial least square 273 (PLS) regression analysis are the most commonly used multivariate data analysis tools for 274 metabolomics. PCA is used for visualizing groupings in the complete data set where all 275 samples are grouped with maximum separation [92]; whereas PLS is used to predict the 276 fundamental connection of a set of dependent variables from a large set of independent 277 variables [93].

278 Research in plant metabolomics shows a very significant increase during the last few years, 279 not only in phytochemical metabolism and biosynthesis pathway studies, but also for 280 potential bioactive phytochemical compound screening and assessment of the biological 281 effects of phytochemicals. The metabolomic study has been applied to biological fluid for 282 investigation of the bioavailability of phytochemicals [94, 95]. Metabolomic analysis has 283 been used as a tool for quality control of plants by optimizing clustering patterns according to 284 the phytochemical fingerprint of the plants [96, 97]. In addition, it has been applied in 285 profiling of species difference according to the production countries and cultivars [98-101]. 286 An investigation showed that NMR-based metabolomic profiling was able to evaluate

287 biosynthesis of antioxidant phenolics in fungus effectively [102]. Multivariate data analysis 288 of NMR data was able to distinguish and predict the antioxidant capacity of sour cherry 289 cultivars (Paper 1). Furthermore, metabolism of nitrogen in plants was studied with different 290 metabolomic approaches, that may contribute to the regulation of plant growth and 291 improvement of crop quality [103].

292 Metabolomics as a new promising tool in biological science has notably improved the 293 emerging of systems biology. The research in metabolomics will continue to evolve, however 294 there are still a number of challenges in the development, e.g. lack of quantitative assessment 295 for individual metabolites and the difficulties in detection of the specific metabolites in the 296 system [88, 89, 104].

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313 4. Rose hip

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315 Rose hip is a pseudo fruit of the rose plant Rosaceae family. It is commonly derived from 316 Rosa canina , Rosa rugosa , Rosa acicularis or Rosa cinnamomea [105]. Rose hip is native to 317 Europe, northern Africa and central Asia. In this chapter we reviewed the Canina section rose 318 hip varieties found mostly in northern and central Europe, on its chemical content, biological 319 performance and perspectives. Rosa canina also named as dog rose, or hyben in Danish.

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321 4.1 Folk remedy

322 Rose hip is consumed in a processed form presented as finished products like jam, cakes, 323 bread, syrup, soup and wine. In Hungary, rose hip is used in the making of Palinka; in 324 Denmark, dry rose hip powder is commercially available in the market; in Sweden, rose hip 325 is mainly used for the production of commercial soups. Rose hip fragrance can be found in 326 some cosmetic shops. Rose hip was once used as a folk remedy that was popular in the 327 Middle Ages for chest ailments [106]. It is known to have been used in the eighteenth and 328 nineteenth centuries to treat the bite of rabid dogs, and the name dog rose may result from 329 this [107]. Rose hip is used to treat gastrititis, flatulence, hiatal hernia, heartburn or 330 stomachache [108]. In Italy and Albania, rose hip is used to treat coughing, stomachache, evil 331 eyes, insect bites and acts as anti-depressive drug [109]. In Turkey, decoction of rose hip 332 mature fruit is used as medicine against colds, flu and diabetes, and the raw material is 333 applied externally as an antiseptic agent [110]. In addition, the decoction of the leafs is used 334 in treating diabetes [111].

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336 4.2 Phytochemical content and biological activities

337 4.2.1 Ascorbic acid and carotenoids

338 Ascorbic acid and carotenoids in rose hip were investigated extensively. Ascorbic acid and 339 carotenoids both are essential vitamins for human diet and are strong antioxidants. Rose hip 340 is well known as a rich source of ascorbic acid. It came into widespread use as a source of 341 ascorbic acid during World War II when Britain was unable to import citrus fruits, and the

342 government organized the collecting of rose hip fruits and processed them into syrup, to help 343 children from preventing scurvy [112]. The ascorbic acid content in rose hip varies according 344 to varieties and origin country (Table 1).

345 Table 1. Ascorbic acid content of rose hip from different varieties

Country Ascorbic acid content Reference Denmark 410 to 2310 mg/100g [113] Sweden, Denmark 330 to 535 mg/100g [114] Chile 3.89 to 24.92 mg/g [115] Sweden 10.81 to 57.63 mg/g [115] Turkey 1072 to 1682 mg/100g [116] Turkey 2365 to 2712mg/100g [117] 346

347 A study with thin layer chromatography and high-performance liquid chromatography 348 determines that total carotenoid in rose hip was 78.5 µg/g dry weight and the major 349 composition of carotenoids was β-carotene, lycopene, β-chryptoxanthin, rubixanthin, 350 zeaxanthin and lutein [118]. Another study includes that rose hip contained remarkable 351 amounts of lycopene ranging from 12.9 to 35.2 mg/100g lycopene [119]. The reported 352 content is similar to that of fresh tomatoes [120, 121], and thus rose hip might be a new 353 potential source of lycopene. The carotenoid content in rose hip varies according to the 354 growing country. Total carotenoid content in the ethanol extracts of rose hip from Chile and 355 Sweden ranged from 0.03 to 0.33 mg/g and 0.12 to 0.34 mg/g, respectively [115].

356

357 4.2.2 Pharmacological properties

358 Apart from the vitamins, rose hip contains a number of other phytochemicals which are 359 considered to be potential health promoting substances displaying different biological activity. 360 Table 2 shows the phytonutrient content and antioxidant activities of different rose hip 361 varieties. Rose hip phenolics displayed chemical defense against galling insect damage, as 362 genotypes without galling insect damage contained significantly higher total phenolics [122]. 363 The remaining water fraction of rose hip ethanol extract after removing the non-polar 364 compounds was able to reduce the blood glucose level in rats, while the non-polar fraction 365 had no correlation with the anti-diabetic effects [123]. Oral administration of the combination 366 of rose hip and Lactobacillus plantarum DSM9843 was able to protect mouse colon from 367 ischaemia/reperfusion oxidative injury by reducing the level of reactive species

368 malondialdehyde [124]. Quercitin isolated from rose hip down-regulated the tyrosinase 369 activity in mouse melanoma cells in a dose depending manner, suggesting that rose hip 370 quercitin might be able to inhibit the melanogenesis [125]. Tiliroside and cis -tiliroside from 371 rose hip seeds displayed antioxidative effects and were cytotoxic to brine shrimp and bacteria 372 [126].

373 Table 2. Phytonutrient test and antioxidant test of different rose hip extracts Source Extract Phytonutrient test a Antioxidant activity b Reference Fruit powder Ethanol TP, AA, TC FRAP, TEAC [115] Fruit powder TP. AA DPPH [127] Fruit, seeds, Methanol TP, TF, TT, TFA DPPH, reducing power, β-carotene [128] petals, bleaching inhibition, lipid flowers, gall peroxidation

Dried skin Boiling water TP Reducing power, H 2O2 scavenging [129] activity, superoxide anion radical scavenging activity, DPPH. Leaf Hydrodistillation, EO, TP, TF, TC DPPH, TEAC [130] hexane, dichloromethane, methanol a: TP: total phenolics, AA: ascorbic acid/ascorbate/vitamin C, TC: total carotenoids, TF: total flavanoids, TT: total tocopherol, TFA: total fatty acids, EO: essential oil b: FRAP: ferric ion reducing antioxidant power ,TEAC: trolox equivalent antioxidant capacity, DPPH:diphenyl-1-picrylhydrazyl assay 374

375 The investigation of rose hip was mostly concentrated on its effect against oxidative stress 376 induced inflammation due to historical usage in the folk remedies. Rose hip powder reduced 377 the release of reactive oxygen species from polymorphonuclear neutrophils in osteoarthritis 378 patients and healthy humans without any allergic reactions or gastrointestinal disturbance, 379 suggesting that rose hip displays a protective effect on different tissues [131, 132]. The 380 effects of rose hip extract on neutrophil respiratory burst not only depend on ascorbic acid, 381 but also on polyphenolics and other compounds [133]. 3-β-D-galactopyranosyloxy-2- 382 (octadeca-9Z,12Z,15Z-trienoyloxy)propanyl octadeca-9Z,12Z,15Z-trienoate was a 383 galactolipid discovered from rose hip with inhibitory effect against the chemotaxis of human 384 peripheral blood neutrophils and later was patented in several countries and registered under 385 the trade name GOPO ® [134, 135]. This compound was proven to be one of the antioxidative 386 and anti-inflammatory compounds of rose hip powder and was non-toxic to 387 polymorphonuclear leukocytes [136]. Ethanol extract of rose hip from Turkey displayed 388 significant inhibition against mouse inflammatory and pain models, and further analysis

389 showed that flavonoids and tannins were the dominant compounds in the bioactive fraction 390 [137]. Jäger discovered that all organic solvent extracts of rose hip displayed good inhibitory 391 effect against both COX-1 and COX-2 [138]. Later, linoleic acid and α-linolenic acid which 392 displayed strong inhibitory effect against COX-2 were isolated from rose hip [56]. However, 393 another investigation on two rose hip varieties found that only dichloromethane extract and 394 hexane extracts from rose hip displayed inhibitory effect against COX enzymes and

395 leukotriene B 4 (LTB 4) and the active fractions contain not only linoleic acid and α-linolenic 396 acid, but also three triterpene acids: betulinic acid, oleanolic acid and ursolic acid [127]. The 397 mixture of the three triterpene acids was further confirmed displaying immunomodulatory 398 effects by inhibiting lipopolysaccharide-induced interleukin-6 release in Mono Mac 6 cells 399 [139].

400

401 4.2.3 Clinical trials

402 As shown in table 3, several clinical studies investigated the effects of rose hip powder 403 against arthritis and chronic pain. These studies proved that rose hip reduces the arthritis pain 404 effectively during the treatment period and the effect was similar to that of analgesic drugs, 405 NSAIDs, steroids or anti-arthritis drugs. Furthermore, no serious adverse effect was observed 406 during the studies indicating that rose hip might be considerably safe for arthritis patients to 407 use in treatment of chronic pains.

408 Table 3. Clinical efficacy test of rose hip powder Symptom Type of test Dose, Time length Effectiveness Reference Osteoarthritis Randomized, 5g/day vs. placebo Significant reduction of pain and [140] knee and hip double-blind, over 4 months improved joint mobility placebo-controlled, parallel trial Osteoarthritis Randomized, 5g/day vs. placebo Significant reduction of pain after [141] knee and hip double-blind, over 3 months 3 weeks treatment. placebo-controlled, cross-over trial Osteoarthritis Randomized, 5g/day vs. placebo Moderate reduction on joint pain, [142] multiple sites double-blind, over 3 months improve general wellbeing, sleep placebo-controlled, quality and mood cross-over trial Osteoarthritis Pilot surveillance 5g/day Appreciable overall improvement [143] multiple sites study over 1 year in relief of pain Rheumatoid Randomized, 5g/day vs. placebo Patien ts had significant [144] arthritis double-blind, over 6 months improvement physically, pain placebo-controlled, relief effect was same as parallel trial analgesics, NSAIDs , steroids and anti-rheumatic drugs

409

410 Fewer investigations were done on the anti-carcinogenesis effects of rose hip. Previously, 411 petroleum ether and ethanol extracts of dried rose hip seed demonstrated significant 412 cytotoxicity to Yoshida ascites sarcoma cells [145]. Another research on rose hip showed that 413 after boiling and stewing, the extracts showed no anti-mutagenicity in Salmonella 414 typhimurium TA 100, but raw rose hip decreased sodium azide mutagenicity [146]. A 5 % 415 solution of the rose hip tea extract displayed moderate inhibition of sulfoconjugation and a 416 slight reduction of glucuronidation in Caco-2 cells [147].

417

418 4.2.4 Cancer cell proliferation activity

419 In part of my PhD study, preliminary investigations were made on rose hip hexane (Hex) 420 fractions, dichloromethane (DCM) fractions, methanol (MeOH) fractions, and remaining

421 aqueous (H 2O) fraction and tetrahydrofuran (THF) extracts against HT-29, Caco-2 and SW- 422 480 in vitro cancer cell proliferation activities (Material and methods section in Appendix). 423 Figure 5 shows the inhibitory effects of all rose hip fractions and extracts. In general, all 424 fractions and extracts showed only moderate or weak to no inhibition against cancer cells. 425 The inhibitory effect of all fractions and extracts was significantly different for all cancer cell 426 lines (P < 0.001). However, no difference was observed in any cancer cell line when 427 comparing between the fractions of rose hip with and without artificial gastric acid juice 428 treatment (P > 0.05). Figure 6 shows the PCA score plot on all fractions against cancer cell 429 proliferation. As shown in figure 6(a), the score plot was not able to distinguish the difference 430 of inhibitory effect between extracts of rose hip with and without artificial gastric acid juice

431 treatment; in figure 6(b), DCM fractions and the remaining H 2O fractions were independent 432 from the others, indicating these two fractions may be interesting for further study.

433 As described before, the DCM fraction of rose hip contained a number of bioactive

434 compounds including the triterpenoids and lipids; the remaining H2O fraction of rose hip 435 contained the most polar compounds including monosaccharide, oligosaccharide and pectins 436 [148]. Compared to the inhibitory effect of triterpene acids against cancer cells (paper 4), rose 437 hip crude extracts were not highly cytotoxic to the cancer cells. Ingestion of rose hip is not 438 generally associated with toxicity [105, 148], therefore it may be related to the antagonistic 439 effects between the phytochemicals [149, 150]. Pure compounds might not behave in the

440 same way when they are in the crude extract or in the whole food [14]. My results show that 441 cancer cells reacted differently towards the extracts and pure compounds, stating the 442 behavioral difference of cancer. An investigation on 14 human colon cancer cell lines showed 443 that the protein and gene expression of each cell line was different [151]. Furthermore, 444 subpopulations within the same human colorectal cancer cell line were distinct 445 morphologically and genetically [152, 153].

446 Figure 5. Inhibitory effects of rose hip extracts against (a) HT-29, (b) Caco-2, and (c) SW-480 cancer cell 447 proliferation activities. The inhibition presented is the result comparison to negative control without 448 sample treatment. AGJ: Artificial gastric acid juice pretreated samples before solvent extraction; RAW: 449 raw rose hip powder without pretreatment. Hex: hexane fraction; DCM: dichloromethane fraction; 450 MeOH: methanol fraction; H2O: remaining aqueous fraction; THF: tetrahydrofuran extract. 451

452

453 454 Figure 6. PCA score plot varied according to (a) the pretreatment method, (b) different solvent extraction. 455

456

457

458

459

460

461

462

463

464

465

466

467

468

19 469 5. Bioactive Pure Compounds

470

471 5.1 Triterpene acids

472 Triterpenoids are terpenes consisting of six isoprene units and with C30 skeleton. They are 473 widely found in nature with diverse backbone structures and comprise the largest group of 474 plant natural products with over 20,000 known members [154, 155]. Recently, pentacyclic 475 triterpenoids have been discovered displaying diverse biological and pharmaceutical 476 activities, including antibacterial, antifungal, anti-inflammatory, anti-HIV, hepatoprotective, 477 anticancer activity, etc. [156-164]. Betulinic acid, oleanolic acid and ursolic acid are 478 pentacyclic triterpene acids that are found in a number of plants, especially plants used in folk 479 medicine. These three triterpene acids have been isolated from rose hip and the mixture 480 displays immunomodulatory effects [139].

481 Table 4. In vitro cytotoxic effect of betulinic acid (BA), oleanolic acid (OA) and ursolic acid (UA) on 482 human cancer cell lines. Triterpenoid Cancer type References BA Pancreatic carcinoma [165, 166] Gastric carcinoma [166] Prostate cancer [167-169] Colon cancer [168, 170, 171], paper 4 Leukemia [168, 171, 172] Lung cancer [168, 171] Breast cancer [168, 171] Cervical carcinoma [168, 171] Ovarian carcinoma [171] Brain tumor [171, 173] Head and neck cancer [174] Skin cancer [175] OA Ovarian carcinoma [176] Breast cancer [176, 177] Bone osteosacorma [178] Colon cancer [179, 180] , paper 4 Liver cancer [181] UA Liver cancer [181] Breast cancer [182-184] Prostate cancer [185 -187] Skin cancer [188] Colon cancer [180, 189], paper 4 Melanoma [190] Brain tumor [190] Thyroid anaplastic carcinoma [190] Gastric cancer [191] Lung cancer [192] Ovarian carcinoma [193] 483

484 A great number of studies point out that these three triterpene acids are potential antitumor 485 agents, because they demonstrate good inhibitory effects against different cancers. 486 Furthermore, normal human cells are highly resistant to these triterpene acids compared to 487 cancer cells [168, 181, 194, 195]. Table 4 includes in vitro anticancer assays which have been 488 conducted on the triterpene acids, and proved that these triterpene acids were inhibiting a 489 number of different cancer cells. Oleanolic acid was considerably less cyctotoxic to cancer, 490 however strong inhibitory effect against cancer was found in its derivatives [178, 196].

491 492 Table 5. Molecular targets of betulinic acid (BA), oleanolic acid (OA) and ursolic acid (UA) in anti- 493 inflammatory and anticancer activities. Triterpenoid Target Reference BA Bax, Bcl-2, Bcl-xL, caspase-3, -8, and -9, CD95, [165, 167-171, 173, 174, 197- cyclin D1, JNK, Mcl-1, NF-κB, PARP, p53, Sp, 199] topoisomerase I, VEGF

OA Na +/K + ATPase, caspase-3 and -8, ICAM-1, MMP, [177, 179, 181, 200, 201] MRP1, mTOR, NF-κB, VEGF

UA Na +/K + ATPase, AIF, Akt, Bax, Bcl-2, Bcl-xL, [181, 183-193, 202-204] caspase-3, -8, and -9, ERK, COX-2, Fas/FasL, GR, ICAM, I κBα, iNOS, JNK, MMP, MKP-1, mTOR, NF-κB, p65, PARP,PI3K, RT, uPA, VEGF, STAT

494 495 AIF: Apoptosis-inducing factor; Akt: serine/threonine protein kinase; Bax: Bcl-2-associated X protein; Bcl: B- 496 cell lymphoma, CD95: classical death receptor protein; FasL: Fas ligand; GR: glucorticoid receptor; ICAM-1: 497 Inter-cellular adhesion molecule 1; I κBα: I-κB protein; iNOS: inducible nitric oxide synthase; JNK: c-Jun n- 498 terminal kinase; Mcl-1: Myeloid cell leukemia 1; MKP-1: Mitogen-activated protein kinase phosphatase 1; 499 MMP: Matrix metalloproteinase; MRP1: multidrug resistance protein; mTOR: mammalian target of rapamycin; 500 NF-κB: nuclear faktor-κB; PARP: Poly(ADP-ribose) polymerase; PI3K: phosphoinositide-3 kinase; RT: 501 Receptor tyrosine; Sp: Specificity protein; uPA: urokinase-type plasminogen activator; VEGF: vascular 502 endothelial growth factor; STAT: Signal Transducers and Activators of Transcription protein 503

504 Research points out that triterpene acids triggered cancer cells toward apoptosis by various 505 mechanisms such as regulation of transcription factors, cell proliferation promoters and anti- 506 apoptotic proteins. Table 5 shows the molecular targets of the triterpene acids in anti- 507 inflammatory and anticancer activities.

508 As shown in figure 7 (unpublished data), betulinic acid, oleanolic acid and ursolic acid had 509 no inhibitory effects against COX-2 directly or PGE2 production in Caco-2 cancer cells. 510 Similarly previous investigation conducted by Su et al. indicated that betulinic acid was not

511 able to inhibit COX-2 [205]. However, betulinic acid was able to indirectly modulate the 512 COX-2 expression in colon carcinoma cells through inhibition of I κBα kinase and p65 513 phosphorylation [206]. In addition, betulinic acid was reported to down-regulate the PGE2 514 production in human peripheral blood mononuclear cells [207] and macrophage cells [208]. 515 The level of PGE2 in murine macrophage cells was reduced by oleanolic acid saponin, by 516 down-regulating the NF-κB level in cells. On the other hand, oleanolic acid up-regulated the 517 COX-2 expression in human coronary smooth muscle cells and further increased the 518 prostacyclin expression that might contribute to cardio-protection [209]. Previously, 519 investigations on ursolic acid claimed that it was able to activate the apoptosis of cancer cells 520 by inhibiting the COX-2 and Bcl-2 expression in cancer cells [210, 211]. Ringbom et al.

521 indicated that ursolic acid exhibited direct COX-2 inhibition with a IC 50 of 130µM [212], 522 however the concentration is almost 4 times higher than in my study in paper 4. Shishodia 523 indicated that the COX-2 inhibitory effect of ursolic acid might be due to the down-regulation 524 of NF-κB [204]. In addition, Wang pointed out that the down-regulation of COX-2 in lung 525 cancer cells was due to the inhibition of ERK activation [213].

526 Figure 7. COX-2 inhibitory effects or rose hip monocompounds based on enzyme-based assay and Caco-2 527 cell based assay (unpublished data), Columns labeled with different alphabet are significantly different (P 528 < 0.05). 529

530 5.2 Linoleic acid and α-linolenic acid

531 Linoleic acid is a C18 fatty acid with double bond at the n-6 and n-9 positions. α-linolenic 532 acid is a C18 fatty acid with 3 double bond at the n-3, n-6 and n-9 positions. Linoleic acid is 533 used in the biosynthesis of arachidonic acid, which is found in cell membrane lipids, and is

22 534 the source of a number of prostaglandins. Both linoleic acid and α-linolenic acid are essential 535 fatty acids that must be consumed to maintain proper health. In my study, linoleic acid and 536 α-linolenic acid showed no inhibition against Caco-2, HT-29 and SW-480 cancer cells at a 537 test concentration of 10µg/ml (approximately 36 µM). Similar to previous research, both 538 linoleic acid and α-linolenic acid showed no inhibition against Caco-2 at 80 µM, and 539 inhibition was observed at a concentration of 160 µM. Furthermore, linoleic acid and α- 540 linolenic acid had no effect on cell proliferation of HT-29 [214].

541 As shown in figure 7, linoleic acid and α-linolenic acid showed significant inhibition against 542 COX-2 when tested with the COX inhibitor screening assay kit, and the inhibitory effect is as 543 good as non-selective COX inhibitor indomethacin, but neither of them inhibited the PGE2 544 production in Caco-2 cells. Referring back to the research study that claimed that crude 545 extracts, linoleic acid and α-linolenic acid from rose hip which display inhibitory effect 546 against COX activity, the assays applied in the study were not associated with any cell line 547 [56, 127, 138]. Linoleic acid was reported to induce the expression of COX-2 and the release 548 of PGE2 in retinal pigment epithelial cells [215], endothelial cells [216] and intestinal smooth 549 muscle cells [217]. Furthermore, experiments proved that elevated consumption of dietary 550 linoleic acid increased the synthesis of cyclooxygenase products, thereby stimulated human 551 breast cancer metastasis [218] and gastric carcinoma metastasis in mice [219]. On the other 552 hand, dietary α-linolenic acid reduced oxidative stress and displayed anti-inflammatory effect 553 by decreased COX-2 expression in murine macrophage cells [220], hepatoma cells [221] and 554 Colitis mice [222].

555 Enzyme-based and cell-based COX-2 assays gave distinct result in my study. The most 556 significant is the COX-2 selective inhibitor NS-398, which supposed to be a COX-2 selective 557 inhibitor, but it shows no inhibition in the enzyme-based assay. Compared to the enzyme- 558 based assay which compounds reacted against the enzyme directly, the cell-based or animal- 559 based assay might be more reliable for screening of bioactive compounds, because the 560 microenvironment in living cells and animals is far more complicated than in pure enzyme. 561 Bioactive compounds might react with different targets in the cell microenvironment at the 562 same time and eventually lead to a reduction of the enzyme expression.

563

564

565 6. Conclusions

566 Bioactive phytochemical content varies in different plant foods, even in the same plant 567 species but different cultivars. Therefore selection of cultivars that are suitable for the local 568 growth is very important, in order to produce crops with high content of bioactive 569 phytochemicals that are potentially benefit for human health in preventing a number of 570 diseases.

571 Metabolomic study is an application driven science, where the modeling system is strongly 572 dependent on the specific individual dataset. PCA in multivariate data analysis is a useful tool 573 for predicting and screening for the potential health promoting cultivars of certain plant foods, 574 for example sour cherries but not blackcurrants. However it could be used to distinguish the 575 blackcurrants with or without pesticides treatment.

576 Blackcurrants grown without pesticide treatment contained significantly higher levels 577 ascorbic acid and displayed better inhibition against cancer cell proliferation, when compared 578 to the blackcurrants with pesticides treatment, indicating that blackcurrants grown without 579 pesticides treatment may be potentially healthier for human consumption. In addition, the 580 growth and physical condition of plant and the total anthocyanin are depending on individual 581 cultivars.

582 Rose hip extracts did not display strong inhibitory effects against cancer cell proliferation 583 activity, but the pure compounds from rose hip displayed strong inhibitory activity against 584 cancer cell proliferation. Combination of betulinic acid, oleanolic acid and ursolic acid 585 showed no synergistic effect when they were mixed and tested against Caco-2 cancer cell line. 586 However combination of oleanolic acid and ursolic acid displayed additive effect against HT- 587 29 and SW-480 cancer cell lines.

588 Linoleic acid and α-linolenic acid showed strong inhibition on COX-2 when tested with 589 COX-2 inhibitor enzyme immunoassay, but showed no inhibition against COX-2 in Caco-2 590 cell-based PGE2 production assay. The difference of result suggested that difference of 591 microenvironment might have effect on the biological performance of the pure compounds.

592

593

594 7. Perspectives

595 Although frequent consumption of plant foods with bioactive phytochemical content can help 596 to prevent a number of diseases, the risk associated with high consumption level of these 597 plants should be evaluated carefully, as these bioactive phytochemicals originally are a part 598 of the normal plant defense system and may be toxic in high doses to humans.

599 Unlike rose hip, sour cherries and blackcurrants in Denmark have no specific or national 600 breeds for large scale cultivation. Metabolomic study with multivariate data analysis on the 601 NMR data may be applied to speed up the sourcing of potential health promoting cultivars for 602 sour cherries but not blackcurrants. However, this tool could be applied in the process of 603 quality control monitoring the application of pesticides in blackcurrants.

604 In relation to human health and nature, reduction in the use of pesticides in agriculture 605 farming may lower the environment agrochemicals contamination; furthermore it may reduce 606 the risk of cancer incidences in the society and minimize the financial input of the farmers. 607 However pesticide free treatment is more suitable for crops with high tolerance of leaf 608 damage, such as blackcurrants. There are other sustainable agricultural methods that are 609 suitable for other crops that fit the requirement of the plant characteristics.

610 Further investigation is necessary to optimize the degradation process of these plant foods in 611 gastrointestinal environment and the phytochemical absorption and metabolism. The future 612 trend may likely be a combination of the “omics” studies, aiming to overview and fully 613 understand the functional mechanism of phytochemicals against human diseases.

614 Selection of a proper assay for experiments is important to confirm the accuracy of the result. 615 In vitro cell-based assay is a more ideal tool for primary screening compared to the enzymatic 616 assay because the microenvironment in cell is more complicated.

617

618

619

620

621

622 Appendix

623

624 Material and methods for:

625 (a) Inhibitory effects of rose hip extracts against cancer cell proliferation activity;

626 (b) Inhibitory effects of pure compounds against COX inhibitor screening immunoenzyme 627 assay (enzyme-based assay) and PGE2 assay (cell-based assay).

628

629 1. Chemicals 630 Hydrochloride acid, pepsin, sodium chloride, hexane, dichloromethane, methanol, 631 tetrahydrofuran, dimethyl sulfide oxide (DMSO) and β-cyclodextrin were obtained from 632 Sigma-Aldrich (Missouri, USA). Dulbecco's Modified Eagle Medium (DMEM) was obtained 633 from Invitrogen (Paisley, UK). Trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) and 634 phosphate buffer saline-ethylenediaminetetraacetic acid (PBS-EDTA) were obtained from 635 Lonza (Braine, Belgium). Penicillin G-potassium salt and streptomycin sulfate were obtained 636 from Serva (Heidelberg, Germany). Fetal calf serum (FCS) was obtained from PAA 637 Laboratories (Pasching, Austria). WST-1 cell proliferation reagent was obtained from Roche 638 Diagnostics (Mannheim, Germany). Lipopolysaccharide (LPS) was obtained from 639 Calbiochem, Merck (Damstadt, Germany). COX inhibitor screening assay kit and 640 monoclonal PGE2 enzyme immunoassay kit (PGE2 EIA kit) were purchased from Cayman 641 Europe (Tallinn, Estonia). 642 643 2. Inhibitory effects of rose hip extracts against cancer cell proliferation activity 644 2.1 Preparation of rose hip (Rosa canina) extracts 645 Rose hip seeds and rose hip hair were homogenized and blended in a blender. Rose hip skin 646 powder was provided by Hyben Vital A/S. 647 The rose hip skin, seed and hair were individually divided into 2 portions. The first portion 648 remained untreated, while the second portion was pretreated with artificial gastric acid juice 649 (AGJ) before extraction. The AGJ is prepared according to Hayashiba [223]. In brief, sample 650 was mixed with of AGJ and reflux at 37 ºC for 3 hours. The residue was filtered and washed 651 with water until neutral ph and were subsequently freeze dried and ready for extraction.

652 10 g of sample with or without AGJ treatment was mixed with water and was extracted three 653 times in a sequence of hexane, dichloromethane and methanol, with a volume of 30 mL 654 solvent each time. Another 10 g of sample was extracted with THF for 3 times, in a volume 655 of 30 mL each time. The same fraction or extraction was combined together and concentrated 656 with vacuum evaporator, later dried thoroughly with nitrogen flow.

657 Dried extracts were dissolved in different solvents according to their solubility forming a 658 concentration of 20 mg/mL: hexane and dichloromethane fractions were then subsequently 659 dissolved in DMSO containing 1 % (w/v) β-cyclodextrin; methanol fraction and THF extracts 660 were dissolved in DMSO; remain aqueous fraction was dissolved in water. The samples were 661 kept in -20 ºC until execution of the experiment.

662 2.2 Anticancer cell proliferation activity

663 Human colon cancer cell lines HT-29, Caco-2 and SW-480 (European Collection of Cell 664 Cultures, Salisbury, UK) were used in the experiment. Cells were grown in DMEM medium 665 supplemented with 10 % FCS, 100 IU/mL penicillin and 100 µg/mL streptomycin. The 666 medium was changed every second day and the cells were passaged every fourth day. 667 Trypsin-EDTA was used for detachment of cells from culture flask. Cells were incubated in

668 8000 WJ CO 2 incubator (Thermo Fischer Scientific, Leicestershire, UK) at 37 ºC with 5 %

669 humidified CO 2.

670 Cells were seeded into Nunc sterile transparent 96-well plate at a density of 1x10 4 cells per 671 well and incubated for 24 hours. Samples were added into the 96-well plate and the cells 672 were incubated for another 72 hours [224]. Proliferation assay was determined using WST-1, 673 based on the cleavage of tetrazolium salt to formazan by the mitochondrial dehydrogenase. 674 After 3 hours of incubation with WST-1, the absorbance was detected at a wavelength of 450 675 nm and a reference wavelength of 630 nm using BioTek Synergy 2 multi-mode microplate 676 reader. The absorbance was corrected by subtracting the value at 630 nm from the value at 677 450 nm. All samples were tested in triplicate in two independent experiments, and related 678 solvents for dissolving the samples were used as control. Wells without sample were used as 679 control and inhibition was calculated relative to the control for each sample. For rose hip 680 extracts, test concentration was 100 µg/mL.

681

682 3. Inhibitory effects of pure compounds against COX inhibitor screening 683 immunoenzyme assay (enzyme-based assay) and PGE2 assay (cell-based assay) 684 3.1 Pure compounds

685 Pure compounds tested in this experiment were: Betulinic acid, oleanolic acid, ursolic acid, 686 linoleic acid and α-linolenic acid. DMSO was used to dilute the compounds separately to 1.0 687 mg/mL.

688 3.2 COX inhibitor screening immunoenzyme assay

689 COX inhibitor screening assay was run according the provided protocol from the COX 690 inhibitor screening kit set purchased from Cayman Europe. All monocompound samples were 691 tested in a concentration of 5, 1.67 and 0.56 µg/mL. DMSO and ethanol were used as blank 692 control for the related sample. Indomethacin and NS-398 were used as positive control.

693 3.3 PGE2 assay

694 Caco-2 cells were grown in DMEM medium supplemented with 10 % FCS, 100 IU/mL 695 penicillin and 100 µg/mL streptomycin. The medium was changed every second day and the 696 cells were passaged every fourth day. Trypsin-EDTA was used for detachment of cells from 4 697 culture flask and cells were cultivated at 37 ºC 5 % CO 2. A total of 1x10 cells were seeded 698 into each well of Nunc sterile transparent 96-well plate and incubated for 48 hours. Cells 699 were then incubated with 500 µM aspirin for 3 hours to inactivate the endogenous COX-1 700 [225]. Cells were washed twice with PBS-EDTA, and 200 ng/mL LPS was added with or 701 without samples treatment. After incubation for 4 hours, media were collected and 702 centrifuged. All media were tested according to the given protocol from the PGE2 EIA kit 703 set. Test concentration for monocompound was 5µg/mL.

704

705

706

707

708

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Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original article Bioactivity and total phenolic content of 34 sour cherry cultivars

Gaik Ming Khoo a, Morten Rahr Clausen a, Bjarne Hjelmsted Pedersen b, Erik Larsen a,* a Department of Food Science, Faculty of Agricultural Sciences, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark b Department of Horticulture, Faculty of Agricultural Sciences, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark

ARTICLE INFO

Article history: Received 30 September 2010 ABSTRACT Received in revised form 4 March 2011 Accepted 5 March 2011 Thirty four sour cherry cultivars were investigated for their total phenolic content, total anthocyanin content, total antioxidant capacity and cancer cell proliferation inhibition activity. Birgitte Bo¨ttermo¨ Keywords: and Fanal, which contained the highest total phenolics (754 13.4 and 596 5.7 mg gallic acid Prunus cerasus Sour cherry equivalent/100 g, respectively), displayed the strongest total antioxidant capacity (63 7.5 and 52 6.9 Phenolics mmol trolox equivalent/g, respectively) and cancer cell proliferation inhibition activity (63 1.7 and 70 Anthocyanins 1.6%, respectively). Sureﬁre and Favorit were the cultivars with lowest total phenolic content (111 2.2 and HT-29 74 2.5 mg gallic acid equivalent/100 g, respectively) and total anthocyanin content (40 1.1 and 21 0.5 Antioxidant malvin equivalent/g, respectively), total antioxidant capacity (12 2.3 and 9 3.1 mmol trolox equivalent/g, Proton nuclear magnetic resonance respectively) and cancer cell inhibition activity (7 3.5 and 2 0.2%, respectively). Multivariate data Multivariate statistical analysis analysis on proton nuclear magnetic resonance (1H-NMR) data showed discrimination of the cultivars Food analysis according to their bioactivity. The present study suggests that combination of 1H-NMR and multivariate Food composition statistical analysis can be used to predict and discriminate total phenolic content, total anthocyanin content, total antioxidant capacity and cancer cell proliferation inhibition activity of sour cherry cultivars. ß 2011 Elsevier Inc. All rights reserved.

1. Introduction 2009; Seeram et al., 2001). Whole fruit products of sour cherry and the secondary metabolite mixtures could be biologically more active than Sour cherries (Prunus cerasus L.) are rich in phenolic compounds pure compounds (Seymour et al., 2008), where pure compounds which display a broad spectrum of health promoting benefits (King might not behave in the same manner as in whole foods, resulting in and Youez, 1996; Stoner et al., 2008; Yao et al., 2004). Sour cherries inconsistency in clinical trials (Liu, 2004). contain significant levels of anthocyanins that posses strong Metabolomics is a branch of system biology, analyzing the antioxidant and anti-inflammatory activities (Wang et al., 1999b; metabolomes which is the complete set of metabolites in cells, Mulabagal et al., 2009; Seeram et al., 2001); inhibited intestinal tumor tissues, organ, biological fluids or extracts. It is an application – development in ApcMin mice and reduced proliferation of human driven science, which relies on analytical techniques and data colon cancer cells HT-29 and HCT-116 (Kang et al., 2003). Water handling system. Metabolomic techniques produce complex data extracts of sour cherry and cyanidin aglycon inhibited cyclooxy- sets and a vast amount of information. One sample contains huge genases (Wang et al., 1999b; Mulabagal et al., 2009). Recent research amounts of variables, and thus multivariate modeling is useful for showed that consumption of sour cherry anthocyanins increased the interpreting the NMR data. level of superoxide dismutase in serum, reduced the level of tumor In this study, total phenolic content, total anthocyanin content, necrosis factor-a, interleukin-6 and malondialdehyde (MDA) in total antioxidant capacity and cancer cell proliferation inhibition serum and PGE-2 in paw of Freund’s adjuvant-induced arthritis in rats activity of 34 different sour cherry cultivars were determined. A (He et al., 2006, 2005). In the past decade, sour cherry products have metabolomic approach using nuclear magnetic resonance was had an increased utilization in the food market because of their investigated, for the possibility of developing an effective potential health benefit (Kirakosyan et al., 2009). Most of the screening method for health promoting sour cherry cultivars. investigations have focused on Montmorency and Balaton cultivars, due to their high antioxidant activities and economical value 2. Materials and methods (Kirakosyan et al., 2009; Wang et al., 1999a; Mulabagal et al., 2.1. Plant materials

* Corresponding author. Tel.: +45 89993367; fax: +45 89993495. The 34 sour cherry cultivars used in this study were Stevnsbaer E-mail address: [email protected] (E. Larsen). Birgitte, Stevnsbaer Viki, Tiki, M7, Recta, Zigeunerkirschen, K27/2,

Please cite this article in press as: Khoo, G.M., et al., Bioactivity and total phenolic content of 34 sour cherry cultivars. J. Food Compos. Anal. (2011), doi:10.1016/j.jfca.2011.03.004 G Model YJFCA-2111; No. of Pages 5

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Nefris, Fanal, Bofa, Kelleris 16, Skyggemorel Hannover, Birgit- 2.6. Antioxidant assay te Bo¨ttermo¨, Ungarische Traubige, Cigganymeggy 7, Erdi Bo¨t- termo¨, Surefire, Favorit, Oblachinska Holo, Safir, Vytenu Star, Total antioxidant capacity of sour cherries was measured using Zagarvysne, Gerema, Pernillla, Aarslev 2510, Aarslev 2403, Aarslev trolox equivalent antioxidant capacity (TEAC) assay. The experi- 1803, Aarslev 2504, Dana x 1, Stevnsbaer PH, Sumadinka, ments were performed in triplicate in 96-well plate with two Heimanns Rubin 4, Lutovka and Nadwislanka. Ripen sour cherries independent tests. Trolox was used for standard curve calibration. were harvested in Aarslev (Aarhus University, Faculty of Agricul- In brief, 50 ml of filtrated samples were mixed with 200 ml of ABTS tural Sciences, Aarslev, Denmark) in the period from July to August radical buffer solution and the absorbance was immediately read 2009. Sour cherries were stored at 24 8C immediately after after 1 min at a wavelength of 734 nm using BioTek Synergy 2 harvested. multi-mode microplate reader. Solution without sample was used as blank. Mean value and standard deviation were calculated. 2.2. Chemicals Antioxidant capacity was expressed as trolox equivalent per gram (TE/g) pitted fresh cherry. 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), sodium dihydrophosphate, methanol, sulfuric acid, 2,20- 2.7. Cell proliferation assay azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), ammonium persulfate, Folin–Ciocalteu reagent, gallic acid, sodium Human colon cancer cell line HT-29 (European Collection of Cell hydroxide and deuterium oxide 99.9 atom% D containing 0.05% Cultures, Salisbury, UK) was used in the experiment. Cells were

(w/v) 3-(trimethylsilyl) propionic 2,2,3,3-d4 acid sodium salt (D2O grown in DMEM medium supplemented with 10% FCS, 100 IU/ml containing 0.05% TSP) were purchased from Sigma-Aldrich, penicillin and 100 mg/ml streptomycin. Medium was changed Missouri, USA. Dulbecco’s Modiﬁed Eagle Medium (DMEM) was every second day and cells were passaged every fourth day. purchased from Invitrogen, Paisley, UK. Trypsin–EDTA and PBS– Trypsin–EDTA was used for detachment of cells from culture ﬂask.

EDTA were from Lonza, Braine, Belgium. Penicillin G.K salt and Cells were incubated at 37 8C5%CO2 in 8000 WJ CO2 incubator streptomycin sulfate were purchased from Serva, Heidelberg, from Thermo Scientific (Leicestershire, UK). Germany. Fetal calf serum (FCS) was from PAA Laboratories, Cells were seeded into 96-well plate at a density of 1 104 cells Pasching, Austria. WST-1 cell proliferation reagent was from Roche per well and incubated for 24 h. Filtrated samples were added into Diagnostics, Mannheim, Germany. 96-well plate with concentration of 50 ml/ml and the cells were incubated for another 72 h. Proliferation assay was determined 2.3. Sample preparation using WST-1, based on the cleavage of tetrazolium salt to formazan by the mitochondrial dehydrogenase. After 3 h of incubation with Fresh frozen cherries were removed from the freezer and pitted. WST-1, the absorbance was detected at a wavelength of 450 nm Approximately 200 g of pitted cherry from each cultivar was and a reference wavelength of 630 nm using BioTek Synergy 2 homogenized in a ratio of 2 g cherry with 1 ml water, using a food multi-mode microplate reader. The absorbance was corrected by blender. The homogenized samples were centrifuged for 30 min at subtracting the value of 630 nm from 450 nm. All samples were 12,000 rpm at 4 8C. Supernatants were collected and filtered with tested in triplicate in two independent experiments. Wells without 0.45 mm filter. Filtrates were kept at 80 8C until use. sample were used as control and inhibition was calculated relative to the control for each sample. 2.4. Total phenolic content 2.8. NMR experiment for the sour cherries extracts Total phenolic content of sour cherries were measured with rapid Folin–Ciocalteu method (Magalha˜es et al., 2010). The 500 ml filtered sample was mixed with 100 mlD2O containing experiments were performed triplicate in 96-well plate with 0.05% TSP. The 1H-NMR spectra were recorded at 25 8C on a Bruker two independent tests. Gallic acid was used for standard curve Avance 600 spectrometer operating at a proton frequency of calibration. In brief, 50 ml of standard or filtered samples were 600.13 MHz, equipped with a 5 mm 1H-TXI probe (Bruker Biospin, mixed with 50 ml Folin–Ciocalteu reagent (1:5, v/v, diluted with Rheinstetten, Germany). The spectra were acquired using a single water) followed by adding 100 ml of 0.3 M sodium hydroxide. After 908 pulse experiment with relaxation decay of 5 s. Water 3 min, the absorbance was detected under the wavelength of suppression was achieved by irradiating the water peak during 760 nm using BioTek Synergy 2 multi-code microplate reader from relaxation decay. 32 K data points spanning a spectral width of BioTek (Vermont, USA). Water was used as blank in the 12.1 ppm were collected. All spectra were referenced to TSP at experiment. Mean value and standard deviation were calculated. 0 ppm. The spectra were corrected manually using Topspin 2.1 Total phenolic content was expressed as milligram (Bruker Biospin, Rheinstetten, Germany). gallic acid equivalent per 100 g pitted fresh cherry (mg GAE/100 g). 2.9. Data processing and statistical analysis 2.5. Total anthocyanin content 1H-NMR spectra were subdivided into 0.074 ppm intergral Total anthocyanin content was measured spectrophotomet- regions and intergrated, reducing each spectrum into 1146 rically (Krawczyk and Petri, 1992). Homogenized samples were independent variables in the region 0.5–9.0 ppm. Further analysis extracted with acidified methanol and ultraviolet absorption was performed using SIMCA P+ 12.0.1.0 (Umetrics, Umea˚ , was measured at a wavelength of 526 nm with MPS-2000 Sweden). NMR data was scaled using pareto scaling, while all multipurpose recording spectrophotometer from Shimadzu other data were scaled to unit variance. Principal component (Kyoto, Japan). Each sample was measured in replicates. Malvin analysis (PCA) was applied to observe clustering behavior of the was used as standard calibration (Molar absorptivity of malvin, samples and PLS regressions with full cross validation were used to e =37700M1 cm1). Total anthocyanin content was investigate the correlation between NMR spectra and total calculated as malvin equivalent per 100 g pitted fresh cherry phenolic content, total anthocyanin content, total antioxidant (ME/100g). The mean value and standard deviation were capacity and HT-29 cancer cell proliferation inhibition activity. The calculated. correlation coefficient of total phenolic content, total anthocyanin

G.M. Khoo et al. / Journal of Food Composition and Analysis xxx (2011) xxx–xxx 3 content, total antioxidant capacity and HT-29 cancer cell research groups to be 160, 27 and 109 mg/100 g, respectively proliferation inhibition activity were calculated in Microsoft Office (Pedisic´ et al., 2007; Veres et al., 2008; Kim et al., 2005). In this Excel. study, their total anthocyanin contents were 214, 98 and 131 mg ME/100 g, respectively. These differences in total anthocyanin 3. Results and discussion content showed that the plant growth region and the harvest period might have an impact on plant growth and metabolite 3.1. Total phenolic content and total anthocyanin content concentration (Premier, 2002). Sour cherries from the cultivars that are abundant in total Table 1 shows the total phenolic content and the total phenolic content contained more total anthocyanin. The sour anthocyanin content of the 34 sour cherry cultivars. Total phenolic cherry cultivars that showed high content of total phenolics and content and total anthocyanin content varies among sour cherry total anthocyanin in this study mostly were cultivars belonging to cultivars analyzed in the present study. stevnsbaer, Fanal type, or cross breeding cultivars. Stevnsbaer is Previous reported total phenolic content of sour cherries were the most important sour cherry variety in Denmark, producing varied from 78 to 500 mg GAE/100 g fresh cherry (Bonerz et al., high quality of sour cherries with very dark red color. Fanal and 2007; Kim et al., 2005; Dragovic´-Uzelac et al., 2007). Total phenolic Nefris were very similar varieties from Germany and Poland, content of sour cherry cultivars in this study were varied between respectively, both belonging to the Fanal type sour cherry variety, 74 and 754 mg GAE/100 g. Birgitte Bo¨ttermo¨ had the highest and sharing similar characteristics and are well known in amount of total phenolic content among sour cherry cultivars, producing high quality juice (Christensen, 1986, 1988, 1990). followed by Fanal and Tiki which contain more than 500 mg GAE/ Birgitte Bo¨ttermo¨ is a cross breeding cultivar between Stevns- 100 g of total phenolics. Other cultivars that contained total baer Birgitte and Erdi Bo¨ttermo¨, which latter is a popular cultivar in phenolics more than 400 mg GAE/100 g were Aarslev 1803, Hungary. Tiki is the result of cross breeding between stevnbaers Heimann Rubin 4, Stevnsbaer Viki and Bofa. Cultivars that and Fanal, which characteristics are similar to stevnbaer, with a contained the lowest total phenolics were Favorit and Surefire. very dark red color appearance. Aarslev 1803, Aarslev 2510, Total anthocyanin content of sour cherries were between 285 Aarslev 2403 and Aarslev 2504 are new cross breeding cultivars in and 21 mg ME/100 g. Aarslev 1803, Birgitte Bo¨ttermo¨ and our research center. In contrast to the dark red color sour cherries Aarslev 2403 were the three highest among all cultivars. Other which contain high total phenolics and total anthocyanin, Favorit cultivars with total anthocyanin content more than 200 mg ME/ and Surefire which were the two cultivars that had the lowest total 100 g were Tiki, Recta, Nefris, Fanal, Bofa, Aarslev 2510 and phenolic and total anthocyanin content, share light pink color in Sumadinka. Surefire and Favorit were the cultivars with the lowest appearance. K27/2 and Kelleris 16 were the only two cultivars in content of anthocyanins. Total anthocyanin content of Recta, Erdi this study with considerably high content of total phenolics but Bo¨ttermo¨ and Sumadinka were previously reported by other low concentration in total anthocyanin.

Table 1 Total phenolic content (TP), total anthocyanin contents (TA), total antioxidant capacity (TEAC) and HT-29 cancer cell proliferation inhibition activity of 34 sour cherry cultivars and the standard deviation (SD).

Cultivar TP, mg GAE/100 g SD TA, mg ME/100 g SD TEAC, mmol TE/g SD HT-29 inhibition, % SD

Birgitte Bo¨ttermo¨ 754 13.4 272 4.1 63 7.5 63 1.7 Fanal 596 5.7 250 1.2 52 6.9 70 1.6 Tiki 515 11.4 264 4.4 42 4.4 65 0.9 Aarslev 1803 465 18.7 285 3.1 47 7.5 47 1.6 Heimanns Rubin 4 443 5.2 237 0.2 49 7.7 63 8.8 Stevnsbaer, Viki 403 9.8 178 3.7 33 2.7 62 1.6 Bofa 400 8.8 228 2.5 37 3.4 51 2.0 Nefris 399 13.5 202 2.6 39 5.8 57 1.0 Stevnsbaer, Birgitte 373 3.4 175 1.8 34 3.0 43 3.1 K27/2 373 11.1 81 1.1 27 2.1 62 1.9 Recta 359 9.2 214 3.2 28 2.7 38 3.4 Aarslev 2510 358 8.4 256 4.9 39 6.8 25 9.1 Aarslev 2403 340 18.1 266 5.6 38 5.2 21 8.6 Saﬁr 336 4.7 200 3.0 32 3.7 26 12.2 Stevnsbær, PH 333 10.0 185 3.6 35 4.3 40 0.9 Aarslev 2504 301 13.1 176 1.6 31 2.6 32 0.9 Nadwislanka 293 27.2 183 2.7 38 6.3 51 3.6 Kelleris 16 240 4.8 60 0.6 22 2.8 18 3.5 Dana x 1 227 22.5 146 3.4 26 4.3 15 4.7 Erdi Bo¨ttermo¨ 222 7.0 98 1.0 18 3.4 14 5.3 Pernilla 213 15.3 133 0.7 20 2.3 15 3.5 Sumadinka 211 3.8 131 1.2 21 1.9 3 4.2 Lutovka 209 10.6 102 0.1 24 2.4 8 3.4 Cigganymeggy 7 205 4.9 122 3.0 19 1.8 2 6.8 Zigeunerkirschen 200 14.7 107 0.1 18 2.1 13 1.4 M7 199 6.3 109 0.2 15 2.2 24 5.9 Oblachinska Holo 188 8.4 105 3.2 18 1.8 4 6.3 Ungarische Traubige 186 28.2 85 0.6 14 1.8 9 6.4 Skyggemorel Hannover 182 2.7 61 0.2 17 1.9 0 4.2 Vytenu Star 155 4.6 92 2.1 13 2.2 1 3.6 Gerema 146 14.0 95 0.8 15 2.2 3 2.3 Zagarvysne 129 3.4 83 3.7 15 1.9 0 2.1 Favorit 111 2.2 40 1.1 12 2.3 7 3.5 Sureﬁre 74 2.5 21 0.5 9 3.1 2 0.2

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Table 2 Correlation coefﬁcient between total phenolic content (TP), total anthocyanin content (TA), total antioxidant capacity (TEAC) and HT-29 cancer cell proliferation inhibition activity.

TP TA TEAC

TA 0.84 – – TEAC 0.95 0.90 – HT-29 0.87 0.71 0.84

3.2. Total antioxidant capacity

The total antioxidant capacity of sour cherries and their food products have been investigated extensively, but mostly focused on the Balaton and Montmorency cultivars (Seeram et al., 2001; Wang et al., 1999a,b; Pedisic´ et al., 2007; Kirakosyan et al., 2009). Water soluble and lipid soluble antioxidants of some Hungarian sour cherries have been studied (Veres et al., 2008). The total antioxidant from different sour cherry cultivars showed signiﬁcant difference (Bonerz et al., 2007; Veres et al., 2008; Blando et al., 2004). As shown in Table 1, total antioxidant capacity of sour cherries in this study was between 9 and 63 mmol TE/g. Among the sour cherry cultivars analyzed, Birgitte Bo¨ttermo¨ exhibited the strongest antioxidant capacity followed by Fanal and Heimanns Rubin 4. Other cultivars that displayed antioxidant capacity greater than 40 mmol TE/g were Tiki and Aarslev 1803. Sureﬁre and Favorit cultivars showed the lowest antioxidant capacity among all cultivars. Similar to the trend in total phenolic content and total anthocyanin content, most of the sour cherries from stevnsbaer, Fanal type and cross breeding cultivars displayed highest total antioxidant capacity.

3.3. Cancer cell proliferation assay

Anthocyanins from sour cherries have been reported to reduce the proliferation of human colon cancer cell HT-29 and HCT116 in a Fig. 1. PCA score scatter plot of NMR data, colored according to level of total antioxidant capacity, mmol TE/g (A); level of HT-29 cancer cell proliferation dose-dependent manner (Kang et al., 2003). Cyanidin-3-O-gluco- inhibition activity, % (B). side, one of the major pigments of sour cherries have been shown to inhibit growth of a lung cancer cell MCF7 (Reddy et al., 2005). The present study is the largest investigation of the inhibitory PCA modeling was conducted on 1H-NMR data of sour cherries activity against HT-29 colon cancer cell proliferation of sour cherry to observe the discrimination and correlation of the variances. Sour cultivars. As shown in Table 1, the inhibition was different from cherries contain high amounts of sugar in the aqueous extracts that cultivars. Fanal, Tiki and Heimann Rubin 4 displayed the strongest might interfere on the results in the modeling. Furthermore, taking inhibition against HT-29 cell proliferation. Skyggemorel Hannover, into consideration that biological activity of sour cherries is Cigganymeggy 7, Surefire, Favorit, Oblachinska Holo, Vytenu Star, regarded to be related to the phenolic compounds, only 1H-NMR Zagarvysne, Gerema and Sumadinka showed weak or no inhibition data above 5 ppm which belong to the aromatic region was applied against the cancer cell proliferation, while others showed in the multivariate statistical analysis; 1H-NMR data below 5 ppm moderate inhibition against the cancer cell proliferation. which are related to carbohydrates and aliphatic compounds were excluded from the data. PCA modeling is good in explaining 3.4. Correlation study difference between observations with common variances. An observation with different variance will affect the modeling by Table 2 shows the correlation coefficient between total phenolic triggering the model towards itself. Surefire was the sample that content, total anthocyanin content, total antioxidant capacity and contained the lowest total phenolics, total anthocyanin and HT-29 cancer cell proliferation inhibition activity. Total phenolic displayed the lowest bioactivity. It was a strong outlier and has content and total anthocyanin content were highly correlated, shifted PC1 with respect to itself. Thus it was removed from the showing that sour cherry contained high level of anthocyanins. data set because it only contributed to noise in the models. Total antioxidant capacity was highly correlated to total phenolic content and total anthocyanin content as well as HT-29 cancer cell Table 3 1 proliferation inhibition activity, pointing out that not only sour Summary of fits of PLS modeling for H-NMR data and total phenolic content (TP), total anthocyanin content (TA), total antioxidant capacity (TEAC) and HT-29 cancer cherry anthocyanins were biologically active antioxidants, color- cell proliferation inhibition activity. less phenolics in sour cherries have important impact to the 2 2 antioxidative effect and biological performances (Piccolella et al., Variable R Q RMSEE Component 2008). The interaction between phytochemicals have to be taken TP 0.975 0.815 23.688 3 into account, as there might be synergic effect, additive effect of TA 0.957 0.796 12.499 3 TEAC 0.921 0.765 3.736 2 negative effects between the sour cherry antioxidants (Kirakosyan HT-29 0.784 0.689 11.302 1 et al., 2010).

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Fig. 1(A) shows the PCA score scatter plot of 1H-NMR spectra of five sour cherry cultivars. European Food Research and Technology 224, 355–364. sour cherry colored according to the level of total antioxidant Christensen, J.V., 1986. Evaluation of characteristics of 18 sour cherry cultivars. capacity. Cultivars that displayed higher and lower antioxidant Tidsskrift for Planteavl 90, 339–347. capacity were clustered separately and situated oppositely along Christensen, J.V., 1988. Evaluation of 14 sour cherry sultivars. Tidsskrift for Plan- teavl 92, 345–349. PC1 and PC2. The result was similar when the PCA score scatter plot Christensen, J.V., 1990. A review of an evaluation of 95 cultivars of sour cherry. was colored according to the level of total phenolic content and total Tidsskrift for Planteavl 94, 51–63. anthocyanin content, since both of them and total antioxidant Dragovic´-Uzelac, V., Levaj, B., Bursac´, D., Pedisic´, S., Radojcˇic´, I., Bisˇko, A., 2007. 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In general, cultivars with higher total content of phenolics and Piccolella, S., Fiorentino, A., Pacifico, S., D’Abrosca, B., Uzzo, P., Monaco, P., 2008. anthocyanins displayed better total antioxidant capacity and Antioxidant properties of sour cherries (Prunus cerasus L.): role of colorless cancer cell proliferation inhibition activity, although a lower phytochemicals from the methanolic extract of ripe fruits. Journal of Agricul- tural and Food Chemistry 56, 1928–1935. correlation was observed between total anthocyanin content and Premier, R., 2002. Phytochemical composition: a paradigm shift for food-health cancer cell proliferation inhibition. Total phenolic content, total considerations. Asia Pacific Journal of Clinical Nutrition 11 (SUPPL. 6), anthocyanin content and total antioxidant capacity of sour cherries S197–S201. could be predicted by applying multivariate data analysis to 1H- Reddy, M.K., exander-Lindo, R.L., Nair, M.G., 2005. Relative inhibition of lipid peroxidation, cyclooxygenase enzymes, and human tumor cell proliferation NMR data. This approach could be useful for large scale screening by natural food colors. Journal of Agricultural and Food Chemistry 53, 9268– of sour cherry cultivars and other berries with potential health 9273. promoting effects. Seeram, N.P., Momin, R.A., Nair, M.G., Bourquin, L.D., 2001. Cyclooxygenase inhibitory and antioxidant cyanidin glycosides in cherries and berries. Phytomedicine 8, 362–369. Acknowledgements Seymour, E.M., Singer, A.A.M., Kirakosyan, A., Urcuyo-Llanes, D.E., Kaufman, P.B., Bolling, S.F., 2008. Altered hyperlipidemia, hepatic steatosis, and hepatic per- oxisome proliferator-activated receptors in rats with intake of tart cherry. The Danish Research Council FTP is acknowledged for financial Journal of Medicinal Food 11, 252–259. support through the project ‘‘Advances in Food quality and Stoner, G.D, Wang, L.S., Casto, B.C., 2008. Laboratory and clinical studies of cancer Nutrition Research through implementation of metabolomic chemoprevention by antioxidants in berries. Carcinogenesis 29, 1665–1674. Veres, Z., Holb, I., Nye´ki, J., Szabo´ , Z., Szabo´ , T., Remenyik, J., Fa´ri, M.G., 2008. strategies’’. The authors thank Nina Eggers for her help on Antioxidant and anthocyanin contents of sour cherry cultivars. Acta Horticul- conducting NMR experiment and Mette Marie Løkke on her turae (ISHS), 795 PART 2, 787–792. assistance on multivariate statistical analysis. Wang, H., Nair, M.G., Strasburg, G.M., Booren, A.M., Gray, J.I., 1999a. Antioxidant polyphenols from tart cherries (Prunus cerasus). Journal of Agricultural and Food Chemistry 47, 840–844. References Wang, H., Nair, M.G., Strasburg, G.M., Chang, Y.C., Booren, A.M., Gray, J.I., DeWitt, D.L., 1999b. Antioxidant and antiinflammatory activities of anthocyanins and Blando, F., Gerardi, C., Nicoletti, I., 2004. Sour cherry (Prunus cerasus L.) anthocya- their aglycon, cyanidin, from tart cherries. Journal of Natural 62, nins as ingredients for functional foods. Journal of Biomedicine and Biotech- 294–296. nology 2004, 253–258. Yao, L.H., Jiang, Y.M., Shi, J., Toma´s-Barberań, F.A., Datta, N., Singanusong, R., Chen, Bonerz, D., Wu¨ rth, K., Dietrich, H., Will, F., 2007. Analytical characterization and the S.S., 2004. Flavonoids in food and their health benefits. Plant Foods for Human impact of ageing on anthocyanin composition and degradation in juices from Nutrition 59, 113–122.

Manuscript Number:

Title: Bioactivity and chemical composition of blackcurrant (Ribes nigrum) cultivars with and without pesticide treatment

Article Type: Research Article (max 7,500 words)

Keywords: Ribes nigrum; pesticide treatment; anthocyanin; ascorbic acid; antioxidant; anticancer; prostaglandin E2

Corresponding Author: Dr Erik Larsen,

Corresponding Author's Institution:

First Author: Gaik Ming Khoo

Order of Authors: Gaik Ming Khoo; Morten R Clausen; Hanne L Pedersen; Erik Larsen

Abstract: Eleven blackcurrant cultivars grown with pesticide (PT) and without pesticide treatment (PF) were evaluated to compare the differences in plant growth and physical condition, total anthocyanin content, ascorbic acid content, total antioxidant capacity, effect on prostaglandin E2(PGE- 2) production and anticancer cell proliferation activities. Results showed that the yield and growth of PT blackcurrants was higher. However, PF blackcurrants contained a higher amount of ascorbic acid, and displayed an increased inhibition against cancer cells compared to PT blackcurrants, indicated that PF blackcurrants have an increased potential to deliver health promoting benefit for consumers. Significant differences were observed between blackcurrant cultivars in relation to plant growth and physical condition, total anthocyanin content and PGE-2 assay highlighting the importance of cultivar selection.

Cover Letter

Aarhus University Faculty of Agricultural Sciences Department of Food Science Kirstinebjergvej 10 DK-5792 Aarslev Denmark.

Dear Editor

Please consider our enclosed manuscript “Bioactivity and chemical composition of blackcurrant (Ribes nigrum) cultivars with and without pesticide treatment” for publication in the journal of Food Chemistry.

The paper constitutes the investigation of eleven blackcurrant cultivars harvested from pesticide treated and pesticide free plants. This is for the first time the bioactivities of blackcurrants with different in pesticide treatment being investigated. Our results indicated that pesticide free black currants may be potentially more beneficial to human health.

With the submission of this manuscript I would like to undertake that the above mentioned manuscript has not been published, accepted or under editorial review elsewhere.

Thank you.

Sincerely,

Erik Larsen *Highlights

Research highlights:

- Blackcurrant cultivars with and without pesticide treatment were evaluated.

- Yield and growth of pesticide treated blackcurrants were higher.

- Pesticide free blackcurrants contain higher amount of ascorbic acid.

- Pesticide free blackcurrants show better inhibition of cancer cell proliferation.

- Anthocyanin content depends on individual cultivars.

*Manuscript Click here to view linked References

1 Bioactivity and chemical composition of blackcurrant (Ribes nigrum) cultivars with and

2 without pesticide treatment

3 Gaik Ming Khooa, Morten Rahr Clausena, Hanne Lindhard Pedersenb and Erik Larsena*

4 a Department of Food Science, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev,

5 Denmark.

6 b Department of Horticulture, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev,

7 Denmark.

8 *Corresponding Author: Tel: +45-89993367, Fax: +45-89993495, e-mail address:

9 [email protected]

11 ABSTRACT

12 Eleven blackcurrant cultivars grown with pesticide (PT) and without pesticide treatment (PF)

13 were evaluated to compare the differences in plant growth and physical condition, total

14 anthocyanin content, ascorbic acid content, total antioxidant capacity, effect on prostaglandin

15 E2(PGE-2) production and anticancer cell proliferation activities. Results showed that the

16 yield and growth of PT blackcurrants was higher. However, PF blackcurrants contained a

17 higher amount of ascorbic acid, and displayed an increased inhibition against cancer cells

18 compared to PT blackcurrants, indicated that PF blackcurrants have an increased potential to

19 deliver health promoting benefit for consumers. Significant differences were observed

20 between blackcurrant cultivars in relation to plant growth and physical condition, total

21 anthocyanin content and PGE-2 assay highlighting the importance of cultivar selection. 22 KEYWORDS: Ribes nigrum; pesticide treatment; anthocyanin; ascorbic acid; antioxidant;

23 anticancer; prostaglandin E2

25 1 INTRODUCTION

26 Synthetic agrochemicals are extensively used in agricultural systems, mainly as pesticides

27 and fertilizers. Agrochemical residues are a major source of environmental contamination in

28 soil and ground water; agrochemical residues the found in crops may lead to accumulation in

29 human and animals, causing public health risk problems, including an increasing numbers of

30 different type of oncogenic risk and cancer incidences in communities (Weichenthal, Moase,

31 & Chan, 2010; Gilden, Huffling, & Sattler, 2010). Thus, restriction of agrochemical

32 application and increased governmental support for sustainable agricultural production

33 systems have encouraged the growth of the organic sector (Phillips, 2009; Blumenthal, 2010).

34 Blackcurrants (Ribes nigrum), a species native to central and northern Europe and northern

35 Asia is recognized as one of the major edible berries that are consumed in processed form.

36 The plant is highly tolerant to leaf damage (Pedersen, Toldam-Andersen, Funke, Hann, &

37 Wünsche, 2002) and therefore it is suited for pesticide free or organic farming (Kahu, Jänes,

38 Luik, & Klaas, 2009). Blackcurrants contain high levels of ascorbic acid and are rich sources

39 of phenolic compounds. Ascorbic acid is an antioxidant and acts as a cofactor for various

40 enzymes in diverse metabolic pathways. Phenolic compounds are strong antioxidants and

41 display a diverse range of biological activities. Blackcurrant phenolic extracts displayed

42 effective protection against oxidative stress induced neuronal damage in human cells (Ghosh,

43 McGhie, Zhang, Adaim, & Skinner, 2006). Blackcurrant anthocyanins increased blood flow

44 and enhanced peripheral circulation as well as reduced muscle fatigue in humans (Matsumoto

45 et al., 2005). Another study showed that blackcurrant juice induced peripheral vasodilatation, 46 increased blood flow and decreased blood pressure in healthy women (Yonei et al., 2009).

47 Oral intake of blackcurrant anthocyanins relaxed the bovine ciliary smooth muscles and

48 improved the visual function of animal (Matsumoto, Kamm, Stull, & Azuma, 2005). Crude

49 extract of blackcurrants inhibited influenza virus type A and B in a high temperature

50 environment (Knox, Suzutani, Yosida, & Azuma, 2003) and inhibited herpes simplex virus

51 type-1 and type-2 by inhibiting protein synthesis of infected cells (Suzutani, Ogasawara,

52 Yoshida, Azuma, & Knox, 2003).

53 Due to increased public awareness on pesticide contamination in foods, an increasing number

54 of research studies are emerging on the nutritional quality and phytochemical content in

55 organic compared to conventional food crops (Worthington, 2001; Crinnion, 2010). The

56 present study is the first report on the comparison of blackcurrants grown with pesticide (PT)

57 and without pesticide (PF) treatment. Furthermore, the differences between individual

58 cultivars have also been compared. The impact on yield, berry size, vegetative growth,

59 damage caused by diseases and aphids, total anthocyanin content, ascorbic acid content, total

60 antioxidant capacity, prostaglandin E2 (PGE-2) assay and anticancer cell proliferation

61 activities were investigated.

62 2 MATERIALS AND METHODS

63 2.1 Plant materials

64 Eleven cultivars of blackcurrants were used in this study: „Baldwin‟, „Ben Alder‟, „Ben

65 Dorain‟, „Ben Gairn‟, „Ben Hope‟, „Ben Lomond‟, „Narve Viking‟, „Tiben‟, „Titania‟, plus

66 the breeding selections 8944-4 and 8944-13. In 2003 at Department of Horticulture, Aarhus

67 University, Aarslev, Denmark, the blackcurrant plants were planted as one-year-old plants at

68 a planting distance of 3.5 x 0.5 m. Plots consisted of 6 bushes planted in 4 blocks per cultivar:

69 one block of plant with pesticide treatment (PT) and three blocks of plants without pesticide 70 treatments (PF). All plants were randomized within each block and were grown in MypexTM

71 within the bush row. Additional weeds were removed mechanically or by hand. Organic

72 poultry manure pellets were used as fertilizers in both PT and PF plants. No pesticides were

73 used on the PF plants. For PT plants, the pesticide treatment consisted of mancozeb,

74 boscalid+pyraclostrobin and kresoxim-methyl for the control of American gooseberry

75 mildew (mildew) caused by Sphaerothega mors-uvae (Schweinitz), white pine blister rust

76 (rust) caused by Cronartium ribicola (J. C. Fischer) and leaf spot caused by Gloeosporidiella

77 ribis (Libert). Propiconazol was used for the control of grey mould caused by Botrytis

78 cinerea in the flowers and pirimicarb was used for the control of aphids. All blackcurrants

79 were harvested mechanically within the period of July to August 2009, and samples were

80 stored at -24 ˚C immediately after harvested.

81 2.2 Plant growth and physical condition

82 Vegetative growth was assessed in June by giving a score from 1 to 9, where 1 = no growth.

83 Yield was recorded during harvest and berry size was calculated from the total weight of 100

84 berries from each cultivar. Damage caused by diseases and aphids was estimated visually and

85 assessed by giving a score from 1 to 9, where 1 = no damage. Damage caused by mildew and

86 aphids was observed in June 2009 and damage caused by leaf spot and rust was observed in

87 August 2009.

88 2.3 Chemicals

89 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), sodium dihydrophosphate,

90 acetonitrile, methanol, sulfuric acid, trifluoroacetic acid (TFA), D,L-homocysteine, oxalic

91 acid, N-ethylmaleimide, fluorescein, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)

92 (ABTS), ammonium persulfate, 2,6-dichlorophenol-indophenols and deuterium oxide 99.9

93 atom% D containing 0.05% (w/v) 3-(trimethylsilyl) propionic 2,2,3,3-d4 acid sodium salt 94 (D2O containing 0.05% TSP) were purchased from Sigma-Aldrich (Missouri, USA).

95 Dulbecco's Modified Eagle Medium-GlutamaxTM (DMEM) was from Invitrogen (Paisley,

96 UK). Trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) and phosphate buffer saline-

97 ethylenediaminetetraacetic acid (PBS-EDTA) were from Lonza (Braine, Belgium). Penicillin

98 G-potassium salt and streptomycin sulfate were from Serva (Heidelberg, Germany). Fetal calf

99 serum (FCS) was from PAA Laboratories (Pasching, Austria). WST-1 cell proliferation

100 reagent was from Roche Diagnostics (Mannheim, Germany). Lipopolysaccharide (LPS) was

101 from Calbiochem, Merck (Damstadt, Germany). ,2'-azobis-2-methyl-propanimidamide,

102 dihydrochloride (AAPH) and monoclonal prostaglandin E2 enzyme immunoassay (PGE-2

103 EIA) kit were purchased from Cayman Europe (Tallinn, Estonia).

104 2.4 Sample preparation

105 Frozen blackcurrants were removed from the freezer and approximately 200 g of frozen

106 blackcurrants were homogenized separately with deionized water (2 g of fruit with 1 mL

107 water), using a food blender. Homogenates were centrifuged at 12,000 rpm for 30 minutes.

108 The supernatants were collected and filtered with MinisartCA 0.45 µm filters (Sartorius,

109 Goettingen, Germany) and the filtrates were kept at -80 ˚C until analysis.

110 2.5 Total anthocyanin content

111 Total anthocyanin content of blackcurrants was measured spectrophotometrically (Wrolstad,

112 1976). The homogenates were extracted with 50 mL acidified methanol (96% methanol: 3%

113 sulfuric acid, w/w) and the ultraviolet absorption was measured at a wavelength of 526 nm

114 with MPS-2000 Shimadzu multipurpose recording spectrophotometer (Shimadzu, Kyoto,

115 Japan). Each sample was measured in two replicates. Total anthocyanin content was

116 calculated as Malvin equivalent per 100 g (ME/100 g) blackcurrants (extinction coefficient, ε

117 = 37700 M-1 cm-1). The mean value and standard deviation were calculated. 118 2.6 Ascorbic acid content

119 The homogenates were extracted with 0.1% aqueous oxalic acid. The mixtures were purged

120 with nitrogen during extraction (Kaack & Austed, 1998). After that, the mixtures were

121 filtered and the aliquot were treated with D, L-homocysteine to reduce dehydroascorbic acid

122 to ascorbic acid. The excess homocysteine was eliminated with N-ethylmaleimide and the

123 extracts were titrated with 2, 6-dichlorophenol-indophenols to determine the ascorbic acid

124 content (Lento, Daugherty, & Denton, 1963). Each sample was measured in duplicates. The

125 mean values and standard deviations were calculated.

126 2.7 Total antioxidant capacity

127 Total antioxidant capacity of blackcurrants was measured using the trolox equivalent

128 antioxidant capacity (TEAC) assay and oxygen radical absorbance capacity (ORAC) assay.

129 For TEAC assay, ABTS (19.4 mM) was dissolved in water and mixed with ammonium

130 persulfate (8.8 mM). The mixture was placed overnight at room temperature to allow the

131 formation of ABTS·+. The mixture was then diluted 100 times with phosphate buffer (75 mM,

132 pH 7.42) prior to use. The experiment was performed in triplicate in a Nunc transparent 96-

133 well plate (Thermo Fischer Scientific, Leicestershire, UK) with two independent tests. Trolox

134 was used for standard curve calibration. A 50 µL subsample of standard or filtrated samples

135 was mixed with 200 µL of ABTS·+ working solution and the absorbance was read at a

136 wavelength of 734 nm using BioTek Synergy 2 multi-mode microplate reader (BioTek,

137 Vermont, USA). Solutions without samples were used as blank. Mean value and standard

138 deviation were calculated. The results were expressed as trolox equivalent per gram (TE/g)

139 blackcurrants. 140 ORAC assay was performed as described by Huang with minor modification (Huang, Ou,

141 Hampsch-Woodill, Flanagan, & Prior, 2002). Fluorescein solution was prepared (1.2 x 10-8

142 mM) in phosphate buffer (75 mM, pH 7.42) and AAPH was dissolved in phosphate buffer at

143 a final concentration of 15 mM. The assay was performed in triplicate in a Nunc black 96-

144 well plate (Thermo Fischer Scientific, Leicestershire, UK) with two independent tests. Trolox

145 was used for standard curve calibration. A 25 µL subsample of standard or filtered samples

146 was mixed with 150 µL of fluorescein solution and incubated at 37ºC for 30 minutes. The

147 assay was initiated by adding 25 µL AAPH solution. Fluorescence was at minute intervals for

148 60 minutes with an excitation wavelength of 485 nm and an emission wavelength of 515 nm

149 using BioTek Synergy 2 multi-mode microplate reader. Mean values and standard deviations

150 were calculated. The results were expressed as trolox equivalent per gram (TE/g)

151 blackcurrants.

152 2.8 Anticancer cell proliferation activities

153 Two human colon cancer cell lines, HT-29 and Caco-2 (European Collection of Cell Cultures,

154 Salisbury, UK) were used in the experiment. Cells were grown in DMEM medium

155 supplemented with 10% FCS, 100 IU/mL penicillin and 100 µg/mL streptomycin. The

156 medium was changed every second day and cells were passaged every fourth day. Trypsin-

157 EDTA was used for detachment of cells from culture flask. Cells were incubated in 8000 WJ

158 CO2 incubator (Thermo Fischer Scientific, Leicestershire, UK) at 37ºC with 5% humidified

159 CO2.

160 Cells were seeded into Nunc sterile transparent 96-well plate (Thermo Fischer Scientific,

161 Leicestershire, UK) at a density of 1x104 cells per well and incubated for 24 hours. Filtrated

162 samples were added into the 96-well plate with final concentration of 50 µL/mL and the cells

163 were incubated for another 72 hours. Proliferation assay was determined using WST-1, based 164 on the cleavage of tetrazolium salt to formazan by the mitochondrial dehydrogenase. After 3

165 hours of incubation with WST-1, the absorbance was detected at a wavelength of 450 nm and

166 a reference wavelength of 630 nm using BioTek Synergy 2 multi-mode microplate reader.

167 The absorbance was corrected by subtracting the value at 630 nm from the value at 450 nm.

168 All samples were tested in triplicate in two independent experiments. Wells without sample

169 were used as control and inhibition was calculated relative to the control for each sample.

170 2.9 PGE-2 assay

171 PGE-2 assay was used to determine the cyclooxyganse-2 inhibitory activity. In brief, a total

172 of 1x104 Caco-2 cells were seeded into each well of Nunc sterile transparent 96-well plate

173 and incubated for 48 hours. Cells were then incubated with 500 µM aspirin for 3 hours to

174 inactivate the endogenous cyclooxygenase-1 (Bang et al., 2002). Cells were washed twice

175 with PBS-EDTA, and 200 ng/mL LPS was added with or without filtrated samples treatment.

176 After incubation for 4 hours, media were collected and centrifuged. All media were tested

177 according to the given protocol for the PGE-2 EIA kit set.

178 2.10 HPLC analysis

179 Filtered samples were diluted 10 times and separated on Kinetex 26 u C18 100Å, 100 x 4.6

180 mm (Phenomenex, California, USA) using Dionex Ultimate 3000 HPLC system (Dionex,

181 California, USA) equipped with Chameleon software program. All samples were measured

182 twice using an isocratic elution with a mobile phase consisting of 0.5% TFA aqueous (solvent

183 A, 95%) and acetonitrile (solvent B, 5%) at a flow rate of 1.0 ml/min for 13 min, followed by

184 1 min column wash with 75% solvent A and 25% solvent B. Measurement of phenolic

185 compounds and anthocyanins were determined at wavelength of 365 nm and 520 nm,

186 respectively. 187 2.11 NMR experiments

1 188 A 500 µL of filtered sample was mixed with 100 µL D2O containing 0.05% TSP. The H-

189 NMR spectra were recorded at 25ºC on a Bruker Avance 600 spectrometer (Bruker Biospin,

190 Rheinstetten, Germany) operating at a proton frequency of 600.13 MHz, equipped with a 5

191 mm 1H-TXI probe (Bruker Biospin, Rheinstetten, Germany). The spectra were acquired using

192 a single 90º pulse experiment with relaxation decay of 5 s. Water suppression was achieved

193 by irradiating the water peak during relaxation decay. A total of 32 K data points spanning a

194 spectral width of 12.1 ppm were collected. All spectra were referenced to TSP at 0 ppm. The

195 spectra were manually phased and baseline corrected using Topspin 2.1 (Bruker Biospin,

196 Rheinstetten, Germany).

197 2.12 Data processing and statistical analysis

198 1H-NMR spectra were subdivided into 0.015 ppm intergral regions and intergrated, reducing

199 each spectrum into 819 independent variables. Further analysis was performed using SIMCA

200 P+ 12.0.1.0 (Umetrics, Umeå, Sweden). HPLC and NMR data were set as X variables; yield,

201 berry size, vegetative growth, damage caused by diseases and aphids, total anthocyanin

202 content, ascorbic acid content, total antioxidant capacity, PGE-2 assay and anticancer cell

203 proliferation activities were set as Y variables. Principal component analysis (PCA) was

204 applied to data to observe clustering behavior of the samples and partial least squares (PLS)

205 regression with full cross validation was used to investigate the correlation between NMR

206 spectra or HPLC data and Y variables. NMR data was scaled with parato scaling; HPLC data

207 was scaled with centering scaling; other data used in SIMCA analysis was scaled with unit

208 variance scaling. 209 The statistical differences between pesticide treatment and individual cultivars were

210 calculated with general linear model in SAS 9.1 software package (SAS Institute, North

211 Carolina, USA). Linear regressions between total anthocyanin content, ascorbic acid content,

212 total antioxidant capacity, PGE-2 assay and anticancer cell proliferation activities were

213 investigated to determine the correlation significance.

214

215 3 RESULTS AND DISCUSSION

216 3.1 Plant growth and physical condition

217 Table 1 shows the average of yield, berry size, scores of vegetative growth and scores of

218 damage caused by diseases and aphids of PT and PF plants and individual cultivars.

219 Significant differences in yield and vegetative growth were found between PT and PF plants;

220 PT plants had a stronger growth than PF plants and there was approximately a 100% increase

221 in yield of PT blackcurrants compared to PF blackcurrants. This is most likely attributed to

222 exposure of the plants to diseases because of the absence of pesticide protection. No

223 significant difference was observed in berry size between two treatments but there were

224 significant differences between cultivars, indicating that berry size is depend on individual

225 cultivars and not pesticide treatment. The incidence of leaf spot was significantly less on PT

226 plants than PF plants but no significant difference were observed in the incidences of mildew

227 and rust between the two treatments. There was a similar level of damage by aphids on both

228 PT and PF plants. Significant difference were found between individual cultivars in all plant

229 growth and physical condition measurements, indicated that indicated that yield and

230 vegetative growth not only depending on pesticide treatment, but on individual cultivars too.

231 In addition, different cultivars had different levels of resistance to diseases and aphids. 232 [Table 1 around here]

233 3.2 Total anthocyanin content

234 Anthocyanins are the major group of phenolics in blackcurrants, accounting for

235 approximately 80% of total phenolics, the four main pigments delphinidin 3-O-glucoside,

236 delphinidin 3-O-rutinoside, cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside contribute

237 up to 97% of the total anthocyanin in blackcurrants (Anttonen & Karjalainen, 2006;

238 Karjalainen et al., 2009). „Baldwin‟, „Ben Alder‟, „Ben Lomond‟, „Ben Dorain‟ and „Titania‟

239 have previously been investigated for their total anthocyanin or total phenolic content

240 (Bordonaba & Terry, 2008; Wu, Gu, Prior, & McKay, 2004; Moyer, Hummer, Finn, Frei, &

241 Wrolstad, 2002). In this study we furthermore included „Ben Gairn‟, „Ben Hope‟, „Narve

242 Viking‟, „Tiben‟ and „8944-4‟ and „8944-13‟. Blackcurrant cultivars grown in Denmark

243 contain 168 to 613 mg ME/100 g, depending on cultivars and harvested year (Pedersen,

244 2008). As shown in Table 2, „Ben Gairn‟ and „Ben Alder‟ were the cultivars containing the

245 highest concentration of total anthocyanin; „Baldwin‟ and „8944-4‟ were the lowest. Research

246 comparing organic and conventional grown blackcurrants reported no significant differences

247 in total phenolic content (Anttonen & Karjalainen, 2006), which is in compliance with our

248 study showing no significant differences found between pesticide treatment. However,

249 significant differences were observed between individual cultivars. This suggested that

250 anthocyanin production in blackcurrants is genetically predetermined and pesticide

251 treatments on plants have little or no effect on the biosynthesis of anthocyanins. Nevertheless,

252 significant differences in total phenolics were found between conventional, organic and

253 sustainable grown marionberries and srawberries (Asami, Hong, Barrett, & Mitchell, 2003).

254 [Table 2 around here]

255 3.3 Ascorbic acid content 256 Blackcurrants are an excellent source of ascorbic acid with an average content of 125 to 151

257 mg/100 g fresh weight (Szajdek & Borowska, 2008). Ascorbic acid content in different

258 blackcurrant cultivars harvested from 2001 to 2005 in Denmark ranged from 96 to 219

259 mg/100g (Pedersen, 2008). As shown in table 2, the cultivar „Baldwin‟ had the highest

260 concentration of ascorbic acid, followed by „Ben Dorain‟. „Titania‟ and „Ben Alder‟ had the

261 lowest concentration of ascorbic acid among the blackcurrant cultivars. However, no

262 significant difference was observed between individual cultivars. In this study we found out

263 that pesticide treatment showed significant impact on ascorbic acid content (Table 2).

264 Previous reports determined higher levels of ascorbic acid in organically grown crops than

265 conventional crops (Worthington, 2001). A three years study showed that blackcurrants from

266 an organic production system had significantly higher contents of ascorbic acid than fruit

267 from a conventional system (Kahu et al., 2009).

268 3.4 Total antioxidant capacity

269 Epidemiological studies have indicated that regular dietary intake of food rich in antioxidants

270 will help to reduce the risk of cancer and cardiovascular diseases, inhibit free radical

271 production and reduce the concentration of reactive oxygen species in the body (Wu et al.,

272 2004; Dragovic-Uzelac et al., 2007). Blackcurrants are rich in antioxidants (Cho et al., 2009;

273 Halvorsen et al., 2002). Previous research showed that ORAC assay of blackcurrant cultivars

274 were between 37 and 93 µmol TE/g fresh-frozen fruit (Moyer et al., 2002). In this study,

275 TEAC values of were ranged from 44 to 55 µmol TE/g. „Ben Gairn‟ and „Ben Dorain‟ had

276 the highest TEAC values. „Narve Viking‟, „Titania‟ and „Ben Hope displayed the lowest

277 TEAC values. In contrast to the results obtained by TEAC assay, cultivars with the highest

278 ORAC value of were „Baldwin‟ and „8944-13‟. „Ben Hope‟ and „Ben Dorain‟ had the lowest 279 ORAC values among all cultivars. Neither TEAC assay nor ORAC assay showed significant

280 differences in relation to cultivation methods or between individual cultivars (Table 2).

281 3.5 Bioassay activities

282 PGE-2 is one of the major metabolites in inflammatory process stimulated by

283 cyclooxygenase-2 (COX-2), as a key enzyme for the inflammatory process, it is a mediator

284 enhancing pain, swelling, fever, vascular permeability and redness during inflammation

285 (Smith, Dewitt, & Garavito, 2000). Thus COX-2 activity in cells can be estimated by

286 measuring the PGE-2 production. Proanthocyanidins in blackcurrants inhibit PGE-2 synthesis

287 in vitro but not in the whole blood assay (Christensen, 1990). In this study, we proved that

288 all blackcurrant extracts inhibited PGE-2 production. After treatment with blackcurrant

289 extracts, PGE-2 production was reduced to between 35 to 54%. The positive control,

290 indomethacin reduced PGE-2 production to 29%. The cultivars „Titania‟and „8944-4‟

291 displayed the highest inhibitory activity against COX-2 enzyme. No significant difference on

292 pesticide treatment was observed, however individual cultivars showed different inhibition

293 against PGE-2 production (Table 2).

294 Blackcurrant extracts has been shown to inhibit the growth of a number of cancer cell lines:

295 Hela, Fem X, LS 174, MCF-7, PC-3 and MDA-MB-231 (Cho et al., 2009; Konic-Ristic et al.,

296 2011). In this study, HT-29 cancer cell proliferation inhibition of black currants was between

297 no inhibition and up to 51%. Caco-2 cancer cell proliferation inhibition was ranged from 10%

298 to 56%. The cultivar „Baldwin‟ and „8944-13‟ displayed the strongest inhibitory effect

299 against both cancer cell lines. „Ben Lomond‟ and „Ben Gairn‟ showed the weakest inhibitory

300 effect in HT-29 and Caco-2, repsctively. PF blackcurrants showed significantly higher

301 inhibitory effect against HT-29 and Caco-2 cancer cell proliferation compared to PT

302 blackcurrants, however no significant difference was observed between cultivars (Table 2). 303 3.6 Correlation analysis

304 Table 3 shows the linear correlation between total anthocyanin content, ascorbic acid content,

305 total antioxidant capacity, PGE-2 assay and anticancer cell proliferation activities. In this

306 study we found that total antioxidant capacities were poorly correlated to total anthocyanin

307 content and ascorbic acid content in blackcurrants, although they are both strong antioxidants.

308 Additionally we found no correlation between TEAC and ORAC assay. As reported

309 previously, blackcurrant anthocyanins have a poor correlation to the total antioxidant capacity

310 (Moyer et al., 2002). Dragović-Uzelac et al. (2007) reported that different total antioxidant

311 methods resulted in different values and might not have correlation. Differences in the results

312 from ORAC and TEAC in this study reflected the presence of several types of antioxidant in

313 blackcurrants with different reactivity towards the radicals. However, we observed

314 significant correlation between ascorbic acid content and anticancer cell proliferation

315 activities. Ascorbic acid exerted anti-proliferative effects by inhibiting the cell cycle of

316 prostate carcinoma cells (Frömberg et al., 2010) and induced apoptosis in human breast

317 cancer cells SK-BR3 and Hs578T (Hong et al., 2007). Although the effectiveness of ascorbic

318 acid in cancer treatment remains controversial, the interest among scientist has not decreased

319 as a number of research results continue to point out that ascorbic acid therapy is effective

320 depending on the protocol used (Padayatty & Levine, 2000; Tamayo & Richardson, 2003;

321 Ohno, Ohno, Suzuki, Soma, & Inoue, 2009).

322 From the results we suggest that in addition to anthocyanins and ascorbic acid, there may

323 well be additional bioactive compounds in blackcurrants present in lower concentrations that

324 are responsible for the biological performance, although, synergistic effects of all

325 phytochemicals should be taken into consideration (Liu, 2004).

326 [Table 3 around here] 327 Multivariate data analyses were applied to investigate correlations between yield, berry size,

328 vegetative growth, damage caused by diseases and aphids, total anthocyanin content, ascorbic

329 acid content, TEAC assay, ORAC assay, PGE-2 assay, anticancer cell proliferation activities,

330 NMR and HPLC data. Figure 1(a) shows the corresponding PCA score plot. PC1 and PC2

331 explained 32% and 16% of the variation, respectively, and could separate two main groups

332 according to pesticide treatment, though not completely. Figure 1(b) shows the corresponding

333 loading plot. Pesticide treatment had a great impact on yield, vegetative growth, ascorbic acid,

334 leaf spot but not PGE-2 assay or aphids on the plants. Although the statistical analysis in

335 Table 1 indicated that there was no significant difference for berry size between cultivation

336 methods, however the multivariate data analysis showed that pesticide treatment has an

337 impact on berry size. Correlation between ascorbic acid and anticancer cell (Caco-2 and HT-

338 29) proliferation activities were confirmed by the close proximity of these three variables in

339 the loading plot. Total anthocyanin content and ascorbic acid content were negatively

340 correlated, however further investigation is needed to confirm this relationship.

341 [Figure 1 around here]

342 In this study, neither PCA modeling of NMR nor HPLC data was able to distinguish the

343 cultivation method used. The results showed that modeling of 1H-NMR data and HPLC data

344 were unable to describe the majority of the Y variables. There was a good correlation

345 between both data and total anthocyanin content, with a linear regression of 0.68 for NMR

346 data and 0.72 for HPLC data, which is not surprising, since anthocyanins are the dominant

347 compounds in blackcurrants fruits and the absorbance specific to phenolics were measured in

348 HPLC analyses. Similarly, modeling of liquid chromatography-mass spectroscopy data of

349 blackcurrants was used to distinguish blackcurrants from different farms but not the

350 cultivation methods (Anttonen & Karjalainen, 2006). 351

352 4 CONCLUSIONS

353 This study showed that pesticide treatment had a significant impact on yield, vegetative

354 growth, leaf spots, ascorbic acid content and anticancer cell proliferation inhibition activities.

355 PF blackcurrants contained higher concentration of ascorbic acid and displayed better

356 inhibitory effects against cancer cell proliferation compared to PT blackcurrants, and thus

357 blackcurrants from plants grown without pesticide treatment is potentially more health

358 promoting. However, anthocyanin content was depending on individual cultivars not

359 pesticide treatment.

360 Acknowledgements

361 The authors are grateful to Dr. Michelle Williams for critical reading of the manuscript and

362 lab technician Nina Eggers for her assistance in the preparation of blackcurrant juices and

363 NMR experiments. The Danish Research Council FTP is acknowledged for financial support

364 through the project “Advances in food quality and nutrition research through implementation

365 of metabolomic strategies”.

366

367

368

369

370

371 372 Reference List

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506

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509

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513

514

515

516

517

518

519 Figure Captions

520

521 Figure 1. (a) PCA score plot and (b) PCA loading plot for yield, berry size, vegetative

522 growth, damage caused by diseases and aphids, total anthocyanin content, ascorbic acid

523 content, TEAC assay, ORAC assay, PGE-2 assay and anticancer cell proliferation activities.

524

525

526

527

528 529

530 Tables

531

532 Table 1. Average of yield, berry size, scores (1-9, where 1 = no growth or no infections) for

533 vegetative growth and damage caused by diseases and aphids for blackcurrant with pesticide

534 treatment (PT) and without pesticide treatment (PF) and average of individual cultivars.

535 Level of significance: N.S. = no significance,* P <0.05, ** P <0.01, *** P <0.001.Pesticide

536 treatment: LSD test; Cultivar: Value within a column with same letter show no significant

537 difference between cultivars.

Pesticide Berry Size, Vegetative Leaf Yield, kg Aphids Mildew Rust Treatment g/100 growth Spot PT 2.03 67 6.6 2.5 1.4 3.8 1.7 PF 1.05 62 5.4 2.5 1.7 5.5 2 LSD 0.56 *** N.S. 0.55 *** N.S. N.S. 1.05 * N.S. Berry Size, Vegetative Leaf Cultivar Yield, kg g/100 Growth Aphids Mildew Spot Rust Baldwin 1.22 ABCD 48 CD 5.1 CD 1.9 B 3.1 A 8.5 A 1.5 DE Ben Alder 1.98 ABC 65 AB 4.8 D 2.8 A 1.4 C 5.3 CD 2.4 BC Ben Dorain 0.92 BCD 59 BC 5.0 CD 2.4 AB 1.3 C 4.3 DE 3.1 A Ben Gairn 1.43 CD 67 AB 5.3 CD 3.0 A 1.0 C 2.9 E 1.4 DE Ben Hope 2.09 ABC 75 A 6.6 AB 1.9 B 1.0 C 4.1 DE 3.1 A Ben Lomond 2.29 AB 75 A 6.0 ABC 2.4 AB 3.5 A 6.9 B 1.9 CD Narve Viking 2.31 A 74 A 7.0 A 2.3 AB 1.0 C 1.2 F 2.7 AB Tiben 1.48 ABCD 72 A 6.7 AB 2.8 A 1.3 C 5.8 BC 1.7 DE Titania 0.35 C 71 AB 6.8 AB 2.8 A 1.0 C 3.7 E 1.0 E 8944-4 0.46 D 45 D 5.0 CD 2.6 A 1.0 C 6.4 BC 1.1 E 8944-13 1.41 ABCD 50 CD 5.8 BCD 2.4 AB 2.0 B 5.5 BCD 1.3 DE P * *** *** *** *** *** *** 538

539

540

541 542

543 Table 2. Average of total anthocyanin content (TA), ascorbic acid content (AA), TEAC assay,

544 ORAC assay, PGE-2 assay and anticancer cell proliferation (HT-29 and Caco-2) activities for

545 blackcurrant with pesticide treatment (PT) and without pesticide treatment (PF) and average

546 of individual cultivars. LSD test, level of significance: N.S. = no significance,* P <0.05, ** P

547 <0.01, *** P <0.001. Cultivar: Value within a column with same letter show no significant

548 difference between cultivars.

549

Peticide TA, AA, TEAC, ORAC, PGE-2 HT-29 Caco-2 Treatment mg ME/100 g mg/100 g µmol TE/g µmol TE/g production, % inhibition, % inhibition, %

PT 321 100 48 84 46 11 17 PF 350 139 51 82 47 33 34 LSD N.S. 26.7** N.S. N.S. N.S. 16.3** 14.3*

TA, AA, TEAC, ORAC, PGE-2 HTs-29 Caco-2 Cultivar mg ME/100 g mg/100 g µmol TE/g µmol TE/g production,% inhibition,% inhibition, %

Baldwin 203 F 176 A 53 ABC 90 A 45 BC 51 A 56 A Ben Alder 408 AB 90 B 49 ABC 81 A 54 A 16 AB 29 AB Ben Dorain 369 BC 152 AB 54 AB 77 A 50 AB 27 AB 30 AB Ben Gairn 445 A 102 B 55 A 89 A 52 AB 23 AB 10 AB Ben Hope 313 CDE 114 AB 46 C 74 A 50 AB 33 AB 20 AB Ben Lomond 274 DE 111 AB 46 BC 84 A 45 BC -9 B 22 AB Narve Viking 359 BC 117 B 44 C 86 A 52 AB 29 AB 22 AB Tiben 411 AB 105 AB 52 ABC 84 A 47 AB 7 AB 23 AB Titania 329 CD 87 B 46 C 78 A 35 D 12 AB 21 AB 8944-4 268 E 125 AB 49 ABC 82 A 38 CD 20 AB 15 AB 8944-13 314 CDE 131 AB 52 ABC 89 A 45 BC 34 AB 33 B P *** N.S. N.S. N.S. ** N.S. N.S. 550

551

552

553

554

555 556

557 Table 3. Linear regression correlation between total anthocyanin content (TA), asocorbic acid

558 content (AA), TEAC assay, ORAC assay, PGE-2 assay and anticancer cell proliferation (HT-

559 29 and Caco-2) activities of blackcurrants. Level of significance: * P < 0.05; ** P < 0.01; ***

560 P < 0.001.

561

TA AA TEAC ORAC HT-29 AA 0.090 TEAC 0.064 0.227 ORAC 0.006 0.028 0.074 PGE-2 0.265* 0.023 0.065 0.029 HT-29 0.008 0.418*** 0.138 <0.001 Caco-2 0.070 0.374** 0.068 0.012 0.424*** 562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577 578

579 Figures

580 Figure 1

581

582

583

584

585

586 Table(s)

Pesticide Berry Size, Vegetative Leaf Yield, kg Aphids Mildew Rust Treatment g/100 growth Spot PT 2.03 67 6.6 2.5 1.4 3.8 1.7 PF 1.05 62 5.4 2.5 1.7 5.5 2 LSD 0.56 *** N.S. 0.55 *** N.S. N.S. 1.05 * N.S. Berry Size, Vegetative Leaf Cultivar Yield, kg Aphids Mildew Rust g/100 Growth Spot Baldwin 1.22 ABCD 48 CD 5.1 CD 1.9 B 3.1 A 8.5 A 1.5 DE Ben Alder 1.98 ABC 65 AB 4.8 D 2.8 A 1.4 C 5.3 CD 2.4 BC Ben Dorain 0.92 BCD 59 BC 5.0 CD 2.4 AB 1.3 C 4.3 DE 3.1 A Ben Gairn 1.43 CD 67 AB 5.3 CD 3.0 A 1.0 C 2.9 E 1.4 DE Ben Hope 2.09 ABC 75 A 6.6 AB 1.9 B 1.0 C 4.1 DE 3.1 A Ben Lomond 2.29 AB 75 A 6.0 ABC 2.4 AB 3.5 A 6.9 B 1.9 CD Narve Viking 2.31 A 74 A 7.0 A 2.3 AB 1.0 C 1.2 F 2.7 AB Tiben 1.48 ABCD 72 A 6.7 AB 2.8 A 1.3 C 5.8 BC 1.7 DE Titania 0.35 C 71 AB 6.8 AB 2.8 A 1.0 C 3.7 E 1.0 E 8944-4 0.46 D 45 D 5.0 CD 2.6 A 1.0 C 6.4 BC 1.1 E 8944-13 1.41 ABCD 50 CD 5.8 BCD 2.4 AB 2.0 B 5.5 BCD 1.3 DE P * *** *** *** *** *** *** Table(s)

Peticide TA, mg AA, TEAC, ORAC, µmol PGE-2 HT-29 Caco-2 Treatment ME/100 g mg/100 g µmol TE/g TE/g production, % inhibition, % inhibition, %

PT 321 100 48 84 46 11 17 PF 350 139 51 82 47 33 34 LSD N.S. 26.7** N.S. N.S. N.S. 16.3** 14.3*

Peticide TA, mg AA, TEAC, ORAC, µmol PGE-2 HT-29 Caco-2 Treatment ME/100 g mg/100 g µmol TE/g TE/g production, % inhibition, % inhibition, %

TA AA TEAC ORAC HT-29 AA 0.09 TEAC 0.064 0.227 ORAC 0.006 0.028 0.074 PGE-2 0.265* 0.023 0.065 0.029 HT-29 0.008 0.418*** 0.138 <0.001 Caco-2 0.07 0.374** 0.068 0.012 0.424*** Figure(s) (a)

4 PT PF

PC 2 PC 0

-2

-4

-6 -4 -2 0 2 4 6 PC 1

(b)

0.6 PGE-2 rust

0.4 TA AA HT-29 berry 0.2 yield size TEAC

Caco-2 PC 2 PC 0 mildew vegetative -0.2 leaf ORAC growth spot aphids -0.4 -0.4 -0.2 0 0.2 0.4 PC 1 Phytotherapy Research

Bioactivity of sour cherry cultivars grown in Denmark

Journal: Phytotherapy Research

Manuscript ID: Draft

Wiley - ManuscriptFor type: Full Peer Paper Review

Date Submitted by the n/a Author:

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

http://mc.manuscriptcentral.com/ptr Page 1 of 12 Phytotherapy Research

1 2 3 4 1 Bioactivity of sour cherry cultivars grown in Denmark 5 6 7 2 Gaik Ming Khoo a, Morten Rahr Clausen a, Bjarne Hjelmsted Pedersen b, and Erik Larsen a* 8 9 10 a 11 3 Department of Food Science, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark. 12 13 14 4 b Department of Horticulture, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark. 15 16 17 18 5 *Corresponding Author: Tel: +45-89993367, Fax: +45-89993495, e-mail address: 19 20 6 [email protected] Peer Review 21 22 23 24 7 Abstract 25 26 27 8 Thirty four varieties of sour cherries ( Prunus cerasus ) were investigated for their total 28 29 9 antioxidant activity, Caco-2 cancer cell proliferation inhibitory activity and effect on 30 31 32 10 prostaglandin E2 (PGE2) production. Total phenolic content, oxygen radical absorbance 33 34 11 capacity (ORAC) and cancer cell proliferation inhibitory activity of sour cherries were 35 36 12 closely correlated but not PGE2 production. The cultivars ‘Birgitte x Böttermö’, ‘Fanal’ and 37 38 39 13 ‘Tiki’ were the three cultivars with the highest ORAC values (180, 147 and 133 µmol TE/g, 40 41 14 respectively) and inhibition against Caco-2 cancer cell proliferation (74, 79 and 73%, 42 43 15 44 respectively). ‘Stevnsbaer Birgitte’ (22%) and ‘Stevnsbaer Viki’ (22%) inhibited PGE2 45 46 16 production with a similar potency as the positive controls indomethacin and NS-398. 47 48 17 Significant differences between cultivars in all bioactivity experiments indicated that 49 50 51 18 selection of cultivars is important to obtain sour cherries with better potential health 52 53 19 promoting effects. 54 55 56 20 Keywords: Prunus cerasus , sour cherries, ORAC, cancer, PGE2 57 58 59 21 60

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1 2 3 22 1 Introduction 4 5 6 7 23 Cyclooxygenase-2 (COX-2) is the key enzyme in the process of inflammation responsible 8 9 24 for the biosynthesis of prostaglandin E2 (PGE2) an important mediator that initiates typical 10 11 25 symptoms of inflammation including fever, swelling and redness (Smith et al. , 2000; Claria, 12 13 14 26 2003). Furthermore it has been shown that COX-2 and PGE2 are involved in cancer 15 16 27 development, since over-expression of COX-2 and high levels of PGE2 were detected in 17 18 28 different cancer cells (Coussens et al. , 2002; Lu et al. , 2006). 19 20 For Peer Review 21 22 29 Sour cherries ( Prunus cerasus ) one of the important fruit crops in Denmark. Sour cherries 23 24 30 have traditionally been used for wine making and liquers. In addtion, there are some 25 26 31 finished products available commercially, such as jam, syrup, sweets and canned fruits. Sour 27 28 29 32 cherries contain a substantial amount of phenolics including anthocyanins which are 30 31 33 reported to have anti-inflammatory and antioxidant properties (Wang et al. , 1999a; Wang et 32 33 34 Min 34 al. , 2000). Sour cherry anthocyanins inhibited tumor development in APC mice and 35 36 35 reduced the growth of human colon cancer cells (Kang et al. , 2003). In addition, the 37 38 36 anthocyanins showed potential anti-inflammatory effects against arthritis in rats, by 39 40 41 37 decreasing the level of tumor necrosis factor-α and PGE2 (He et al. , 2006). Most of the 42 43 38 research on sour cherries focused on ‘Montmorency’ and ‘Balaton’ cultivars because of 44 45 39 their high anthocyanin content. However, ‘Montmorency’ often gives a very low yield and 46 47 48 40 low anthocyanin content when grown in Denmark (Christensen, 1990). 49 50 51 41 Evaluation of sour cherry cultivars that are suitable for growing in Denmark has been 52 53 42 conducted earlier (Christensen, 1986; Christensen, 1988; Christensen, 1990; Christensen, 54 55 56 43 1997). A thorough investigation of sour cherry cultivars grown in Denmark with high 57 58 44 content of anthocyanins and better bioactivity is needed. This study aimed at determining 59 60 45 the bioactivity of selected sour cherry cultivars in Denmark, targeting for cultivars with

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1 2 3 46 potential health promoting effects. Thirty four sour cherry cultivars were evaluated using the 4 5 6 47 oxygen radical absorbance capacity (ORAC) assay, cancer cell proliferation inhibition 7 8 48 activity and PGE2 inhibition activity assays. 9 10 11 49 12 13 14 15 50 2 MATERIALS AND METHODS 16 17 18 51 2.1 Plant materials 19 20 For Peer Review 21 52 The sour cherries from 34 different cultivars were harvested from fields at the Department 22 23 53 of Horticulture, Aarhus University, Aarslev, Denmark, between the period of July and 24 25 26 54 August 2009 depending on the ripening status. Sour cherries were stored at -24 ºC 27 28 55 immediately after harvest. For the experiments, frozen sour cherries were removed from the 29 30 56 31 freezer and pitted. The pitted sour cherries were then homogenized (2 g cherry per 1 mL 32 33 57 water) in a food blender. Homogenized samples were centrifuged for 30 min at 12000 rpm 34 35 58 at 4ºC. The supernatants were collected and filtered through a 0.45 µm filter. Filtered 36 37 38 59 samples were kept at -80 ºC prior to use. 39 40 41 60 2.2 Chemicals 42 43 44 61 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fluorescein, sodium 45 46 62 dihydrophosphate, acetonitrile, trifluoroacetic acid (TFA) and acetylsalicylic acid were 47 48 49 63 purchased from Sigma-Aldrich (Missouri, USA). Dulbecco's Modified Eagle Medium- 50 51 64 Glutamax TM (DMEM) was purchased from Invitrogen (Paisley, UK). Trypsin- 52 53 65 54 ethylenediaminetetraacetic acid (Trypsin-EDTA) and phosphate buffer saline- 55 56 66 ethylenediaminetetraacetic acid (PBS-EDTA) were purchased from Lonza (Braine, 57 58 67 Belgium). Penicillin G-potassium salt and streptomycin sulfate were purchased from Serva 59 60 68 (Heidelberg, Germany). Fetal calf serum (FCS) was purchased from PAA Laboratories

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1 2 3 69 (Pasching, Austria). WST-1 cell proliferation reagent was purchased from Roche 4 5 6 70 Diagnostics (Mannheim, Germany). Lipopolysaccharide (LPS) was purchased from 7 8 71 Calbiochem, Merck (Damstadt, Germany). Monoclonal PGE2 enzyme immunoassay (PGE2 9 10 72 11 EIA) kit and 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) were 12 13 73 purchased from Cayman Europe (Tallinn, Estonia). 14 15 16 74 2.3 Oxygen radical absorbance capacity (ORAC) assay 17 18 19 20 75 The ORAC assay wasFor performed Peer as described byReview Huang with minor modification (Huang et 21 22 76 al. , 2002). The fluorescein solution was prepared (1.2 x 10 -8 mM) in phosphate buffer (75 23 24 77 mM, pH 7.42) and AAPH was dissolved in phosphate buffer to a final concentration of 15 25 26 27 78 mM. The assay was performed in triplicate in a Nunc black 96-well plate (Thermo Fischer 28 29 79 Scientific, Leicestershire, UK) with two independent tests. Trolox was used for standard 30 31 80 32 curve calibration. A 25 µL subsample of standard or filtered samples was mixed with 150 33 34 81 µL of fluorescein solution and incubated at 37 ºC for 30 minutes. The assay was initiated by 35 36 82 adding 25 µL AAPH solution. Fluorescence was read every minute for 60 minutes with an 37 38 39 83 excitation wavelength of 485 nm and an emission wavelength of 515 nm using a BioTek 40 41 84 Synergy 2 multi-mode microplate reader. Mean values and standard deviations were 42 43 85 calculated. The results were expressed as trolox equivalent per gram (TE/g) sour cherry. 44 45 46 47 86 2.4 Caco-2 cancer cell proliferation assay 48 49 50 87 Caco-2 (European Collection of Cell Cultures, Salisbury, UK) were grown in DMEM 51 52 88 medium supplemented with 10% FCS, 100 IU/mL penicillin and 100 µg/mL streptomycin. 53 54 55 89 Medium was changed every second day and cells were passaged every fourth day. Trypsin- 56 57 90 EDTA was used for detachment of cells from culture flask. Cells were incubated in 8000 58 59 60 91 WJ CO 2 incubator (Thermo Scientific, UK) at 37 ºC with 5% humidified CO 2.

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1 2 3 92 Cells were seeded into 96-well plate at a density of 1x10 4 cells per well and incubated for 24 4 5 6 93 hours. Filtered samples were added at a final concentration of 50 µL/mL and the cells were 7 8 94 incubated for another 72 hours. Proliferation assay was performed using WST-1, based on 9 10 95 11 the cleavage of tetrazolium salt to formazan by the mitochondrial dehydrogenase. After 3 12 13 96 hours of incubation with WST-1, the absorbance was detected at a wavelength of 450 nm 14 15 97 and with 630 nm for background correction using BioTek Synergy 2 multi-mode microplate 16 17 18 98 reader. All samples were measured in triplicate in two identical experiments. Wells without 19 20 99 sample were used as Forcontrol and Peer inhibition was Review calculated relative to the control for each 21 22 100 sample. 23 24 25 26 101 2.5 PGE2 assay 27 28 29 102 PGE2 assay was used to determine COX-2 inhibitory activity of sour cherries. In brief, 30 31 103 32 Caco-2 cells were grown in DMEM medium supplemented with 10% FCS, 100 IU/mL 33 34 104 penicillin and 100 µg/mL streptomycin. The medium was changed every second day and the 35 36 105 cells were passaged every fourth day. Trypsin-EDTA was used for detachment of cells from 37 38 4 39 106 culture flask and cells were cultivated at 37 ºC 5% CO 2. 1x10 cells was seeded into each 40 41 107 well of transparent 96-well plate and incubated for 48 hours. Cells were then incubated with 42 43 108 500µM aspirin for 3 hours to inactivate the endogenous cyclooxygenase-1 (Bang et al. , 44 45 46 109 2002). After that, the cells were washed twice with PBS-EDTA, and 200 ng/mL LPS was 47 48 110 added with or without filtered sample. After incubation for 4 hours, media were collected 49 50 111 and centrifuged. All media were tested according to the given protocol for the PGE2 51 52 53 112 content with Cayman PGE2 EIA kit set. 54 55 56 113 2.6 HPLC analysis 57 58 59 60 114 Filtered samples were diluted 10 times and separated on a column (Kinetex 2.6 µm C18

115 100Å, 100 x 4.6 mm, Phenomenex, California, USA) using Dionex Ultimate 3000 HPLC

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1 2 3 116 system (Dionex, California, USA) equipped with Chameleon software program. All samples 4 5 6 117 were measured twice using a linear gradient elution with a mobile phase consisting of 0.5% 7 8 118 TFA aqueous (solvent A) and acetonitrile (solvent B) at a flow rate of 1.0 ml/min. The 9 10 119 11 gradient was 5-25 % B (0-13 min), 25-5% B (13-14 min) and 5% B (14-15 min). 12 13 120 Measurement of phenolic compounds and anthocyanins were determined at 365 nm and 520 14 15 121 nm, respectively. Total phenolic content was quantified with quercitrin standard at the 16 17 18 122 wavelength of 365 nm and the results were expressed as quercitrin equivalent per 100 gram 19 20 123 (QE/100g) sour cherries.For Total anthocyaninPeer content Review was quantified with cyanidin chloride at 21 22 124 the wavelength of 520nm and the results were expressed as cyanidin equivalent per 100 23 24 25 125 gram (CE/100g) sour cherries. 26 27 28 126 2.7 Statistical analysis 29 30 31 127 Analysis of variance and Students T-test were performed using SAS 9.1 software package 32 33 128 34 (SAS Institute, North Carolina, USA) with a confidence level of 95 %. Linear regressions 35 36 129 between total phenolic content total anthocyanin content, ORAC value, PGE2 assay and 37 38 130 anticancer cell proliferation activities were investigated to determine the correlation 39 40 41 131 significance. 42 43 44 132 45 46 47 133 3 RESULTS AND DISCUSSION 48 49 50 134 Table 1 shows the total phenolic and total anthocyanin content, ORAC value, Caco-2 cancer 51 52 53 135 cell proliferation inhibition activity and the PGE2 production assay in the 34 different sour 54 55 136 cherry cultivars. Sour cherry cultivars with high total phenolics and total anthocyanins 56 57 137 content were ‘Tiki’, ‘Aarslev 2510’, ‘Fanal’ and ‘Aarslev 2403’. In contrast ‘Gerema’, 58 59 60

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1 2 3 138 ‘Skyggemorel Hannover’, ‘Zagarvysne’, ‘Vytenu Star’, ‘Surefire’ and ‘Favorite’ contained 4 5 6 139 the lowest amount of total phenolics and anthocyanins. 7 8 9 140 ORAC values, Caco-2 cancer cell inhibitory effect and PGE2 assay of the 34 sour cherry 10 11 141 cultivars were significantly different between varieties (P < 0.001). ORAC values ranged 12 13 14 142 from 15 to 180 µmol TE/g; inhibitory effects against Caco-2 varied from no inhibition to a 15 16 143 strong inhibitory effect up to 79 %. As shown in table 2, total phenolics, total anthocyanin, 17 18 144 ORAC values and Caco-2 cancer cell inhibitory activity are significantly correlated (P< 19 20 For Peer Review 21 145 0.001).‘Birgitte x Böttermö’, ‘Fanal’ and ‘Tiki’ displayed the strongest antioxidant activity 22 23 146 in the ORAC assay; these 3 cultivars also displayed the strongest inhibitory effect against 24 25 26 147 the growth of Caco-2 cancer cell line. In contrast, ‘Surefire’ and ‘Favorite’ had the lowest 27 28 148 ORAC value and also displayed very weak inhibitory effects against Caco-2 cancer cell 29 30 149 proliferation. 31 32 33 150 34 The effect of sour cherry cultivars against PGE2 production were compared against a blank 35 36 151 and two positive controls. The positive controls, indomethacin and NS-398, decreased the 37 38 152 PGE2 production to 24 and 33% respectively. The 34 sour cherry cultivars exhibited 39 40 41 153 different levels of inhibition against PGE2 production. ‘Stevnsbaer Birgitte’ and ‘Stevnsbaer 42 43 154 Viki’ displayed the strongest suppression against PGE2 production which was similar to the 44 45 155 positive control. However, ‘Nefris,’ ‘Sumadinka’, ‘Pernilla’ and ‘Surefire’ showed no 46 47 48 156 inhibitory effect against PGE2 production. However, PGE2 assay was independent and not 49 50 157 correlated with other results in present study (P > 0.05). In vitro anti-inflammatory activity 51 52 158 of sour cherry anthocyanins and their aglycons has been reported and assigned to inhibition 53 54 55 159 of COX-2 (Mulabagal et al. , 2009; Reddy et al. , 2005; Wang et al. , 1999b). Other research 56 57 160 pointed out that sour cherry juice displaying strong anti-inflammatory effect by inhibiting 58 59 60 161 the COX-2 level Freund’s adjuvant mice (Šáric et al. , 2009).

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1 2 3 162 [Table 1 about here] 4 5 6 7 163 The majority of the sour cherry cultivars with high total phenolics, high ORAC values and 8 9 164 better inhibitory effect against Caco-2 cancer cell proliferation were either Stevnsbaer-type, 10 11 165 Fanal-type or new cross breeding cultivars. Stevnbaer-type and Fanal-type sour cherries 12 13 14 166 shared very similar characteristics and produced high quality dark red sour cherries 15 16 167 (Christensen, 1986). ‘Tiki’ is a hybrid between ‘Stevnbaer’ and ‘Fanal’ (DLBR 17 18 168 Landbrugsinfo, 2008), ‘Birgitte × Böttermö’ is a cross breeding cultivar of ‘Stevnbaer 19 20 For Peer Review 21 169 Birgitte’ and ‘Erdi Böttermö’. In the present study we observed that both ‘Tiki’ and ‘Birgitte 22 23 170 x Böttermö’ contained higher amount of phenolics as compared to the parental cultivars. 24 25 26 171 ‘Aarslev 2510’, ‘Aarslev 2403’, ‘Aarslev 1803’ and ‘Aarslev 2504’ are new cross breeding 27 28 172 cultivars; ‘Fanal’ and ‘Nefris’ are sharing a similar characteristic and both belong to Fanal- 29 30 173 type sour cherry cultivars. ‘Heimann Rubin 4’ is a cultivar origin from Germany with large 31 32 33 174 fruits and produce high quality juices, similar to the characteristic of Fanal-type sour 34 35 175 cherries (Christensen, 1990). 36 37 38 176 [Table 2 about here] 39 40 41 177 4 Conclusions 42 43 44 45 178 Sour cherry phenolics are strongly correlated with antioxidative and anticancer effects of 46 47 179 sour cherries. The cultivars that are rich in phenolics display stronger antioxidant activity 48 49 180 and anticancer activity. However, we suggest that there may be some other compounds in 50 51 52 181 sour cherries that are responsible for the COX-2 inhibitory activity beside anthocyanins. In 53 54 182 addition, more in vivo experiments and clinical research on sour cherries is needed to 55 56 183 57 confirm the bioavailability and mechanism. The significant difference of the bioactivity 58 59 184 results suggests that selection of cultivars is an important procedure for growers to obtain 60 185 high quality sour cherries with potential health promoting effects.

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1 2 3 186 4 5 6 7 187 Reference List 8 9 188 10 11 189 Smith WL, Dewitt DL, Garavito RM. 2000. Cyclooxygenases: Structural, cellular, and 12 190 molecular biology. Annual Review of Biochemistry 69: 145-182 13 14 191 Claria J. 2003. Cyclooxygenase-2 biology. Current Pharmaceutical Design 9: 2177-2190 15 16 420: 17 192 Coussens LM, Werb Z. 2002. Inflammation and cancer. Nature 860-867 18 19 193 Lu HT, Ouyang WM, Huang CS. 2006. Inflammation, a key event in cancer development. 20 194 Molecular CancerFor Research Peer 4: 221-233 Review 21 22 195 Wang H, Nair MG, Strasburg GM, Booren AM, Gray JI. 1999a. Antioxidant polyphenols 23 196 from tart cherries ( Prunus cerasus ). Journal of Agricultural and Food Chemistry 47: 24 25 197 840-844 26 27 198 Wang H, Nair MG, Strasburg GM, Booren AM, Gray I, Dewitt DL. 2000. Cyclooxygenase 28 199 active bioflavonoids from Balaton(TM) tart cherry and their structure activity 29 200 relationships 30 201 17. Phytomedicine 7: 15-19 31 32 202 Kang SY, Seeram NP, Nair MG, Bourquin LD. 2003. Tart cherry anthocyanins inhibit 33 Min 34 203 tumor development in Apc mice and reduce proliferation of human colon cancer 35 204 cells. Cancer Letters 194: 13-19 36 37 205 He YH, Zhou J, Wang YS, Xiao C, Tong Y, Tang JCO, Chan ASC, Lu AP. 2006. Anti- 38 206 inflammatory and anti-oxidative effects of cherries on Freund's adjuvant-induced 39 35: 40 207 arthritis in rats. Scandinavian Journal of Rheumatology 356-358 41 42 208 Christensen JV. 1990. A review of an evaluation of 95 cultivars of sour cherry. Tidsskrift for 43 209 Planteavl 94: 51-63 44 45 210 Christensen JV. 1986. Evaluation of characteristics of 18 sour cherry cultivars. Tidsskrift for 46 211 Planteavl 90: 339-347 47 48 92: 49 212 Christensen JV. 1988. Evaluation of 14 sour cherry sultivars. Tidsskrift for Planteavl 50 213 345-349 51 52 214 Christensen JV. 1997. Evaluation of 12 sour cherry varieties. Erwerbsobstbau 39: 38-41 53 54 215 Huang D, Ou B, Hampsch-Woodill M, Flanagan JA, Prior RL. 2002. High-throughput assay 55 216 of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling 56 217 57 system coupled with a microplate fluorescence reader in 96-well format. Journal of 58 218 Agricultural and Food Chemistry 50: 4437-4444 59 60 219 Bang YH, Lee JH, Tae HK, Hang SK, Young SH, Jai SR, Kyong SL, Jung JL. 2002. 220 Furanoligularenone, an eremophilane from Ligularia fischeri , inhibits the LPS-

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1 2 3 221 induced production of nitric oxide and prostaglandin E2 in macrophage RAW264.7 4 68: 5 222 cells. Planta Medica 101-105 6 7 223 Mulabagal V, Lang GA, DeWitt DL, Dalavoy SS, Nair MG. 2009. Anthocyanin content, 8 224 lipid peroxidation and cyclooxygenase enzyme inhibitory activities of sweet and 9 225 sour Cherries. Journal of Agricultural and Food Chemistry 57: 1239-1246 10 11 226 Reddy MK, exander-Lindo RL, Nair MG. 2005. Relative inhibition of lipid peroxidation, 12 13 227 cyclooxygenase enzymes, and human tumor cell proliferation by natural food colors. 14 228 Journal of Agricultural and Food Chemistry 53: 9268-9273 15 16 229 Wang H, Nair MG, Strasburg GM, Chang YC, Booren AM, Gray JI, DeWitt DL. 1999b. 17 230 Antioxidant and antiinflammatory activities of anthocyanins and their aglycon, 18 231 cyanidin, from tart cherries. Journal of Natural Products 62: 294-296 19 20 232 For Peer Review 21 Šáric A, Sobocanec S, Balog T, Kušic B, Šverko V, Dragovic-Uzelac V, Levaj B, Cosic Z, 22 233 Šafranko ZM, Marotti T. 2009. Improved antioxidant and anti-inflammatory 23 234 potential in mice consuming sour cherry juice ( Prunus Cerasus cv. Maraska). Plant 24 235 Foods for Human Nutrition 64: 231-237 25 26 236 DLBR Landbrugsinfo. Sortsliste surkirsebær (Last updated on 1-23-2008). 27 237 http://www.landbrugsinfo.dk/Planteavl/Havebrug/Frugt-og- 28 29 238 baer/Kirsebaer/Sider/Sortsliste_surkirsebaer.aspx (Accessed on 5-24-2011) 30 239 31 240 32 33 34 241 35 36 37 242 38 39 40 41 243 42 43 44 244 45 46 47 245 48 49 50 246 51 52 53 54 247 55 56 57 248 58 59 60 249

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1 2 3 250 Table 1 Results of sour cherry cultivars on their total phenolic (TP, QE = quercitrin 4 5 251 equivalent), total anthocyanin (TA, CE = cyanidin equivalent), oxygen radical absorbance 6 7 252 capacity (ORAC, TE = trolox equivalent), Caco-2 proliferation inhibitory activity, PGE2 8 9 253 assay and the least significant difference (LSD). Results for ORAC, Caco-2 inhibition and 10 254 11 PGE2 assay are expressed in a form of mean±SD. Values in the same column followed by 12 255 identical letters show no significant difference. Level of significance, ***P < 0.001. 13 14 15 TP, TA, ORAC, Caco-2 PGE2 16 Cultivars mg QE/100g mg CE/100g mol TE/g inhibition, % production, % 17 Tiki 140.2 188.6 133 ± 18.6 bc 73 ± 2.1 ab 50 ± 9.2 fghijk 18 Aarslev 2510 133.0 154.8 103 ± 23.7 defg 14 ± 5.1 jk 68 ± 2.6 bcdefghi 19 Fanal 127.3 172.6 147 ± 16.9 b 79 ± 1.3 a 69 ± 24.8 bcdefghi 20 For Peer Review 21 Aarslev 2403 125.2 177.1 97 ± 21.9 defgh 9 ± 5.0 jk 58 ± 6.2 efghij 22 Heimanns Rubin 4 106.2 129.1 115 ± 15.9 cd 72 ± 0.6 ab 85 ± 4.6 abcdef 23 Birgitte x Böttermö 106.1 100.8 180 ± 14.5 a 74 ± 0.7 ab 73 ± 20.4 abcdefgh 24 Bofa 97.2 126.7 112 ± 12.3 cde 65 ± 2.5 bc 76 ± 9.9 abcdefg 25 Aarslev 1803 92.7 103.8 114 ± 10.6 cd 53 ± 3.2 def 58 ± 8.9 efghij 26 27 Nefris 89.5 108.8 115 ± 15.7 cd 70 ± 1.5 bc 101 ± 6.2 abc 28 Recta 85.7 105.0 91 ± 10.1 defghi 29 ± 2.0 i 42 ± 4.9 ghijk 29 Safir 83.1 107.9 72 ± 11.1 hijklm 44 ± 5.1 fg 86 ± 6.6 abcdef 30 Stevnsbaer,PH 82.8 80.6 103 ± 23.8 defg 44 ± 9.2 g 61 ± 7.9 efghi 31 Aarslev 2504 72.9 93.1 66 ± 9.3 ijklm 15 ± 3.2 j 79 ± 8.4 abcdef 32 33 Stevnsbaer Birgitte 70.4 85.3 108 ± 16.5 cdef 50 ± 1.2 efg 22 ± 2.3 k 34 Nadwislanka 67.7 74.7 85 ± 27.8 efghij 55 ± 3.6 de 88 ± 11.4 abcde 35 Stevnsbaer Viki 67.4 80.9 113 ± 18.8 cde 61 ± 0.9 cd 22 ± 3.0 k 36 Sumadinka 66.7 72.2 61 ± 17.3 jklmn -5 ± 4.7 no 103 ± 23.5 ab 37 Danax 1 66.0 88.7 81 ± 26.9 fghijk 14 ± 8.3 j 55 ± 12.4 fghijk 38 39 K27/2 60.2 41.9 79 ± 14.6 ghijkl 68 ± 2.6 bc 64 ± 5.4 defghi 40 Kelleris 16 60.2 45.5 56 ± 12.7 klmn 4 ± 2.1 klm 52 ± 1.2 fghijk 41 M7 55.2 74.6 51 ± 10.5 lmnop 5 ± 2.7 kl 70 ± 5.8 bcdefgh 42 Lutovka 54.9 55.8 65 ± 16.1 ijklmn 3 ± 12.3 klmn 68 ± 4.1 bcdefghi 43 Pernilla 49.0 60.8 72 ± 19.2 hijklm -2 ± 3.6 lmn 106 ± 21.0 a 44 45 Cigganymeggy 7 46.0 67.8 44 ± 10.0 mnopq 2 ± 1.4 klmn 74 ± 12.9 abcdefgh 46 Erdi Böttermö 45.1 38.4 37 ± 6.7 nopqr 2 ± 3.1 klmn 91 ± 6.3 abcde 47 Zigeunerkirschen 44.3 56.2 50 ± 16.0 mnop 10 ± 2.3 jk 58 ± 9.3 efghij 48 Oblachinska Holo 43.1 60.8 49 ± 14.3 mnop 7 ± 9.8 jk 57 ± 7.4 efghijk 49 Ungarische Traubige 41.5 34.4 22 ± 5.5 pqr 4 ± 1.6 klmn 65 ± 13.3 cdefghi 50 51 Gerema 40.8 13.6 53 ± 14.6 klmno -4 ± 3.1 mno 79 ± 14.5 abcdef 52 Skyggemorel Hannover 38.5 24.6 59 ± 7.5 jklmn -11 ± 3.4 o 56 ± 1.7 efghijk 53 Zagarvysne 31.2 23.7 51 ± 14.0 lmno 3 ± 3.9 klmn 40 ± 5.8 hijk 54 Vytenu Star 25.5 15.3 25 ± 8.0 opqr -5 ± 5.9 mno 72 ± 4.4 abcdefgh 55 Surefire 22.4 11.0 15 ± 3.4 r 2 ± 1.9 klmn 101 ± 5.4 ab 56 57 Favorite 16.0 18.2 16 ± 7.7 qr 7 ± 2.1 jk 58 ± 17.9 efghij 58 Blank - - - - 100 ± 0.0 abcd 59 Indomethacin - - - - 24 ± 0.2 jk 60 NS-398 - - - - 33 ± 0.0 ijk LSD (T=95%) - - 28.5*** 8.98*** 35.95***

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1 2 3 256 Table 2. Linear regression (R 2) of total phenolic (TP), total anthocyanin (TA), oxygen 4 5 6 257 radical absorbance capacity (ORAC), Caco-2 proliferation inhibitory activity and PGE2 7 8 258 assay. Level of significance, ***P < 0.001. 9 10 11 TP TA ORAC Caco-2 12 13 TA 0.96*** - - - 14 ORAC 0.85*** 0.77*** - - 15 Caco-2 0.66*** 0.61*** 0.82*** - 16 PGE2 -0.06 -0.08 -0.17 -0.11 17 18 259 19 20 For Peer Review 21 260 22 23 261 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Investigation of three triterpene acids against cancer cell proliferation activity

Journal: Phytotherapy Research ManuscriptFor ID: Draft Peer Review Wiley - Manuscript type: Short Communication

Date Submitted by the n/a Author:

Complete List of Authors: Khoo, Gaik Ming; Aarhus University, Food Science Clausen, Morten; Aarhus University, Food Science Larsen, Erik; Aarhus University, Food Science

Keyword: betulinic acid, oleanolic acid, ursolic acid, combination effect, cancer

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1 2 3 4 5 1 Investigation of three triterpene acids against cancer cell proliferation 6 7 8 2 activity 9 10 11 3 Gaik Ming Khoo, Morten Rahr Clausen, and Erik Larsen* 12 13 4 Department of Food Science, Aarhus University, Kirstinebjergvej 10, DK-5792 Aarslev, Denmark. 14 15 16 5 *Corresponding Author: Tel: +45-89993367, Fax: +45-89993495, e-mail address: 17 18 6 [email protected] 19 20 7 For Peer Review 21 22 23 8 ABSTRACT 24 25 9 The object of this study was to investigate the inhibitory effect of betulinic acid, oleanolic acid and 26 27 10 ursolic acid on cancer cell proliferation activity. Results showed that these three compounds 28 29 30 11 displayed good inhibitory effect against cancer cell proliferation activity at the concentration of 31 32 12 10µg/ml. The combination of oleanolic acid/ursolic acid displayed additive effects against HT-29 33 34 35 13 and SW-480 cancer cell proliferation, but not Caco-2 cancer cell. However, the combination of 36 37 14 betulinic acid/oleanolic acid, betulinic acid/ursolic acid and betulinic acid/oleanolic acid/ursolic 38 39 15 acid showed no inhibitory effects against cancer cell proliferation. 40 41 42 16 43 44 17 KEYWORDS: betulinic acid; oleanolic acid; ursolic acid; combination effect; cancer 45 46 18 47 48 49 19 1 INTRODUCTION 50 51 20 Triterpene acids are members of the terpene family that consist of six isoprene units. A rosehip 52 53 21 fraction containing a mixture of betulinic acid, oleanolic acid and ursolic acid displayed inhibitory 54 55 56 22 effect against the release of pro-inflammatory cytokine interleukin-6 (IL-6) from the Mono Mac 6 57 58 23 cells. Further investigation showed that oleanolic acid/betulinic acid and ursolic acid/betulinic acid 59 60 24 displayed significant inhibitory effect against the release of IL-6 when compared to the pure

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1 2 3 4 5 25 compounds (Saaby et al. , 2011). Betulinic acid is a pentacyclic lupane-type triterpenoid which is 6 7 26 widely distributed in the plant kingdom. It displays anti-inflammatory, anti-HIV and antineoplastic 8 9 27 activities (Alakurtti et al. , 2006). Betulinic acid exhibits antitumor activity against a numbers of 10 11 12 28 cancer-types (Alakurtti et al. , 2006) and possibly through the inhibition against topoisomerase 13 14 29 (Chowdhury et al. , 2002). It is one of the compounds selected for the Rapid Access to Intervention 15 16 30 Development program by National Cancer Institute (Udeani et al. , 1999). Oleanolic acid and its 17 18 19 31 isomer ursolic acid, are widely found in plants, especially in a number of plants used as traditional 20 For Peer Review 21 32 folk medicine (Liu, 1995). Oleanolic acid and ursolic acid are well known for their anti- 22 23 33 inflammatory and hepatoprotective effects (Liu, 1995). Both oleanolic acid and ursolic acid were 24 25 26 34 able to induce apoptosis of liver cancer cells (Yan et al. , 2010) and inhibited tumor growth in mice 27 28 35 (Hsu et al. , 1997). Surprisingly, betulinic acid, oleanolic acid and ursolic acid were relatively non- 29 30 31 36 toxic to healthy cells and animals (Galgon et al. , 2005; Hunan Medical Institute, 1977; Zuco et al. , 32 33 37 2002). 34 35 38 The present study includes the investigation betulinic acid, oleanolic acid and ursolic acid on 36 37 38 39 inhibition of three human colon cancer cell lines. This is the first time that the combination of these 39 40 40 three triterpenoids has been investigated on the inhibitory effect against in vitro cancer cell 41 42 41 proliferation activity. 43 44 45 42 46 47 43 2 MATERIALS AND METHODS 48 49 44 Standard compounds betulinic acid, oleanolic acid and ursolic acid and dimethyl sulfoxide (DMSO) 50 51 52 45 were purchased from Sigma-Aldrich (Missouri, USA). Dulbecco's Modified Eagle Medium 53 54 46 GlutaMAX TM (DMEM) was purchased from Invitrogen (Paisley, UK). Phosphate buffer saline- 55 56 57 47 ethylenediaminetetraacetic acid (PBS-EDTA) and Trypsin-ethylenediaminetetraacetic acid 58 59 48 (Trypsin-EDTA) were purchased from Lonza (Braine, Belgium). Penicillin G-potassium salt and 60

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1 2 3 4 5 49 streptomycin sulfate were purchased from Serva (Heidelberg, Germany). Fetal calf serum (FCS) 6 7 50 was from purchased PAA Laboratories (Pasching, Austria). WST-1 cell proliferation reagent was 8 9 51 purchased from Roche Diagnostics (Mannheim, Germany). 10 11 12 13 52 Betulinic acid, oleanolic acid and ursolic acid were dissolved in DMSO. Human colon cancer cell 14 15 53 lines Caco-2, HT-29 and SW-480 (European Collection of Cell Cultures, Salisbury, UK) were used 16 17 18 54 in the experiments. Growing of cells and the WST-1 method for the determination of cancer cell 19 20 55 viability were performedFor as described Peer (Khoo et al.Review, 2011). All samples were tested in triplicate in 21 22 56 two independent experiments, and DMSO was used as control. Inhibition for each sample was 23 24 25 57 calculated relative to the control. The three triterpene acids were tested individually at 26 27 58 concentrations of 10, 5.0, 2.5, 1.25 and 0.625 µg/ml. For investigation of combination effects, 28 29 59 mixtures of two pure compounds (5.0 µg/ml of each), a mixture of three compounds (3.3 µg/ml of 30 31 32 60 each) and different combination ratios of oleanolic acid/ursolic acid with a total concentration of 33 34 61 10µg/ml were tested with the same method mentioned above. The statistical analysis was carried on 35 36 with SAS 9.1 software package (SAS Institute, North Carolina, USA) with a confidence level of 37 62 38 39 63 95%. 40 41 42 64 43 44 45 46 65 3 RESULTS AND DISCUSSION 47 48 49 50 66 The inhibitory pattern of HT-29 and SW-480 were similar, thus we only show the results of HT-29. 51 52 67 As shown in table 1, at the highest test concentration of 10 µg/ml, both betulinic acid and ursolic 53 54 68 acid displayed strong inhibition against HT-29 (88 % and 79 %, respectively) and SW-480 (76 % 55 56 57 69 and 82 %, respectively), but a moderate to weak inhibition against Caco-2 (19 % and 22 %, 58 59 70 respectively). At the concentration of 10 µg/ml, oleanolic acid displayed only moderate inhibition 60

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1 2 3 4 5 71 against Caco-2 (49 %), very weak inhibition against HT-29 (7 %) and no inhibition against SW- 6 7 72 480. All three compounds displayed very weak or no inhibition against the cancer cell lines at a 8 9 73 concentration of 5.0 µg/ml and below. All combination mixtures had antagonistic effects against 10 11 12 74 Caco-2. An additive effect was observed for the combination of oleanolic acid/ursolic acid against 13 14 75 HT-29. Similar inhibitory effects were observed against SW-480 (23 %). However, the combination 15 16 76 of betulinic acid/ursolic acid showed only weak inhibition against HT-29 and no inhibition against 17 18 19 77 Caco-2 or SW-480. Similarly, the combination of betulinic acid/oleanolic acid, and the combination 20 For Peer Review 21 78 of three triterpene acids, displayed poor inhibitory effect against all cancer cell lines. All 22 23 79 combinations were not inhibiting Caco-2 effectively. Additive effect was observed in the 24 25 26 80 combination of oleanolic acid/ursolic acid against HT-29. However, significant decreases were 27 28 81 observed on the actual inhibitory effects of HT-29 for the combination of betulinic acid/oleanolic 29 30 31 82 acid and betulinic acid/ursolic acid when compared to the theoretical inhibitory effect. 32 33 34 83 [Table 1 about here] 35 36 37 38 84 Figure 1 shows the inhibitory effects of different composition of oleanolic acid and ursolic acid 39 40 85 against cancer cell proliferation. Results showed that different combinations of the mixtures had no 41 42 86 inhibitory effects against Caco-2, but significant differences were found on the inhibitory effect 43 44 45 87 against HT-29 for different compositions of oleanolic acid and ursolic acid. The inhibitory effects 46 47 88 were higher when the combination mixture consisted of higher concentration of ursolic acid and 48 49 50 89 lower concentration of oleanolic acid. 51 52 53 90 [Figure 1 about here] 54 55 56 57 91 Unlike previous study conducted by Saarby et al. (2011), we only found an additive effect in the 58 59 92 combination of oleanolic acid/ursolic acid; antagonistic effects between the pure compounds was 60

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1 2 3 4 5 93 observed when betulinic acid was one of the component in the combination. Antagonistic effects 6 7 94 between closely related compounds such as luteolin and luteolin-7-glucoside have previously been 8 9 95 observed, where both compounds are strong antioxidants but the combination mixture only showed 10 11 12 96 an effect similar to luteolin alone (Hsu et al. , 2005). Synergistic effects have e.g. been observed in a 13 14 97 combination of docosahaxaenoic acid and curcumin against human breast cancer cell line, where 15 16 98 both compounds have no effects when tested individually (Altenburg et al. , 2011). 17 18 19 20 99 In this study the results showedFor that Peer combination Review of oleanolic acid/ursolic acid displayed additive 21 22 100 effect against proliferation of HT-29 but not Caco-2. Although betulinic acid and ursolic acid 23 24 25 101 showed good inhibitory effects against all three cancer cell lines, but their combination displayed 26 27 102 antagonistic effects and showed poor inhibition against cancer cell proliferations. However, these 28 29 103 triterpenic acids and their combination effects should be further investigated in different in vitro or 30 31 32 104 in vivo experiment to confirm their bioactivities and mechanism. 33 34 35 105 36 37 38 106 Reference List 39 40 107 41 42 108 Alakurtti S, Mäkelä T, Koskimies S, Yli-Kauhaluoma J. 2006. Pharmacological properties of the 43 109 ubiquitous natural product betulin. European Journal of Pharmaceutical Sciences 29: 1-13 44 45 46 110 Chowdhury AR, Mandal S, Mittra B, Sharma S, Mukhopadhyay S, Majumder HK. 2002. Betulinic 47 111 acid, a potent inhibitor of eukaryotic topoisomerase I: Identification of the inhibitory step, 48 112 the major functional group responsible and development of more potent derivatives. Medical 49 113 Science Monitor 8: BR254-BR260 50 51 114 Udeani GO, Zhao GM, Shin YG, Cooke BP, Graham J, Beecher CWW, Kinghorn AD, Pezzuto JM. 52 115 1999. Pharmacokinetics and tissue distribution of betulinic acid in CD-1 mice. 53 54 116 Biopharmaceutics and Drug Disposition 20: 379-383 55 56 117 Liu J. 1995. Pharmacology of oleanolic acid and ursolic acid. Journal of Ethnopharmacology 49: 57 118 57-68 58 59 119 Yan S, Huang C, Wu S, Yin M. 2010. Oleanolic acid and ursolic acid induce apoptosis in four 60 120 human liver cancer cell lines. Toxicology in Vitro 24: 842-848

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1 2 3 4 5 121 Hsu HY, Yang JJ, Lin CC. 1997. Effects of oleanolic acid and ursolic acid on inhibiting tumor 6 122 growth and enhancing the recovery of hematopoietic system postirradiation in mice. Cancer 7 123 Letters 111: 7-13 8 9 124 Hunan Medical Institute. 1977. Effects of oleanolic acid on experimental liver injury and 10 125 therapeutic value in human hepatitis. Traditional Medicine (Zhong Cao Yao) 8: 32-37 11 12 13 126 Galgon T, Wohlrab W, Dräger B. 2005. Betulinic acid induces apoptosis in skin cancer cells and 14 127 differentiation in normal human keratinocytes. Experimental Dermatology 14: 736-743 15 16 128 Zuco V, Supino R, Righetti SC, Cleris L, Marchesi E, Gambacorti-Passerini C, Formelli F. 2002. 17 129 Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer 18 130 Letters 175: 17-25 19 20 Saaby L, Moesby L, HansenFor EW, ChristensenPeer SB. Review 2011. Isolation of immunomodulatory triterpene 21 131 22 132 acids from a standardized rose hip powder (Rosa canina L.). Phytotherapy Research 25: 23 133 195-201 24 25 134 Khoo GM, Clausen MR, Pedersen BH, Larsen E. 2011. Bioactivity and total phenolic content of 34 26 135 sour cherry cultivars. Journal of Food Composition and Analysis. 27 136 doi:10.1016/j.jfca.2011.03.004 28 29 30 137 Hsu HF, Houng JY, Chang CL, Wu CC, Chang FR, Wu YC. 2005. Antioxidant activity, 31 138 cytotoxicity, and DNA information of Glossogyne tenuifolia. Journal of Agricultural and 32 139 Food Chemistry 53: 6117-6125 33 34 140 Queirós B, Barreira JCM, Sarmento AC, Ferreira ICFR. 2009. In search of synergistic effects in 35 141 antioxidant capacity of combined edible mushrooms. International Journal of Food 36 37 142 Sciences and Nutrition 60: 160-172 38 39 143 Altenburg J, Bieberich A, Terry C, Harvey K, VanHorn J, Xu Z, Davisson V, Siddiqui R. 2011. A 40 144 synergistic antiproliferation effect of curcumin and docosahexaenoic acid in SK-BR-3 breast 41 145 cancer cells: unique signaling not explained by the effects of either compound alone. BMC 42 146 Cancer 11: 149 43 44 147 45 148 46 47 149 48 49 150 50 51 52 151 53 54 152 55 56 57 153 58 59 154 60

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1 2 3 4 5 155 6 7 8 156 Table 9 10 11 12 157 13 14 15 158 Table 1. Inhibitory effects of betulinic acid (BA), oleanolic acid (OA) and ursolic acid (UA) against 16 17 18 159 Caco-2 and HT-29 cancer cell proliferation activity. The theoretical inhibition % is the sum of 19 20 160 inhibitory activity of the Forpure compounds Peer at 5 µg/ml. Review Significant difference between theoretical and 21 22 161 actual inhibition % were calculated (* P < 0.05; ** P < 0.01). 23 24 25 26 Triterpenic Concentration, Caco-2 inhibition, % HT-29 inhibition, % 27 acid µg/ml Theoretical Actual Theoretical Actual 28 29 BA 10 - 18.9 ± 5.63 - 88.5 ± 0.04 30 OA 10 - 48.9 ± 12.53 - 7.2 ± 2.42 31 32 UA 10 - 22.4 ± 8.35 - 79.4 ± 4.44 33 BA 5 - 0.7 ± 1.95 - 9.1 ± 1.60 34 35 OA 5 - -1.3 ± 2.13 - 9.3 ± 1.68 36 UA 5 - 4.0 ± 0.81 - 15.3 ± 6.89 37 38 BA/OA/UA 3.3/3.3/3.3 - -0.8 ± 1.36 - 5.3 ± 0.40 39 BA/OA 5/5 -0.7 ± 2.89 -3.7 ± 0.48 18.3 ± 2.32 7.9 ± 1.52 ** 40 BA/UA 5/5 4.6 ± 2.12 -2.8 ± 0.89 ** 24.4 ± 7.07 7.2 ± 1.30 * 41 42 OA/UA 5/5 2.6 ±2.28 6.1 ± 1.54 24.6 ± 7.09 32.2 ± 0.83 43 162 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 Inhibitory effect of different composition of the mixture of oleanolic acid (OA) and ursolic acid (UA) 20 against Caco2 and HT29 cancer cell proliferation. Mixtures were tested at a combination 21 concentration of 10g/ml with ursolic acid: oleanolic acid (UA:OA) a ratio of 6:0, 5:1, 4:2, 3:3, 22 2:4, 1:5 and 0:6. Plot of the mixture are according to the concentration of ursolic acid. Plots 23 labeled with different alphabets were significantly different (P < 0.001). 24 178x62mm (150 x 150 DPI) 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/ptr