UNIVERSITA’ POLITECNICA DELLE MARCHE DOTTORATO DI RICERCA IN “ALIMENTI, NUTRIZIONE E SALUTE” Coordinatore: Chiar.mo Prof. Salvatore Amoroso

In vitro effects of Alba strawberry cultivars on two murine breast cancer cell lines

PhD student:

Relatore:

Dott. Luca Mazzoni

Prof. Maurizio Battino Correlatori: Dott.ssa Francesca Giampieri Dott. Josè Miguel Alvarez Suarez

XIII ciclo Triennio Accademico 2012-2014

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A chi più ha creduto in me, maggiore è il mio ringraziamento..

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ABSTRACT The general aim of this PhD course was the evaluation of the effects of strawberry extracts treatment on two in vitro models of murine breast cancer cell line, trying to detect a specific pathway (AMPK) through which strawberries exert their anticancer activity. In the first part of the PhD course 5 different strawberry commercial cultivars, 4 of them resulting from the UNIVPM breeding program, were evaluated for their sensorial and nutritional qualities, in particular soluble solids and total acidity. Regarding the nutritional qualities, the content of total phenolics, flavonoids, anthocyanins, vitamin C and folates were evaluated. The antioxidant capacity was also measured, through 3 different methodologies. After the evaluation of the 5 different cultivars, Alba was chosen for its nutritional value and was used for the second part of PhD course. In this second part, the anticancer activity of methanolic and anthocyanin purified extracts from Alba cultivar on two murine cancer cell lines, N202/1A (with high levels of HER2/neu oncogene) and N202/1E (with low levels of HER2/neu oncogene) were evaluated after 48 and 72 h of treatment. In particular, the viability, apoptosis and intracellular ROS rates were assessed. The cell damage was also estimated through the evaluation of the mitochondrial functionality (respiratory and glycolitic assay) and the oxidative status (measurement of SOD and CAT activity). Finally, Western Blot assays were performed to analyze the expression of several proteins related to apoptosis, autophagy, metastasis, oxidative status, mitochondrial functionality, oncogene expression and AMPK pathway. On this regard, cells were also treated with Compound C, an AMPK inhibitor, and the effects of this compound on cell viability, apoptosis and AMPK expression were assessed, in order to compare them with the results obtained after the strawberry treatment. This study demonstrated that a well characterized strawberry possessed an antiproliferative effect on cancer cells, through the induction of apoptosis and oxidative stress. At the same time, this study is one of the first that have tried to in deep a candidate pathway for the explanation of the strawberry effects on cancer cells, and as a results a relation between the AMPK pathway and the anticancer effects of strawberries was demonstrated.

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ITALIAN SUMMARY Effetti in vitro della cultivar di fragola Alba su due linee cellulari murine di tumore al seno L’obiettivo principale degli studi condotti durante il corso di dottorato è stato quello di valutare gli effetti del trattamento con estratti di fragola su 2 modelli murini in vitro di cancro al seno, cercando di verificare se nel meccanismo di azione tramite il quale le fragole sembrano svolgere un’azione antitumorale sia implicato l’AMPK. Nella prima parte degli studi sono state valutate le caratteristiche sensoriali (solidi solubili e acidità totale) e nutrizionali (contenuto totale di fenoli, di flavonoidi, di antociani, di vitamina C e di folati) e la capacità antiossidante di 5 diverse cultivar commerciali di fragola. Dopo averle valutate, la scelta per effettuare i test in vitro è ricaduta su Alba, per il suo valore nutrizionale. Nella seconda parte del dottorato è stata valutata la capacità antitumorale degli estratti metanolici ed antocianici della cultivar Alba su 2 linee tumorali murine di cancro al seno, N202/1A (alta espressione dell’oncogene HER2/neu) e N202/1E (bassa espressione dell’oncogene HER2/neu), dopo 48 e 72 ore di trattamento. In particolare, sono stati determinati il tasso di vitalità, di apoptosi e di ROS intracellulari. Il danno alle cellule tumorali è stato valutato anche tramite l’analisi della funzionalità mitocondriale (capacità di glicolizzare e di consumare ossigeno) e lo stato ossidativo (misura dell’attività della SOD e della CAT). Infine, è stato usato il Western Blot per l’analisi dell’espressione di proteine correlate all’apoptosi, all’autofagia, alle metastasi, allo stato ossidativo, alla funzionalità mitocondriale, all’espressione dell’oncogene e alla regolazione dell’AMPK. A questo proposito, le cellule sono state anche trattate con il Composto C, un inibitore dell’AMPK, e sono stati valutati i suoi effetti sulla vitalità cellulare, l’apoptosi e l’espressione dell’AMPK rispetto a quelli in presenza di un trattamento con estratti di fragola. Questo studio ha dimostrato che fragole ben caratterizzate possiedono un effetto antiproliferativo su cellule tumorali, tramite l’induzione di apoptosi e di stress ossidativo. Allo stesso tempo, questo lavoro approfondisce le conoscenze sul possibile meccanismo di azione, spiegando in parte l’effetto delle fragole su cellule tumorali e dimostrando l’esistenza di una relazione tra la via dell’AMPK e l’effetto anticancerogeno delle fragole stesse.

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Table of Contents

1.

Introduction ......................................................................................................................... 8 1.1. An overview on breast cancer incidence .................................................................. 8 1.2. Origin and type of breast cancer ............................................................................... 9 1.3. Phytochemicals and cancer ..................................................................................... 11 1.4. Strawberry and cancer ............................................................................................ 11 1.5. Aim of the study ..................................................................................................... 14

2.

Experimental design .......................................................................................................... 16

3.

Part I: Strawberry nutritional quality as a tool for reaching healthy objectives ................ 18 3.1. Origin and evolution ............................................................................................... 18 3.2. Botanical characterization ...................................................................................... 19 3.3. The genetic heritage................................................................................................ 21 3.4. Strawberry in the world .......................................................................................... 22 3.5. Strawberry in Italy .................................................................................................. 23 3.6. The research and genetic improvement .................................................................. 24 3.7. Sensorial quality ..................................................................................................... 25 3.8. Nutritional Aspects ................................................................................................. 26 3.9. Phytochemicals in strawberries .............................................................................. 29 3.10. Factors affecting the concentration of phytochemicals in strawberries ............... 32 3.11. Importance of strawberry to health ....................................................................... 35

4.

Part I: Evaluation for strawberry nutritional quality and bioactive compounds contents. 38 4.1. Materials and methods ............................................................................................ 38 4.2. Results .................................................................................................................... 46 4.3. Discussion............................................................................................................... 52

5.

Part II: Breast cancer cell lines treatment with different strawberry extracts ................... 57 5.1. HER2 ...................................................................................................................... 61 6

5.2. Berries and cancer .................................................................................................. 67 5.3. Materials and methods ............................................................................................ 71 5.4. Results .................................................................................................................... 81 5.5. Discussion............................................................................................................. 102 6.

Conclusion....................................................................................................................... 115

References .............................................................................................................................. 118

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1. Introduction 1.1. An overview on breast cancer incidence Breast cancer represents the most common neoplastic disease among women worldwide, with an estimated 1.67 million new cancer cases diagnosed in 2012 (25% of all cancers), and is the second leading cause of cancer death among women in more developed regions (198,000 dead, 15.4%) after lung cancer (Ferlay et al. 2013). Breast cancer incidence rates clearly vary depending on the world region considered, ranging from 27 cases per 100000 women in Middle Africa and Eastern Asia to 96 cases in Western Europe (Fig. 1). However, the survival of breast cancer is more favorable in high-incidence developed regions, and as a consequence the range in mortality rates among world regions differed from that for incidence, with rates ranging from 6 dead per 100000 women in Eastern Asia to 20 dead in Western Africa (Fig. 2) (Ferlay et al. 2013).

Fig 1 Estimated Breast Cancer Incidence Worldwide in 2012 (Ferlay et al. 2013).

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Fig 2 Estimated Breast Cancer Mortality Worldwide in 2012 (Ferlay et al. 2013)

During the last years, however, the overall mortality rate of breast cancer has decreased, in particular through the increased emphasis on prevention and early detection of this pathology, even if mortality rates of aggressive forms of breast cancer are still high (Jemal et al. 2010a,b,c; De Santis et al. 2008) due to the disease complexity and the lack of effective and safe therapies for this malignant tumors (Thiel et al. 2012). 1.2. Origin and type of breast cancer Several research studies have identified some of the risk factors that are responsible for the expression of breast cancer, including atypical hyperplasia of the breast, family history, early menarche, late-age at first full-term pregnancy and late menopause (Kelsey et al. 1993; Hulka and Stark 1995). The regulation of some of these risk factors can be very difficult (e.g., genetic predisposition), so the implementation of novel strategies for reduction of breast cancer risk is becoming a primary objective and clinically desirable. This goal is partially fulfilled with the utilization of selective estrogen-receptor (ER) modulators (e.g., tamoxifen and raloxifene) (Fisher et al. 1998; Land et al. 2006). Estrogens are a key factor involved both in the initiation and in the proliferation of breast cancer, so a therapeutic effective approach can be based on the inhibition of estrogen formation and action (Faria et al. 2010). However, it has to be taken into account that selective ER modulators have adverse side effects including uterine cancer, thromboembolism, cataracts, and perimenopausal symptoms (Fisher et al. 2008). Furthermore, this pharmacological solution is not suitable for all the breast cancer 9

that are ER negative, so it became of primary importance to develop drugs that can demonstrate an anticancer activity in both positive and negative ER breast cancer (Faria et al. 2010). The 10-15 % of all the breast cancer cases (Cleator et al. 2007) are the so named “triple negative” breast cancer phenotype, due to their lack of expression not only of (ER), but also of progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2) (Reis-Filho and Tutt 2008). These kind of breast cancers are characterized by an aggressive clinical history with low survival rate (van de Rijn et al. 2002; Foulkes et al. 2004) and by a high risk of metastasis to the cerebrum (Tsuda et al. 2000) and visceral sites in respect to other types of breast cancers. Actually the standard treatment for this disease is the traditional chemotherapy, but this kind of cancer is highly resistant, and existed a limit amount of data and information on which to base treatment and prevention strategies for occurrence and recurrence of this disease (Adams et al. 2010). Another key player in breast cancer malignancy is HER2 (Moasser 2007; Sundaresan et al. 1999), overexpressed in 25% to 30% of human breast cancer (Liu et al. 2007). HER2 belongs to the family of epidermal growth factor receptor (EGFR); this family of receptors includes four major proteins named EGFR (also known as HER1 or ErbB1), HER2 (p185 neu/ErbB2), HER3 (ErbB3) and HER4 (ErbB4) (Sundaresan et al. 1999; Yarden 2001; Harari and Yarden 2000; Muthuswami et al. 1999; Karunagaran et al. 1996). Among all EGFR, HER2+-breast cancers results to be more malignant and more resistant to therapies than cancers lacking HER2 expression (Yarden 2001; Harari and Yarden 2000; Chin et al. 2006). The elevated expression of HER2 on the extracellular membrane in some cancer cell lines, together with its overexpression in both primary tumors and metastatic sites, makes HER2 a primary target for the development of novel strategies for cancer prevention or treatment (Davies and Hiscox 2011; Higa et al. 2010). Consequently, inhibition of HER2 expression represents one of the most validated therapeutic strategies for treating many human cancers up to breast tumor, including ovarian (Slamon et al. 1989), gastric (Lemoine et al. 1991; De Vita et al. 2010), bladder (Sauter et al. 1993), salivary (Stenman et al. 1991) and lung carcinoma (Tateishi et al. 1991). Recently, considerable attention has been focused on the role of apoptosis process in mediating the lethal effects of antineoplastic agents in breast cancer cells, and in particular on the sequence of events referred to this process. Apoptosis is characterized by a series of cellular events that are typical of this process, such as membrane blebbing, cell shrinkage, chromatin condensation and formation of a DNA ladder with multiple fragments of 180–200 bp caused by internucleosomal DNA cleavage (Steller 1995). 10

1.3. Phytochemicals and cancer In the last decades, the ability of phytochemicals to modulate apoptosis signaling pathways have attracted a growing attention as anti-cancer agents (Aravindaram and Yang 2010; Tosetti et al. 2009). The richest dietary sources of these antioxidant and bioactive compounds are fruits and vegetables, the basis of the Mediterranean Diet. Their intake has been correlated with a decreased risk of developing several chronic pathologies, including cardiovascular and neurodegenerative diseases (Johnsen et al. 2003; Heinonen et al. 1998; De Ruvo et al. 2000; Vauzour et al. 2010), obesity (Papathanasopuolos and Camilleri 2010), diabetes (Carter et al. 2010), infections (Holt et al. 2009) and cancer (Etminan et al. 2004; Smith-Warner et al. 2003; Yi-Fang et al. 2002; Chu et al. 2002), comprised breast cancer (La Vecchia and Bosetti 2006). Nowadays, convincing evidences are confirming that the combination of antioxidant micronutrients and nonessential phytochemicals, e.g. phenolic compounds, present in fruits and vegetables, play a synergistic and cumulative role in health promotion (Johnsen et al. 2003; Vauzour et al. 2010). Accordingly, the numerous beneficial effects attributed to phenolic compounds, a broad group of biologically active compounds that have many biological potentialities in vivo and in vitro (Giampieri et al. 2012a,b; Tulipani et al. 2008a, 2009a; Hakkinen and Torronen 2000; Azzini et al. 2010), have given rise to a new interest in finding vegetal species with high phenolic content and relevant biological activity. Polyphenols such as flavonoids, including anthocyanins, proanthocyanidins, flavonoids, and flavonols, can act as chemo-protectants, since the different structural components of this wide class of compounds seem (i) to possess complementary and overlapping potential protective actions, such as antioxidant and anti-inflammatory ones, and (ii) to enhance activity and expression of detoxification enzymes (Li et al. 2013). However, the intake of these natural antioxidants through the diet is strictly related to the dietary habits of populations, being abundant in the Mediterranean diet and quite scarce in a traditional Western diet (Draelos 2013). In addition, the low bioavailability of some of these antioxidants present in the diet is another important aspect to be considered, because it can influence their biological effects (Giampieri et al. 2014a). 1.4. Strawberry and cancer Among fruits, an increasing interest is rising around berries, and in particular for the strawberry (Fragaria x ananassa Duch), because of the nutritional quality and the several bioactive compounds that it contains. Moreover, strawberries are economically and commercially important and widely consumed fresh or in processed forms, such as jams, 11

juices and jellies. Therefore, they are among the most studied berries from the agronomic, genomic and nutritional points of view. Compared to other non-berry fruits previously used for human studies (Lotito and Frei 2004), strawberry is a relevant source of folate (Olsson et al. 2004a; Tulipani et al. 2008a), is rich in vitamin C (vit C) and contains several phytochemicals, that can strongly influence the nutritional and organoleptic qualities of this fruit (Scalzo et al. 2005a; Proteggente et al. 2002; Deighton et al. 2000; Tulipani et al. 2009a; Giampieri et al. 2012b). Their health effects are attributed to high levels of antioxidant compounds, most of which are phenolic compounds such as anthocyanins, flavonols, flavanols,

condensed

tannins

(proanthocyanidins,

ellagitannins,

and

gallotannins),

hydroxybenzoic and hydroxycinnamic acid derivatives, and hydrolyzable tannins (Giampieri et al. 2012a,b). These compounds are reported to possess antioxidant, anticancer, antiinflammatory, and antineurodegenerative biological properties (Giampieri et al. 2012a). Various in vitro studies have been conducted using strawberry extracts to evaluate their antiangiogenic and chemopreventive properties on cancer cell lines in a dose-dependent manner (Seeram and Heber 2006) and antiproliferative and anti-inflammatory activities, inducing apoptosis and cell cycle arrest against human stomach, prostate, intestine, and breast cancer cell lines (Boivin et al. 2007). Anthocyanin compounds found in berry fruit play a fundamental role in the antioxidant, chemopreventive, and anti-inflammatory activities (Giampieri et al. 2012a), but in addition to anthocyanins, other phenolic compounds, such as ellagitannins and flavonols, seem to exert a protective role in carcinogenesis by reducing the bioavailability of carcinogens (Carter et al. 2010). Many studies have also confirmed the influence of several factors on the anthocyanin profile of strawberry, depending on cultivars (Maatta et al. 2004; Lopes-da-Silva et al. 2007; Alvarez-Suarez et al. 2011), the genetic background (Diamanti et al. 2010, 2012a; Battino and Mezzetti 2006), the degree of ripeness, the postharvest storage of the fruits, and the climatic factors (Romandini et al. 2013; AlvarezSuarez et al. 2014a). So that it is possible to find new genotypes and cultivars producing fruit with higher contents of bioactive compounds, explore new genetic resources, or develop tailored breeding programs (Diamanti et al. 2010, 2012a,b). The breeding process can be successful if the variability and heritability of bioactive compounds is assured for the progenies derived from parental fruits. The biotechnological approach is a methodology that is able to provide a genetic improvement through modification of specific biosynthetic pathways (Ulrich et al. 2007). The application of such technology is still mostly limited to functional studies on specific pathways (Della Penna 2001), while the commercial exploitation of new products is highly limited by public concerns and biosafety rules now 12

standing for the commercial release of new genetically modified products. In any case, a deep knowledge of both the cultivated and wild genetic resources, which may be utilized for genetic and genomic studies, is an essential prerequisite for obtaining new cultivars of high interest both for the fresh market and processing industry. Recently the possibility to improve the fruit content of such phytochemicals by traditional breeding programs has been demonstrated (Whitaker 2011) and, to this end, the detailed characterization of the genetic resources to be used in the cross combination has been an important component. The inclusion in breeding programs of wild species with a genetic background able to produce progeny that have increased health related phytochemicals is also important (Capocasa et al. 2008a). Furthermore, if the nutritional quality of strawberry is enhanced in association with favourable sensorial parameters (such as titratable acidity, sugar content, fruit firmness and aroma), the consumption of fruit by consumers will be encouraged, leading to strawberries having a positive effect on health. Consequently, the biochemical characterization and the biomedical health validation of new products resulting from new genetic material, characterized by increased content of health-related phytochemicals, is an important issue (Diamanti et al. 2014). 1.4.1. Strawberry mechanisms of action against cancer The role of strawberry bioactive compounds on the cancer prevention seems to involve different mechanism, even if mechanisms of action are still unclear. As previously stated, the antioxidant capacity has been considered for years as the first line defense against the earlier stages of the mutagenesis process, through the capacity of these compounds to: (i) scavenge ROS species and decreasing the DNA oxidative damage; (ii) stimulate antioxidant enzymes; (iii) enhance DNA repairing. The antioxidant effect of these bioactive compounds is a crucial step for the antitumoral capacity of strawberry treatment, but several studies has recently underlined the ability of these compounds to modulate the cellular processes linked to the cancer progression, such as the cell proliferation, differentiation, apoptosis, cell cycle arrest, intracellular communication, inflammation and angiogenesis. Nevertheless, only few studies are still available on the anticancer effect of strawberry against breast cancer. For example, Olsson et al. found that for MCF-7 breast cancer cells, a high ratio of ascorbate to dehydroascorbate correlated with a higher inhibition of cell proliferation at the second highest concentration of strawberry extract. The significance of the effect of ascorbate on cancer cell proliferation might lie in a synergistic action with other compounds (Olsson et al. 2006). On the same cell lines, different berry extracts, including strawberry extracts, have demonstrated 13

an antiproliferative activity after 48h of cell incubation with the berry extract (Seeram et al. 2006a). Similarly, strawberry extract is capable to inhibit cell viability in different cancer cell lines at different concentrations, but the mechanism of action is to be determined yet (Weaver et al. 2009). Only recently, in a study of Somasagara et al., nevertheless the mechanism of action of methanolic strawberry extract is still unclear, the authors found that strawberry can modulate the expression of p73, when p53 is mutated in cancers like breast cancer and can activate the mitochondrial pathway of apoptosis to abrogate cancer cell proliferation (Somasagara et al. 2012). In this study also in vivo strawberry treatment on mice bearing tumor resulted in significant reduction in tumor volume without affecting the function of other organs, and also the histological evaluation showed that morphology and cellular architecture of the tissues was unaffected by the strawberry treatment. Immunohistochemical studies also confirmed a decrease in cell proliferation as well as activation of apoptosis following treatment with strawberry, suggesting regression of tumor in mice models (Somasagara et al. 2012). As previously occurred, rodent models of chemical carcinogenesis have provided essential information for better understanding of the biological, cellular and molecular disturbances involving the development of mammary gland neoplasm (Medina 2007). This model is similar to human mammary gland neoplasm from the standpoint of morphology, histology, molecular background and the biochemical markers expressed (Costa et al. 2002; Russo et al. 1990), and in its response to hormones, age, genetic factors (Shull 2007; Kubatka et al. 2002; Russo and Russo 1996; Russo et al. 2005), diet (Costa et al. 2004), etc., making this a highly valuable model for testing the effects of dietary components on breast cancer (Kumaraguruparan et al. 2007). 1.5. Aim of the study In the present study, the effect of a strawberry methanolic extract (Alba cultivar) and its anthocyanin-rich fraction on two different mice breast cancer cell lines has been evaluated. In particular, N202/1A and N202/1E cell lines were treated for 24, 48 and 72 hours with extracts of Alba cultivar and cell viability, apoptosis rate, ROS concentration, antioxidant activity and mitochondrial functionality have been evaluated. After that, a Western Blot analysis after treatment with methanolic extract for 48 hours was performed to detect the expression of different proteins related to several pathways that can be eligible for explaining the mechanism of action of strawberry treatment on cancer cells. Furthermore, an apoptosis assay after treatment with Compound C and strawberry methanolic extract was performed to better

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understand strawberry mechanism of action against cancer cells, including the AMP-activated protein kinase (AMPK) pathway. Before cell treatment, a prior characterization of 5 different strawberry cultivars was performed, evaluating the sensorial quality, the antioxidant capacity and the nutritional value through the detection of the main bioactive compounds they contained. The best cultivar resulting from this previous study was then selected for being tested on the two murine cancer cell lines.

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2. Experimental design In this three-years PhD course, a project on the evaluation of strawberry treatment on two different lines of murine breast cancer cells has been organized and applied. In the first year, a preliminary evaluation of strawberry sensorial and nutritional value was performed. In particular, total sugar content, total acidity, firmness, total antioxidant capacity (TAC), total phenolic content (TPH), total anthocyanin content (ACY) and vit C content were measured in five different commercial varieties of strawberry: Adria, Alba, Cristina, Romina and Sveva. In the second year of the course, more in-depth and accurate analysis on fruits of the same varieties were conducted, to confirm the results obtained in the first year. The same sensorial and nutritional analysis were performed on these varieties, with the adding of the measure of total flavonoids content (TFC) and the measure of folate content. In this case, a new HPLC methodology was developed for the first time in order to identify and quantify what kind of folic acid-derivatives were present in the analyzed strawberries. Then, Alba cultivar was chosen for the cancer cell treatment, because it demonstrated the best sensorial and nutritional profile in both the years of study. In the same year, the study of the effect of strawberry treatment on two murine breast cancer cell lines was started. In particular, different concentrations of total methanolic extract and anthocyanin-rich extract of Alba cultivar were tested at different incubation time on N202/1A and N202/1E mouse breast cancer cells for the evaluation of cell viability, cell apoptosis rate, intracellular ROS concentration, antioxidant capacity and mitochondrial functionality. The two breast cancer cell lines were different for the expression of Her2 oncogene: N202/1A presented high levels of surface Her2 oncoprotein, while N202/1E presented low levels of surface Her2. Results obtained in the second year were of great importance for complete the analysis in the third and last year of the course. On the basis of the previous results, one concentration of the methanolic extract was chosen to perform the Western Blot analysis over the expression of several candidate genes for an attempt of the understanding of the mechanism of actions through which the strawberries act against the development of cancer cells. With the Western blot analysis, the expression of some genes related to the oxidative status, apoptosis, autophagy, mitochondrial functionality and in particular to AMPK pathway were evaluated. In particular, we believed that the AMPK pathway could be of central importance in the knowledge and explanation of the mechanism of action of strawberries in cancer. For this reason, a last analysis was conducted on the apoptotic rate of cancer cells treated with strawberry, Compound C and strawberry together with Compound C. Compound C is a 16

pharmacological compound with the important function of blocking the phosphorylation of AMPK and, as a consequence, to inactivate this protein. If our theory is right, the effect of strawberry should be similar to that of Compound C, and in particular of inhibition of AMPK phosphorylation, leading cancer cells to the same fate that they reach with the Compound C treatment.

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3. Part I: Strawberry nutritional quality as a tool for reaching healthy objectives The modification of the lifestyle in the modern society has underlined new crucial aspects from the nutritional point of view. What we call “food” is not only a simple instrument for satisfying the appetite of the consumer, but is becoming a central factor to which the human health is strictly related. The exaggerated or the inadequate food assumption could be one of the causes of the insurgence of several illness and diseases, and at the same time the careful choice of food can help to prevent or limit the insurgence of these pathologies (Testoni et al. 2004). The increasing interest for these high-nutritional foods stimulated the scientific interest towards the bioactive molecules of plant origin which seems to demonstrate a central role in the prevention of several diseases. In this context, fruits and vegetables represent the foods with the higher amount of bioactive compounds, demonstrating the best beneficial effects on human health. Among fruits, as previously stated, strawberry received in the last years an increasing attention and a growing number of scientific evidences demonstrated how a short- or a long-term assumption of strawberries could be beneficial for the consumers (Baruzzi et al. 1998). The nutritional properties of strawberry are depending on the amount and the profile of the bioactive and antioxidant compounds it contains (e.g. polyphenols, vitamins and folates). Several research studies, some of which at the D3A Department of Università Politecnica delle Marche, have the final aim of obtaining strawberry fruits with high nutritional quality, that correspond to high concentrations of antioxidant compounds in the fruits. In this context, the programs of genetic improvement represent one of the main instrument for the realization of this objective, and the breeding of cultivated germplasm with wild germplasm seems to be a solution that can allow to obtain fruits with good commercial characteristics and with an high amount of bioactive compounds. 3.1. Origin and evolution The name Fragaria vesca, attributed to the wild strawberry, derived from the Latin Fragus, that means fragrance, aroma, and Vescus that denotes what is soft. This small drop of red nectar was already known and appreciated by prehistoric man, as evidenced by some artifacts found in mountainous and lakeside areas in Central Western Europe. In the Bible, in fairy tales, in the mythology and in some of the oldest treatises on medicine and botany, there are praises and commendations of this delicious fruit. This plant was not subjected to cultivation by the Greeks nor by the Romans, since any memory reported this aspect. It was then known as spontaneous fruit plant, typical of forested areas, but was not regarded as food. Also, no 18

notes were found on the consumption of these fruits in the most important treatises on Latin cuisine, nor in the writings of Greek medicine; similarly, this plant was not found in the lists of crops and agriculture, nor in the volumes of the four Latin writers Cato, Varro, Columella and Palladio. It seems, however, that the Ancient Romans loved this wild fruit, so that in the Middle Ages the strawberry became the symbol of temptation. The mythology recounts that it was often found on the tables of the Romans, especially during festivals in honor of Adonis, at the death of which, as the legend of Ovid tells, Venus, goddess of love, wept copious tears that arrived on earth and were transformed into small and juicy red fruits with heart form: the strawberries (Roudeillac and Veschambre 1987)! Before 1600 a.d., the strawberry wasn’t recognized in a specific systematic or agronomic collocation yet. Only from the end of 1600, the strawberry began to be considered as an horticultural plant, and we can consider that its cultivation started from this point. The most important event that strongly influenced the development and the evolution of the cultivation of strawberry was the interest that the European farmers shown on the Chilean strawberry, the so called Fragaria chiloensis, instead of the spontaneous European species (F. vesca, F. moschata and F. viridis). The first researcher that deeply investigated this species of strawberry was the French Antoine Nicolas Duchesne (Andreotti et al. 2010). In his studies, Duchesne identified unisexual characters in both F. moschata and F.chiloensis plants. He possessed only female plants of F. chiloensis, so he placed these plants near male plants of F. moschata and obtained the first fruits that were not derived from male flowers of F. chiloensis (Andreotti et al. 2010). In this way, Duchesne was the first that used the intraspecific crossing for the propagation of strawberry, laying the foundations of the modern strawberry cultivation. In fact, the modern strawberry derives from the casual hybridization of F. virginiana plants from West United States with F. chiloensis plants from pacific coast of Chile, in 1776. The species so obtained was Fragaria x ananassa, and presented big fruits with perfectly germinable seeds that produced easy-pollination flowers (Andreotti et al. 2010). 3.2. Botanical characterization The strawberry is a herbaceous plant belonging to the Rosaceae family, Rosoideae subfamily, Fragaria gender. The strawberry is a perennial plant, and is often regarded as herbaceous. It consists of a root system, a short stem (rhizome or crown) and a leaf system. The collated roots originate from the crown, near the surface of the ground; their function is to absorb the nutrient compounds and also to storage the reserve substances, as well as the rhizome, which 19

generates other shoots with their own roots. The root system is constituted by primary and secondary roots in variable numbers depending on the species, the cultivar and the amplitude of the crown. Most of the roots are distributed around 30 cm under the ground level, but can go deeper in sandy soils and remain near the surface in clay soils. Leaves are usually three-lobed, oval-shaped, more or less elongated and toothed, placed on a petiole of variable length. Stolons derive from leaves axillary buds: they are stems creeping on the ground capable of giving new seedlings at each node. This type of propagation, taking place in a vegetative way, allows to obtain plants that are homogeneous with the same characteristics of the parent plant (Avanzato et al. 1991). The flowers of the strawberry plant are borne on inflorescences inserted on the stem and on the axillary buds of the leaves; these inflorescences are composed of a unique primary axis and many secondary axes ending with the flower. The strawberry varieties actually cultivated present a perfect flower (hermaphrodite), but there are still examples of imperfect flowers with only male or female organs. Each perfect flower is composed of i) a calix consisting of several sepals (5 or more), ii) a corolla formed by 5 or more petals, iii) many male organs (stamens) each of them constituted by a filament, of variable length, bearing the anthers that contain pollen, iiii) and a receptacle located internally to the crown bounded by stamens, where the female organs (pistil) are placed, each of them composed of an ovary containing an ovum, that will give rise to an achene when fertilized. In order to obtain fruits of regular shape is necessary that all the pistils are fertilized. When conditions are not favorable for pollination, a part of the pistils cannot been fertilized, giving rise to deformed or malformed fruits (Andreotti et al. 2010). The edible fruit in reality is a false fruit formed by the enlargement of the receptacle tissues. The infructescence possesses numerous achenes at the level of the outer surface, that constitute the real fruit which arise from the fertilization of the ovules contained in each pistil. The first fruit that reach maturation is the one originated from the inflorescence on the primary axis, and is generally larger in size but of more irregular shape than those of the secondary axes. The weight of the fruit is typical for the different cultivars and therefore extremely variable: it ranges from a few grams to about 160 grams. The color of the fruit is also influenced by the genotype: it ranges from light red to dark shades of red, with lighter areas sometimes. The fruit shape can be oblate, spherical, conical, oval, heart-shaped, spherical, sub-spherical, kidney-shaped, double cone, wedge and wedge-shaped elongated. The strawberry cultivars are generally classified on the basis of their ability to produce fruits during the year: 20

• floricanes: differentiation of flowers with a light period of less than twelve hours and with a sufficient thermoperiod from September until the occurrence of the first frost; production of big fruits once a year during the spring, in about four weeks; • primocanes: differentiation of flowers with a light period of more than fourteen hours and production of smaller fruits from spring until autumn. They are not widely used at the industrial level, but are utilized almost exclusively at family level for their slow reproduction; • day length neutral: differentiation of flower buds with various lighting conditions, provided that the thermoperiod is respected. Therefore, the strawberry harvest time depends on timing of fruit ripening and changes depending on the variety, the type of growing system (protected or open field) and the latitude at which the fruit is grown. On the basis of ripening stage instead, strawberries are recognized as early, intermediate, late and very late varieties. 3.3. The genetic heritage The various species of Fragaria differ each other from the genetic heritage point of view: the main difference is constituted by the ploidy level, i.e. by the number of chromosomes in the nucleus of eukaryotic cells. According to these differences, it is possible to distinguish about 16 species, with four different levels of ploidy (diploidy, tetraploidy, exaploidy and octoploidy) with a base number of chromosomes equal to 7. Depending on the ploidy level, it is therefore possible to distinguish: • Diploid Species → species characterized by an even number of morphologically identical chromosomes two by two (2n = 2x = 14 chromosomes) in the cell nucleus. Among the diploid species, it is possible to distinguish between F. vesca and F. viridis. 

F. vesca, or wild strawberry, is the species that has the highest global distribution. Its plants are erect and stolonant; they have very small leaves and hermaphrodite flowers. The fruits are small, hemispherical, red and very aromatic.



F. viridis is native of Europe and Western Asia. It has an high resistant fruit, so it possesses a high commercial value, and is very resilient to heat. The plants are poorly developed, with few stolons; the inflorescences are small and erect; the flowers are greenish-yellow color.

• Tetraploid Species → species characterized by a number of chromosomes equal to 28 at the cell nucleus level (2n = 4x = 28 chromosomes). The known tetraploid species are F. moupinensis and F.orientalis.

21



F. moupinensis is a spontaneous species founded in eastern Tibet and eastern China. The plants have short stolons and trifoliate leaves; the floral scapes typically carry two to four flowers; the fruit is small, round and of poor taste.



F. orientalis is a spontaneous species of the western region of Siberia, Mongolia, Macedonia and Korea. The plants are small, erect, with long and thin stolons, and with oval leaves. The inflorescences have large flowers, but reduced in number. The fruit is soft, faintly aromatic, and of variable shape from conical to rounded.

• Exaploid species → species characterized by 42 chromosomes at the nuclear level: 2n = 6x = 42 chromosomes. 

F. moschata is the only existing hexaploid species; it is widespread in northern and central Europe, up to Russia. The plant is dioecious, very vigorous, with broad leaves; inflorescences bear large flowers, unisexual in spontaneous varieties and hermaphrodites in those cultivated. The fruit is rather large, with dark red color and the aroma of muscat.

• Octoploid species → species characterized by 56 chromosomes in the nucleus: 2n = 8x = 56. The octoploid species, F. ovalis, F. chiloensis and F. virginiana, are characterized by bigsized fruits, and the hybrid cultivated species F. x ananassa was generated from their hybridization. 

F. chiloensis, as the name implies, it is native to Chile. Plants of this variety are usually dioecious, but plants monoecious and polygamous were also found. The leaves are large, dark green, slightly pubescent; the flowers are large, unisexual or, occasionally, hermaphrodites. The fruit is large with glass fitting, dark red with white flesh, a little spicy.



F. virginiana is widespread in North America; the plant is usually dioecious, rarely flourishing again, and has a sparse foliage with large, thin and pubescent leaves. The flowers are unisexual and give fruits of considerable size, although smaller than the F. chiloensis, rounded, pink or dark red, with white flesh and aromatic.



F. ovalis shows very similar characteristics to F. virginiana, so much so that some scholars even consider it as a subspecies of F. virginiana.

3.4. Strawberry in the world The strawberry is a widespread species all around the world, and it occupies a prominent role in leading fruit areal. Since 1980, world production has increased more than 50% (Baruzzi et al. 1998). 22

The 36% of strawberries are produced in the Americas and, in particular, in the United States, confirming this country as the first manufacturer in the world, followed by Spain (10%), Japan (8%), Italy, Korea and Poland (all with 6% ). The increase in world production is the result not only of a limited increase in the planted area, but even and especially of the intense research activity taking place in the scientific community on this species. In fact, many institutions are focusing their research on the strawberry fruit; these institutions are located in more than 50 countries, but most of them are located in Europe, on the Atlantic Coast of the United States and in California (Baruzzi et al. 1998). The global cultivation of the strawberry has experienced a downturn in 2002, but showed a subsequent recovery and finally a significant increase. Spain, for example, has consolidated its role as the second world largest producer, with an offer that currently exceeds the threshold of

300,000

t,

after

having

reduced

its

production

for

a

few

years

(http://agronotizie.imagelinenetwork.com/vivaismo-e-sementi/2008/03/10/il-mondo-dellafragola-la-fragola-nel-mondo/5032). 3.5. Strawberry in Italy The comparison between the 2012 and the 2013 strawberry campaign in Italy in terms of acreage devoted to the cultivation of strawberry, shows that the current season is in line with the previous one, which in turn had recorded an increase of 4% compared to the 2011 season. The Campania region is the main producing region of Italy, with its 890 dedicated hectares, followed by Veneto, Basilicata and Sicilia regions. With regard to the regional distribution of areas dedicated to the cultivation of strawberries, the comparison between the average value of the 2000-2002 period with that of the 2011-2013 period indicates that there was an overall increase for the regions of South Italy (Campania, Basilicata, Calabria and Sicilia) from 45% up to 54%. On the contrary, there has been a decline in the northern regions (EmiliaRomagna, Veneto, Piemonte, Trentino Alto Adige), from 46% down to 34%. In Campania, the region with the largest area devoted to the cultivation of the strawberry, the annual distribution of the amount collected in 2012 can be summarized as follows: 20% from February to March, 40% in April, 40% in May-June. Despite some fruit were slightly bleached because of cold and rain, in general there were strawberries with good size, color, consistency and quality. The average production has held steady at 37-39 ton/ha, but the average selling price of the fruit has been affected by competition from the foreign producers. In Basilicata, there has been a delay in the maturation of the fruit with a concentration of production in late April/early May, resulting in an average production of 31.1 ton/ha. In 23

Emilia Romagna, the quality and the quantity of the entire production were generally good, in particular for the production in the open field. In the greenhouses some production difficulties were recorded due to the presence of deformed fruits and to the floral abortions, determined from excessively high temperatures. The average production was of 33-34 ton/ha. In Veneto, almost all of the production is predominantly derived from greenhouses cultivations. The production quality was overall very good, with a high sugar content since the beginning of the selling season. There were no damages caused by misshapen and diseases. The average production has held steady at 24-26 tons/ha. Finally, one aspect to consider in the market of strawberry is the export of fruits to foreign countries. The 76% of Italian exports of strawberries in recent years has involved three countries: Germany with 44% of the total exports (average 2007-2011), Austria with 17% of the total export (average 2007-2011) and Switzerland with 16% of the total export (average 2007-2011). The strawberry export from Italy is highly concentrated in the second quarter of the year, from April to June (85% in 2011), followed by the first quarter with 11% (in 2011) and the third and fourth quarter, both with 2% (http://www.freshplaza.it/article/52080/I-datisulla-produzione-e-sul-commercio-di-fragole-in-Italia,-Spagna-e-Francia-per-lanno-2012-e2013). 3.6. The research and genetic improvement The genetic improvement (breeding) is one of biotechnological tools most commonly used, especially in Europe, for the production of fruits that possess certain qualitative and quantitative characteristics. The considerable variability and combining ability of this technology, favored by the octoploid genetic basis of strawberry, rendered the breeding research area a field of particular interest. The genetic improvement programs have often very general objectives, that are common to almost all countries, regardless of the area which is considered (Baruzzi et al. 1998). However, it may happen, and not too seldom, that some specific researches can be developed focusing on local characteristics for the obtaining of species with certain agronomic characteristics: for example, the new plants can perfectly adapt to a specific climate rather than a specific type of terrain, or that are resistant to a typical disease of a certain habitats rather than another. Today, the breeding process is a tool for the meeting of the needs of consumers, which are always more demanding, but is also involved in improving the nutritional quality of the strawberry, in particular regarding the content in bioactive compounds such as minerals, vitamins and polyphenols. The genetic improvement, for example, allows to increase the level 24

of antioxidant compounds in strawberries, and this could be an important opportunity to obtain a higher intake of antioxidants even when the consumption of fruit is low. So the genetic improvement today not only aims to select plants with high productivity, adequate resistance to diseases and insects, and perfect flower, but also tries to get a good texture, size and color of the fruit that is consistent with market demands; moreover, the need for qualitative and organoleptic parameters that satisfy the consumer, and a high nutritional value that increases their added value is required. 3.7. Sensorial quality If the purpose of genetic improvement in the past was to ensure a maximum yield to the producer, trying to obtain cultivars with agronomic and phytosanitary traits suitable for this purpose, today the research is looking toward a better quality product, from the point of view of the consumer, which is the final evaluator. The quality for the consumer includes three aspects: the appearance (uniformity of size, shape and color), the organoleptic qualities (taste and aroma), the nutritional characteristics (nutraceutical properties due to the presence of compounds with antioxidant activity). The sensorial analysis evaluates the flavor, bringing together the answers from the five senses evaluation and the popularity of the products. Trained specialists assess the products with sensory testing, evaluating both the overall enjoyment and each single attribute bound to touch (hardness-juiciness), to taste (sweetnessacidity) and to smell (aroma). Judgments will be recorded in a card, properly graded on a scale. The sensory responses are then converted into scores. The averages of the obtained results are used to build the quality profile, generally represented with star-like diagrams. Sweetness and aroma represent the most appreciated features in strawberries. The analytical methods for the quantification of the sweetness is the measurement of soluble solids, mainly consisting of sugars, while the total acidity, another attribute of great impact on the strawberry taste, is measured by titration. These tests are rapid and inexpensive, in contrast to those relating to the aroma, that are however very important, given the great importance of aromaticity in the strawberry acceptance. The aroma of strawberries consists of a combination of several chemical compounds included in the categories of esters, aldehydes, alcohols, furans and sulfur compounds. The aromatic composition of the current cultivars is determined by their genetic origin. The typical aroma, very difficult to find in the regularly consumed strawberries, is characteristic of wild strawberries, among which the most common is the F. vesca, with furaneol as the main volatile compound.

25

3.8. Nutritional Aspects The ripened fruit of strawberry is red on the outside while the interior has a color from white to dark red depending on the variety. The red color of the fruit is mainly due to anthocyanins, particularly the pelargonidin-3-glucoside. The shape and size of the fruit depends on the variety, environmental conditions, the technique of cultivation and the quality of soil. The fruits of plants of the Fragaria genus can be eaten fresh, preserved in jams or in spirit (and remained stable over time) and as processed products (juices, jellies, jams). The phytochemicals present in the strawberry determine its nutritional profile by giving different nutraceutical properties; the quantity and quality of these various substances affect the Nutritional Quality (QN) of the fruit and its preventive effect against many chronic diseases (Tulipani et al. 2011a). First, the content of vegetable fiber (1.6 g/100 g fresh weight (FW)) has a number of positive action on health: their satiating effect, as well as the contribution to the reduction of calorie intake in the diet (important to note that the strawberry is a food with a rather low calorie intake: a dose of about 100 g provides just 32 kcal), also allows for adjustment of the glycemic index, slowing the rate of digestion. This effect of improvement in glycemic control is further accentuated by the high concentration of fructose (> 50% of total sugars). To a lesser extent, strawberries are a natural source of essential fatty acids: in particular, the seeds are rich in unsaturated fatty acids (of which over 95% is represented by monounsaturated or MUFA). The high content of vit C (54 mg/100 g FW) makes the strawberry one of the main dietary sources of this compound and make it particularly important from the nutritional point of view. The presence of vit C is in fact a determining factor in the assessment of QN of the fruit and its concentration is a benchmark for comparison and genetic improvement of commercial varieties. Besides the presence of vit C, also the high concentration of folates (20-25 mg/100 g FW, one of the highest concentrations observed among fruits) assumes a preponderant role in the definition of QN. Despite the attention is directed mainly to the potential benefits of these two abundant compounds, other vitamins, albeit to a lesser extent, are present in strawberry, such as thiamine (B1), riboflavin (B12), niacin (B3), vitamin B6, vitamin K, vitamin A and vitamin E. The strawberry is also an excellent source of microelements, especially manganese: in fact, a portion of about 150 g of strawberry can provide more than 20% of the daily requirement; the same strawberry amount is able to cover the 5% of the required potassium (present in an amount of about 160 mg/100 g FW) and appears to be also a good source of iodine, magnesium, iron and phosphorus.

26

CHEMICAL COMPOSITION OF STRAWBERRY Strawberry nutrient (Fragaria vesca)

Amount

Water

(g) 90.5

Protein

(g) 0.9

Lipid

(g) 0.4

Sugars

(g) 5.3

Fiber

(g) 1.6 soluble 0.45 insoluble 1.13

Energy

(Kcal) 27

Sodium

(mg) 2

Potassium

(mg) 160

Iron

(mg) 0.8

Calcium

(mg) 35

Phosphorus

(mg) 28

Thiamin

(mg) 0.02

Riboflavin

(mg) 0.04

Niacin

(mg) 0.50

Folate

(mg) 20.0

Vit. A

(mg) Tr

Vit. C

(mg) 54

Tab. 1 Chemical composition of strawberry (Source INRAN). The data are expressed per 100 g of FW

In addition to traditional nutrients, strawberries are one of the richest dietary sources of phytochemicals known as non-nutritional compounds, represented in particular by the class of polyphenols (Mazzoni et al. 2013), that give the fruit color and aroma characteristic and have a high antioxidant power. 3.8.1. Folates The term folates refers to all derivatives of tetrahydrofolic acid, which are present in food in about 150 different chemical forms, and to the metabolically active forms in the human body (Giampieri et al. 2012b).

27

These compounds, belonging to the water-soluble vitamins of the B group, differ one to another in three points inside the molecule: i) the state of reduction of the pteridinic ring, ii) the type of mono carbonaceous unit bound to it, iii) and the number of glutamic acid residues. In many foods, folates are present in conjugated form to one or more residues of glutamic acid (Bates and Heseker 1994). The essential biological function of folate is to act as a coenzyme in transferring one-carbon units to a variety of target molecules. In particular folates act as acceptors and/or donors of one-carbon units in two fundamental biological processes: • the synthesis of nucleic acids (eg. Purine synthesis); • the re-methylation of homocysteine to methionine. Folate is particularly important to prevent the onset of diseases related to their deficiency, such as megaloblastic anemia and some types of cancer. Their assumption is also strongly recommended for women of childbearing age to prevent the tragic consequences that can be recorded in the fetus in the early stages of pregnancy in the case of a deficiency of this compound (Giampieri et al. 2012b). Together with vit C, folates play a key role in enhancing the nutritional quality of strawberries considering that, among fruits, they are one of the richest natural resources of these essential micronutrients. However, there are still few studies focused on the evaluation of folate content in strawberries, their stability during storage, as well as their maintenance after the processing of the fruit. Based on the available data, the dietary intake of folate by eating strawberries is high, since 250 g of strawberries provide 30% of the recommended daily allowance (RDA) (Giampieri et al. 2012b). 3.8.2. Vit C Vit C is found in high quantities in the strawberry, making it one of the fruits with the highest concentration (even more than citrus fruits such as lemon and orange); a handful of strawberries may be able to meet in fact the recommended daily dose of vit C (Mazzoni et al. 2013). Sources of vit C

Amount

strawberries

54 mg

apricots

13 mg

oranges

50 mg

28

bananas

16 mg

cherries

11 mg

kiwi

85 mg

apples

5 mg

peaches

4 mg

Tab. 2 Contents of Strawberry vit.C (mg/100g of FW) compared to other fruits (Source INRAN)

The presence of this element gives to strawberry a high antioxidant power and a high nutritional value, and the new research lines in the agronomic field are pushing more and more towards the selection of varieties rich in vit C (Mazzoni et al. 2013). Vit C, also known as ascorbic acid, is a water soluble vitamin which has important biological functions in humans (Giampieri et al. 2012b). Firstly, it is a very reactive and effective antioxidant, which acts either directly or indirectly by neutralizing reactive oxygen species through enzymatic and not-enzimatic reactions (Giampieri et al. 2012b). Even when present in small amounts, it plays an important role in the protection of essential biomolecules in the human body (proteins, lipids, carbohydrates, DNA and RNA) from damage by free radicals, that are generated during normal metabolism or in pathological conditions or through exposure to toxins and pollutants (Giampieri et al. 2012b). Secondly, vit C is also required as an essential cofactor for the synthesis of collagen, hormones, and neurotransmitters. Furthermore, its presence seems to favor the activity of the immune system. In Europe, RDA has been recently revised and increased to 60 mg per day; it is important to note that the RDA is still calculated as the daily amount needed to prevent disorders related to a deficiency of vit C rather than the defense from chronic diseases or the improvement of the health status of the individual (Giampieri et al. 2012). 3.9. Phytochemicals in strawberries Polyphenols are a wide and heterogeneous class of non-nutritional bioactive compounds; recently, a great number of studies have been directed to the identification and quantification of phenolic compounds in strawberries, showing a relevant variation in the phytochemical content and, consequently, in the NQ of the fruits, depending on genetic and several pre- and postharvest factors. (Olsson et al. 2004a; Santos-Buelga and Scalbert 2000). Anyway, strawberry phenolics are well known for their antioxidant and anti-inflammatory actions, and possess directly and indirectly antimicrobial, anti-allergy and anti-hypertensive properties, as well as the capacity of inhibiting the activities of some physiological enzymes and receptors 29

(Alvarez-Suarez et al. 2011; Wang et al. 1996). Flavonoids (mainly anthocyanins, secondly flavonols and flavanols) are the most present in strawberry, followed by the hydrolysable tannins (ellagitannins and gallotannins) and minor constituents such as phenolic acids (hydroxybenzoic and hydroxycinnamic acid) and condensed tannins (proanthocyanidins) (Mazzoni et al. 2013). 3.9.1. Anthocyanin Interest in anthocyanins, the flavonoids that provide much of the flavor and color to strawberries, has increased immensely during the past decade as epidemiological evidence hints at their chemo-preventive activity (Wang and Stoner 2008). In earlier studies, it has been shown that anthocyanin and other phenolic compounds may exert their beneficial effects, including reducing the risk of cardiovascular diseases and cancers, through their antioxidant, antiinflammatory and chemoprotective properties (Mazza 2007; Wang and Stoner 2008). The exact mechanisms related to their beneficial effects are still uncertain; however, their ability to suppress proliferation (Li et al. 2009) and angiogenesis (Matsubara et al. 2005), in addition to the induction of cancer cell apoptosis (Shin et al. 2009) may have contributed to these positive effects. These compounds have also been reported to scavenge reactive oxygen species, inhibit in vitro low-density lipoprotein oxidation, prevent platelet aggregation and decrease serum lipids (Dell’Agli et al. 2004; Han et al. 2007; Liu et al. 2008). Few recent studies of anthocyanins have demonstrated a significant growth inhibition of some tumour cells including human colon cancer, human cervical carcinoma, human leukaemia, and prostate cancer cells (Wang et al. 1999; Kuo 1996; Agarwal 2000; McDougall et al. 2008). 3.9.2. Ellagitannins The ellagitannins (ETs), along with anthocyanins, are the most abundant phenolic compounds in strawberries. From the chemical point of view, the ETs are esters of glucose and hexahydroxydiphenic acid, and may occur in different structures: as monomers (such as ellagic acid glycosides), as oligomers (sanguiin H-6, the most common ET in strawberry) and as complex polymers. The ETs, along with gallotannins, constitute the group of hydrolysable tannins; following the hydrolysis reaction, these compounds release ellagic acid (EA) allowing the formation of different metabolites. Despite the ETs were often described as active principles in the plants, their content and their composition were analyzed only recently in foods. In strawberries, some studies have reported that the content of ETs is between 25 and 59 mg/100 g FW (Mazzoni et al. 2013). 30

3.9.3. Ellagic Acid Ellagic acid is the polyphenol present in higher concentration in strawberry, representing about 50% of the total phenolic content. The content of ellagic acid found in strawberries is around 39.6 and 52.2 mg/100 g FW and decreases during the maturation process of strawberry. Due to its antioxidant properties, in some countries ellagic acid is used as a food additive. Furthermore, ellagic acid is a strong anticancer molecule: in fact, in a recent American research on cancer, published in the journal International Journal of Cancer in 2008, the strawberries are among the best fruits and vegetables related to lower rates of death from cancer. The biological and pharmacological properties of ellagic acid are due, at least in part, to the ability to interact with biomolecules such as proteins and DNA. Its action has been demonstrated against cervical cancer as well as colon cancer cells. A diet rich in foods that contain high concentrations, such as strawberries, blackberries, raspberries and nuts, it is definitely recommended, even though the doses to which ellagic acid should be taken for exerting its real benefits are still unknown (Freedman et al. 2008). 3.9.4. P-coumaric acid The concentration of p-coumaric, an important antioxidant flavonoid, in strawberries increases up to full ripening of the fruit and can even reach values close to 6 mg/100 g of FW. The concentration of this compound also varies in different cultivars, with average values between 0.9 and 4.1 mg/100 g of FW (Mattila et al. 2006). 3.9.5. Other polyphenolic compounds In addition to the already mentioned compounds, the strawberry also contains other polyphenolic substances. The content in flavonols and their composition, for example, has been subjected to various studies in strawberry; flavonols derived from kaempferol and, especially, from quercetin have been identified: their average content is between 0.5 and 1.4 mg/100 g of FW. In some cultivars, also acylated flavonols such as quercetin-3malonylglucoside, kaempferol-3-malonylglucoside and kaempferol-coumaroylglucoside have been identified. The flavonols are the only class of flavonoids that do not occur naturally in the glycosylated form. In strawberries, they are present in monomeric form (such as flavan-3ols) or polymer, such as condensed tannins or procyanidins (PCs). PCs has often been identified in the pulp and in the achenes of the fruit, even if in small quantities. They have a number of physiological activities such as antioxidant properties (both direct and indirect), antimicrobial, anti-allergic, anti-hypertensive and inhibitory activity against some enzymes 31

and receptors. The content of flavan-3-ols ranges from 6 to 126 mg/g of fresh fruit (Heinonen et al. 1998; Maatta et al. 2004). Finally, phenolic acids occur as derivatives of hydroxycinnamic acid (i.e. caffeic acid) and hydroxybenzoic acid (i.e. gallic acid) (Giampieri et al. 2012a). 3.10.

Factors affecting the concentration of phytochemicals in strawberries

3.10.1. Genetic Factors The phenolic content and TAC in fruits and vegetables are subject to variations due to numerous genetic, environmental and technological factors, some of which can be controlled to optimize the QN. In particular, the genetic background has a very important role in the determination of QN in strawberries, since the content of micronutrients and phytochemicals can greatly vary among different cultivars. For example, in a study of Tulipani et al. nine different genotypes of strawberries were compared, and a difference of 1.8 times between the highest and the lowest value of the total content of polyphenols was detected (Tulipani et al. 2008b). This type of variability has also been observed by Scalzo et al. in a study comparing the TAC of wild strawberry (Fragaria vesca) with six different varieties of cultivated strawberry (Fragaria x ananassa) (Scalzo et al. 2005a). The results confirmed that the TAC, anthocyanins content, polyphenols content and the concentration of other substances that contribute to the antioxidant activity are strongly affected both by the species and the cultivars, also highlighting that the values of the wild species are about 2.5 times higher compared to the average values of the commercial cultivars. Moreover the study of Diamanti et al. has shown that the varieties deriving from local germplasm may be an important source of useful genes for improving the nutritional characteristics of the fruit (Diamanti et al. 2012a). The role of genetic resources and genetic improvement for the signing of new strawberry varieties that combine sensory and nutritional quality has been shown by the Polytechnic University of Marche through a long breeding program, including the assessment of progeny derived from combinations of inter-specific (F. x ananassa x F. virginiana glauca) and intraspecific (cultivated varieties) crossing (Mazzoni et al. 2013). 3.10.2. Environmental Factors The nutritional value of strawberries may also be affected by the climatic conditions of cultivation, such as soil type, exposure to sunlight and moisture levels, which can affect the amount of micronutrients and phytochemicals. In addition, it needs to consider the different applied cultivation methods in the evaluation of the polyphenol content and the TAC of fruit. 32

As shown in several studies (Lopes da Silva et al. 2007), the different varieties of strawberry in the different harvest years can cause considerable variability, especially in the concentrations of the main anthocyanins, which vary from 200 to 600 mg/kg FW. Thus, in evaluating the polyphenolic compounds and anthocyanins content as possible indicators of QN of the fruit, it is essential to take into account their variability, even within the same variety. There is also a relationship between the difficulty of plant growth and the amount of antioxidant molecules inside the fruit: in fact, the higher concentration of these compounds is found in fruits of wild species, which cannot easily find the nutrients, the micro-elements, the macro-elements and the water available for their development, differently from cropped strawberries, which are supported with fertilizers, manuring and watering at the right time. 3.10.3. Agronomic techniques A study from Nagy showed that low levels of vit C in fruits were related to the use of high levels of nitrogen fertilizer on crops, and potassium fertilization increased the ascorbic acid (AA) (Nagy 1980). Based on this report, it was found that nitrogen fertilizers, especially at high values, is capable to decrease the vit C in fruits. The nitrogen fertilization also increases the amount of foliage in the plant and this reduces the light intensity and the accumulation of AA in the shadowed parts. Since the overuse of nitrogenous fertilizers increases the concentration of NO3 and simultaneously decreases the level of vit C, thus it has a double negative effect on the quality of the fruit. Another agronomic technique that ensures high yields and productivity and a certain quality standard is the cultivation under tunnels (more or less wide) covered with plastic film. This system is recently widely used in Spain, and ensures the productivity aspect; however, it is negatively correlated to the presence of antioxidants, which are produced by the plant mainly as a defense against pests or any kind of stress (related to the climate, the environment, etc.); this culture system resets almost every type of stress for the plant, and the nutritional quality of the fruit will always be lower than the plants grown in the wild. 3.10.4. Ripening degree The polyphenolic composition of strawberries varies according to the ripening degree and the harvesting period which is considered. For example, in the pulp of unripe fruits there is usually a concentration of polyphenolic compounds and an antioxidant capacity greater than in a ripe fruit. In a recent study (Tulipani et al. 2011b), four cultivars in three different harvest times (unripe fruit green, pink fruits and ripe red fruits) have been compared and it was found 33

that the unripe strawberries of each genotype had a higher phenolic compounds content than the pink and the red fruits. In contrast, the levels of vit C can record an increase in dark red strawberries, although not always statistically significant. Even the anthocyanin profile varies in the course of maturation, but with an opposite tendency in respect to the other phenolic compounds. In fact, in all cultivars anthocyanins accumulated when the strawberry turns to red, and were found in much smaller quantities inside pink fruits, down to a total absence in the green fruits. Finally, also the antioxidant capacity changes during the ripening period and storage, with the same trend of the changes in phenolic compounds concentrations. As some studies confirm, in fact, the strawberry TAC gradually decreases during ripening; this reduction was related to the strong decrease of the tannins, while the slight increase in the concentration of polar non-phenolic antioxidants, such as vit C, cannot counteract this negative trend (Mazzoni et al. 2013). 3.10.5. Storage The short-term storage greatly influences both the QN and the profiles strawberries phytochemical compounds; the storage temperature seems to be one of the key factors in the modification of the stability of phenolic antioxidants in fruits during post-harvest. Some authors have studied the changes in anthocyanin and phenolic composition and antioxidant capacity of berries during storage (Tulipani et al. 2008c; Piljac-Zegarac and Samec 2011). In particular, it has been shown that the main effect of conservation (two days at 4 ° C and one day at room temperature, in the dark) in the analyzed cultivars was registered on folates concentration, which increased in all studied genotypes. Regarding the QN, the content of flavonoids is significantly higher in fruit after short term storage. In addition, the total phenolic and anthocyanins content in strawberries can increase with increasing temperatures and storage times. Kalt et al. have shown that strawberries stored for eight days at 30°C showed an increase of anthocyanins up to 6.8 times compared to those stored at lower temperatures (Kalt et al. 1999). Similarly, other authors have shown that maintaining the strawberries for 7 days at 10°C rather than at 0°C or 5°C, the amounts of both total phenols and anthocyanins were higher (Jin et al. 2011; Ayala-Zavala et al. 2004). The short-term storage affects in a positive manner also on the strawberry TAC, because the reactions that occur in the fruit during the post-harvest period can facilitate the formation of compounds with higher antioxidant capacity, even when characteristics such as taste and smell are particularly impaired. The longer the duration and temperature of storage and the greater the increase of the antioxidant capacity of the fruit. A high storage temperature (10°C) for seven 34

days or more, for example, seems to improve the TAC, although this high temperature not allows to obtain strawberries that are optimal for the commercial quality. From these studies resulted that the effect of storage practices may affect the chemical composition of the fruits and, therefore, the antioxidant capacity. These works suggested as a room or higher storage temperature, in particular of short duration, positively influences the phenolic metabolism, by increasing the antioxidant capacity and therefore, ameliorating the beneficial effect of these fruits in respect of human health. In these cases, however, the high storage temperature adversely affects the commercial characteristics of the fruit, as the smell and the taste, making them not pleasing to the consumer. The best solution is therefore to apply a refrigerated storage temperature, which keeps the flavor and aroma of the fruit also improving the nutritional quality (Alvarez Suarez et al. 2014). 3.11.

Importance of strawberry to health

The strawberries have been appreciated since ancient times for their therapeutic properties. The energy provided by these fruits is extremely low, since they are constituted for the 90% only by water. Their excellent nutritional profile is linked to their high concentrations of minerals and vitamins, in particular to the significant content of vit C and folic acid, and the high concentration and variety of phenolic compounds. The beneficial effects of strawberries are mainly due to the presence of antioxidants, that brake and retard the chemical weathering processes such as oxidation related to external factors like air, heat, light and the presence of metals. The international scientific community has been working for years on the increasing knowledge about the potential health benefits of strawberries: recent studies on the molecular nature of antioxidant compounds of these fruits have allowed to discover that they contain flavonoids such as catechin, quercetin, kaempferol and anthocyanins, but also polyphenols, such as ellagic acid and stilbenes, in particular resveratrol (Wang et al. 2007). In past decades, much attention has been paid to the TAC of fruit as an eligible parameter for quality and as an indicator of bioactive compounds present in foodstuffs and, therefore, of their healthfulness. This parameter is strictly correlated to the presence of efficient oxygen radical scavengers and strawberries are a rich source of some of the most powerful dietary antioxidants, such as vit C and polyphenols, especially anthocyanin and ellagic acid. Moreover, it should be keep in mind that even if the highest antioxidant potential of strawberries has been often proved, only recently it has been demonstrated that the high TAC of strawberries is strongly influenced by species and cultivar (Capocasa et al. 2008b; Tulipani et al. 2011b; Tulipani et al. 2008b; Tulipani et al. 2008a; Tulipani et al. 2009b). Several 35

studies suggest that strawberry phenolics possess a wide range of biological activities in the prevention of many chronic diseases, such as inflammation, oxidative stress, cardiovascular disease (CVD), certain types of cancers, type 2 diabetes and obesity. For instance, the dietary strawberry consumption can positively affect risk factors for CVD by inhibiting inflammation, improving endothelial function, inhibiting platelet aggregation, improving plasma lipid profile, modulating the eicosanoid metabolism, scavenging free radicals and increasing LDL resistance to oxidation (Basu et al. 2010; Mazza 2007; Youdim et al. 2000). There are several mechanisms by which strawberries exert these capacities, and they are not yet completely known. Certainly, anti-oxidation is one possible and relevant mechanism, since strawberry supplementation significantly decreases oxidative stress, by decreasing malondialdehyde formation, protecting LDL from oxidation and protecting mononuclear blood cells against increased DNA damage (Azzini et al. 2010; Tulipani et al. 2011a). Beside antioxidant effects, other mechanisms could also be involved in the health-promoting effects of strawberries. In fact, it has been found that the relatively long-term consumption of moderate amounts of mixed berries increased HDL cholesterol, reduced blood pressure and resulted in positive changes in platelet function, indicating that some of the constituents of berries, alone or in combination, play a role in the reduction of CVD risks at normal amounts (Erlund et al. 2008; Giongo et al. 2010; Alvarez-Suarez et al. 2014b). Moreover, the strawberry has also been recently investigated for its potential contribution to the dietary management of hyperglycemia linked to type 2 diabetes and to the related complications of hypertension. Regarding cancer prevention, some of the known chemopreventive agents present in berries include vitamins (vitamins A, C and E and folic acid), minerals such as calcium and selenium, dietary fibre, carotenoids, phytosterols such as sitosterol and stigmasterol, triterpene esters and phenolic compounds such as anthocyanins, flavonols, flavanols, proanthocyanidins, ETs, and phenolic acids (Duthie 2007; Seeram 2008). Several in vitro studies showed that strawberry phenolics may have anti-inflammatory effects (Wang et al. 2005), and suppress mutagenesis through antioxidative and genoprotective properties (Xue et al. 2001). Strawberry extracts also seem to modulate cell signalling in cancer cells and inhibited cell proliferation of several type of cancers (Zhang et al. 2008), inducing cell cycle arrest and apoptosis (Boivin et al. 2007; Seeram et al. 2006a), and suppressing tumour angiogenesis (Atalay et al. 2003). However, most of these exciting findings come from in vitro studies, so that new in vivo investigations focused in particular on patients with precancerous conditions are strongly aimed. Regarding ETs, only few investigations have been specifically focused on the bioavailability of ETs from whole strawberries. A recent 36

study investigated the metabolism of ETs from different dietary sources by evaluating the urinary excretion of ETs metabolite derivatives in humans after consumption of a single dose of strawberries, red raspberries, walnuts and oak-aged red wine. It is important to note that neither ellagitannins nor their metabolic derivates were detected in the urine samples after consumption of the four ET-containing foodstuffs, suggesting that the ET structure, or differences in the food matrix and other constituents of the food source, may contribute to differences in bioavailability (Giampieri et al. 2012a).

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4. Part I: Evaluation for strawberry nutritional quality and bioactive compounds contents. 4.1. Materials and methods 4.1.1. Plant material and sampling method The methods to evaluate strawberry fruit nutritional value were tested by analyzing fruits of 5 different varieties available in the experimental collections of Agricultural Faculty of Università Politecnica Marche (UNIVPM) located in Agugliano (AN), in central east of Italy (43°31'60" N - 13°22'60" E). Fruit samples from the selected varieties included in the germplasm collection of the experimental center, were hand-picked at the same day-time in different days, corresponding to the ripening times of the selected clones, from the second to the fourth picking, in the 2012 and 2013 cultivation years. Fruit samples were selected for homogenous fruit, avoiding unripe, wounded or shriveled fruits. The selected cultivars were 4 from Università Politecnica delle Marche license (Adria, Cristina, Romina and Sveva) and 1 from New Fruits license (Alba). At second, third and fourth harvest about 10 fruits were selected and pulled together to create homogenous fruit samples, of approximately 600 g each. Within 2 h after harvest, whole fruits were stored at -20°C before analyses. All the fruit samples have been analyzed at UNIVPM laboratory. 4.1.2. Extraction method For the evaluation of antioxidant capacity, total phenolic content, total anthocyanin content and total flavonoids content, the methanolic extract was prepared via homogenization. Frozen strawberries were thawed for 60 min at 4°C. Ten grams aliquots of the fruits were added to 100 mL of the extraction solution, consisting of methanol/milliQ water/concentrated formic acid (80:20:0.1 v/v), and fruits were homogenized using an Ultraturrax T25 homogeniser (Janke & Kunkel, IKA Labortechnik) at medium-high speed for 2 min. Extraction was maximized by stirring the suspension for 2 h in the dark at room temperature, then tubes were centrifuged at 3500 rpm for 15 min in two sequential times, to sediment solids. Supernatants were filtered through a 0.45 μm Minisart filter (PBI International), transferred to 5.0 ml amber glass vials and stored at –20°C until analysis. For vit C quantification, the extracting solution consisted in MilliQ water containing 5% meta-phosphoric acid and 1 mM EDTA. Vit C was extracted by sonication of 1 g of freeze strawberries in 4 ml of extracting solution, during 5 minutes, after a previous homogenization 38

using an Ultraturrax T25 homogenizer (Janke & Kunkel, IKA Labortechnik) at medium-high speed for 2 min. After the ultra-sound assisted extraction, the cell walls and proteins were precipitated by centrifugation at 2500 rpm for 10 min at 4°C, the surnatant was filtered through a 0.45 μm PTFE filter into 1.8 mL HPLC vials, and immediately analysed. For folate extraction, 5 g FW frozen strawberries were added to 15 ml of extraction buffer (0.1 M phosphate buffer containing 1.0% of L(+)-ascorbic acid (w/v) and 0.1% 2,3Dimercapto-1-propanol (v/v) at pH 6.5, freshly prepared) in a 50 ml plastic centrifuge tube, and homogenized using an Ultraturrax T25 homogenizer (Janke & Kunkel, IKA Labortechnik) at medium-high speed for 2 min. The capped tube was then placed on a water bath at 100°C for 10 min, and then rapid cooled on ice. Tubes were then centrifuged at 4696g for 20 min at 4°C. the supernatants were filled to an exact volume in 25 ml volumetric flasks with extraction buffer. For deconjugation of polyglutamylated folates, 175 μl of folate conjugase from rat serum was added to the 5 ml of extraction solution to another centrifuge tube, which was then incubated on a shaking oven at 37°C for 2h. Rat serum (10 ml) for folate conjugase was dialyzed in three steps (40 min each) by using 800 ml of 50 mM phosphate buffer, pH 6.1, containing 0.1% 2,3-Dimercapto-1-propanol in each step. The dialysis was performed with stirring at 4 °C. Folate conjugase activity was checked using PteGlu3 as substrate in 0.1 M phosphate buffer, pH 6.1, containing 1% sodium ascorbate at 37 °C as described by Pfeiffer et al. (Pfeiffer et al. 1997). Concentrations of PteGlu3 and produced folic acid were measured by means of UV detection at 290 nm. The dialyzed rat serum was stored at -80 °C. To avoid the possible adverse effects of refreezing and rethawing on enzyme activity, rat serum was frozen in small portions (0.5 ml). The enzyme activity was always checked prior to use (Patring et al. 2005). Then, an additional treatment of 5 min at 100°C was carried out to inactivate the enzyme, again followed by cooling on ice. The samples were then centrifuged again at 4696g for 20 min at 4°C. The final surnatant was then filtered through 0.45 μm filter pore size, 25 mm inner diameter, nylon disposable syringe filters, and the filtrates were purificated through solid-phase extraction (SPE) on strong anion-exchange (SAX) Isolute cartridges (3 ml/500 mg of quaternary amine N+, counter ion Cl-, Supelco, Bellefonte, PA) as described by Jastrebova et al. and Iniesta et al. (Jastrebova et al. 2003; Iniesta et al. 2009). The cartridges were conditioned by rinsing with methanol (2.5 ml, 2 times) and water (2.5 ml, 2 times), followed by purification buffer (0.01 M dibasic potassium phosphate containing 1% L(+)-ascorbic acid (w/v) at pH 7.0, 2.5 mL, 2 times). Aliquots (2.5 ml) of the sample extracts were applied to the cartridges and passed slowly with a flow rate not exceeding 1 drop/s. The elution of retained folates was performed slowly (flow rate not 39

exceeding 1 drop/s) with 0.1M sodium acetate containing 10% sodium chloride (w/v), 1% L(+)-ascorbic acid (w/v), and 0.1% 2,3-Dimercapto-1-propanol (v/v). The first portion (0.7 ml) of eluate was discarded, and the second portion (3.8 ml) was collected and injected in HPLC (Shohag et al. 2011). Anthocyanin extraction was performed as previously described (Alvarez-Suarez et al. 2011). Frozen strawberries (50 g) were homogenized in methanol containing 0.1% HCl, kept overnight (~14 h) at 3–5 °C and later filtered through a Büchner funnel under vacuum. The solid residue was exhaustively washed with methanol; the filtrates obtained were centrifuged (4000 x g, 15 min, 21 °C) and the solid residue further submitted to the same process for the number of times necessary to complete color extraction. The aqueous extract obtained was washed with n-hexane to remove liposoluble substances and then an aliquot (2 ml) of the aqueous phase was carefully deposited onto a C-18 SepPaks Vac 6cc cartridge (Waters). Sugars and more polar substances were removed by passing 15 ml of ultrapure water and anthocyanin pigments further eluted with 5 ml of methanol: 0.1% trifluoroacetic acid (95:5). 4.1.3. Sensorial quality Fruit Flesh Firmness was measured by using a hand-held penetrometer with an 8-10 mm piston, depending to the fruit. Soluble Sugars (SS) were determined using a hand-held refractometer and results are reported as °Brix. Total Acidity (TA) was determined from 10 ml of juice diluted with distilled water (1:2 v/v) and titrated with 0.1 N NaOH, to pH 8.2, and expressed in mEq of NaOH per 100 g of fruit. These quality parameters were studied on undamaged fruit samples, harvested at ripening stage (for each fruit type a proper ripening stage and parameter has to be identified to better standardize the sampling), including pooled fruit of at least 2 of the main harvest. 4.1.4. Total antioxidant capacity Three methods were used for the determination of the TAC of strawberry extracts: the Trolox Equivalent Antioxidant Capacity (TEAC), the Ferric Reducing Antioxidant Power (FRAP), and the DPPH free radical method. 4.1.4.1.

TEAC

TEAC assay consists in the quantification of strawberry extract free radical scavenging activity challenged against 2,2‟-azinobis-(3-ethylbenz-thiazoline-6-sulfonate) radical cation (ABTS·+) and compared to Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic 40

acid) as standard reference product (Re et al. 1999; Pellegrini et al. 1999). Briefly, 1 ml of the ABTS·+ radical solution is added to 10 μl of reagent, that can be ethanol for the blank of the standard curve, trolox solution for the standard curve, methanol:water 80:20 for the blank of the strawberry extracts, or strawberry extract for the analysis. After this step, the analysis solution must be vortexed for 20 seconds, and after 1-3 minutes must be red in the spectrophotometer at 734 nm, measuring the color inhibition of the ABTS·+ radical. The % of inhibition must be calculated following this formula: % color inhibition = (blank Abs – sample Abs)/blank Abs x 100 TEAC value will be determined comparing the % color inhibition with the standard calibration curve of trolox. The antioxidant capacity values are expressed as mmol Trolox equivalents/kg FW. 4.1.4.2.

FRAP

The FRAP assay was carried out according to the protocol proposed by Deighton and coworkers (Deighton et al. 2000), with slight modifications from the original method (Benzie and Strain 1996). The antioxidant capacity of the sample solution is determined by its ability to reduce ferric to ferrous ion. When iron is complexed with 2,4,6-tripyridyl-s-trizine (TPTZ) in sodium acetate solution at an acidic pH, its reduction results in a colour change of the solution, from pale rust to blue. The absorbance of the solution at 593 nm reflects the extent of reduction. The reducing power may be compared to that of the ferrous sulphate or of other aqueous antioxidants such as Trolox, often used as alternative standard. The FRAP reagent solution was daily prepared immediately prior to procedure, by combining ten volumes of sodium acetate (300 mM, pH 3.6) with one volume of TPTZ (10 mM in HCl 40 mM) and one volume of ferric chloride (20 mM) aqueous solutions. The sodium acetate and TPTZ solutions were prepared in advance and stored in (dark) glass bottles at room temperature, whereas the ferric chloride solution must be freshly prepared immediately prior to use; the day of analysis, the FRAP reagent is stable for at least 2 hours at room temperature. Blank for zeroing the spectrophotometer consisted in milliQ water, since interference from the extraction solvent was not suspected (interference doesn’t occur with methanol or ethanol/water when dilute). Briefly, 100 μL of alternatively blank, Trolox standard or 10-fold milliQ water diluted strawberry extract were added to 900 μL FRAP reagent into 1.5 ml eppendorfs. The mixture was then quickly vortexed for 15 seconds (starting the timer immediately) and allowed to stay until 4 min have passed. After exactly 4 min, the absorbance of the solution was read at 593 nm (Beckman spectrophotometer, DU644 model) against blank. Trolox aqueous dilutions 41

were used for calibration. Each sample was analyzed in eight replicates and FRAP results were expressed as plasma micromolar concentration (μM) of Trolox equivalents. 4.1.4.3.

DPPH

This assay is based on the ability of diphenilpicrilidazile (DPPH) to react with the phenolic compounds present in strawberry extracts. The DPPH radical is a persistent molecule, characterized by a violet color, which has an absorption peak at 515 nm. As in the case of ABTS, also here, when the radical species is added to the sample, a reaction of reduction is carried out in respect of the antioxidant species present; in this way a discoloration proportional to the antioxidant activity of the sample will be detected. For this reason, also in this assay, the antioxidant activity is expressed as percentage of decrease in absorbance at 515 nm of DPPH, in the presence of antioxidant molecules. This percentage is calculated by the equation: % inhibition A515= (1-Asample / Acontrol) x 100 The evaluation of the antioxidant activity is always made through the construction of a calibration line with the Trolox; from the values of reduction of absorbance (in %) of the samples, it is possible to go back to their unknown antioxidant ability, which will be expressed in terms of Trolox equivalents, i.e. mmol TXeq on kg of strawberries. The preparation of the solution of DPPH is required fresh made. To prepare 5 ml of 3 mM DPPH solution in methanol, weigh accurately about 0.00591 g of DPPH, to which are added 5 ml of methanol. The obtained solution is stirred with a vortex and is kept in the dark until the moment of its use. To perform the assay it is necessary to dilute the concentrated solution of DPPH with methanol, in a way that gives an absorbance of 0.6 to 0.7. At this point the samples are prepared in a test tube, putting 1.450 mL of DPPH diluted solution and 50 μl of sample, that could be water (for the control), strawberry extract, or Trolox solution (for the standards). All the resulting solutions are stirred by vortexing and left in the dark for 1 hour. Then samples absorbance are read at 515 nm against the blank. From the absorbance values obtained through the spectrophotometric reading at 515 nm for each sample analyzed the percent inhibition was then calculated. With the values of % inhibition obtained within various concentrations of the standard (Trolox), it has been constructed a calibration line, and comparing the samples results with the calibration line, it was possible to quantify the strawberry antioxidant capacity. 42

4.1.5. Total Phenolic Content The TPH of the hydrophilic extracts was determined using the Folin-Ciocalteu colorimetric method, as modified by Slinkard and Singleton (Slinkard and Singleton 1977). Briefly, 100 μL of alternatively blank (milliQ water), water diluted strawberry extracts or gallic acid standard solutions (1/10) were added to 500 μL of Folin-Ciocalteau reagent previously water diluted (1/10) and kept at 4°C, in the dark. The mixture was incubated for 1 to 8 min at room temperature, then 400 μL of 0.7 M sodium carbonate (CNa2O3) was added and the mixture vortexed well. The solution was incubated for 2 h at room temperature (~23°C), in the dark, then the specific absorbance at 760 nm was read, after zeroing the spectrophotometer with blank. A methanol:water (80:20, v/v) solution of 6 mM gallic acid (GA) was prepared, and stored at 4°C for at maximum one week. Serial standard dilutions (0.5 - 3.0 mM) were daily prepared from the stock solution, for quantifications. Calibration was obtained by plotting the known GA concentrations versus the corresponding absorbance760, and final results were expressed as milligrams of gallic acid equivalents per gram of fresh weight of strawberry (mg GAEq/g FW). Data were generally reported as a mean value ± standard deviation (SD). 4.1.6. Total Anthocyanin Content The total anthocyanin content of the hydroalcoholic strawberry extracts was determined using a modified pH differential method previously described (Giusti and Wrolstad 2001), with some modifications. The method relies on the reversible structural transformations of the anthocyanin chromophore as a function of pH, which are manifested by strikingly different absorbance spectra and thus can be measured using optical spectroscopy. On the basis of these anthocyanin-specific reactions, the pH-differential method permits accurate and rapid measurement of the total anthocyanins, even in the presence of polymerized degraded pigments and other interfering compounds. Briefly, 0.025 M potassium chloride (KCl) buffer, pH 1.0 (buffer 1), and 0.4 M sodium acetate buffer (CH3CO2Na), pH 4.5 (buffer 2) were prepared. Then, two dilutions of the strawberry extracts and of the standard solutions were prepared, one with buffer 1, pH 1.0 (1/10, v/v), and the other with buffer 2, pH 4.5 (1/10). These dilutions were let to equilibrate for 15 min, before measuring the absorbance of each dilution at 500 nm, i.e. the visible wavelength of maximum absorbance for pelargonidin-like anthocyanins (Aλvis-max = 500 nm), and at 700 nm (A700) to correct for haze, against a blank cell filled with milliQ water. The final absorbance of the diluted samples (A) was calculated as follows: A = (Aλvis-max– A700) pH 1.0 – (A λvis-max– A700) pH 4.5 43

A pelargonidin-3-glucoside (Pg-glc) stock solution was prepared by diluting 0.002 g of Pg-glc into 10 ml methanol:water 80:20, v/v (0.2 mg/ml). The stock solution was checked to be around pH = 4, then was aliquoted in amber glass vials and stored at –80°C until the analysis. Absorbance readings were converted to quantifications through a calibration curve, obtained by plotting known concentrations of Pg-glc serial dilutions (0.02 – 0.1 mg/ml) versus the corresponding absorbance calculated. Results were expressed as milligrams of Pg-glc equivalents per gram of fresh weight of strawberry [mg Pg-glcEq/g FW] ± SD. 4.1.7. Total Flavonoids Content Total flavonoid content was determined by using a colorimetric method described previously (Jia et al. 1998; Dewanto et al. 2002). Briefly, 250 μl of alternatively blank (water), strawberry hydrophylic extract (via homogenization) or (+)-Catechin standard solution was mixed to 1.25 ml of MilliQ water in a test tube, following by addition of 75 μl of a 5% sodium nitrate (NaNO2) solution. After 6 min, 150 μl of a 10% aluminium chloride hexahydrate (AlCl3 *6H2O) solution was added to the mixture, and allowed to stand for 5 min. Then, 500 μl 1M sodium hydroxide (NaOH) were added, the mixture was brought to 2,5 mL with MilliQ water and mixed well, and the absorbance was immediately read at 510 nm against blank. For quantitative results, from a methanol:water (80:20, v/v) stock solution of (+)-Catechin (1 mg/ml), serial standard dilutions were prepared (0.0125-0.1 mg/ml), and their known concentrations versus the corresponding absorbance were plotted. Results are expressed as mg of catechin equivalents per gram of fresh weight of strawberry [mg CEq/g FW] ± SD. 4.1.8. Vit C content Vit C (ascorbic acid) was measured as described by Helsper and co-workers (Helsper et al. 2003). Strawberry extracts were subjected to HPLC analysis immediately after the extraction procedure (via sonication). The HPLC system comprised a Jasco PU-2089 Plus controller, a Jasco UV-2070 Plus ultraviolet (UV) detector set at absorbances of 262 and 244 nm, and a column incubator at 30 °C. The HPLC column used was a Supelcosil LC8 150x4.6mm. The elution was isocratic with 50 mM potassium phosphate (KH2PO4) in MQ water, leading to pH 3.2 (below the pKa of the ascorbic acid) by adding orthophosphoric acid, and analysis consisted in a 10 minutes run, after which the column was cleaned with 50% acetonitrile. Vit C eluted at RT ≈ 5.3 min. Quantification of the vit C content was carried out through a calibration curve prepared by running standard concentrations of vit C similarly prepared in 44

respect to the extracts, and measured in duplicate at the beginning and the end of the analysis. Results are expressed as mg vit C per gram of fresh weight of strawberry [mg vit C/g FW] ± SD. 4.1.9. Folates identification and quantification The chromatographic separation method previously described by Patring et al. and Jastrebova et al. (Patring et al. 2005; Jastrebova et al. 2003) was used to determine individual folate from the previously described strawberry folate extract, with slight modification. An HPLC system (Jasco PU-2089 Plus) was used, consisted of a gradient binary pump, an UV detector (Jasco UV-2070 Plus), a fluorescence detector (FLD) (Jasco FP-2020 Plus), and a computer running ChromNAV software. The separation of folates was performed on a Mediterranea Sea18 250x4.6mm, at room temperature. The flow rate was 0.4 ml/min. The injection volume was 20 μl, with a total running time of 42 min. For the detection and quantification of folates, a FLD (excitation/emission = 290/360 nm for reduced folates and 360/460 nm for 10-HCOfolic acid) and an UV detector were used (290 nm). Peak purity and identity were confirmed by a comparison of relative peak areas in both detectors. The mobile phase was a binary gradient mixture of 30 mM potassium phosphate buffer at pH 2.3 and acetonitrile. The gradient started at 6% (v/v) acetonitrile and was maintained isocratically for the first 5 min. Thereafter, the acetonitrile content was raised linearly to 25% within 20 min and was kept constant for 2 min. Thereafter, it was decreased linearly to 6% acetonitrile for 1 min and was applied for 14 min to re-equilibrate the column. Quantification was based on an external standard method, in which the peak area was plotted against the concentration and the leastsquares regression analysis was used to fit lines to the data. Peak purity and identity were confirmed by a comparison of relative peak areas in both detectors. Results are expressed as mg of each folate per gram of fresh weight of strawberry [mg folate/g FW] ± SD. 4.1.10. Statistical analysis Statistical analyses were performed using STATISTICA software (Statsoft Inc., Tulsa, OK, USA). Data were subjected to one-way analysis of variance for mean comparison, and intergenotypes significant differences were calculated according to HSD Tukey’s multiple range test. Data are reported as mean ± SD. Correlations were calculated on a genotype mean basis, according to Pearson’s Test. Differences at p < 0.05 were considered statistically significant. All the analysis were performed in triplicate.

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4.2. Results Genetic background seemed to significantly affect all the studied parameters of fruit quality of the five strawberry clones analyzed. Figures below show the sensorial quality (Fig. 3), the TAC (Fig. 4) and the concentration of bioactive compounds, in particular phenolics, anthocyanins, flavonoids, vit C content (Fig. 5) and folate content (Fig. 7-8). 4.2.1. Sensorial quality The highest total sugar content was observed for the variety Romina (averaging 7.62 °Brix), followed by Cristina (7.52 °Brix) and Sveva (6.9 °Brix), that were statistically similar to Romina. The highest total acidity value was instead detected for Alba (13.15 meqNaOH/100g FW), Sveva (12.94 meqNaOH/100g FW) and Adria (11.30 meqNaOH/100g FW), while regarding the firmness, again Alba showed the highest value (4.77 hg/N), followed by Romina (4.66 hg/N). in respect to the higher values of each sensorial parameters, Adria showed the lower sugar content value (6,17°Brix), while Romina showed the lower acidity (10.38 meqNaOH/100g FW) and Cristina the lowest firmness (3.14 hg/N) (Fig. 3).

Fig. 3 Total sugar content, total acidity and firmness of the five strawberry genotypes tested. Data are expressed as mean values ± SD among the two fruiting seasons in study. Columns belonging to the same set of data with different superscript letters are significantly different (p