Corylus Avellana: a New Biotechnological Source of Anticancer Agents

Corylus avellana: a new biotechnological source of anticancer agents

Ana Gallego Palacios

TESI DOCTORAL UPF / 2015

Thesis Directors:

Dr. Elisabet Moyano Claramunt, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra.

Dr. Mercedes Bonfill Baldrich, Departament de Productes Naturals, Biologia Vegetal i Edafologia, Universitat de Barcelona.

DEPARTAMENT DE CIENCIES EXPERIMENTALS I DE LA SALUT

“Mira a la derecha y a la izquierda del tiempo y que tu corazón aprenda a estar tranquilo” Federico García Lorca

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Agraïments/Agradecimientos/Acknowledgement

En primer lugar quería agradecer a los organismos que han apoyado económicamente el trabajo plasmado en esta tesis, al Ministerio de Educación y Ciencia de España (BIO2011-29856-C02-1) y a Generalitat de Catalunya (2014SGR215). También a la Universidad Pompeu Fabra por proporcionarme la beca pre-doctoral que me ha permitido realizar este doctorado y la Universidad de Barcelona donde he realizado todo el trabajo experimental. Hay muchas personas que han contribuido, tanto directa como indirectamente, en este proyecto y a las cuales quiero agradecer. En primer lloc volia agrair a les meves directores de tesi, la Dr. Elisabeth Moyano i la Dr. Mercedes Bonfill per l’ajuda, la dedicació i el suport que m’han donat durant tots aquest anys. Eli he pogut aprendre molt de tu, no només a nivell científic sinó també a nivell personal. Gracies pel teu temps, pel teu positivisme i per les teves reflexions. A la Mercè perquè sempre has tingut la porta del teu despatx oberta per qualsevol dubte i perquè sempre m’has donat la llibertat i la independència necessària per millorar. Al Dr. Javier Palazón y a la Dr. Rosa Cusidó porque aunque oficialmente no son los directores de esta tesis han contribuido enormemente a ella, aconsejándome y ayudándome en todo momento. Especialmente al Dr. Palazón por valorarme y por todas las oportunidades que me ha ofrecido a nivel formativo, ya sea asistiendo a congresos o mediante estancias en diversas universidades. Todo ello, junto con el conocimiento que todos ellos me han aportado, me ha permitido aprender muchísimo y madurar

v como científica. También, gracias a todos ellos por el buen ambiente de trabajo que han creado durante todo este tiempo. During these years I had the chance work and collaborate with some departments around Europe and I would like to acknowledge to all of them. They kindly collaborate with us, and they help me so much to improved my knowledge: to Dr. Paul Christou and Dr Teresa Capell from the University of Lleida, to Dr. Yamal Ouazzani and Dr. Emilie Adelin from the CNRS (Centre national de la recherche scientifique, Gif-sur Yvette, France) to Dr Regine Eibl and Nicole Imseng from the University of Zurich and to Dr Isidoro Mentón and Dr. M Isabel V. Baanante from the University of Barcelona. All of them and all the students from their groups were really friendly and professionals and it was a pleasure for me works with them and learns from their experience. También quería agradecerle al Dr. Manuel Pastor su paciencia y ayuda con el diseño del experimento fraccional factorial y con los análisis estadísticos. A los servicios científicotécnicos de la UB, especialmente al Dr Isidre Casals, a la Dr. Olga Jáuregui, al Dr. Albert Adeva y al Dr. David Bellido por su apoyo. Me gustaría recalcar la ayuda de Olga, de la que he podido aprender mucho y la cual me ha enseñado la importancia de trabajar con seriedad y rigor, siendo siempre lo más crítica posible con todo aquello obtenido. Durante estos años he podido trabajar con muchas personas de todo el mundo, creándose un ámbito multicultural que me ha permitido aprender muchas culturas y abrir mi mente. Con muchos de ellos he podido compartir grandes momentos y experiencias como con Miriam, Karla, Rafa, Liliana, Diego, Raúl. Gracias por todo y por

vi todo lo que vendrá. También he tenido la oportunidad de trabajar con muchas personas que han hecho estancias en nuestro laboratorio. Algunos de ellos han colaborado activamente con esta tesis, como Kristina, Lucie, Victor, Xavi, David, Paula y Gemma. Con todos ellos tuve la posibilidad de trabajar mano a mano, formando un equipo y aprendiendo el uno del otro continuamente. Fuisteis de gran apoyo en los momentos difíciles. También hubieron otras personas que aunque no trabajaron directamente conmigo, amenizaron el trabajo y con los que también compartí grandes momentos: Oscar, Jannet, Dulce, Alejandra, Morteza, Ana Belen, Maryam, Esther, Mohamad, Nadia, Suellen, Marta, Maria, Ana Luisa, Farnoosh, Marta, Fran, Adrian, Andrea, Said, Arif, Burhan, Sara, Hajar, Virginie, Ozçag, Aida, Ali, David, Carmen…A Yaiza, a la que conocí en el laboratorio y se convirtió en una de mis mejores amigas y en la mejor compañera de piso que alguien puede tener. Por nuestras noches de terapia, nuestras cervezas de consuelo, por nuestra peculiar visión del mundo y por el gran apoyo que nos hemos dado mutuamente durante todos estos años. A los de arriba: Nati, Laura, Jean, Ying, Carmellina, Patricia, Olivia, Carolina, Lucia, Gabriela, Javier, Luciana, y a los de al lado: Kosta, Miren, Joan, Xavi, Marta, Ambra…porque siempre hemos sido una gran familia y hemos compartido muchas comidas, salidas y grandes charlas. A mis amigos de toda la vida Juana y Jordi, y a todo el grupo de querer es poder: Calero, Xavi, Javi, Laura, Joan, Adrià, Sandra, David, Iván y Diego. Siempre me habéis apoyado y animado, me habéis mantenido con los pies en la tierra alejándome de mi

vii pequeño mundo y haciéndome ver que siempre hay otro punto de vista en la vida. Gracias por todo lo que hemos compartido, y por todo aquello que vendrá. A Oscar porque es difícil que la vida nos junte, pero por mucho tiempo que pasemos sin vernos siempre tendremos una gran amistad. A les unifriends, perquè des del principi hem estat juntes i encara ho estem, tot i que algunes hagin marxat ben lluny. Pels nostres sopars japonesos, les nostres converses i el suport que sempre ens hem donat: Mireia, Monica, Miriam, Laura, Anna, Angela i Cris. Y por último, uno de los pilares más importantes de mi vida, a mi familia. Porque siempre habéis estado allí, en los momentos más felices y en los momentos más tristes. Siempre me habéis apoyado y aconsejado en todo. A mi padre porque siempre me has ayudado, apoyado y comprendido (aunque no siempre ha sido fácil), queriendo lo mejor para mí. A mi madre porque me ha guiado y ha hecho que hoy sea lo que soy, porque me enseñó a ser crítica y a luchar. A Raquel, Sergio y Eloy y a David, Mónica y Lucas porque siempre estáis ahí, porque pese a ser la freak de la familia, siempre me habéis apoyado y comprendido. Siempre contaremos los unos con los otros. También, a Laura por nuestras comidas de primas, por tu gran apoyo y comprensión y por nuestra complicidad. Siempre estaremos allí, la una para la otra.

A todos vosotros, y a muchos más, muchas gracias por haber contribuido a este periodo de mi vida, sin vosotros nada hubiese sido igual.

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Abstract

The difficulty of increasing taxane production and reducing its costs has prompted a search for new biotechnological sources. The unexpected discovery of taxanes in Corylus avellana has generated considerable interest in studying this plant and its derived cell cultures. We therefore focused this work on the study of cell suspension cultures of C. avellana as a new biotechnological approach to the production of taxol and related taxanes. With this goal, we optimized a scale-up process, the type of elicitor and moment of elicitation, cell culture growth, and an analytical technique to detect the compounds of interest. Also, C. avellana tree extracts were analyzed, leading to the identification of compounds with antiproliferative activity against cancer cell lines. These results provide new insights into cell suspension cultures of C. avellana and how to increase taxane production.

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Resum

La dificultat d’incrementar la producció de taxans i de reduir els seus costos ha promogut la cerca de noves fonts biotecnològiques. El fet de trobar taxol i taxans a Corylus avellana ha incrementat l’ interès en el seu estudi. Per aquesta raó, hem focalitzat aquest treball en trobar noves estratègies per la producció de taxans en suspensions cel·lulars de C. avellana. Amb aquest objectiu, hem optimitzat el procés d’escalat, l’elicitació i el creixement dels cultius, així com la tècnica analítica per identificar i quantificar els compostos d’interès. A més, hem estudiat diversos extractes de l’arbre, fet que ens ha permès trobar altres compostos amb activitat antiproliferativa en línies cel·lulars cancerígenes. Tots els resultats proporcionen noves eines pel cultiu de suspensions cel·lulars de C. avellana i per a l’increment de la producció de taxans.

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Index Pages Acknowledgments...... v-viii Abstract...... xi Resum...... xiii

Abbreviations…………………………………………. 19, 20 Justification and aims…………………………………. 23, 24 1. Introduction...... 27-58 1.1 Corylus avellana L………………………... 29-33 1.1.1 Botanical description…………….... 29-31 1.1.2 Nutritional profile...... 31, 32 1.1.3 Phytochemical profile...... 32, 33 1.2 Taxol and related taxanes...... 33-40 1.2.1 Mechanism of action...... 36,37 1.2.2 Difficulties in taxol production...... 37-40 1.3 Empirical approach to increase high-value secondary metabolites in plant culture...... 40-51 1.3.1 Significance of the medium composition in cell growth...... 42-44 1.3.2 Significance of the elicitation in secondary metabolite production..... 44-49 1.3.3 Scale-up...... 49-51 1.4 Improving taxane analytical detection...... 51-55 1.5 Drug discovery from medicinal plants...... 55-58

2. Chapter I: Development of a hazel cell culture- based paclitaxel and baccatin III production process on a benchtop scale ...... 61-74

3. Chapter II: Assessing factors that affect the growth of C. avellana cell suspension cultures: a statistical approach...... 77-110

4. Chapter III: Optimization of a liquid chromatography-tandem mass spectrometry method for the quantification of traces of taxanes in a Corylus avellana cell suspension medium………………………………………... 113-140

5. Chapter IV: Antiproliferative activity of C.avellana plant extracts in HeLA, HepG2 and MCF-7 cells…………………………………… 143-181

6. Discussion...... 185-199 7. Conclusions...... 201-204 8. References...... 205-223 9. Annex..….…………………………..………… 225-241 10. Appendix………………….…….……..……… 243-246

Abbreviations

µ : Specific growth rate. GDP: Guanosine-5'- 2,4D: 2,4- diphosphate Dichlorophenoxyacetic acid GTP: Guanosine-5'- ACN: Acetonitrile triphosphate

B: Baccatin III HgCl2: Mercury (II) chloride B5: Gamborg B5 medium HPLC: High performance BAP: 6-Benzylaminopurine liquid chromatography C.avellana: Corylus avellana HPLC-MS/MS: High CE: Collision energy performance liquid CF: Cephalomannine chromatography tandem mass Cor: Coronatine spectrometry CS: Chitosan HPLC-MS: High DAD: Diode Array Detector performance liquid DB: Deacetylbaccatin III chromatography mass DBP: Dibutyl phthalate spectrometry DCM: Dichloromethane IAA indole acetic acid DCW: Dry cell weight IC50: half maximal inhibitory DMSO: Dimethylsulfoxide concentration DT: Deacetyltaxol IP: propidium iodide DTX: Docetaxel IS: Internal standard DW: Dry weight JA: Jasmonates E: Extraction JAR1: jasmonate resistant1 ESI: Electrospray ionization JAZ: Jasmonate ZIM domain. EtOH: Ethanol Kin: kinetin FCW: Fresh cell weight FDA: Fluorescein diacetate

LC/MS/MS: Liquid PGR: Plant Growht regulator chromatography tandem mass PUFAs: Polyunsaturated fatty spectrometry acids LDL: low density proteins rcf:Relative centrifugarl force LOD: Limits of detection rpm: Revolurions per minute LOQ: Limits of RSD: Relative standard quantification deviation M: Maceration S/N: Signal to noise MeJA: Methyl jasmonate SA: Salicylic acid MeOD: Deuterated methanol SCF: Skip/Cullin/F-box MeOH: Methanol SD: Standard deviation MRM: Multiple-reaction T: Taxol monitoring Td: Doubling time MS: Murashige and Skoog Th.C: Theoretical MUFAs: Monounsaturated concentration fatty acids US: Ultrasounds MW: Molecular weight

NAA: 1-Naphthaleneacetic acid

NaClO: Sodium hypochlorite

NINJA: Novel interactor of

JAZ

NMR: Nuclear magnetic resonance

NT: Not treated pcv: packed cell volume

Justification and aims

Recent studies have suggested that taxol and taxanes can be produced by plants and organisms other than Taxus spp. Since the discovery of taxol in C. avellana and derived cell suspension cultures, it has been considered as a potential alternative source of taxol and taxane production. The focus of our work was therefore to develop a new biotechnological approach for the scaled up production of this anticancer agent or its semisynthetic precursors. Other factors that can affect growth were also assayed to increase productivity even further. As the work progressed, it became necessary to develop a reliable and sensitive analytical technique to precisely detect taxol and taxanes released into the culture medium. Additionally, it was proposed to screen for new compounds produced or accumulated in the C. avellana tree, with the possibility of finding new taxane-like compounds.

As a consequence, we defined 4 aims:

1. Improve taxane production in cell suspension cultures of C. avellana by elicitation with 100µM methyl jasmonate or 1 µM coronatine in a cell suspension culture, and scale up the optimized process to benchtop bioreactors.

2. Determine factors affecting growth of cell suspension cultures of C.avellana to establish the best conditions for their growth and increase the productivity.

3. Develop a reliable and sensitive analytical method to determine released taxanes in cell cultures of C.avellana.

4. Bioprospect C.avellana by bioassay-guided and characterization of new compounds with antiproliferative activity.

Introduction

1.1 Corylus avellana L

1.1.1 Botanical description

Corylus avellana L. is an angiosperm dicotyledonous plant from the Betulaceae family originary from Eurasia (Figure 1). It is a monoecious plant, self-incompatible and wind-pollinated. This family has other genera like Betula, Alnus, Carpinus and Ostrya which include trees and bushes. Some authors place Corylus as a separate family, Corylaceae, between Abedulaceae and Betulaceae, but recent molecular analysis reveals that Corylus belongs to the Betulaceae family (APGII system).

Kingdom Plantae Division Angiospermae Class Magnoliopsida Order Fagales Family Betulaceae Genus Corylus Species avellana Figure 1. Taxonomic description of C. avellana. The picture details the seeds and their involucre of C. avellana.

Corylus is widely spread around the world with around 18 species, among them C. americana (USA), C. avellana (Europe and Western Asia), C. cornuta (USA), C. maxima (South-West of Europa, South-East of Asia), C. jacquemontii (India), C. chinensis (China) and C. fenox (Himalaya, Tibet). According to the localization, there are differences in the involucre: while C.

americana and C. cavellana have an involucre of seed size (Figure 1), C. cornuta and C. maxima have an involucre longer than the seed, C. jaquemontii has a thorny and rigid involucre with glandular fuzz and C. fenox has a very thorny involucre similar to chestnut (Rieger, 2006; Kole, 2011). Taking into account the distribution of C. avellana around the world, it is possible to infer that C. avellana needs a temperate climate, and grows better in areas with high ratios of precipitation (Girona, 1987; Girona et al., 1994). For this reason, it is mainly found on the coast of the Black Sea, southern Europe and some areas of the United States. Turkey is the main hazelnut producing country in the world, providing 74% of the world production, followed by Italy, Spain and USA, and they are principally produced for human consumption (Shahidi and Alasalvar, 2008).

C. avellana is a deciduous bush or a small tree that can grow up to 8 meters. The bark is brown to red and smooth, with a tendency to crack and turn gray with age. Leaves measure between 5-10 cm, are coarse and hairy on both sides, with a double-serrate margin, from subobicular to oval shape. Leafs are alternate with a short petiole. Male flowers appear in groups of 2-3 green to yellow hanging aments (Figure 2A). The cylindrical aments are from 3 to 8 cm long and 5mm in diameter. Pollen measures between 18-26 microns and is trizonoporate, each pore measuring about 2 microns of diameter. In an equatorial view, the pollen is circular and isopolar but in a polar view it is angleperturate. Each bract from the aments has two bracteoles and the flowers have neither calyx nor corolla,

being reduced to one filament divided A in four branches with two stamens and with a hairy crown on the apex of the anthers. Female flowers are in clusters of 2 to 6 flowers hidden by the breacteas. The ovary is inferior and bicarpelar; only the red forked styles are visible, allowing the reception of the pollen (Figure 2B). Hazel trees B bloom from December to February and bear fruit from August to September. The hazelnut fruits are achenes with a brown pericarp that Figure 2. C. avellana surrounds a single seed, which is L. male flowers (A) and surrounded by an involucre with a female flowers (B, laciniate border (Rieger, 2006; Kole, author Petr Novotný). 2011).

1.1.2 Nutritional profile

As is well known, hazelnuts have long been used in the human diet. It was recently recognized as a heart-healthy food by the Food and Drug Administration (FDA) (Alasavar et al., 2006), and plays an important role in human nutrition. It is very healthy due to the presence of fat, carbohydrates, proteins, minerals, vitamins, amino acids and dietary fiber. Regarding their nutritional aspects, hazelnuts have been studied for their capacity to reduce plasma cholesterol, an anti-obesity effect and antioxidant effect. Hazelnut

fatty acid composition, with a high content of monounsaturated (MUFAs) and polyunsaturated (PUFAs) fatty acids, the presence of phytosterols and phytostanols, tocopherol and tocotrienol and the high levels of squalene bring health benefits related to the blood serum profile and protecting low density proteins (LDL) from oxidation and decreasing plasma oxidized LDL levels. Their MUFA and PUFA content is also related with a protective effect against ischaemic cardiovascular disease (Kris-Etherton et al., 2008). Also, it has been demonstrated that hazelnut supplementation can reduce oxidative stress, reducing injury induced by inflammatory diseases (Durak et al., 1999; Oliveira et al., 2008; Shahidi and Alasalvar, 2008).

1.1.3 Phytochemical profile

Hazelnuts have been widely studied for their nutritional components. It has also been demonstrated that the hazelnut tree is an important source of phytochemicals. Phytochemicals are defined as plant-derived compounds that have an effect on the human health, such as antioxidant, anti-inflammatory, anticancer, antimutagenic or antiproliferative effects. One of the most important groups of phytochemicals are phenolic compounds, extensively studied due to their antioxidant properties. It is possible to quantify phenolic compounds in seed skin, shell, leaves, involucre and seed. The content and type of phenolic acids vary depending on the tissue, the extraction methodology and the solvents used, but gallic acid, caffeic acid, p-coumaric acid, ferulic

acid and sinapic acid are the most important. Also, flavonoids like anthocyanidin, flavan-3-ol, proanthocyanidins and pytoestrogens have been detected in different tissues of C. avellana trees. These compounds are related with a reduction in the risk of cardiovascular diseases, cancer and stroke mainly due to their antioxidant properties (Shahidi and Alasalvar, 2008). Finally, Hoffman et al., 1998 found taxol and related taxanes in C. avellana bark, leaves, limbs, shells and seed. They were analyzing methanol extracts with the aim of finding compounds that give resistance to Eastern Filbert blight (C. avellana disease caused by a fungus, Anisogramma anomala), but when they analyzed the MS pattern of the compounds, some peaks seemed to correspond to taxol and taxol fragments. After the analysis of different hazelnuts, they confirmed the presence of taxol in C. avellana, although variations in the accumulation were observed regarding the cultivar analyzed and the season. They cultivated and indentified endophytes from the most productive tree, C. avellana Gasaway, and some fungi were also found to be taxol producers.

1.2 Taxol and related taxanes

Taxanes, and particularly taxol, are phytochemicals with high added value. Since their discovery in 1967 in Taxus brevifolia (Wani et al., 1971), more than 500 natural taxanes have been characterized in different Taxus spp. (Wang et al., 2011), as well as a huge number of synthetic taxol analogues (Yared et al., 2012). Taxanes have an

Figure 3. Structure of taxol. important effect against several types of human cancer, including ovarian, breast, head, neck and small cell and non-small cell lung cancers, AIDS-related Kaposi’s sarcoma, lymphoma, prostate cancer, gastric cancer, and bladder cancer, among others (McGuire et al., 1989; Holmes et al., 1991; Saville et al., 1995; Eisenhauer et al., 1998). Taxol is a diterpene alkaloid chemically named tax-11-en-9-one, 5β, 20-epoxy-1,2α, 4, 7β, 10β, 13α-hexahydroxy-4,10-diacetate-2- benzoate-(α-phenylhippurate), its molecular weight is 853.9 and its molecular formula is C47H51NO14 (Figure 3). Taxol is characterized by the taxane skeleton, consisting in three aromatic rings substituted with two hydroxyl groups (C1, C7), a benzoyl group (C2), two acetoxy groups (C4, C10), an oxetane ring (C4-C5) and the lateral chain (C13), giving to the structure a total of eleven stereocenters (Kingston et al., 1993). Biological activity is carried out principally by the lateral chain, the oxetane ring and the benzoyl group, since modification of these parts reduces the activity of this compound (Kingston, 2000). While the taxane skeleton is always constant, 34

modifications in the functional groups and in the lateral chain have been described, leading to the identification of a huge number of taxanes. However, the most commercially important taxanes are baccatin III, deacetylbaccatin III, 10-deacetyltaxol and cephalomannine (Figure 4). Their importance lies in the possibility of using them in semi-synthesis processes to produce taxol or other highly active taxanes.

O O OH O OH HO O

O O H O H O HO O O HO HO HO O O O O

Baccatin III 10-Deacetylbaccatin III

O OH O O OH HO O O

O O NH O NH O O H H O O O O O HO O H O OH O O H O O O

10-Deacetyltaxol Cephalomannine Figure 4. Structures of the most commercially important taxanes: baccatin III, 10-deacetylbaccatin III, 10-deacetyltaxol and cephalomannine.

1.2.1 Mechanism of action

Taxol is classified as a microtubule-interfering agent because it is able to bind to the microtubules and block the cell cycle. Microtubules are intracellular filaments constituted by heterodimers of α- tubulin and β- tubulin. Heterodimers polymerize longitudinally in a specific head-to-tail orientation to form protofilaments. 13 protofilaments associated laterally are necessary to assembly a microtubule. α- tubulin is placed always on the + end (polymerization end), and β- tubulin on the –end (depolymerization end). Microtubules are essential for the cytoskeleton machinery, the metabolism, the cell division and the cell morphology (Schiff et al., 1979; Alushin et al., 2014).

Tubulin dimmers and microtubules are in dynamic equilibrium, which means that a continuous depolymerization and repolymerization is necessary, a process known as dynamic instability, to ensure a correct function. Dynamic instability is carried out by the binding and hydrolysis of GTP to the tubulin dimmer. Bound GTP is incorporated at the + end, contributing to the polymerization, but when GTP is hydrolyzed to GDP, the tubulin conformation changes and depolymerization is carried out. Taxol is able to join to a specific region in the β-tubulin, on the interior lumen of the microtubule, blocking the depolymerization and promoting the polymerization, even in the absence of the GTP. Therefore, highly stable microtubules are formed, leading to the stopping of the cell cycle in the metaphase-anaphase transition (Figure 5) (Schiff et al., 1979; Gornstein and Schwarz 2014).

+ end

α Microtubules β

Tubulin Tubulin - end dimmers protofilaments

Taxol

Figure 5. Microtubule formation and taxol mechanism of action.

1.2.2 Difficulties in taxol production

Cancer is a term that encompasses a huge number of diseases that can involve any part of the body. It is characterized by an uncontrolled growth of abnormal cells in a definite point (tumor), which can colonize other tissues and organs (metastasis). Metastasis is one of the major causes of death associated with cancer (World Health Organization [WHO], 2010). Cancer risk is associated with a certain genetic background and external agents and is a leading cause of death worldwide, causing about 8.2 million deaths in 2012 (Globocan, 2012). It is expected that annual cancer cases will increase to 22 million in the next 20 years.

Taxol, as described before, is one of the most interesting natural anticancer drugs, due to its wide spectrum of antineoplastic action. Its use is restricted because of a limited supply from its natural source, the inner bark of Taxus spp. Only one cancer treatment

requires about 2.5-3 grams of taxol, consequently it is necessary to harvest the inner bark of eight 60-year-old yews (Bedi et al., 1994). As the global demand for taxol is now higher than 10 years ago, but its natural extraction is still not efficient, alternative sources to satisfy the demand for this compound are being sought. These include total synthesis (economically unfeasible because of its highly complex structure and specific stereochemical requirements), semi-synthetic processes from more abundant taxanes (such as baccatin III, 10 deacetylbaccatin III, 10 deacetyltaxol and cephalomannine) (Wang et al., 2011), and biotechnological production using cell suspension cultures of Taxus spp. Biotechnological production using cell cultures of Taxus spp. have been successfully developed for large-scale paclitaxel production, even though complex and specialized techniques, with long incubation times, are required (Cusido et al., 2010).

Scientists all over the world are currently researching new ways to obtain taxol at an industrial level. Besides C. avellana, other alternative sources of taxane production, fungi and bacteria are also a focus of interest. In 1993, it was shown that the endophytic fungus Taxomyces andreanae found in T. bevifolia was able to produce taxol (Stierle et al., 1993). Since then, more than 40 fungi able to produce taxol have been described, the majority of them endophytic fungi from Taxus spp. and including different genera like Pestalotipsis, Pestalotia, Sporormia, Trichothecium, Tubercularia, Seimatoanthlerium, Alternaria, Penicillium, Fusarium, among others (Flores-Bustamante et al., 2010). A high variation in the quantitative data is observed regarding the concentrations. A range 38

between 0.001 ng/mL in Colletotrichum spp. (Strobel et al., 1999) to 800 ng/mL in C. cladosporioides (Zhang et al., 2009) was reported. On the other hand, several genera of bacteria have also been reported as paclitaxel producers, including Pantoea spp., bacillus spp., Curtobacterium spp., Sphingomonas, Bacillus spp., Erwinia taxi, Kitasatospora sp. and Streptomyces sp. (Page and Landry, 1996; Page et al., 2000; Caruso et al., 2000). The concentrations reported ranged between 0.01 ng/mL in Streptomyces spp. (Caruso et al., 2000) and 25 ng/mL in B. Subtilis, Pantoea spp. and Curtobacterium spp. (Page et al., 2000).

All these findings are now being called into question, due to highly inconsistent information, mainly regarding the existence of the secondary metabolite pathways in bacteria and fungi. Heinig et al., 2013, postulated that multiple horizontal gene transfer is difficult and a rare evolutionary event. They isolated different endophytic fungi from Taxus spp., corroborating taxane production by CIEIA (competitive inhibition enzyme inmunoassay) and LC/MS/MS in two strains, but all the molecular assays carried out to obtain genes homologous to the taxol pathway in Taxus spp. failed. It was concluded that taxanes, being highly lipophilic compounds, can accumulate in endophyte cell wall structures. This finding was supported by the fact that after the second subculture no taxanes were detected by LC-MS/MS. Moreover, Sachin et al., 2013, also reviewed that no STR gene (Strictosidinne synthase, an important gene in the alkaloid pathway) is found in any fungus species, but they hypothesized the presence of extra-chromosomal elements (in plasmid or endohypal bacteria) in the fungal cytoplasma. 39

Following this line, several studies have demonstrated that Saccharomyces cerevisiae and Escherichia coli can be used to produce taxol and metabolic intermediates by genetic engineering, introducing sets of genes involved in this pathway (DeJong et al., 2006; Ajikumar et al., 2010; Ding et al., 2014; Lv et al., 2014). At the moment, only a few enzymes have been functionally expressed in yeast. Some of them are related with the production of isoprene in Saccharomyces cerevisiae and Escherichia coli (Lv et al., 2014; Ajikumar et al., 2010). Others are related with the production of taxadiene, the first committed taxol intermediate, obtaining a concentration of 1 mg/L in Escherichia coli (Ajikuar et al., 2010) and 72 mg/L in Saccharomyces cerevisiae (Ding et al., 2014). Fermentation as a potential source of taxol is a very important biotechnological challenge, but the lack of availability of a complete set of genes involved in paclitaxel biosynthesis is at present a limiting factor in microorganism production (Flores- Bustamante et al., 2010).

1.3 Empirical approach to increasing high-value secondary metabolites in plant cultures

In vitro cultures are considered as a reasonable source of high-value secondary metabolites, allowing a sustainable production that is not restricted by the low yields of natural extraction or by the high cost associated with chemical synthesis (Wilson and Roberts, 2012). Biotechnology can exploit the cell, tissue, organ or the entire plant, and culture them to obtain the desired compounds. These cultures

are independent of geographical/season variations, are politically independent, the production is fast, downstream processes and recoveries are more efficient than in the plant, it is possible to obtain compounds that are not commonly found in the plant, and it is possible to develop a defined process to produce compounds in a continuous and uniform way ensuring quality and high yields (Orhan, 2012). As mentioned before, the growing commercial importance of paclitaxel and taxanes has created great interest in enhancing their production through biotechnological systems.

Under specific culture conditions, stems, leaves and seeds can undergo dedifferentiation to form calli, and cell suspension cultures can be obtained by the desegregation of the calli. This process is usually achieved by transferring a sterile plant explant onto an appropriate solid medium with a determinate ratio of plant growth regulators (PGR) and incubated at 25 ºC in darkness. This process can be easily carried out in a few steps with a model plant, but it can be difficult for recalcitrant plant species. It is therefore necessary to evaluate different sterilization processes, culture media and plant growth regulators at various concentrations to obtain plant dedifferentation. In the first reported study using a C. avellana cell suspension culture (Bestoso et al., 2006), calli were successfully induced from leaves, seeds and stems in MS medium supplemented with different PGR, concluding that 2,4-dichlorophenoxyacetic acid (2,4D), used alone or in combination with benzyladenine (BAP), is the best way of inducing callus formation in hazelnut explants. Callus induction from seeds was also high using naphtalene acetic acid (NAA) in combination with BAP. Highly variable calli were

obtained when different explants and PGR combinations were used, but seeds produced white and friable calli that could be maintained and subcultured over long periods of time. Once a cell suspension culture is obtained, the next step is to increase the yields of the desired secondary metabolites. Usually, the methods are based on empirical approaches and involve the study of culture parameters such as screening for fast growing/highly productive cell lines, cell immobilization, media optimization, elicitors, membrane permeation and scaling up the process (Onrubia et al., 2013a).

1.3.1 The significance of medium composition in cell growth

Nutrients provided to the culture medium are one of the most important parameters for obtaining a highly efficient cell suspension culture, characterized by a higher growth ratio and a higher secondary metabolite production. Plant cell culture media include basal medium (with a specific concentration of macronutrients, micronutrients and vitamins), carbon source (usually sucrose), and plant growth regulators (auxins and cytokinins). Regarding the basal medium, most plant cell cultures grow well in MS medium (Murashige and Skoog, 1973), but due to the growing interest in developing new plant cell cultures and an increase in the study of the plant cell requirements, different basal media have been developed: MS medium variations, B5 medium (Gamborg B5 medium, Gamborg et al., 1968), B5 medium variations, Kao and Michayluk medium (Kao and Michayluk, 1975), Litvay medium 42

(Litvay et al., 1985), ER (Eriksson medium; Erikson, 1965), among others. There are several contradictory indications regarding the optimum carbon source and PGR used, even with different cell lines from the same species. The plant cell culture medium is usually supplemented with a single simple sugar or with a combination of simple sugars (sucrose, glucose, fructose, maltose), but depending on the species considered there are differences in the type and concentration used (Gertlowski and Pettersen et al., 1993; Oksman- Caldentey et al., 1994; Chattopadhyay et al., 2003; Nagella and Murthy, 2011). PGR are crucial factors in cell growth and metabolite production. The types and concentrations of auxins (like Indole acetic acid [IAA], 2,4D or NAA) and cytokinins (like Kinetin [Kin], Bap or Zeatin [Zea]), or the ratio between them, strictly control plant cell growth and division. Therefore, plant cell growth and secondary metabolite production are strongly influenced by modifications in the carbon source, basal medium and PGR, making it possible to increase them by an optimization of the culture medium.

The literature reveals differences in the type of PGR, their concentration, and even the basal medium used to culture cell suspensions of C. avellana. MS supplemented with 2.4D (4 - 0.5 mg/L) and BAP (4 - 0.5 mg/L) has been widely used to culture cell suspension cultures of C. avellana (Bestoso et al., 2006; Rezaei et al., 2011, 2013; Qaderi et al., 2013), but MS supplemented with NAA (3 mg/L) and IAA (3 mg/L) (Safari et al., 2012), and B5 modified medium with 0.2 mg/L BAP and 1.86 NAA (Bemani et al., 2013) have also been reported. In all cases, the carbon source

was sucrose, usually at 20-30 g/L. Taking into account all the data, a media optimization study was mandatory to determine the best conditions to grow the cells. When optimizing culture conditions, the different parameters can be analyzed following two basic strategies: either modifying one factor at a time, or using fractional factorial designs that allow different variables to be studied in one run, thereby reducing both cost and time. The aim of our work was the study of 2 basal media (B5 and MS), 2 carbon sources (30 g/L sucrose, and sucrose plus fructose at 5 g/L each), 2 types of auxins (2,4D and NAA), 2 types of cytokinins (KIN and BAP) and 2 amounts of PGR (2mg/L or 1 mg/L auxins and 1mg/L or 0.5 mg/L cytokinins).

1.3.2 The significance of elicitation in secondary metabolite production

The use of elicitors with the aim of increasing secondary metabolite production in plant cell cultures has been extensively described (Sevon, 1997; Lin et al., 2001; Kim et al., 2002; Palazon et al., 2003; Bentebibel et al., 2005; Expósito et al., 2010; Onrubia et al., 2010, 2013b). Elicitors are biotic or abiotic factors that can stimulate different signaling pathways that lead to an increase in the production of phytoalexins (generally secondary metabolites). Examples of abiotic stress include light, pH, and osmotic stress, while biotic stress can be applied with methyl jasmonate (MeJA) salicylic acid (SA), coronatine (Cor) or wall fungus extracts, all of which have proven to be effective in increasing secondary metabolism in different plant cell cultures. Elicitation is an effective 44

strategy, but the response of cell cultures to elicitation depends not only on factors related to the elicitor itself (such as type, concentration, duration of elicitation, etc.) but also on the plant species, cell line and state of development of the cell culture (Namdeo, 2007). Previous studies in Taxus spp. have shown that methyl jasmonate increases taxane production when compared with the control (Yukimune et al., 1996; Bonfill et al., 2007; Expósito et al., 2010), but more recently coronatine has been found to be more effective than methyl jasmonate in increasing taxane production (Onrubia et al., 2013b).

A B

Figure 6. Structures of methyl jasmonate (A) and coronatine (B).

Jasmonates (JA), especially methyl jasmonate (Figure 6), are oxylipins, a group of compounds that encompass compounds derived from -linoleic acid. Jasmonate biosynthesis was elucidated in 1984 (Vick and Zimmermann, 1984) but its mechanism of action is not fully understood. A suggested downstream signaling has been described by Fontseca et al. (2009), and Pauwels and Gossens, (2011) (Figure 7).

In normal conditions, JAZ (JAsmonate ZIM domain) binds to MYC2, avoiding the gene expression involved in jasmonate pathways. JA are able to interact with the JAR1 receptor (jasmonate resistant1), allowing the activation of MeJA due to its binding to isoleucine (JA-Ile). JA-Ile can interact and activate the SCFCOI1 (SKIP-CULLIN-F-Box) complex with an E3 ubiquitin ligase activity. This activation leads to the translocation of SCFCOI1 to the nucleus and the polyubiquitination of JAZ (usually binding to TOPLESS and NINJA), promoting its degradation by the proteosome and triggering MYC2 activation. MYC2 activation leads to the expression of early gene targets of the JA pathway, but other transcriptional factors have been described in the specific activation by JA-signalling (ERF family, WRKYs, MYBs, NACs…).

Figure 7. Methyl jasmonate pathway. MeJA: Methyl jasmonate; Ile: Isoleucine; SCFCOI: SCF complex; JAZ: Myc repressor; Myc2: Transcriptional factor involved in the MeJA mode of action; ub: ubiquitin.

On the other hand, coronatine (Figure 6) is also a phytoalexine, but it is produced by Pseudomonas syringae. It is a polyketide of coronafacic acid and an analog of methyl jasmonate and coronamic acid. Although its mode of action is still unknown, high structural and functional homologies have been demonstrated between COR and MeJA (Feys et al., 1994; Bender et al., 1999; Uppalapali et al., 2005). Taxane production in cell suspension cultures of C. avellana has been studied by different elicitation methods, listed in Table 1. In the first study (Bestoso et al., 2006), which confirmed that taxane production was not due to fungal contamination, elicitation with MeJA and MeJA plus chitosan achieved a 2- to 4-fold increase compared with control conditions. Similarly, treatment with short periods of ultrasound (US) (40kHz for 2, 3, 5 and 10 min) and/or salicylic acid (SA: 25, 50 mg/L) increased taxane production compared with the control, especially with two 3-min US treatments at days 10 and 12 combined with 50 mg/L SA, which led to a 14- fold increase (Rezaei et al., 2011). The effect on taxol production and cell viability of low-intensity US (4-455 mW) in treatments of 4-40 min was evaluated. Low power for short and long periods, and high power for short periods did not change cell viability significantly, but total amounts of each target taxane increased significantly (Safari et al., 2012). The effect of SA, alone or in combination with dibutyl phthalate (DBP), has been corroborated (Rezaei et al., 2013). DBP was used to remove taxanes

Table 1. Taxane production in cell suspension cultures of C. avellana obtained after different elicitation treatments by different authors. MeJA: Methyl jasmonate; CS: Chitosan; SA: Salicylic acid; US: Ultrasound; DBP: Dibutyl phthalate; TTC: Total taxane content; T: taxol; DB: 10 deacetylbaccatin III; B: baccatin III. * Referred as a taxol, 10-deacetylbaccatin III, 10-deacetyltaxol and 7-xylosyltaxol. All the data represent the total amount (intracellular and extracellular) and the quantification was carried out by HPLC-DAD using each author’s methodology.

Treatment Taxanes Concentration Reference Elicitor Concentration MeJA 200µM 2 mg/L TTC* Bestoso et al. MeJA + 200µM + 182µ/L 3.5 mg/L (2006) CS SA 25 mg/L 0.2 mg/L SA 50 mg/L 0.25 mg/L 40 KHz US 0.072 mg/L 1 time 3 min 40 KHz US 0.101 mg/L Rezaei et al. 2 times 3 min T (2011) SA: 25 mg/L

SA+US US: 2 times 3 min; 0.220 mg/L 40 KHz SA: 50 mg/L SA+US US: 2x3 min; 40 0.700 mg/L KHz

US 20 min 4 mW DB 0.46 mg/L Safari et al. B (2012) US 20 min 455 mW T 0.75 mg/L

0.183 mg/L SA 50 mg/L

Rezaei et al. DBP 10% (v/v) T 1.991 mg/L (2012) SA + 50 mg/L + 10% 2.989 mg/L DBP (v/v)

(in situ solvent extraction) and avoid the downstream process that can lead to a reduction in production. Although cell viability was

affected by the SA and DBP treatment, taxane production was significantly enhanced after the addition of 50 mg/L SA at day 8 and 10 % (v/v) of DBP at day 10, obtaining an increase of up to 42- fold compared with the control. Specifically, extracellular taxol accumulation was improved. DBP increased the effect of SA, suggesting a synergistic accumulative effect. Regarding this topic, the aim of our work was to study methyl jasmonate and coronatine as elicitors, and perform optimizing assays to establish which elicitor has the most enhancing effect on taxane production. Moreover, the point of elicitation was assayed, which depended on the cell density measured as packed cell volume (pcv).

1.3.3 Scale-up

Cell suspension cultures are the most suitable plant culture for scale-up, offering the chance to increase growth ratio and secondary metabolite production (Eibl et al., 2009a). In plant cell cultures, the scale-up process from mL-scale until bioreactor level is complicated. Extracellular availability of the target compounds is preferred to facilitate the in situ recovery of the product and promote a continuous production by the cells. Therefore, it is necessary to design and select the most suitable bioreactor for plant cell culture depending on the cell-specific demands (cell density, aeration, agitation, optimum medium, etc.) and on the engineering aspects (mixing, type of aeration, culture rheology, etc.). Aeration and agitation are among the most critical parameters for plant culture. Agitation promotes an effective distribution of nutrients and 49

oxygen, but plant cells are highly sensitive to shear forces. Therefore, to reduce shear sensitivity it is necessary to limit the mechanical agitation, which can lead to an inefficient oxygen and nutrient distribution. However, the cell culture medium usually behaves like a Newtonian fluid, and metabolite excretion, cell aggregation and biomass concentration can increase medium viscosity, resulting in diffusion and mixing problems (Eibl et al., 2009b; Wilson and Roberts, 2012). A stirred bioreactor is commonly used for all cell types, since its geometrical similarities with the shake flask make scale-up easier, ensuring a good mixing and oxygen transfer, and offering a good alternative to high- viscosity cell culture (Wilson and Roberts, 2012). For these reasons, plant cell cultures usually grow better in stirred bioreactors at low agitation speeds. Also, wave and orbitally shaken bioreactors offer the possibility of reducing shear forces, with homogeneous oxygen and nutrient distribution.

Due to a growing interest in scaling up processes, disposable or single-use bioreactor systems are becoming increasingly available on the market, including wave-mixed, stirred and bubble column bioreactors. Reduction of cross-contamination, high flexibility, easy handling, and being less time- and cost-consuming are the main advantages of single-use bioreactors (Eibl et al., 2009b; Wilson and Roberts, 2012). These advantages have led to a growing acceptance of single-use bioreactors in the last 15 years (Eibl et al., 2012; Georgiev et al., 2013). The aim of our work was scale up the taxane contents in hazelnut cell cultures from mL to benchtop bioreator level, and

improve production by supplementation with elicitors such as coronatine and methyl jasmonate. Culture conditions were optimized using orbitally shaken flasks and the TubeSpin® Bioreactor 50, and then scaled up to benchtop stirred bioreactors BIOSTAT® B plus and the single-use UniVessel®, which are suitable for plant suspension cell cultures (Eibl et al. 2009b).

1.4 Improving taxanes analytical detection

Chromatographic techniques were developed in the early 1900s, and since then a high number of methods have been developed to achieve the separation, identification and quantification of different chemicals from a wide range of sample mixtures. Chromatographic techniques are based on a mobile phase that transports the compound mixture through a stationary phase, where the separation of the compounds takes place due to the differential affinity with the stationary phase. Gas chromatography (GC), liquid chromatography (LC), capillary electrophoresis (CE) or thin-layer chromatography (TLC) are examples of chromatographic techniques (Settle, 1997).

High performance liquid chromatography (HPLC) is one of the most used analytical separation techniques for the quantitative and qualitative analysis of unknown mixtures. High pressure delivered by the pump impels the mobile phase through the column (stationary phase). A small volume of liquid sample is injected into the system and once it moves down into the stationary phase, those

compounds with a high affinity pass through more slowly than those with lower affinity (Figure 8). There are four major separation modes: reversed-phase chromatography, normal-phase, adsorption chromatography, ion exchange chromatography and size exclusion chromatography.

Mobil phase

A B

A B C Pump Column

ABC Detector Abs

A B C Retention time

Sample Mixture A+B+C

Figure 8. Schematic representation of HPLC system.

Reversed-phase chromatography is the most widely used mode of HPLC (over 90% use this mode) due to the high resolution obtained in complex matrices for a wide range of compounds. Column packing is non-polar (like C18, C3, C18...) and the mobile phase is water with different proportions of a water-miscible organic solvent (like acetonitrile or methanol). There are two types of pump: isocratic pump (constant mobile phase) and gradient pump (variable mobile phase composition), the latter being the most commonly preferred mode for complex samples or mixtures with diverse polarities, but it is difficult to develop, since retention and

selectivity are affected by many factors. The retention time is a critical parameter in compound identification, but depending on the detector used, other parameters such as molecular weight or absorption in UV can also be used. There is a broad range of detectors that can help to identify and quantify the eluting compounds from the mobile phase. As there is no universal detector, it is necessary to find the best one for the different sample requirements. Some of the most commonly used are UV-VIS, photo diode array (DAD), evaporative light scattering (ELSD), fluorescence, and mass spectroscopic detectors. A UV-Vis detector is used to measure compounds that absorb the spectrum in the UV or visible region, the analysis being performed using a fixed wavelength. The photo diode array is also a UV-Vis detector, but a wide range of wavelengths can be monitored simultaneously. Evaporative light scattering detectors are used for non-volatile compounds such as lipids, sugar and compounds with a high molecular weight. Fluorescence detectors offer a higher sensitivity but it is limited to naturally fluorescent compounds. Finally, an MS detector, which uses molecular weight to identify the compounds and has a very low limit of detection, is the most sensitive and selective detector. Table 2 lists the main advantages and limitations of HPLC methodologies. (Settle, 1997; Dong, 2013; Agilent technology, 2011) Analyte concentration is also important to develop a high sensitive and high resolution HPLC method. When the concentration is less than 1% by weight or at the µg/mL level it is typically referred to

Table 2. The main advantages and limitations of HPLC systems.

Advantages Limitations Fast Low sensitivity for some compounds High resolution Coelution Accuracy Lack of universal detector Automation Arduous (for regulated testing) as a trace analysis. In these cases, identification and quantification can be extremely difficult. With the aim of increasing sensitivity and specificity, HPLC can be coupled to a triple quadrupole mass spectrometer, combining the excellent separation capability of the HPLC with the high sensitivity and selectivity of the mass spectrometer. MS spectrometer allows the independent MS analysis of the parent ion, which can undergo a selective fragmentation, monitoring fragments and improving selectivity and sensitivity. This methodology allows us to detect compounds at trace-level, as in the case of taxanes in C. avellana, with a high conviction. As the taxane content in the C. avellana tree is ten times lower than in Taxus spp. (Hoffman et al., 1998), it is necessary to develop a suitable sensitive method, including extraction and quantification. Different methodologies to determine taxol and other taxanes are available, most of them using HPLC or Enzyme-Linked ImmunoSorbent Assays (ELISA). HPLC-MS has also been used to determine taxanes in plant samples and cell suspension cultures, but with the aim of identification and characterization rather than quantification (Kerns et al., 1994; Hoffman et al., 1998). Although

an HPLC-MS/MS method has been described for taxane analysis in complex matrices such as Taxus spp. plant extracts (Li et al., 2009), no HPLC-MS/MS method has been available to determine the low content of taxol and taxanes found in cell suspension cultures of C. avellana extracts. The aim of our work was to develop a sensitive HPLC-MS/MS method to quantify, in the medium of C. avellana cell suspension cultures, five of the most commercially important taxanes: the therapeutic compound taxol, and baccatin III, 10- deacetylbaccatin III, 10-deacetyltaxol and cephalomannine, which can be used for the semi-synthetic production of taxol.

1.5 Drug discovery from medicinal plants.

For a long time, plants have been used to treat different diseases, including cancer, diabetes and infections. Natural products can play an important role in drug discovery, constituting a source of inspiration and innovation (Cragg and Newman, 2005). Since 1994, almost half of the drugs were based on natural products (Harvey et al., 2008) and between 2005 and 2007 thirteen new natural-product drugs were approved (Butler, 2008). If we focus on cancer, more than 60% of the drugs used in its treatment have a natural origin (Newman and Cragg, 2007). For example, Silix alba (Willow) bark was used in traditional medicine to treat fever and pain. At the end of the 18th century, Felix Hoffmann working with the Bayer Company synthesized aspirin from salicylic acid in willow bark, reducing its side effects by structural modification. Other well- known plant-derived drugs are morphine from Papaver sonniferum,

vinblastine and vicristine from Catharanthus roseus, Digoxin from Digitalis spp. or taxol from Taxus spp.

Drug discovery in medicinal plants continues today and involves multidisciplinary work that may combine ethnobotanical, phytochemical, biological and molecular techniques. The basic procedure to analyze crude plant extracts is represented in Figure 9. Firstly, it is necessary to separate the compounds from the plant material (leaves, bark, roots…) by using a selective solvent and a specific extraction procedure. This methodology results in the crude extract, which contains all the compounds soluble in the solvent. These extracts can be screened by functional biological assays (for example, for antimicrobial, antitumour, antiparasitic or antiinflammatory activity), and once the activity is confirmed, an activity-guided fractionation can be carried out to separate plant compounds. Fractions can be also screened by functional assays using a simplified mixture of the extract, which makes it possible to isolate only those compounds showing the desired activity. The main challenge with drug discovery from a natural source is to separate and purify lead compounds from the crude extract, so fractionation is repeated until the pure compound is obtained. All of these procedures are extremely difficult and time-consuming, since there is no information about what specific characteristics are being looked for. Once the compound has been purified, as the probability of obtaining a known compound is high, spectroscopy techniques like MS and MS/MS can determine the exact mass and molecule fragmentation, which can be compared with the literature or with different databases to identify the compound. If there is no 56

information about this compound, it is necessary to characterize it structurally by nuclear magnetic resonance (NMR), infrared spectroscopy (IR) or other newly combined spectroscopy techniques like LC-NMR-MS or LC-UV-NMR-MS-FTIR (Balunas and Kinghorn, 2005; McRae et al., 2007; Rishton, 2008).

Biological assays: Lead fraction/s Antimicrobial Antiparasitic Antiinflammatory

Antiproliferative Abs

Retention time Lead extract Compound isolation Silica gel chromatography Crude Extracts

Compound identification: HPLC/MS; HPLC-MS/MS

Compound characterization: NMR; LC-NMR-MS; LC-UV-NMR- MS-FTIR Fractionation

Figure 9. Schematic procedure for the isolation and identification of new natural compounds from a crude plant extract.

As mentioned before, there has been an increase in cancer incidence and mortality in recent years, so there is a growing interest in finding new active compounds that can be used to fight against cancer. The discovery of taxanes in C. avellana plant extracts has prompted the study of this plant. Previous experiments have reported cytotoxicity activity in cell suspension cultures extracts, but the effect was attributed to taxol. SK-Mes-1 cells (derived from lung cancer) treated with an extract from the culture medium of cell

suspension cultures of C. avellana blocked cells at the metaphase/anaphase transition and inhibited the cell cycle, with a higher effect than yew extraction (Bestoso et al., 2008). Very recently, the effect of a hazel cell suspension culture was evaluated in the MCF-7 cancer cell line (derived from breast cancer), concluding that the hazel cell extract was more effective than pure taxol, even if its taxol content was the same as the pure compound. Consequently, it was hypothesized that hazel extracts may have other taxol-like compounds that can enhance the effect (Bemani et al., 2012).

The aim of our work was to evaluate the anti-proliferative effect on cells of C. avellana leaves and stems by analyzing crude extract fractions, with the ultimate goal of identifying and characterizing compounds with antiproliferative activity in C. avellana plant materials.

Chapter I:

Development of a hazel cell culture-based paclitaxel and baccatin III production process on a benchtop scale

Gallego A, Imseng N, Bonfill M, Cusido RM, Palazon J, Eibl R, Moyano E. Development of a hazel cell culture-based paclitaxel and baccatin III production process on a benchtop scale.Journal of Biotechnology. 2015; 195: 93-102.

DOI:10.1016/j.jbiotec.2014.12.023

Status: Published.

Chapter II:

Assessing factors that affect the growth of C. avellana cell suspension cultures: a statistical approach

Gallego A, Bonfill M, Cusido RM, Pastor M, Palazon J,

Moyano E. Assessing factors that affect the growth of C. avellana cell suspension cultures: a statistical approach.

In vitro cellular and developmental biology-plant

Status: In press. Ref.: Ms. No. IVPL-D-14-00337R1

Abstract

The detection of taxol and related taxanes in Corylus avellana has generated considerable interest in studying in vitro cell cultures of this plant. Cell suspensions are a sustainable and rational option for obtaining a continuous and reliable source of secondary metabolites in large-scale processes. We therefore focused our study on the main factors that affect the growth of C. avellana cell suspensions as a key approach to improving the culture productivity. In this work, calli were successfully induced from C. avellana seeds, leaves and stems, and the efficiency of different sterilization methods was analyzed. The effects of the basal medium, carbon source, and the type and quantity of plant growth regulators on culture growth were studied. A fractional factorial design allowed us to reduce the number of experiments and analyze all the combinations in one run, thereby reducing time, variability and costs. Statistical analysis (ANOVA) revealed that 1- Naphthaleneacetic acid (NAA) and sucrose are mandatory for the growth of C. avellana cell suspension cultures, with no interactions detected between the parameters analyzed, while growth did not depend on the addition of cytokinins. The secondary metabolism was not inhibited, detecting 1175.45 ng/L of baccatin III and traces of taxol, deacetyltaxol and cephalomannine. Additionally, prompted by the high growth rate of the C. avellana calli, we assayed a new cold temperature-based method to maintain a stock of calli using half-strength MS solid medium, concluding that up to 5 months at 4ºC is optimal to ensure white friable calli.

Keywords: Corylus avellana; fractional factorial design; growth optimization; in vitro culture; taxanes.

Introduction

Corylus avellana is a dicotyledoneous plant from the Betulaceae family, originating from Eurasia and widely spread around the world. The Corylus genus includes 18 species, among them C. americana (USA), C. avellana (Europe and Western Asia), C. cornuta (USA), C. maxima (South-West of Europe, South-East of Asia), C. jacquemontii (India), C. chinensis (China) and C. fenox (Himalaya, Tibet). The fruits of C. avellana, the hazelnuts, are well established in the human diet, but the plant has not been considered as a producer of medicinal compounds. Recent studies revealed that C. avellana produces taxol and taxanes, even though its phylogenetic origin is very far from the main taxane producers, the Taxus species. Hoffman et al. (1998) determined taxol and taxanes in C. avellana plant extracts. More recently, the production of taxol in C. avellana leaves and hazelnut shells was studied by Ottaggio et al. (2008), accumulation being 3-fold higher in leaves. C. avellana cell cultures have also been shown to produce taxol and other taxanes (Bestoso et al., 2006; Rezaei et al., 2011, 2013; Safari et al., 2012; Bemani et al., 2013).

Taxanes, particularly taxol, are high-value phytochemicals with an important effect against several types of human cancer, including ovarian, breast, head, neck and small cell and non-small cell lung cancers, AIDS-related Kaposi’s sarcoma, lymphoma, prostate 82 cancer, gastric cancer, and bladder cancer, among others (McGuire et al., 1989; Holmes et al., 1991; Saville et al., 1995; Eisenhauer et al., 1998). The extraction of medicinal compounds from their natural source is limited by environmental and economic risks and difficulties. The use of taxol is severely restricted by its limited supply from the inner bark of Taxus spp. Consequently, as natural extraction is unfeasible to meet the growing global demand for taxol and structural complexity hampers its chemical synthesis, there is a growing interest to find alternative sources of taxane production that can be biotechnologically enhanced. In vitro plant cell cultures are an excellent option for obtaining important secondary metabolites without endangering biodiversity (Charlwood and Rhodes, 1990). Cell suspension cultures are particularly suitable for large-scale processes, providing a reliable source of natural products (Navia-Osorio et al., 2002; Bentebibel et al., 2005; Mustafa et al., 2011). Although taxol production using Taxus cell suspension cultures is a commercial reality, it is still not sufficient to meet the demand, so alternative natural sources of taxol production are being studied, including Corylus avellana, the hazelnut tree. A highly efficient cell suspension culture with enhanced growth and secondary metabolite production can be obtained by manipulating the culture conditions. Different approaches focus on the nutrients present in the medium, plant growth regulators, precursor feeding, elicitation, or metabolic engineering. (Sung and Huang, 2000; Rao et al., 2002; Kajani et al., 2012; Karwasara and Dixit, 2012; 83

Sivanandhan et al., 2013; Vasilev et al., 2013; Farjaminezhad et al., 2013). When optimizing culture conditions, the different parameters can be analyzed following two basic strategies: either modifying one factor at a time, or using fractional factorial designs that allow different variables to be studied in one run, thereby reducing both cost and time. In this work we studied the factors affecting the growth of cell suspension cultures of C. avellana with the aim of establishing the best conditions for their culture. With this goal, different explants were used to induce callus formation. Cell suspension cultures were obtained after selecting the most friable and fast-growing calli, and the growth dynamics were determined. The effect of the basal medium, carbon source, and different levels of auxins and cytokinins on cell growth was analyzed using a fractional factorial design. We also studied the effect of 4 ºC treatment on callus growth to obtain a maintenance protocol.

Material and Methods

Plant material and sterilization

Different explants were assayed to establish callus cultures. Stems, leaves and seeds derived from adult C. avellana trees cultivated in Catalonia were used to obtain in vitro cultures. According to their origin, different sterilization methods were used for the explants

(Table 1), namely ethanol (EtOH), mercury (II) chloride (HgCl2), sodium hypochlorite (NaClO) plus 3-4 drops of tween20 with differing times and proportions. Washing with distilled water was

performed before each step, with 3-4 washes at the end. Two different concentrations and times of Captan ® were evaluated.

Table 1. Sterilization methods for hazelnuts (H), leaves (L) and stems (S) of adult trees of C. avellana. The duration of each treatment is indicated in brackets. 1: Fornale et al. (2002) 2: Based on Sanchez-Oleate et al. (2004)

Sterilization steps 1st 2nd 3th 4th 5th 6th NaClO + Captan EtOH HgCl H O Captan 2 Tween20 2 2 ® (min) (s) (min) (min) ® (min) (min) 70% 0.1% 3% Hazelnuts H11 (30) (15) (30) 0.1% 3% L1 (6) (15) 0.25% (10) 70% 0.1% 3% Leaves L2 + 4ºC (30) (10) (15) o/n 70% 0.1% 3% 10% L3 (30) (10) (15) (15) 70% 0.1% S1 (30) (5) S2 = L2 Stems 0.8% 85% 12% S32 (3) (300) (10) + let dry

Callus induction and cell suspension culture

Mature hazelnuts (endosperm containing embryo), leaves and stems were cut in pieces (4 pieces for seeds and pieces of 2-3 cm for leaves and stems) and cultured in Murashige and Skoog solid 85 medium (MS medium; Murashige and Skoog, 1962) supplemented with 30g sucrose/L, 2,4 Dicholophenoxiacetic acid (2,4D) at 2 mg/L and Kinetin (Kin) at 0.4 mg/L to induce calli (callus induction medium). These calli were routinely subcultured every 4 weeks. 4 g of white and friable calli was inoculated in 30 mL of MS medium supplemented as above to establish the C. avellana cell suspension cultures. 200 mL shake flasks were used and the cultures were maintained at 25 ºC in the dark, on an orbital shaker at 110 rpm and were routinely subcultured every 12 days.

Growth studies in sub-optimal conditions

Lag, exponential and stationary phases were determined in cell suspension cultures of C. avellana. 4 g of cells were inoculated and cultured under the conditions described above using the callus induction medium. Cell suspensions were maintained for 24 days, taking samples every 4 days. Viability and cell growth measured as fresh cell weight (FCW) and dry cell weight (DCW) was determined for each sample. Cell viability was determined using fluorescein diacetate (FDA) and propidium iodide (IP), both at 0.01% (w/v) (Pollard and Walker, 1990). FCW was obtained by filtering with nylon filters of 35 μm diameter, and then cells were lyophilized to obtain the DCW.

Fractional factorial design

64 different media would be produced by a 26 factorial design that fixed at two values the following variables: 2 basal media (Gamborg B5 medium [B5 medium] and MS), 2 carbon sources (30 g/L sucrose, and sucrose plus fructose at 5 g/L each), 2 types of auxins (2,4 dichlorophenoxyacetic acid [2,4D] and 1- naphthaleneacetic acid [NAA]), 2 types of cytokinins (kinetin [Kin] and 6-benzylaminopurine [BAP]) and 2 amounts of phytohormones (2mg/L or 1 mg/L auxins and 1mg/L or 0.5 mg/L cytokinins). After application of a fractional factorial design, the 64 possible media obtained by combining the six aforementioned factors were halved to 32 for testing (Table 2). 4 g of cells were inoculated in 30 mL of culture medium and were maintained in the conditions described above for 15 days. At that point, fresh and dry weight was determined. Triplicates for each medium were analyzed.

Table 2. Composition of 32 media resulting from the combination of two basal media (B5 and MS), two carbon sources (S and S/F), two auxins at two concentrations (2,4-D and NAA, 1 mg/L and 2 mg/L), and two cytokinin at two concentrations (Kin and BAP, 0.5 mg/L and 1 mg/L).

Nº Medium Carbon source Auxins Aux level Cytokins Cyt level (mg/L) (mg/L) 1 B5 F/S 2,4D 1 KIN 0,5 2 B5 F/S 2,4D 1 BAP 1 3 B5 F/S 2,4D 2 KIN 1 4 B5 F/S 2,4D 2 BAP 0,5 5 B5 F/S NAA 1 BAP 0,5 6 B5 F/S NAA 1 KIN 1 7 B5 F/S NAA 2 KIN 0,5 8 B5 F/S NAA 2 BAP 1 9 B5 S 2,4D 1 KIN 1 10 B5 S 2,4D 1 BAP 0,5 11 B5 S 2,4D 2 KIN 0,5 12 B5 S 2,4D 2 BAP 1 13 B5 S NAA 1 BAP 1 14 B5 S NAA 1 KIN 0,5 15 B5 S NAA 2 BAP 0,5 16 B5 S NAA 2 KIN 1 17 MS F/S 2,4D 1 KIN 1 18 MS F/S 2,4D 1 BAP 0,5 19 MS F/S 2,4D 2 KIN 0,5 20 MS F/S 2,4D 2 BAP 1 21 MS F/S NAA 2 KIN 1 22 MS F/S NAA 2 BAP 0,5 23 MS F/S NAA 1 KIN 0,5 24 MS F/S NAA 1 BAP 1 25 MS S 2,4D 1 KIN 0,5 26 MS S 2,4D 1 BAP 1 27 MS S 2,4D 2 KIN 1 28 MS S 2,4D 2 BAP 0,5 29 MS S NAA 1 KIN 1 30 MS S NAA 1 BAP 0,5 31 MS S NAA 2 BAP 1 32 MS S NAA 2 KIN 0,5

HPLC taxane analysis

Taxanes were extracted from freezer-dried cells and media as described by Cusidó et al. (1999). HPLC-DAD was used to detect and quantify taxol and related taxanes using the method described in Richheimer et al. (1992). Taxol, baccatin III, cephalomannine, 10-deacetylbaccatin III and 10-deacetyltaxol provided by ChromaDex® were used as standards.

NAA and cytokinin effect on cell suspension culture

After the factorial fractional experiments, the effect of NAA and Kin on the growth of the C. avellana suspension culture was analyzed in more depth. NAA added at 5 different concentrations (0.5, 1, 1.5, 2 and 4 mg/L) without Kin or with 0.5 mg Kin/L was tested. Cells were maintained in the conditions described above taking samples at day 15 (triplicates). Viability and cell growth (FCW and DCW) were determined for each medium.

Statistical analysis

The statistical analysis was performed with a SPSS statistical package (16.0 version). Each triplicate was evaluated as one run. In order to estimate the effect of individual media characteristics on the cell growth, ANOVA analysis was used. When computing the effect of the different factors (media conditions) on the cell growth, only those with p-values lower than 0.05 were considered statistically significant. 89

4 ºC treatment study in callus culture

3-4 g of calli were subcultured in half-strength MS solid medium supplemented with 30 g/L of sucrose (Mirjalili et al., 2009) and were maintained at 4 ºC in darkness for up to 6 months. Every month, 18 replicates were taken and cultivated at 25 ºC in darkness in the MS callus induction medium, evaluating the doubling time after the cold treatment.

Results

Callus induction and cell suspension culture

Hazelnuts, leaves and buds as primary sources of explants were evaluated. Due to the endogenous contamination observed in leaves and stems, three different methods were studied. Table 3 shows the percentage of survival after sterilization and the capacity of each explant to develop calli after 15 days of culture. Overall, hazelnuts sterilized with the H1 method proved to be the best option for callus induction due to high rates of survival (100%), callus formation, and low contamination. In leaves, L3 was the best method, with the percentage of survival (75%) being considerably higher than L2 (37%). In stems, the tissue with the highest rate of contamination, the most effective method was S1, which performed better than Captan ®. After 15 days of culture, calli emerged in all the explant types analyzed, but hazelnuts were more responsive to treatment or prone to callus initiation (> 90%) than leaves and stems. It should also be noted that hazelnut-derived calli were whiter and more

90 friable that those obtained from leaves and stems. Also, stems were less prone to callus induction than leaves.

Table 3. Sterilization study. (a) % of explants without contamination after 15 days of culture, (b) % of explants that survive the sterilization method after one month of culture (c) Calli induction in the different kinds of explants studied after 15 days of culture: (+) low rates of induction, (++) medium rates of induction, (+++) high rates of induction.

% Callus Sterilization % sterilization survival induction methods effectiveness (a) (b) (c) Hazelnuts H1 90% 100% +++ L1 50% 0% Leaves ++ L2 45% 37%

L3 28% 75% S1 61% 45% Stems S2 23% 0% + S3 65% 30%

Growth study in sub-optimal conditions

Figure 1 shows the growth dynamics of C. avellana cell suspension culture expressed as FCW (A) and DCW (B). A short lag phase occurred before day 4, when viability was 70%, showing that the cells were in adaptation. FCW and DCW increased until day 12, after which growth declined slowly until day 24. During the exponential growth phase, FCW increased up to 7.5-fold, and DCW up to 4-fold, and viability was never lower than 85%. The maximum FCW at day 12 was 747 g/L corresponding to 16.27 g/L 91 of DCW. The exponential phase was characterized by a biomass productivity of 53.93 g/L/day and a doubling time of 4.13 days. Day 12 was the starting point of the stationary phase, after which growth declined about 1.5-fold until day 24. Nevertheless, viability decreased slowly from 90% at day 12 until 70% at day 24.

Figure 1. Time course of biomass accumulation (fresh (a) and dry weight (b)) by a cell suspension of C. avellana cultured for 24 days in MS medium supplemented with 30 g/L sucrose, 2 mg/L of 2,4D and 0.4 mg/L of KIN. Data represent average values from four replicates ± S.D.

Fractional factorial study: improving the culture medium

Different media have been reported for the cultivation of C. avellana cell suspension cultures, most of them based on MS supplemented with 30 g/L sucrose, but using different plant regulators and concentrations. Bestoso et al. (2006) used MS medium supplemented with 2.4D at 0.5 mg/L + BAP at 1 mg/L; Razeai et al. (2012 and 2013) used MS medium supplemented with 2.4D at 1 mg/L + BAP at 0.5 mg/L; Safari et al. (2012) used modified MS supplemented with NAA at 3 mg/L + IAA at 3 mg/L, and Bemani et al. (2013) used B5 supplemented with NAA at 1.86 mg/L + BAP at 0.2 mg/L. Due to the variability of these references regarding the culture medium, an experiment to optimize the culture conditions of C. avellana suspension cell cultures for growth was carried out. A fractional factorial design was used to evaluate the influence of the basal medium, carbon source, and different phytohormone concentrations in one run. Each variable was analyzed at two levels, and therefore the estimated effects of every variable reflected the growth differences that can be attributed to the change of this variable from the value arbitrarily labelled -1 to the value labelled +1. In the case of qualitative variables, such as the carbon source, this choice requires no further explanation; for quantitative variables, such as the phytohormone levels, the values chosen were extrapolated from other studies in similar species (Bestoso et al., 2006; Guizhi et al., 2009; Hong et al., 2010). This approach allowed us to reduce the number of experiments from 64 to 32. The assay was conducted for 15 days to observe the cultures

93 in the stationary phase, when secondary metabolite production increases (Verpoorte, 2000; Dixon, 2001).

Figure 2. Biomass accumulation (measured as dry weight) of C. avellana cell suspensions after 15 days of growth in the different media studied. Data represent average values from three replicates ± S.D.

Figure 2 shows the DCW for all the media tested at day 15, at which point viability was never lower than 80% in any media. ANOVA was performed to estimate the influence of each variable on the growth (measured as DW). In a global model, considering all the variables studied, only the effect of the carbon source and auxins can be considered statistically significant (p<0.001 and p=0.001 respectively). The importance of these effects was further confirmed with a model including only these two variables, explaining over 88% of the sum of the squares. In Figure 2 large differences between bar clusters can be observed, the highest bars corresponding to media supplemented with 30 g/L sucrose and the lowest bars to media supplemented with 5 g/L sucrose plus 5 g/L

94 fructose. Also, interactions between the parameters were evaluated, revealing no interactions between them. This model indicated that sucrose and NAA have a statistically significant positive effect on growth in hazel cell culture. Conversely, the basal medium and cytokinin effect on growth was not statistically significant.

Taxane production

Taxane production in the C. avellana cell suspension culture (intracellular and extracellular) was evaluated using medium 29, which showed the highest growth among the assayed media. The main taxane detected were baccatin III (1175.45 ng/L), but traces of taxol, deacetyltaxol and cephalomannine were also detected. The taxanes detected were present in the intracellular extractions.

Effect of NAA and Kin on C. avellana growth

Taking into account the results obtained in the fractional factorial design, a second study was undertaken using a wider range of NAA concentrations, with or without cytokinins in the medium. In this assay, five NAA concentrations were assayed (0.5, 1, 1.5, 2, 4 mg/L) in the presence or absence of 0.5 mg/L kin (Figure 3A). Figure 3B shows the DCW at day 15 in the different media (A-J), being similar in all the media tested. Interestingly, the maximum DCW of 18.7 g/L was obtained in both medium C (supplemented with 1.5 mg/L of NAA) and H (supplemented with 1.5 mg/L of NAA plus 0.5 mg/L kinetin), achieving an increase of 27%

95 compared with the DCW obtained at day 15 in the growth study (Figure 1B). The ANOVA study revealed no correlation between growth and concentrations of NAA, so we used a range of concentrations between 0.5 and 4 mg/L of NAA, obtaining a high growth in each case. Also, ANOVA revealed no differences between NAA used with or without cytokinins. These results were in line with those obtained in the fractional factorial assay.

Figure 3. NAA and Kin effect on the growth of cell cultures of C. avellana. The basal medium utilized was MS supplemented with sucrose 30 g/L and different NAA concentrations, with or without the addition of Kin, each media was named from A-J (A). Dry weight obtained in each media it is represented in (B). Data represent average values from three replicates ± S.D. 96

4 ºC treatment of callus culture

With the aim of conserving a stock of C. avellana calli, and reducing the number of subcultures, contamination rate, costs and epigenetic changes, a 4 ºC conservation study was undertaken. Doubling time at 25 ºC was evaluated during the subcultures to determine the growth ratio of cultures subjected to various months at 4 ºC. After 4 months at 4 ºC and one subculture, the weight of 10- 15% of the calli doubled; after two subcultures this percentage increased to 50-60%, and after three subcultures to 70-100%, being 100% for the calli stored at 4 ºC for two months. Table 4 shows the percentage of calli that doubled their weight after 3 subcultures at 25 ºC in MS callus induction medium after being maintained for 2 to 6 months at 4 ºC in half-strength MS medium in darkness. It was noticed that increasing the storage time at 4 ºC reduced callus growth: after 6 months, only 13.3% of the calli were able to grow. However, it is possible to maintain a sizeable stock of C. avellana calli at 4 ºC for 5 months with good growth rates in the subsequent subcultures (68.75% doubled the weight in three subcultures). Up to 5 months at 4 ºC the calli were white and friable, but after longer periods they turned yellow and showed poor growth.

Table 4. Percentage of calli that were able to growth (doubled the inoculum) after three subcultures at 25 ºC in MS medium supplemented with 2.4D (2 mg/L) and kin (0.4 mg/L) after 2, 3, 4, 5 and 6 months at 4 ºC in half strength MS medium.

Months at 4 ºC 2 3 4 5 6 % of calli doubled 100% 83.30% 73.33% 68.75% 13.33% weight

Discussion

C. avellana is a taxane-producing plant with potential to be an alternative source of these valuable secondary metabolites due to its high growth ratios and easy in vitro culture compared to the yew. In this work we successfully established a C. avellana cell culture, observing that hazelnuts are more prone to callus induction than leaves and stems, although calli were obtained from all the explants tested. In contrast, Bestoso et al. (2006) reported a comparable rate of callus induction from stems and hazelnuts. Variable genotypes and slight differences in in vitro procedures can result in different responses to the same process (Yu and Reed, 1995; Nas, 2004; Baccheta et al., 2008; Contesa et al., 2011).

Although mature tissues of C. avellana trees have high ratios of endogenous contamination (Diaz-Sala et al., 1990; Reed et al., 1998; Nas and Read, 2004; Baccetta et al., 2008), hazelnut sterilization with the H1 method, using solutions of EtOH, HgCl2 and NaClO, proved to be an efficient way of establishing in vitro

98 cultures of C. avellana, with high rates of explant survival and callus formation. Although S3, using the potent fungicide Captan ®, was expected to be the most efficient method for callus induction from stems, considering these tissues are the most prone to contamination, the S1 method gave better results. Our sterilization methodology was least effective with leaves, but calli were more easily induced from the surviving leaf explants than from stems. The growth curve of cell suspension cultures established from hazelnut-derived white and friable calli was characterized by a short lag phase and an exponential phase from days 4 to 12. Rezaei et al. (2011) published a similar growth curve of C. avellana cell cultures, although the lag phase was longer and exponential phase shorter (from days 8 to day 13). The study of the parameters affecting growth, carried out using a fractional factorial design, indicated that MS and B5 basal medium are both equally good for culturing C. avellana cell suspensions. In contrast, the choice of hormones and carbon source had a variable affect on growth. ANOVA analysis showed that NAA (rather than 2,4D) and sucrose (rather than sucrose plus fructose) were mandatory for achieving a high growth of C. avellana cell suspension cultures. As is well known, the easily transported and inter-converted sucrose and fructose are the sugars plants commonly use for metabolism. Sugars also play a role in signal transduction, gene regulation and plant development (Rolland et al., 2002). In our experiment, sucrose at 30 g/L substantially increased growth in cell suspension cultures of C. avellana with a high significance. It has been previously reported that growth and 99 production in cell suspension cultures depend greatly on the type of carbohydrates used in the medium. Many studies on a variety of plant species have found sucrose to be the most effective carbon source (Nagella and Murphy, 2011; Suehara et al., 2012; Singh and Chaturvedi, 2012; Karwasara and Dixit, 2012 and 2013; Sivanandhan et al., 2013), although others describe a better growth with a combination of sucrose plus fructose (Cusido et al., 1999). One of the most important factors affecting cell growth in cell suspension cultures is considered to be the use of plant cell regulators, but the type and concentration need to be optimized for each plant, or even for each genotype. Optimizing plant cell regulators may involve the application of an individual auxin or cytokinin, or a combination of both (Mustafa et al., 2011). Among the auxins tested, we observed that NAA had a significant stimulatory growth effect on cell suspension cultures of C. avellana, being more effective than 2,4D. On the other hand, the use of cytokinins in cell suspension cultures of C. avellana had no observable effect. Cell cycles are promoted by fluctuations in cytokinin concentrations (Redig et al., 1996), and can be artificially altered by applying exogenous cytokinins (Hartig and Beck, 2005). It has also been reported that the cytokinin concentration required for cell cycle progression is extremely low (10-7-10-5 M) (Mustafa et al., 2011). Different processes have been described to obtain habituated cell suspension cultures that do not require the addition of these phytohormones to the media, such as tobacco (Binns and Meins, 1973), sugarbeet (Kevers et al., 1981), soybean (Miura and Miller, 1969; Wyndaele et al., 1988) and strawberry (Asano et al., 100

2002). On the other hand, cytokinin autonomy has been observed in some strains of tobacco (XD6X and TBY-2) cells, linked to the capacity of these cells to synthesize enough of the plant hormone by themselves (Nishinari and Syono, 1980; Nagata et al., 1992; Redig et al., 1996). Our results suggested that cell suspension cultures of C. avellana may be cytokinin-autonomous, since no habituation process was applied. To study the role of NAA and cytokinins in our cell suspension culture in more depth, a second assay was carried out, applying different levels of NAA, with or without cytokinins. A wide range of NAA concentrations was found to be suitable for the growth of C. avellana suspension cultures (from 0.5 mg/L to 4 mg/L), suggesting that the culture is sensitive to low levels of NAA, while higher doses are not toxic. The results of the fractional factorial design were confirmed when ANOVA revealed no statistically significant differences between the use of NAA with or without cytokinins. It can be concluded that these plant hormones (Kin, BAP) can be omitted from the growth media for cell suspension cultures of C. avellana. We also explored if the assay conditions leading to the maximum growth suppressed taxane production. Our analysis showed a lower taxane yield than those reported in the literature (Bestoso et al., 2006; Rezaei et al., 2011, 2013; Safari et al., 2012; Bemani et al., 2013), reflecting that the type and concentration of plant regulators used can dramatically modify growth and secondary metabolite production in cultured plants. Nonetheless, our study confirms that despite an increase in primary metabolism, the secondary 101 metabolite pathway of the target compounds was not inhibited. Cusido et al. (2002) described a lower taxane production in Taxus spp. when the growth was enhanced, circumventing this effect with a two-stage culture. This approach, using a medium to obtain the maximum growth and another to stimulate taxane production, could also be applied in future studies with C .avellana. Further studies could also consider other parameters than those described in this work. Bearing in mind a future scale-up of these cell suspension cultures, a stock of callus biomass, needs to be maintained, which can be costly and time-consuming. To the best of our knowledge, this is the first time it has been reported that C. avellana calli can be maintained at 4 ºC for 5 months in half-strength MS, and then cultured in the routine medium at 25 ºC with good ratios of growth. This is an important strategy to reduce the number of subcultures, contamination rate, cost and epigenetic changes (Mustafa et al., 2011). Although this process is not as reliable as cryopreservation, it is an easy and fast way to routinely maintain a stock of C. avellana calli, and may also be a good strategy for plants recalcitrant to cryopreservation. Nevertheless, further research is required to determine the putative metabolic changes that these calli may undergo.

Acknowledgements

Work in the Plant Physiology Laboratory (University of Barcelona) was financially supported by the Spanish MEC (BIO2011-29856-

102

C02-1) and the Generalitat de Catalunya (2014SGR215). A. Gallego held a grant from the Universitat Pompeu Fabra.

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46. Sanchez-Olate M, Rios D, Rodriguez R, Materan ME, Pereira G (2004) Duration of the reinvigorating effect of severe pruning of mature European hazelnut plants (Corylus avellana L.) cv. Negretta with in vitro cultivation. Agric Tec 64(4): 338-346. 47. Saville M, Lietzau J, Pluda J, Feuerstein I, Odom J, Wilson W, Humphrey R, Feigal E, Steinberg S, Broder S (1995) Treatment of HIV-associated Kaposi's sarcoma with paclitaxel. Lancet 346:26-28. 48. Singh M, Chaturvedi R (2012) Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthes acmella Murr. Bioproc Biosyst Eng 35(6):943-951. 49. Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Muthuselvam M, Selvaraj N, Manickavasagam M, Ganapathi A (2013) A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal. Protoplasma 250(4):885-898. 50. Suehara K, Kameoka T, Hashimoto A (2012) Sugar uptake analysis of suspension Arabidopsis, tobacco, and rice cells in various media using an FT-IR/ATR method. Bioproc Biosyst Eng 35(8):1259-68. 51. Sung LS, Huang SY (2000) Medium optimization of transformed root cultures of Stizolobium hassjoo producing L-DOPA with response surface methodology. Biotechnol Progr 16(6):1135- 1140. 52. Vasilev N, Grömping U, Lipperts A, Raven N, Fischer R, Schillberg S (2013) Optimization of BY-2 cell suspension 109

culture medium for the production of a human antibody using a combination of fractional factorial designs and the response surface method. Plant Biotechnol J (7):867-74. 53. Verpoorte R; 2000; Secondary metabolisms; Verpoorte R, Alfermann AW; Metabolic engineering of plant secondary metabolism. Kluwer Academic publishers, London, 1-29. 54. Wyndaele R, Christiansen J, Horseele R, Rüdelsheim P, Van Onckelen H (1988) Functional correlation between endogenous phytohormone levels and hormone autotrophy of transformed and habituated soybean cell lines. Plant Cell Physiol 29 (7): 1095-1101. 55. Yu X, Reed BM (1995) A micropropagation system for hazelnuts (Corylus species). HortScience 30(1):120-123.

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Chapter III:

Optimization of a liquid chromatography-tandem mass spectrometry method for the quantification of traces of taxanes in a Corylus avellana cell suspension medium

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Gallego A, Jáuregui O, Moyano E, Palazon J, Casals I, Bonfill

M. Optimization of a liquid chromatography-tandem mass spectrometry method for the quantification of traces of taxanes in a Corylus avellana cell suspension medium. RSC Advances.

Status: Submitted in January 2015. ID: RA-ART-01-2015-

000803.

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Abstract

Since the recent discovery of taxol and other taxanes in Corylus avellana, this plant species has attracted interest as a potential new source of these compounds. However, its low taxane content in comparison with Taxus spp. has restricted research to analytical identification or global quantitation. A feasible and sensitive method based on liquid chromatography-tandem mass spectrometry using a triple quadrupole analyzer was developed for the analysis of taxol and four other taxanes in a Corylus avellana cell suspension medium. Taxanes were extracted from the cell culture medium with dichloromethane and analyzed using electrospray ionization and quantified by multiple-reaction monitoring mode. Methanol and matrix-matching calibration curves using docetaxel as the internal standard were analyzed. Linearity was confirmed over the whole calibration range (0.3-2.1µg mL-1). The inter- and intra-day precision of taxanes ranged from 80% to 120% and the recovery rates were higher than 80%. Limits of detection were between 0.24- 38 ng mL-1 and the limits of quantification were between 0.8-125 ng mL-1. The low detection and quantitation values obtained allowed us to detect small quantities of the released taxanes (120 ng mL-1 of B, 151 ng mL-1 of CF and 105 ng mL-1 of T), which correspond to about 0.5 ng mL-1 of each taxane, in the 20mL Corylus avellana cell suspension culture medium extracted, even at the beginning of the culture. These results were confirmed by high resolution mass spectrometry.

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Keywords; Corylus avellana cell suspension cultures; high resolution mass spectrometry; liquid chromatography; tandem mass spectrometry; taxanes; taxol.

1 Introduction

Taxanes, particularly taxol, are phytochemicals with high added value. Since the discovery of taxol in 1967 in Taxus brevifolia, more than 500 natural taxanes have been characterized in different Taxus spp., as well as a huge number of synthetic taxol analogues.1-3

Taxanes have an important effect against several types of human cancer, including ovarian, breast, head, neck and small cell and non- small cell lung cancers. Their effectiveness in treating AIDS-related Kaposi’s sarcoma, lymphoma, and prostate, gastric and bladder cancers has also been recently shown.4-7 Taxanes are classified as microtubule-interfering agents because they are able to bind with the microtubules and block the cell cycle in the metaphase-anaphase transition, forming highly stable microtubules by suppressing their depolymerization.8 Due to the high commercial demand for taxol and its low content in Taxus spp., there is a growing interest in exploring new strategies for its production at an industrial level. These include total synthesis, semi-synthetic processes from more abundant taxanes (such as baccatin III, 10-deacetylbaccatin III, 10-deacetyltaxol and cephalomannine), and biotechnological production using cell suspension cultures of Taxus spp. or other taxane-producing species such as Corylus avellana or fungi.2 Plant cell cultures constitute an

118 emerging technology for the production of high-value secondary metabolites, which can be scaled up to bioreactor level. They also avoid supply problems associated with the natural sources of these compounds or traditional plant cultivation. An interesting feature of in vitro plant cells is their ability to release secondary metabolites into the culture medium, thereby facilitating the in situ recovery of the product and promoting a continuous production by the cells. Thus, extracellular availability of the target compounds is preferable to their intracellular extraction from the cells. To our knowledge, taxol was first found in C. avellana trees by Hoffman et al. (1998), and since then, the advantages of plant cell cultures have been harnessed to enhance taxol yields in C. avellana cell suspensions by different strategies, including elicitation.9-12 Different methodologies to determine taxol and other taxanes are available, most of them using high performance liquid chromatography (HPLC) or Enzyme-Linked ImmunoSorbent Assays (ELISA). HPLC-mass spectrometry (HPLC-MS) has also been used to identify and characterize taxanes in plant samples and cell suspension cultures but not to quantify them, so it was necessary to develop a new reliable methodology to quantify these compounds.13, 9 HPLC-MS/MS is a powerful technique to quantify and determine drugs and metabolites from biological samples, due to its inherent specificity, sensitivity and speed. The HPLC system allows us to separate all the compounds present in the sample extracts, permitting independent MS analysis of the parent ion, which undergoes a selective fragmentation that can be monitored.

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Moreover, this methodology allows compounds to be detected at trace levels with high reliability. Although an HPLC-MS/MS method has been described for taxane analysis in Taxus spp. plant extracts, no HPLC-MS/MS method has been available to determine the low content of taxol and taxanes found in cell suspension cultures or in C. avellana extracts.14 In addition to the problems arising from matrix complexity, the taxane content in the C. avellana tree is ten times lower than in Taxus spp.9 It was therefore necessary to develop a suitable method, including extraction and quantification, able to overcome the matrix issues and detect small quantities of each taxane. The high sensitivity of HPLC-MS/MS methods, especially in multiple-reaction monitoring mode (MRM), indicated it was a suitable technique for taxane analysis in C. avellana samples. In the present work we developed a sensitive HPLC-MS/MS method to quantify five of the most commercially important taxanes: the therapeutic compound taxol, and baccatin III, 10- deacetyl baccatin III, 10-deacetyltaxol and cephalomannine, which can be used for the semi-synthetic production of taxol. Multiple- reaction monitoring mode was used to maximize sensitivity and a positive identification of these compounds by high resolution mass spectrometry (HPLC-ITD-FTMS) was achieved. C. avellana cell suspensions were established and the taxanes released into the culture medium were determined and quantified using the reliable analytical method developed. This constitutes the first step in the scale-up process of continuous cultures associated with in situ product removal. 120

2 Experimental

2.1 Chemicals and reagents

All the reagents used were HPLC grade. Methanol, hexane and dichloromethane were purchased from Teknokroma (Sant Cugat, Barcelona, Spain), acetonitrile LC-MS from Sigma (Madrid, Spain), and MilliQ water was obtained using a Milli-Q system (Millipore). Standards of taxanes, 10-deacetylbaccatin III (DB), baccatin III (B), 10-deacetyltaxol (DT), cephalomannine (CF) and taxol (T), from ChromaDex™ were used to prepare the calibration curves. Docetaxel (DTX), used as an internal standard (IS), was also obtained from ChromaDex™. PVDF syringe filters (13 mm, 0.22 µM pore size) from Teknokroma were used to filter standard solutions and samples.

2.2 Preparation of standard solutions

Standard stock solutions were prepared in methanol at 50 µg ml-1 and kept at -20ºC until use. For the working standard solutions, 10- deacetylbaccatin III, baccatin III, 10-deacetyltaxol, cephalomannine and taxol were dissolved in methanol (MeOH) to obtain seven standard solutions with concentrations in the range of 0.3-2.1 µg ml- 1. A stock solution of 50 µg ml-1 docetaxel (IS) was prepared in MeOH, and further dilutions were also made in methanol. To carry out the analysis, 5 µL of stock solution was added to samples before the extraction to obtain a final concentration of 1 µg ml-1.

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2.3 HPLC-MS/MS conditions

2.3.1 HPLC conditions

The HPLC system consisted of an Agilent 1100 chromatograph fitted with a quaternary pump, a refrigerated autosampler and a UV detector. A Supelcosil ™ LC-F 5 µm (25 cm x 4.6 mm) column (Supelco, Bellefonte, USA) was used. The mobile phase consisted of water (A) and acetonitrile (B) with the following gradient (t(min), %B): (0, 25), (28, 50), (28.5, 90), (32, 90), (32.5, 25), (40, 25). The flow rate was 1.00 ml min-1, and a 1/3 split was done before the MS. The column was maintained at room temperature and the wavelength was set at 225 nm. Injection volume was 10 µL.

2.3.2 MS/MS conditions

The HPLC system was coupled to a triple quadrupole mass spectrometer API 3000 (AB Sciex, Ontario, CA) fitted with a TurboIon spray source working in positive ion mode and using the following settings: nebulizer gas (N2) at 9 (arbitrary units), curtain 3 -1 gas (N2) at 10 (arbitrary units), auxiliary gas (N2) at 8000 cm min , o heated at 400 C, ion spray voltage at + 5 kV, CAD gas (N2) at 5 (arbitrary units), declustering potential DP at +60 V, focusing potential FP at +200 V, entrance potential at +10 V, collision energy at +20 V, and collision cell exit potential CXP at +15 V. MS and MS/MS parameters have been established through infusion experiments using a Harvard syringe pump at 10 µL min-1 (individual standard solution at 1mg l-1) in acetonitrile/water 1:1.

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In order to achieve the highest sensitivity, MRM was the acquisition method of choice, all the transitions (Table 1) having a dwell time of 80 msec. Identification and confirmation transitions were determined for each compound. Taxanes were identified by comparing retention times with the standards in MRM trace chromatograms. Data were acquired and analyzed using analyst 1.4.2 software.

2.4 HPLC-ITD-FTMS

An Accela chromatograph (Thermo Scientific, San Jose; CA,USA) was coupled to an LTQ-Orbitrap Velos instrument (Thermo Scientific, San Jose; CA,USA). A Supelcosil™ LC-F 5 µm (25 cm x 4.6 mm) column (Supelco, Bellefonte, USA) was used and the chromatographic conditions were the same as for the triple quadrupole system. All the analyses were done in positive mode with the following MS conditions: full scan analysis from m/z 100 to 1100 at 30000 resolution using the FTMS.

2.5 Plant Material

C. avellana cell suspensions were established from selected white and friable callus induced from hazelnuts. 3 g of calli were inoculated in 30 mL of Murashige and Skoog medium,15 supplemented with sucrose at 30 g L-1, 2,4-dichlorophenoxyacetic acid 2 mg L-1 and kinetin 0.4 mg L-1. The cell cultures were maintained in 200 mL flasks at 25 ºC in the dark on an orbital shaker at 110 rpm and were routinely subcultured every 15 days 123 until a fine suspension was obtained, establishing different cell suspension lines. The different cell lines were preliminarily screened for their taxane content. The cultures without taxanes, or with a taxane content below the limit of detection, were used for optimization purposes (calculations of the limit of detection (LOD), limit of quantitation (LOQ), recovery, and matrix effect) and also to obtain the matrix-matching calibration curve, while those containing taxanes were analyzed with the optimized method.

2.6 Sample preparation

Taxanes in the C. avellana cell culture medium (20 mL) were extracted using 2 mL of dichloromethane for each 10 mL of medium, vortexed for 2 minutes and sonicated (40kHz, Branson) for 1 hour. The organic phase was dried with nitrogen gas, dissolved in 250 µL methanol and filtered through a PVDF 0.22 µM filter for subsequent analysis. Samples used for optimization purposes, from cell lines without taxanes, were extracted using the same procedure.

2.7 Quality parameters

2.7.1 Linearity: limits of detection and quantification

Calibration curves were done by plotting the analyte area/IS area versus the analyte concentration/IS concentration and weighted using 1/x2, obtaining correlation coefficients (r2) higher than 0.99 and an accuracy in the determination of standard concentration 124 between 80-120%.16 The standard curve was run on three different days in order to evaluate the stability and linearity of our method. Also, the LOD as the concentration with a signal-to-noise (S/N) of 3 and LOQ as the concentration with a signal to noise (S/N) of 10 were determined.

2.7.2 Precision and accuracy

An intermediate concentration of standard solution (1.2 µg mL-1) was injected eight times to evaluate intra and inter-day accuracy and precision. Quantification was carried out using a calibration curve for the same batch.

2.7.3 Recovery and matrix effect

Recovery and matrix effect were evaluated by the standard addition method, spiking samples pre- and post-extraction, respectively. In the pre-extraction, 25 µL of taxanes stock solution in MeOH at 50 µg ml-1 was added to 20 mL of the C. avellana culture medium to obtain a final concentration of 5 µg ml-1. The taxane extraction was then carried out as described before, and dried down. In the post- extraction addition, the same amount of taxane stock solution in MeOH was added to the dried-down extracts and re-evaporated. 5 µL of the DTX standard solution at 50 µg ml-1 was added before the extraction. Samples were resuspended in 250 µL of MeOH.

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2.8 Matrix-matching calibration curve

The same range of concentrations as in the standard curve in methanol was added to a non-taxane-containing C. avellana cell culture medium extract to obtain the points of the matrix-based calibration curve. The additions were carried out before the extraction. Also, in each case 1 µg mL-1 of the DTX as the internal standard was added before the extraction. Taxane extraction was carried out as described before, and the dried-down extracts were resuspended in 250 µL of MeOH. Extractions were carried out in duplicate for each concentration.

3 Results and discussion

Electrospray ionization (ESI) was tested in both positive and negative ion modes, with a higher response found in the former. Therefore, the ESI source in positive ion mode was chosen for taxane detection. The mass spectra were recorded in the range of m/z 100-1000 amu. The main ions obtained from the standards were as follows: DB: m/z 545.4 [M+H]+, B: m/z 587.3 [M+H]+, DT: m/z 812.5 [M+H]+, CF: m/z 832.4 [M+H]+, T: m/z 854.5 [M+H]+, and DTX: m/z 808.5 [M+H]+. Also, m/z 830.5 [M+Na]+ was observed for DTX. Since declustering potential (DP) and collision energy (CE) played a significant role in generating the final MRM mode, both parameters were optimized in order to have the maximum signal for each analyte in infusion experiments (DP 60V and CE 25- 30V). Identification transitions, which were defined as those with

126 highest intensity, and the confirmation transitions obtained are shown in Table 1.

McClure et al. (1992) and Kerns et al. (1994) reported the fragmentation pattern of taxol and 19 natural taxanes, describing the fragment ion characteristic of the paclitaxel core as m/z 509. The target compounds showed lateral chains with different m/z fragments: m/z 286 in the case of T, m/z 286 and m/z 268 for DT, and m/z 264 for CF. Also, m/z 327 was thought to correspond to the baccatin III core after water loss.17,13 Similarly, we hypothesize that m/z 405 corresponds to the baccatin III core with a carboxylic acid substitution, and m/z 363 to the baccatin III core with water incorporation. The Multiple Reaction Monitoring (MRM) transitions for DTX are m/z 808.5527.0, described by Corona et al. (2011), and m/z 830.5549.4 corresponding to the Na+ adduct.18 Figure 1 depicts the common structure for each compound, and the different fragments used for their identification. Although some m/z fragments were already described, only a descriptive fragmentation was usually reported, and there are no studies available combining these data with the evaluation of the quality parameters and with the quantification using a matrix-matching calibration curve.

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Table 1. Parameters of the HPLC-MS/MS analysis of taxanes: (a) molecular weight (Da), (b) retention time (min); the two MRM transitions for each compound correspond to the identification transition and confirmation transition (*). DP was fixed at 60V for all compounds.

MW Molecular CE RT m/z MRM (a) Ion (V) (b) [M+H]+ 20 545.4 363.1 DB 544 545.4 7.31 [M+H]+ 20 545.4327.4* [M+H]+ 20 587.3405.2 B 586 587.3 11.88 [M+H]+ 20 587.3327.3* [M+H]+ 20 812.5286.2 DT 811 812.5 20.50 [M+H]+ 20 812.5268.3* 808.5 [M+H]+ 35 808.5527.0 DTX 807 22.36 830.5 [M+Na]+ 35 830.5549.4* [M+H]+ 20 832.4509.4 CF 831 832.4 23.02 [M+H]+ 20 832.4264.1* [M+H]+ 20 854.5509.3 T 853 854.5 25.00 [M+H]+ 20 854.5286.3*

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Figure 1. A) The common structure for taxol (T), 10-deacetyltaxol (DT), cephalomannine (CF) and docetaxel (DTX). Fragments for each compound are represented. B) The common structure for baccatin III (B) and 10- deacetylbaccatin III (DB). The fragmentation profile is based on m/z 345 (baccatin core) with the described additions and losses.

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3.1 Linearity

The standard curve analysis before the use of the IS showed an increasing angle of slope during the three days (data not shown). To solve this problem, we searched for a suitable IS. Taking into account that an IS should be a compound of the same chemical class as the analytes but with different molecular weight and different MRM transitions, we chose DTX as an appropriate IS to determine the taxanes in our samples, since it fulfilled the aforementioned requirements. Moreover, as a synthetic analogue, it was not present in our samples.

The linearity of the chromatographic method was determined using a mixed standard solution of DB, B, DT, CF and T at seven concentrations in the range of 0.3-2.1µg mL-1 supplemented with 1µg mL-1 of the IS DTX. The graph therefore represents the analyte area/IS analyte area vs analyte concentration/IS concentration (Figure 2). The regression equation and the correlation coefficients of each compound in each standard curve were determined. R2 was higher than 0.997 for all the compounds in the three different standard curves, showing a good linearity over the measured range and allowing a good extrapolation of our data. Weighted least squares linear regression by a factor of 1/x2 resulted in an accuracy range of 80-120% in terms of RSD.16

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Figure 2. Standard curves of 10-deacetylbaccatin III (A), baccatin III (B), 10- deacetyltaxol (C), cephalomannine (D) and taxol (E) in the range of 0.3-2.1 µg mL-1. 1 µg mL-1 of DTX was added as the internal standard (IS), therefore the analyte area/IS area vs analyte concentration is represented.

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3.2 Limits of detection and quantification

Limits of detection and quantification were determined taking into account the matrix effect. With this aim, C. avellana samples from non-taxane-containing cell lines were considered as blank samples and therefore spiked to obtain a concentration of 0.3 µg mL-1. S/N=3 and S/N=10 were used to determine LOD and LOQ, respectively. The LOD was 37.6 ng mL-1 for DB, 2.42 ng mL-1 for B, 0.24 ng mL-1 for DT, 0.53 ng mL-1 for CF and 0.79 ng mL-1 for T. The LOQ was also determined for all the compounds, being 125.5 ng mL-1 for DB, 8.0 ng mL-1 for B, 0.80 ng mL-1 for DT, 1.8 ng mL-1 for CF and 2.7 ng mL-1 for T. No taxanes were found in the blank sample, although they may have been present below the detection limit.

3.3 Precision and accuracy

Intra- and inter-assay precision and accuracy were determined by assaying eight replicates of an intermediate concentration (1.2 µg mL-1). The calculated concentrations from the appropriate standard curve in each analytical run were used to obtain the relative standard deviation (RSD). Accuracy was assessed as the percentage of RSD from the theoretical concentration. These parameters are described in Table 2. Intra-day repeatability reached a maximum RSD of 7% and accuracy ranged between 82-119% RSD. Inter-day repeatability RSD was always lower than 15% and accuracy was between 98-104%. Taking into account all these parameters, we

132 developed a reliable and reproducible method, with a repeatability not exceeding 15% RSD and an accuracy always within the range of 80-120%. Additionally, the RSD of the retention times was calculated for all the runs and was always lower than 1% for all the compounds, again illustrating the chromatographic robustness of the method.

Table 2. Mean, relative standard deviation (RSD) and accuracy for each compound regarding the three different standard curves (1,2,3). Th.C: Theoretical concentration. DB: deacetylbaccatin III. B: baccatin III. DT: 10-deacetyltaxol. CF: cephalomannine. T: taxol.

Th.C. Mean Intra-day RSD (%) Accuracy (%) (µg mL-1) (n=8) 1 1,26 1,9 96,9 DB 2 1,30 1,13 4,7 86,6 3 1,60 1,5 119,0 1 1,29 2,2 101,3 B 2 1,27 1,12 3,2 88,0 3 1,40 4,6 110,6 1 1,28 1,8 101,6 DT 2 1,26 1,10 4,9 87,3 3 1,38 7,7 109,7 1 1,20 2,5 102,6 CF 2 1,17 1,03 4,7 88,3 3 1,42 5,2 119,3 1 1,28 2,9 101,9 T 2 1,26 1,13 4,1 89,8 3 1,50 5,1 119,2

Inter-day Mean (n=24) RSD (%) Accuracy (%) DB 1,33 15,7 102,2 B 1,27 10,1 99,9 DT 1,25 10,9 98,7 CF 1,22 14,0 104,0 T 1,31 12,7 103,7

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3.4 Recovery accuracy and matrix effect

Many parameters can influence sample analysis, two of the most important being recovery and the matrix effect. Recovery is based on the amount of product collected after the extraction method. In complex matrices, such as plant samples, an extraction method providing pure compounds is extremely difficult to develop, so recoveries are usually low. On the other hand, the matrix effect, ion suppression or ion enhancement are due to other compounds in the sample interfering with the detection of the target compounds. The taxane standard solution was added to 5 µg mL-1 C. avellana culture medium before the extraction to analyze recovery, and then after the extraction to analyze the matrix effect. Calculated concentrations were compared with the theoretical concentration added. Recovery by this methodology was 100% for DB, 86.2% for B, 84.6% for DT, 82.9% for CF and 87.5% for T. These percentages were obtained taking into account the calculated suppression for each compound. The results showed a matrix effect of -44.4% for DB, 49.9% for B, 42.6% for DT, 43.4% for CF and 42.9% for T in comparison with the responses achieved when injecting the methanolic solution. The high efficiency of the extraction method and the sensitivity of the system allowed us to detect our compounds despite a certain degree of suppression in our analytical process.

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3.5 Matrix-matching calibration curve

The same range of taxane concentrations as in the standard curve were added to the C. avellana cell culture medium extracts from non- taxane-containing cell lines to obtain a calibration curve. Linearity of the calibration curve was analyzed for all the compounds, showing an r2 of 0.987 for DB, 0.993 for B, 0.992 for DT, 0.986 for CF and 0.984 for T. Weighting of 1/x2 was applied to obtain an accuracy of 80-120%, while real accuracies were 92.5- 114%. Figure 3 shows the MRM transitions (identification) for standard compounds (A) and for a sample of C. avellana cell culture medium extract from a producer cell line (B), detecting and quantifying B, CF and T. The ion ratios between the confirmation and identification transition were calculated and compared with those obtained in the standard, allowing us to confirm the presence of the compound when the difference was less than 20%. The C. avellana medium extract was quantified using the matrix-matching calibration curve, obtaining a concentration of 120 ng mL-1 for B, 151 ng mL-1 for CF and 105 ng mL-1 for T. Taking into account the original volume of medium extracted (20mL), concentrations of 0.5 ng mL-1 for B, 0.63 ng mL-1 for CF and 0.44 ng mL-1 for T were produced by C. avellana cells and released into the medium. These results had been confirmed in the extract using HPLC-ITD-FTMS by comparing their retention times with those of standards previously injected in the same conditions and by the exact mass [M+H]+ of the ions present in the FTMS spectra with an accuracy of  2 mDa.

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A B DB: 545.4 363.1 7.31

B: 587.3 405.2 11.88 11.85

DT: 812.5 286.2 20.51 19.80

DTX: 808.5 527.0 22.36 22.30

CF: 832.4 509.4 23.02 23.01

 24.90 T: 854.5 509.3 24.95

Figure 3. Identification chromatograms for taxane traces, A: Standard solution at 1.2 µg mL-1 with 1 µg mL-1 DTX as IS. B: C. avellana sample with 1 µg mL-1 DTX. 10-deacetylbaccatin III (DB), baccatin III (B), 10-deacetyltaxol (DT), docetaxel (DTX), cephalomannine (CF) and taxol (T).

It is therefore possible to affirm that C. avellana is a taxane- producing plant, although the concentrations obtained were very low in comparison with those obtained in Taxus spp.19 The analysis was carried out at day 3 of the cell suspension culture because of the low taxane concentration obtained at that point. The developed method is therefore sufficiently sensitive to detect taxane production from the beginning of the culture, when the production is usually extremely low. Razaei et al. (2011) reported a taxol

136 content of 13 µg L-1 in the medium of a C avellana cell suspension culture growing in control conditions at day 12 by HPLC-UV analysis.12 Bemani et al. (2013) reported 16 µg L-1 of extracellular taxol at day 14, quantified by HPLC-UV and confirmed by HPLC- MS, but no information about taxol production in the early stages of culture and quantitation by HPLC-MS/MS has been previously reported.20

4 Conclusions

We have developed and optimized a reliable and sensitive HPLC- MS/MS method to determine five of the most commercially important taxanes (taxol, baccatin III, 10-deacetyl baccatin III, 10- deacetyltaxol and cephalomannine) present at a nanogram level in a Corylus avellana cell suspension culture medium. The culture media of C. avellana, a potential new source of taxanes, is of particular interest, as it facilitates the in situ recovery of the product and promotes a continuous production by the cells. In addition, extracellular availability of the target compounds is crucial for scaling up the process.

This new analytical procedure is able to detect secreted taxanes even at the beginning of the culture, when the production is usually extremely low, and allows the production profile to be followed throughout the assay. To the best of our knowledge, this is the first reported application of a liquid chromatography-tandem mass spectrometry method to quantify individual taxanes in Corylus avellana, and to evaluate linearity, precision, accuracy, recovery,

137 and the matrix effect, as well as to develop a matrix-matching calibration curve to quantify more precisely the lower concentration of these compounds.

Acknowledgements

We thank the Centres Científics i Tecnològics of the Universitat de Barcelona (CCiTUB) for their help. This research has been supported by a grant from the Spanish MEC (BIO2011-29856-CO2- 01) and a grant from the Catalan Government (2014SGR215). A. Gallego was supported by a fellowship from the University Pompeu Fabra.

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2. Y.F. Wang, Q.W. Shi, M. Dong, H. Kiyota, Y.C. Gu and B. Cong, Chem. Rev., 2011, 111, 7652-709. 3. J.A. Yared and K.H.R. Tkaczuk, Drug Des. Dev. Ther., 2012, 6, 371-384. 4. W.P. McGuire, E.K. Rowinsky, N.B. Rosenshein, F.C. Grumbine, D.S. Ettinger,D.K. Amstrong and R.C. Donehower Ann. Intern. Med., 1989, 111, 4, 273-279. 5. F.A. Holmes, R.S. Walters, R.L. Theriault, A.U. Buzdar, D.K. Frye, G.N. Hortobagyi, A.D. Forman, L.K. Newton and M.N. Raber, J. Natl. Cancer. Inst., 1991, 83, 1797-1805.

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6. M.W. Saville, J. Lietzau, J.M. Pluda, W.H. Wilson, R.W. Humphrey, E. Feigel, S.M. Steinberg, S. Broder, R. Yarchoan, J. Odom and I. Feuersten, Lancet, 1995, 346, 26-28. 7. E.A. Eisenhauer and J.B. Vermorken, Drugs, 1998, 55, 5-30. 8. P.B. Schiff, J. Fant and S.B. Horwitz, Nature, 1979, 277, 665- 667. 9. A. Hoffman, W. Khan, J. Worapong, G. Strobel, D. Griffin, B. Arbogast, D. Barofsky, R. Boone, L. Ning, P. Zheng and P. Daley, Spectroscopy, 1998, 13, 6, 22-32. 10. L.Ottaggio, F. Bestoso, A. Armirotti, A. Balbi, G. Damonte, M. Mazzei, M. Sancandi and M. Miele, J. Nat. Prod., 2008, 71, 58- 60. 11. F. Bestoso, L. Ottaggio, A. Armirotti, A. Balbi, G. Damonte, P. Degan, M. Mazzei, F. Cavalli, B. Ledda and M. Miele, BMC Biotechnol., 2006, 6,45. 12. A. Rezaei, F. Ghanati, M. Behmanesh, M. Mokhtari-Dizaji, Ultrasound Med. Biol., 2011, 37,1938-47. 13. E.H. Kerns, K.J. Volk, S.E. Hill and M.S. Lee. J. Nat. Prod., 1994, 57, 1391-1403. 14. S. Li, Y. Fu, Y. Zu, R. Sun, Y. Wang, L. Zhang, H. Luo, C. Gu and T. Efferth, J. Pharm. Biomed. Anal., 2009, 49, 81-9. 15. T. Murashige and F. Skoog, Physiol. Plant, 1962, 15, 473-497. 16. M.M. Kiser and J.W. Dolan, LC Trobleshooting, 2004, 17, 138- 143. 17. T.D. McClure, K.H. Schram and M.L.J. Reimer, J. Am. Soc. Mass Spectom., 1992, 3, 672-679.

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18. G. Corona, C. Elia, B. Casetta, S. Frustaci and G. Toffoli, Clin. Chim. Acta., 2011, 412, 358–364. 19. S. Malik, R.M. Cusidó, M.H. Mirjalili, E. Moyano, J. Palazon and M. Bonfill, Process Biochem., 2011, 46, 23-34. 20. E. Bemani, F. Ghanati, A. Rezaei and M. Jamshidi, J. Nat. Med., 2013, 67, 446-451.

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141

142

Chapter IV:

Antiproliferative activity of C. avellana plant extracts in HeLA, HepG2 and MCF-7 cells.

143

144

1 Introduction

Drug development has a long background in assaying traditional medicine, searching for active natural compounds in a wide range of plants. Plants have traditionally been used to treat different diseases such as cancer, diabetes or several infections. After compiling all the data published about new drugs approved since 1994, it was concluded that almost half the drugs were based on natural products (Harvey, 2008), and during the period of 2005- 2007 thirteen natural-product drugs were approved (Butler, 2008). If we focus on cancer, more than 60% of the drugs have a natural origin (Newman and Cragg, 2007). For these reasons, natural products play an important role in drug discovery, being a source of inspiration and innovation (Cragg and Newman, 2005).

The discovery of taxanes in C. avellana plant extracts has promoted the study of this plant and its secondary metabolites. Over 100 compounds have been described in C. avellana, classified as organic acids, triacylglycerols, phytosterols, tocols, phenolic acids, diarylheptanoids, flavonoids, tannins, isoflavones, lignans, terpenes and taxanes (see Annex I). This powerful information can be used to compare new data obtained by analytical techniques with those already described, making it easy to identify and characterize new lead compounds. Previous experiments have reported antiproliferative activity in extracts of C. avellana cell cultures, which was attributed to taxol. SK-Mes-1 cells (derived from lung cancer) treated with an extract from the medium of C. avellana cell cultures blocked cancer cells at the metaphase/anaphase transition,

145 avoiding cell cycle progression, with a higher effect than yew extract (Bestoso et al., 2008). Recently, the effect of a hazel cell extract was evaluated in the MCF-7 cancer cell line (derived from breast cancer), confirming the aforementioned activity and concluding that hazel cell extracts were more effective than pure taxol, even when the taxol content was the same as the pure compound. It was therefore hypothesized that hazel extracts may have other taxol-like compounds that could increase this effect (Bemani et al., 2012). On the other hand, the antiproliferative activity from extracts of the C. avellana trees has not been studied yet.

With the aim of isolating and characterizing new compounds with antiproliferative activity, bioassay-guided experiments were carried out. The main disadvantages in the study of new secondary metabolites is the high complexity arising from the need to work in a multidisciplinary environment, being mandatory knowledge of ethnobotany, phytochemistry, chemistry and molecular biology techniques. For our purpose, compounds from leaves and stems of C. avellana were extracted following different methodologies and the aforementioned activity was evaluated by a MTT assay in three different cell lines: HeLA (derived from cervical cancer cells) HepG2 (derived from liver hepatocarcinoma) and MCF7. The MTT assay is a colorimetric technique based on the ability of the living cells to transform tetrazolium salt MTT (3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide) by the mitochondria into a formazan, a characteristic purple precipitate (Figure 1). 146

Br - Tetrazolium briomide Formazan Figure 1. Tetrazodium conversion to formazan.

Formazan can be dissolved in dimethyl sulfoxide (DMSO), among other solvents, and the optical density of the resulting solution can be measured using a spectrophotometer. The amount of formazan produced is proportional to the number of living cells. This assay is very useful to study the antiproliferative effect of natural compounds. Since the discovery of the MTT assay by Mosmann in 1983, several modifications have been reported, involving different times of incubation, proportions and conditions (Maehara et al., 1988; Sladowski et al., 1993; Hussain et al., 1993; Hamid et al., 2004). In this work, we studied the antiproliferative activity of leaves and stems of C. avellana extracts. With this goal, the MTT assay was used to screen the activity of the initial extract as well as each purification step. Moreover, the identification by HPLC-MS and RMN of the compounds that could have a high antiproliferative activity against different cancer cell lines (HeLA, HepG2 and MCF- 7) was also performed.

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2 Materials and methods

2.1 Mammalian cell lines

HeLA, MCF-7 and HepG2 cell lines were obtained from ATCC (ATCC numbers: CCL-2™, HTB-22™ and HB-8065™ respectively). The cells were cultured in DMEM medium without pyruvate, supplemented with 1 % of penicillin/streptomycin and 10 % of heat inactivate fetal bobine serum. All the components were purchased from Gibco (Invitrogen, Barcelona, Spain). The cells were incubated with 5% CO2 at 37 ºC.

2.2 Plant material and extractions

Stems and leaves were collected in October from three adult trees in Catalonia: (I) wild type from Barcelona, (II) wild type from Terrassa, and (III) a cultivar in Tarragona. Plant material was air- dried over night at 60 ºC, and powdered.

2.2.1 Screening extraction

Two different extraction procedures were tested in the screening assays: 1) maceration of 1 g of freeze-dried cells in Methanol

(MeOH) 90%:H2O 10% (40 mL, 24 h). The process was repeated twice and both methanolic phases were evaporated. 2) Taxane extraction based on Onrubia et al., (2013): 1gr freeze-dried cells was extracted with 40 mL of MeOH:water (9:1, v/v), heated for 8 min at 80 W (microwave-assisted extraction) and filtered through nylon (0.3 µM). This process was repeated twice, and both 148 methanolic extracts were combined. Then, an equal volume of hexane was added to the samples, mixed and centrifuged at 2500 g for 20 min at room temperature. The aqueous phase was recovered and the compounds were extracted using 20 mL of dichloromethane (DCM): water (2:1, v/v). This step was repeated twice. Organic phases were collected, combined and evaporated.

2.2.2 Large-scale extractions

Once screening was performed, the tree that showed the highest antiproliferative activity was extracted using an accelerate solvent system, such as Zippertex® (Ouazzani et al., 2007). Zippertex® extractions apply high temperatures and pressures, increasing the penetration of the solvents into the matter and promoting a higher solubilization of the target compounds. Zippertex® also performs a filtration of the extracts, recovering more than the 80% of the solvent. 25 g of stems and 40 g of leaves were extracted with DCM (2x100 mL) followed by MeOH (2x100 mL) at 100 bars. Afterwards the solvents were evaporated.

2.2.3 Sample preparation for the MTT assay

All the extractions were evaporated in a rotavapor (Buchi Labortechnik AG, Switzerland). The conditions were established for each solvent. Each dry extract was resuspended in DMSO at 1 g DW/mL for maceration extracts, and 2 g DW/mL for the taxane

149 extracts (screening and Zippertex®). All the samples were filtered through a DMSO safe filter (22 µM; Tecknochroma).

2.3 Experimental design

2.3.1 Vehicle validation

HeLA cells were used to validate the vehicle. DMSO and EtOH were analyzed to determine the most suitable solvent and the appropriate volume for the experiments. The concentrations tested were: 0.01%, 0.02%, 0.1%, 0.2% and 1% (v/v). Cells were seeded at 3.4 x104 cells/well in 12 well plates and after 24 h of growth, DMSO or EtOH was added to the medium, and the assay was carried out for 48 h. All conditions were run in triplicates. Cells without vehicle treatment were included to be considered as a control.

2.3.2 Taxol toxicity

The effect of taxol was assayed in the three cell lines, determining

IC50 for each cell line. A DMSO solution of the commercial taxol and different dilutions were done to evaluate the following concentration: 0.5 nM, 5 nM, 50 nM, 500 nM, 1500 nM. 3.4x104 cells/well were seeded for HeLA and HepG2 cells and 7x104 cells/well for the MCF-7 cells in a 12 well plate. Cells were grown for 24 h, and then 9 µL of taxol solution was added to the medium, analyzing the viability after 48 h. All the conditions were run in triplicate. A control without drug treatment and a control where the 150 cells were only treated with the vehicle were included in all the assays.

2.3.3 Plant extract assays

Similar cell densities as used for the taxol experiment were seeded. Two different volumes were tested, 9 µL or 2 µL, for each extraction (obtained through maceration, extraction, Zippertex® and fractionation). Each extract was added to the culture in the exponential phase and the effect was measured 48 h later. All the conditions were run three times. A control without treatment and a control treated only with the vehicle were tested in all the assays.

2.4 Cytotoxicity assay

The viability of the cells was determined by the MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay based on Rodríguez et al. (2013). The growth medium was removed and 0.63 mM of MTT and 18.4 mM of sodium succinate (from Sigma- Aldrich, Madrid, Spain) were added to 1 mL of fresh culture medium and the cells were incubated for 3 h at 37 ºC. Then, the medium was removed and the formazan was resuspended in DMSO supplemented with 0.57% CH3COOH and 10% SDS (from Sigma- Aldrich, Madrid, Spain). Absorbance was measured at 570 nm in a UV2310 spectrophotometer (Dinko, Barcelona, Spain).

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2.5 Analytical procedures

2.5.1 Plant extract fractionation

The MeOH extract from leaves was subjected to an additional fractionation procedure. Compounds of the extract were first separated by Silica gel chromatography using a Combiflash- companion chromatograph (Serlabo) and ready-to-use RediSep column (40 g), obtaining 8 fractions. The mobile phase consisted of DCM (A) and MeOH (B) with the following increasing polarity gradient (t (min), %B): (5, 0), (7-17, 5), (18-28, 10), (32-42, 15), (44-55, 20) in 60 min at 30 mL/min.

Flash chromatography was performed with the most active fractions to purify the lead compounds. A Sunfire C18 III (10 x 250 mm) 5 µm column was used and the mobile phase consisted of water (A): acetonitrile (B), both added with 0.1% formic acid (v/v). A linear gradient of 100% A to 100% B was established in 20 minutes with a flow of 30 mL/min.

2.5.2 HPLC-MS conditions

An Alliance Waters 2695 HPLC instrument (Waters) including autosample 717, a pump 600 equipped with a photodiode array 2998 detector and an evaporative light-scattering detector 2420 was used. The HPLC analytical column used was Sunfire C18 III (4.6 x 150 mm) 3.5 µm operating at 0.7 ml/min. The mobile phase was water (A) and acetonitrile (B), both containing 0.1% of formic acid. A linear gradient from 100% of A to 100% of B was performed in

152

50 minutes. HPLC was fitted to a simple quadrupole Waters- Micromass®ZQ 2000 mass spectrometer. All the analyses were done in positive and negative mode with the following MS conditions: capillary voltage: 3.5; cone voltage: 25 (+), 45(-); extractor voltage: 1; source temperature: 110; desolvation temperature: 250; RF Lens: 0.1; desolvation gas flow: 450; cone gas flow: 50; LM1/HM1 resolution: 15; ion Energy: 0.5 and Multiplier: 600.

2.5.4 Nuclear magnetic resonance

1H spectra were recorded using a Brüker Avance-500 instrument operating at 500 MHz. Deuterated methanol (MeOD) was used as a solvent. The obtained 1H chromatograms for each compound were compared with the corresponding simulated chromatogram estimated by ChemNMR software.

2.6 Statistical analysis

Triplicates were considered for the statistical analysis. The average and standard deviation were calculated for all the measurements and treatment results were compared with the control conditions run in each assay. The statistical analysis was performed with SPSS (version 22.0). In order to estimate the effect of each treatment on cell viability, ANOVA analysis was performed and only p-values lower than 0.05 (*) or 0.01 (**) were considered statistically significant.

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3 Results

3.1 Validation of the vehicle

Figure 2 shows the effect of different concentrations of DMSO or EtOH in the viability of HeLA cells. The cell treatment was done at 24h of seeding, making sure that cells were in the exponential phase. Then, the cells where incubated with 0.01%, 0.02%, 0.1%, 0.2% and 1% (v/v) of DMSO or EtOH during the next 48 h. EtOH denoted a significant reduction in cell viability compared with the control cells (p<0.05) in all conditions assayed. In contrast, DMSO did not show a statistically significant reduction of cell viability. In the presence of DMSO the % of viability was never lower than 93%, while EtOH decreased the viability by up to 36%. EtOH proved to be more cytotoxic than DMSO at any of the concentrations tested. Therefore, DMSO was selected as the solvent to carry out the next experiments. A concentration of DMSO up to 1% (v/v) can be used, but we chose 9 µL of DMSO as a suitable volume for all the treatments (0.75% v/v).

154

HeLA DMSO EtOH

100

80 * * 60 * * *

% Viability % 40

0 NT 0,01% 0,02% 0,10% 0,20% 1% Figure 2. Effect of DMSO (dark color) and EtOH (light color) on the cell viability of HeLA cells. The concentrations studied were between 0.01% and 1% and the cell viability is expressed as the % of values found in the non-treated cells (NT). The results are presented as a mean ± standard deviation (SD) of the triplicates. Statistically significant differences compared to their respective control (NT: no treated) are indicated by *p<0.05; **<0.01.

3.2 Cytotoxicity of taxol

The effect of different concentrations of taxol on cell viability was tested (Figure 3). HeLA and HepG2 cells showed a dose-response effect, since a higher taxol concentration increased cytotoxicity. The HeLA cell line showed a significantly greater reduction in cell viability than HepG2 at all the taxol concentrations. But both cell lines treated with 5 nM taxol showed a statistically significant reduction of cell viability compared with the treatment with DMSO. Although MCF-7 showed a 50% reduction of viability at 5 nM, it was not statistically significant due to the large standard deviation value. Besides, MCF-7 treatment with higher concentrations of taxol did not show any further decrease in cell viability. The IC50 determined was 5 nM for HeLA and HepG2. This concentration 155 was also used for MCF-7 since it was enough to determine an effect.

HeLA HEPG2 MCF-7

100

80 *

60 * * *

%Viability 40 * * * * * * 20 * * * * * * 0 NT DMSO 0,5 nM 5 nM 50 nM 500 nM 1500 nM

Figure 3. Effect of taxol on cell viability of HeLA, HepG2 and MCF7 cells. The taxol was tested in the range of 0.5-1500 nM. The cell viability is expressed as % of values found in the DMSO-treated cells. Each bar represents the mean of three independent experimental values ± standard deviation (SD). Statistically significant differences between treatments and DMSO-treated cells (control) is presented as *p<0.05; **<0.01.

3.3 Effect of the plant extracts

Screening experiments were carried out analyzing the leaves and stems extracts obtained from three different trees: I and II are wild type trees from Barcelona and Terrassa, respectively, and tree III is from a Tarragona cultivar. The dry extracts were resuspended in DMSO at a concentration of 1 g DW/mL in the case of maceration extracts and 2 g DW/mL after the taxane extraction procedure. The

156 cell treatment was performed in the three cell lines at the exponential phase and the incubation period was 48 h.

Figure 4 show the effects of the different plant extracts in HeLA cells. 9 µL of each extract was analyzed and all of them caused a dramatical reduction in cell viability. In all cases the reduction was statistically significant (p<0.01) compared with DMSO-treated cells. Due to the unexpected high effect of the extracts on cell viability, a dose-response assay was next carried out with the maceration extract obtained from leaves of tree III on HeLA cells (Figure 5). The reduction of the extract volume added to the cell culture from 9 to 2 µL resulted in an increase in living cells. Therefore, 2 µL of extract was set for further assays as an appropriate concentration to visualize effects on the viability of the cell population. Figure 6 shows the effect of the treatment of HepG2 and MCF-7 cells with 2 µL of each plant extract. In HepG2 the stem extracts showed higher toxicity than the leaf extracts. Maceration extracts obtained from leaves showed a higher effect than stem extracts. MCF-7 studies showed viabilities above 50% in most cases, with the exception of the stem extract treatments and with the leaf maceration treatment obtained from tree III.

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HeLA

100

40 % Viability % 20

0 NT DMSO P E M E M E M E M E M E M Stems Leaves Stems Leaves Stems Leaves I II III Figure 4. Cell growth inhibitory activity of extracts prepared from three different trees (I, II, III) in HeLA cells. The cell viability is expressed as % of values found in the DMSO-treated cells. 9 µL of the extracts from stems and leaves obtained by taxane extraction (E) or by maceration (M) were studied. The results presented correspond to the mean ± standard deviation (SD) of three independent experiments. NT: no treated cells; DMSO: cells treated with DMSO; P: paclitaxel 5nM.

HeLA

100 80 60

40 % viability % * 20 * * * * * 0 * * NT DMSO P 9µL 4,5µL 2µL Leaves maceration extract (tree III)

Figure 5. Dose-response assay of maceration extract from leaves (tree III) in HeLA cells. The cell viability is expressed as % of values found in the DMSO- treated cells. The results are presented as mean values ± standard deviation (SD) of three independent experiments. Statistically significant differences are presented as *p<0.05; **<0.01. NT: not treated; P: paclitaxel 500nM.

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HepG2 MCF-7

100

40 % Viability % 20

0 NT DMSO P E M E M E M E M E M E M Stems Leaves Stems Leaves Stems Leaves I II III Figure 6. Cell growth inhibitory activity of different extracts obtained from three different trees (I, II, III) in HepG2 and MC7-7 cells. Cell viability is expressed as % of values found in the DMSO-treated cells. 2 µL of the extracts from stems and leaves obtained by taxane extraction (E) or by maceration (M) were studied. The results are presented as mean values ± standard deviation (SD) from three independent experiments. NT: not treated cells; P: paclitaxel 5nM.

The statistical analysis revealed no significance differences between the extracts of tree II and tree III. However, less effect was seen when the cells were incubated in presence of extracts from tree I. Thus, subsequent studies were carried out with extracts from tree III. There were no statistically significant differences between the effect of leaves or stems from tree III in the three cell lines, but maceration extraction showed a significantly higher effect than taxane extraction (p<0.01). Taking into account all of these results, high amounts of C. avellana leaves and stems were extracted in a maceration-like process but with higher efficiency, using Zippertex® technology to perform a more in-depth analysis of the compounds produced or accumulated by the tree.

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3.4 Bioassay-guided: Fractionation and characterization

Extracts from C. avellana markedly reduce viability of human cancer cell lines but there are no reports about the cytotoxic effects of C. avellana tree extracts. With the aim of identifying the main compounds responsible for the growth inhibitory activity described in point 3.3, 25 g of stems and 40 g of leaves (tree III) were extracted with Zippertex® technology using two different solvents, MeOH and DCM. The extracts obtained were evaporated and resuspended in DMSO at a concentration of 2 g DW/mL. Firstly, the antiproliferative activity using this new procedure was confirmed (Figure 7) in the three cell lines with a treatment of 2µl of each extract. Stem extracts and MeOH leaf extracts showed a statistically significant reduction of the cell viability in the three cell lines tested compared with DMSO treatment (*p<0.05, **p<0.01). While both extracts from stems (extraction with MeOH or DCM) showed a similar growth cell reduction, MeOH extraction from leaves had a higher toxicity compared with DCM extraction.

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HeLA HEPG2 MCF-7 120

100 * * 80 * 60 * * * % Viability % 40 * * * * * * * * * * 20 * * * * * * * * 0 NT DMSO P DCM MeOH DCM MeOH Stems Leaves Zippertex® Figure 7. Cell growth inhibitory activity of Zippertex® extracts on HeLA, HepG2 and MCF-7. The cell viability is expressed as % of values found in the DMSO-treated cells. DCM and MeOH correspond to the evaporated extracts resuspended in DMSO. The results are presented as mean values ± standard deviation (SD) from three independent experiments. Statistically significant differences compared to the respective control (DMSO) are presented as *p<0.05; **<0.01. NT: Not treated; P: Paclitaxel 5nM.

Thereafter, fractionation of MeOH extraction with Zippertex® from leaves was carried out by a Combiflash instrument, obtaining 8 fractions. These fractions were resuspended in 50 µL of DMSO, obtaining a concentration of 0.4 g DW /mL (diluted 5 times compared with the initial extraction), and were tested in HeLA cell culture. With the aim of observing a dose-response effect, two concentrations, 9 µL and 2 µL, were assayed (Figure 8). Fractions 1, 2, 6 and 7 showed a statistically significant reduction of the cell viability at both concentrations assayed (p<0.01). Specifically, for fractions 6 and 7 the effect observed depends on the concentration

161 assayed, while both fractions 1 and 2 promoted a dramatic reduction in cell viability. Fractions 3, 4 and 5 showed a statistically significant effect compared to DMSO treatment when using 9 µL for the cell treatment, while 2 µL has no effect on cell growth. This allowed us to identify those fractions containing the highly active compounds.

HeLA 2 µL 9µL

100

* * 40 * * % Viability % * * * * * 20 * * * * * * * * * * * * * 0 * * NT DMSO F1 F2 F3 F4 F5 F6 F7 F8 Figure 8. Effect of the fractions obtained by the Combiflash instrument (F1-F8) from the leaf MeOH extracts using Zippertex® technology on HeLA cells. The cell viability is expressed as % of values found in the DMSO-treated cells. The results are presented as mean values ± standard deviation (SD) from three independent experiments. Statistically significant differences compared to the respective control (DMSO) are presented as *p<0.05; **<0.01. NT: Not Treated.

Due to the dose-response activity showed by fractions 6 and 7, HPLC-preparative, HPLC-MS and NMR were used to isolate, identify and characterize the compounds present in these fractions. Figure 9 showed the chromatograms obtained by HPLC-MS for fraction 6 (a) and fraction 7(b), m/z 478 and m/z 448 being a common mass in both fractions.

162

ELSD Signal - ELSD Signal A 4

100.00

LSU 50.00 1 2 3 0.00 1. m/z 386 Cholesterol 2. m/z 388 Cholestanol 3. m/z 478 Hydroxyphenyl-4-hepten-3-one hexoside familly 4. m/z 448 Quercetin 3-O-Rhamnoside

ELSD Signal - ELSD Signal B 3 600.00

400.00

LSU 200.00 4 0.00 3. m/z 478 Hydroxyphenyl-4-hepten-3-one hexoside familly 4. m/z 448 Quercetin 3-O-Rhamnoside

15.534 Peak 1 -M-F7-P7-2 - ZQ F3 Scan 100.00-1200.00 ES-, Centroid, CV=25

477.1 2.5x10 6 C - 2.0x10 6 [M-H]

1.5x10 6 [2M-H]- Intensity 478.1 1.0x10 6 955.5 956.5 5.0x10 5 479.1 957.5 437.1 474.9 591.4 941.3 953.4 0.0 200.00 400.00 600.00 800.00 1000.00 1200.00 m/z

16.474 Peak 1 -M-F7-P8-2 - ZQ F3 Scan 100.00-1200.00 ES-, Centroid, CV=25 1.6x106 447.0 - 1.4x106 D [M -H] 1.2x106 6 1.0x10 896.3 448.0 895.3 8.0x105 - Intensity [2M-H] 6.0x105

4.0x105 897.3 449.1 2.0x105 898.3 417.2 450.1 879.4 899.3 0.0 16.497 Peak 1 -M-F7-P8-2 - ZQ F1 Scan 100.00-1200.00 ES+, Centroid, CV=25 449.1 6x106 Figure 9. HPLC-MS profile303.0 of fraction 6 (A) and fraction 7 (B) of Zippertex® 5x106

4x106 - leaf extracts with MeOH. MS450.2 chromatograms (ESI ) of the compound 3, 433.1

3x106 Intensity Hydroxyphenyl-4-heptene304.0-3-one (hexoside) (C) and 4, Quercetin O-rhamnoside 2x106

1x106 451.1 (D) are shown. 305.0 147.0 211.1 408.2 452.1493.2 595.2 0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00 1100.00 1200.00 163 m/z

Taking into account the compounds included in Annex I, it was possible to infer the presence of a quercetin-3-O-rhamnoside (m/z 448) and a compound from the diarylheptanoid family (m/z 478), but cholesteranol and cholesterol were also detected in fraction 6. HPLC-preparative and 1H NMR was carried out to obtain pure compounds and to elucidate their chemical structure. Based on the chemical shift, electrostatic screening, intensity and the area at half- height, it was possible to characterize the structure of the compounds isolated. Figures 10 and 11 reveal the 1H NMR chromatogram of the quercetin-3-O-rhamnoside and (3R,5R)-3,5-dihydroxy-1,7-bis(4- hydroxyphenyl)heptanes 3-O-ß-D-glucopyranoside.

164

a a a

a a

b c

c d c

a c b

Figure 10. 1H NMR chromatogram of quercetin-3-O-rhamnoside. Peaks correspond to the signals of a) methine protons, b) spiroacetal carbon, c) oxy- methine protons and d) methyl protons.

165

c c c

a a a d d a d d d a a a a

c a d

Figure 11. 1H NMR chromatogram of (3R,5R)-3,5-dihydroxy-1,7-bis(4- hydroxyphenyl)heptane 3-O-β-D-glucopyranoside. Peaks correspond to the signals of a) methine protons, c) oxy-methine protons and d) methyl protons.

In order to assess the compound identification, a prediction of the chemical shift using ChemNMR software was done. In both cases, chemical shifts of the assigned nodes were similar to the predicted chromatograms. Therefore, based on the literature, the HPLC-MS profile and the data obtained by NMR and data simulation, we can reliably conclude that the main compounds characterized in 166 fractions 6 and 7 are quercetin-3-O-rhamnoside and (3R,5R)-3,5- dihydroxy-1,7-bis(4-hydroxyphenyl) heptane 3-O-β-D- glucopyranoside.

4 Discussion

In this work we studied the anticancer activity of C. avellana tree extracts. Firstly, it was confirmed that DMSO is a suitable vehicle for testing the obtained C. avellana extracts, or the compounds isolated from the extracts, with HeLA, HepG2 and MCF-7 cancer cells in culture. Since DMSO is an amphypathic molecule, it is extensively used as a solvent for drug testing (Modesitta and Parsons, 2010; de Ménorval MA et al., 2012; Galvao et al., 2014). Also, during the subsequent studies, treatment with DMSO alone never gave statistically significant differences.

Due to the variability of the references regarding effects of taxol on cancer cell lines (Liebmann et al., 1993; Gagandeep et al., 1999; Brenes et al., 2007; Chae et al., 2012), we determined the half maximal inhibitory concentration (IC50) for each cell line in culture under our working conditions. As cells are more sensitive to treatment during the exponential phase, all treatments were performed 24 h after seeding (Liebmann et al., 1993). The IC50 values obtained were 5 nM for HeLA and HepG2. 5 nM was the amount that caused maximum effect in MCF-7 cells, with a growth inhibition of about 50%. Concentrations higher than 5 nM did not increase cell death. IC50 of 2.5-7.5 nM were reported by Liebmann et al., 1993 for MCF-7 and HeLA, so our results are in agreement

167 with those already published. Regarding the HepG2 cell line, IC50 of 800 nM (Gagandeep et al., 1999) and 10µg/mL (8 nM) (Brenes et al., 2007) have been described, although Chae et al., (2012) reported that HepG2 cells are very resistant to paclitaxel treatment. Under our work conditions, HepG2 was very sensitive to paclitaxel, which at 5 nM showed a strong effect, reducing cell viability to 50%. In our studies, taxol was used as a positive control due to the fact that some studies describe taxol and taxol-like compounds as responsible for the cytotoxic effects of C. avellana extracts on cell suspension cultures (Bestoso et al., 2006; Bemani et al., 2012, 2013). It is well known that secondary metabolite production and its accumulation is dramatically affected by the season, plant variety and other environmental conditions (Hoffamn et al., 1998; Bacchetta et al., 2008; Cristofori et al., 2010; Amaral et al., 2010). For this reason, stems and leaves were collected from three different locations in Catalonia in the same month (October). All the trees evaluated showed a similar activity against the three cell lines tested, although tree I extracts was generally less toxic than the other two trees. There were no statistical differences between extracts from tree II and tree III, so due to its greater availability we decided to use tree III for a more in-depth analysis of the compounds produced by C. avellana leaves and stems. As taxol and related taxanes were determined in C. avellana plants, we initially hypothesized that we would find taxane-like compounds. The taxane extraction method used was developed by Onrubia et al. (2013) in Taxus spp. Our results suggested that this specific 168 extraction method is more suitable for the isolation of the active compounds from stems, while maceration extraction is more suitable for leaves. Overall, the results showed no statistical differences between maceration and taxane extraction procedures, so we decided to use the Zippertex® extraction method in order to isolate enough quantity of compounds to identify and characterize those with higher activity. Zippertex® improves the Dionex ASE extraction technology because it is possible to modify the pressure from 1 to 150 bars and the temperature from -10º to 250 ºC. Additionally, it is possible to extract a large amount of plant material offering high yields of the extract, thus reducing time and costs (Ouazzani et al., 2007). For these reasons, this technology offered us an ideal procedure to obtain high yields of the compounds produced or accumulated in C. avellana tree III. The extract growth inhibitory activity was comparable to that obtained during the screening experiments, given that the extracts from leaves extracted with MeOH promoted a huge reduction of cell viability in all the cell lines tested (p<0.01). This result allowed us to select the lead extract for in-depth study of the active compounds. Flash chromatography was used to obtain 8 fractions of this sample and two concentrations of each were evaluated in HeLA cells, allowing us to detect those fractions showing dose-response activity. Fractions, 1, 2, 6 and 7 showed high activity at both concentrations. Specifically, fractions 6 and 7 had a dose-response effect, while fractions 1 and 2 caused a huge reduction of cell viability at both concentrations studied. Fraction 1 and 2 were not 169 analyzed further due to their high toxicity and the possible lipophilic nature of their compounds. These fractions were eluted in the first steps of the HPLC-preparative and may have contained lipophilic compounds that would hamper pharmaceutical formulation. On the other hand, a more polar gradient was used to obtain fractions 6 and 7 and both showed a dose-response activity. Consequently, these fractions were further analyzed by HPLC-MS and RMN. HPLC-MS analysis revealed the presence of two polyphenols, quercetin-3-O-rhamnoside (also named quercitrin), a flavonoid, and (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl) heptanes 3-O-ß-D-glucopyranoside, a diarylheptanoid. These results were confirmed by RMN. Moreover, HPLC-MS studies showed that taxanes were not present in the most active fractions, although the presence of taxanes in C. avellana has been widely reported (Hoffman et al., 1998; Bestoso et al., 2006; Bemani et al., 2012, 2013). To our knowledge, this is the first report indicating that compounds other than taxanes, with a high antiproliferative activity, can accumulate in C. avellana tree extracts. As mentioned before, both compounds isolated from the active fractions are polyphenols. This family is a large group of natural compounds that are ubiquitous, abundant, are characterized by a low toxicity and high structural diversity, and have a wide range of biological activities. One of them, quercitrin (quercetin 3-O- rhamnose), is the rhamnose C-3 glycosylate form of quercetin, a flavonoid widely produced by a huge number of plants, which can be found in different glycoside forms, and shows potential 170 anticancer activity (Materska, 2008; Cheng et al., 2010; Deng et al., 2013; Sak, 2014; Sudan et al., 2014). Flavonoids are one of the most studied and abundant groups of polyphenols and have been used since ancient times for their medicinal properties, including anti-inflammatory, cardioprotective, antiallergic or anticancer. Flavonoids are chemically defined by a phenylchromanone structure (C6-C3-C6) with at least one hydroxyl substituent (Sak, 2014; Lewandowska et al., 2014). Several studies have reported a significant role of flavonoids in growth inhibition of breast, colon, prostate, ovary, endometrium and lung cancer cell lines (Danihelova et al., 2013; Sak, 2014). It has been reported that small modifications in the chemical structure can lead to a dramatic change in the activity. Consequently, flavonoids with a similar structure could produce different biological responses, complicating functional analysis (Sak, 2014). Caco-2 cells (derived from human epithelial colorectal adenocarcinoma cells) treated with quercetin showed downregulation of genes involved in the cell cycle and cell proliferation, as well as an upregulation of several tumour suppressor genes, thus disclosing the anticancer activity of quercetin-like compounds (van Erk et al., 2005). Specifically, quercitrin is more soluble in water than the respective aglycone and, besides other actions such as chemopreventive or antibacterial, its antiproliferative activity has been widely studied. An IC50 of approximately 200 µM has been described in different cell lines, like breast, colon, lung, liver, melanoma or prostate (Sudan et al., 2014; Sak, 2014).

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The reported concentration of quercitrin in leaves of C. avellana is about 3235.5 mg/Kg DW, with the appropriate methodology to extract flavonoids at high rates (Oliveira et al., 2008). If we obtained a similar extraction yield of this compound using our extraction methodology, we were assaying a concentration of approximately 24 nM. Although this calculation is not accurate, it allowed us to confirm that we were testing very low concentrations, being far below the IC50 of the compound. On the other hand, (3R,5R)-3,5-dihydroxy-1,7-bis(4- hydroxyphenyl) heptane 3-O-ß-D-glucopyranoside is a diarylheptanoid. These compounds are characterized by two aromatic rings linked by a seven-carbon aliphatic chain. This group is mainly found in species from the Betulaceae, Zingiberaceae, Leguminoseae and Taccaceae families. The potential activity of these compounds has been described as antinflammatory, antioxidant, antitumor, estrogenic, hepatoprotective and neurprotective processes (Keserü and Nógradi 1995; Lee et al., 2005; Jin et al., 2007; Mshvildadze et al., 2007; Lv and She, 2011; Dinic et al., 2014). Specifically, (3R,5R)-3,5-dihydroxy-1,7-bis(4- hydroxyphenyl) heptanes 3-O-ß-D-glucopyranoside has been described before by Yokosuka et al. (2002), in Tacca chantrieri, by Quang et al., (2012) in Tacca plantaginea and by Riethmüller et al., (2013) in C. avellana. Although other diarylheptanoids showed activity, the compound isolated showed no cytotoxic activity at 10 µg/ mL (21nM) in HL-60 cells (human promyelocytic leukemia cells), a IC50 of 157 µg/mL (384 nM) in HSC-2 (oral squamous cell carcinoma) and 213µg/mL (445 nM) in HGF (normal human 172 gingival fibroblast) (Yokosuka et al., 2002). Quang et al. (2012) reported that (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl) heptanes 3-O-ß-D-glucopyranoside inhibits TNF--induced NF-кβ transcriptional activity and activates the transcriptional activity of PPARs, both in a dose-dependent manner in the HepG2 cell line, suggesting diarylheptanoids as a new target for the prevention and treatment of metabolic and inflammatory diseases. Finally, Riethmüller et al. (2013) developed an analytical technique to analyze and quantify diarylheptanoids in C. avellana tree extracts. Leaves were extracted with MeOH and ethyl acetate, but (3R,5R)- 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl) heptanes 3-O-ß-D- glucopyranoside were only detected in the ethyl acetate extract at 5.19 µg/mg DW. Assuming that with our methodology we were able to obtain a similar concentration, although our extract was obtained with MeOH, we were assaying a concentration of about 35 nM.

Taking into account this recent information, it is possible to infer that the activity we have observed may be derived from a synergic effect of different compounds. Synergic effects of different polyphenols, combining a polyphenol compound and a commercial drug, have been previously reported (Sharma et al., 2007; Ali et al., 2009, Montopoli et al., 2009; Lang et al., 2009; Kuhar et al., 2007; Kweon et al., 2010). This is noteworthy, as in recent years it has been reported that multi-drug therapies improve the effectiveness of treatment in many diseases. In Figure 8, we can basically observe quercetin-3-O-rhamnose and (3R,5R)-3,5-dihydroxy-1,7-bis(4- hydroxyphenyl) heptanes 3-O-ß-D-glucopyranoside in fractions 6 173 and 7, among some traces of cholesterol and cholestanol. Although we have not yet confirmed the results, we hypothesized that both compounds could have a synergic antiproliferative effect due to the lower concentrations that are apparently present in fraction 6 and 7. This hypothesis and the specific molecular mechanisms involved in the activity being evaluated are currently under study. The results and outcomes achieved to date open new insights into the antiprolifetative activity of C. avellana in human cancer cell lines and promote the study of this plant.

Acknowledgments

This work was performed in collaboration with Dr. Isidoro Metón and Dr. M Isabel V. Baanante from the Departament de Bioquímica i Biologia Molecular of the Facultat de Farmàcia (Universitat de Barcelona), and Dr. Jamal Ouazzani and Dr. Emilie Adelin from the Pilot Unit (UP) Department at the Institut de Chimie des Substances Naturelle (Centre national de la recherche scientifique, France). Work in the Plant Physiology Laboratory (University of Barcelona) was financially supported by the Spanish MEC (BIO2011-29856- C02-1) and the Generalitat de Catalunya (2014SGR215). A. Gallego was supported by a fellowship from the University Pompeu Fabra.

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Discussion

Our work was focused on the study of C. avellana as a taxol- producer species. Like all plants, C. avellana is capable of synthesizing an overwhelming variety of low-molecular-weight organic compounds called secondary metabolites. Usually, these compounds have a unique and complex structure. As has been well documented, plant secondary metabolites possess interesting biological activities and have many applications, such as pharmaceuticals, insecticides, dyes, flavours and fragrances. Traditionally, these compounds were extracted from the whole plant, but due to their low content this process leads to over- exploitation and drives up the production costs. Biotechnology offers an opportunity to improve production of the target compounds by using in vitro culture, exploiting the cell, organ and entire plant organism and avoiding most of the problems related with the traditional process. We have demonstrated the great potential of biotechnogical approaches to obtain high growth and production, specifically of taxanes.

The first chapter describes the process development from mL scale (using shake flasks and tubeSpin® Bioreactors 50) to L scale (in BIOSTAT® B plus and UniVessel® SU bioreactors). Screening experiments (mL scale) showed the greater efficacy of eliciting the culture with1µM Cor compared to 100 µM of MeJA in enhancing taxane production. Cor-elicitated cultures showed a 403-fold increase of baccatin III and 17-fold increase of taxol production, whereas MeJA increased baccatin III production 2-fold and taxol

187 production 4.7-fold, both compared with the control conditions. The greater effect of Cor has been previously described in Taxus cell suspension cultures by Onrubia et al., 2013b. TubeSpin® Bioreactor 50 was used to determine the best moment of elicitation regarding the culture cell density (measured as pcv), obtaining a maximum taxane concentration of 8.5 mg/L at 50% pcv. In all cases, cell viability was affected by the addition of the elicitors to the medium. Cor has been described as less cytotoxic for Taxus cells than MeJA (Onrubia et al., 2013b), but in C. avellana it showed more cytoxicity than MeJA. It was previously reported that the effect of the elicitor depends not only on the elicitor itself (type, concentration, duration of elicitation, etc.) but also the cell line, plant species and state of development of the culture (Namdeo, 2007). Finally, the two aforementioned bioreactors were evaluated for cell growth, and higher rates of growth were obtained in the UniVessel®SU bioreactors. Consequently, elicitation with 1 µM Cor at 50% pcv was assayed and we were able to obtain over 12 mg of taxanes (baccatin III + taxol) measured by HPLC/DAD. While taxane production obtained in C. avellana in the screening experiments and scaling-up was 10 times lower than in preliminary studies of Taxus spp., this production is still far from being competitive at an industrial level. Therefore, chapter 2 focuses on the study of several factors potentially involved in cell growth, with the aim of increasing productivity even more. Basal medium, carbon source and the type and quantity of PGR were evaluated regarding the growth of our cell suspension culture. A fractional 188 factorial design was used to reduce the number of experiments and to analyze all the parameters in one run. Due to the variability in the culture media used in previous studies, especially in the PGR (Bestoso et al., 2006; Rezeai et al., 2012 and 2013; Safari et al., 2012 and Bemani et al., 2013), it was necessary to assess the medium composition for the growth of C. avellana cell suspension cultures. After this assay, we concluded that NAA and sucrose had a statistically significant positive effect on growth in the C. avellana cell culture. This is in agreement with the literature, in which sucrose is reported to be the most effective carbon source in a wide range of plants (Nagella and Murphy, 2010; Suehara et al., 2012; Singh and Chaturvedi 2012; Karwasara and Dixit 2012 and 2013 and Sivanandhan et al., 2013). PGR plays a crucial role in the growth and production of cell suspensions, and although our routine medium, MS supplemented with 30 g/L sucrose and 2 mg/L of 2.4D and 0.4 mg/L of kin as the PGR, showed good rates of growth, after this assay we determined that NAA is more effective than 2.4D in stimulating the growth of C. avellana. Optimizing the PGR may involve medium supplementation with an individual auxin or cytokinin, or a combination of both (Mustafa et al., 2011). In our study, neither cytokinins nor the basal medium had a statistically significant effect on the growth, so only auxins were necessary for the cell cycle regulation. The literature contains some examples about the cytokinin autonomy of cell suspension culture, which could be linked to a habituation process (Binns and Meins, 1973; Kevers et al., 1981; Miura and Miller, 1969 and Asano et al., 2002) or to the capacity of these cells to synthesize enough 189 hormone by themselves (Nishinari and Syono, 1980; Nagata et al., 1992 and Redig et al., 1996). Since no habituation process was applied, our C. avellana cell suspension line may be cytokinin- autonomous, but further studies are necessary to test this hypothesis. Also, a concentration of 1175.45 ng/L of baccatin III and traces of other taxanes were determined by HPLC /DAD, confirming that, in spite of an increase in primary metabolism, secondary metabolism was not inhibited. Nevertheless, the yield was lower than values reported in the literature (Bestoso et al., 2006; Rezaei et al., 2011 and 2013; Safari et al., 2012; Bemani et al., 2013). Finally, a new methodology was described to maintain big stocks of callus biomass at 4 ºC, which made it possible to reduce the number of subcultures and therefore also the contamination rates and epigenetic changes (Mustafa et al., 2011). We determined that calli can be maintained at 4 ºC for 5 months in half-strength MS medium, and we obtained good ratios of growth in the subsequent subcultures using the routine medium at 25 ºC. Although this process is not as solid as cryopreservation, it is easier and faster to implement, and may be an alternative for recalcitrant plants. Both chapters describe the importance of the medium and culture conditions for increasing growth and consequently the productivity in cell suspension cultures. In our first assay with bioreactors, we optimized the type of elicitor and point of elicitation using our routine culture medium, which showed good rates of callus induction and good rates of growth in the derived cell suspension culture. After the second assay, it was possible to infer that using 190

NAA instead of 2.4D would increase growth rates in a bioreactor, and consequently the productivity of our cultures. Following our main aim of increasing taxol production, key to developing a good methodology was being able to identify and quantify the target compounds in a precise way. The first study discussed before only deals with the quantification of baccatin III and taxol, common taxanes produced by cell cultures. These two taxanes were chosen because of their commercial importance and the correct identification and separation by HPLC-DAD methodologies. But other compounds, like 10-deacetylbaccatin III, 10-deacetyltaxol and cephalomannine, are also important due to the growing interest in using them as precursors for semisynthesis processes. Taxane identification and quantification in cell suspension cultures of C.avellana and Taxus spp. used to be routinely done by HPLC-DAD (Bestoso et al., 2006; Rezaei et al., 2011 and 2012; Safari et al., 2012; Onrubia et al., 2013b; Sabater- Jara et al., 2014). However, in our second assay, although we were able to detect traces of deacetyltaxol and cephalomannine by HPLC/DAD, due to the lower production of these compounds in our cultures, we could not quantify them. The analysis of plant extracts is really complex, basically due to the difficulty of correctly purifying the compounds of interest from other compounds that are also produced. Consequently, HPLC chromatograms are highly complex and usually full of unidentified peaks, giving rise to unintentional errors or a peak being mistaken as positive when in fact it is a mixture of two or more compounds with similar chemical characteristics (coelution). Considering this, 191

HPLC-MS/MS offers a higher specificity and sensitivity, making it possible to identify and quantify traces of compounds with a high degree of conviction. For this reason, as described in the third chapter, a methodology to determine five of the most commercial taxanes (baccatin III, 10-deacetylbacctin III, 10-deacetyltaxol, cephalomannine and taxol) by HPLC-MS/MS was developed. On the other hand, the extracellular availability of the target compounds is crucial for scaling-up the process because it facilitates the in situ recovery of the product and promotes a continuous production by the cells. Therefore, bearing in mind a future scale-up process, we optimized a liquid chromatography tandem mass spectrometry methodology to determine the aforementioned taxanes released to the medium. The combination of a matrix-matched calibration curve and the MRM mode maximized the sensitivity and reliability of quantification. Although some of the m/z fragments for the structural identification of our compounds were already described (McClure et al., 1992; Kerns et al., 1994 and Corona et al., 2011), we reported, for the first time, a methodology to quantify (not only to identify or characterize) released taxanes in C. avellana using the MRM mode and with the evaluation of quality parameters like linearity, limits of quantification, limits of identification, accuracy, precision, recovery and matrix effect. Due to the linearity of the MeOH curve and the matrix-matching calibration curve and the low LOD value (between 0.24-37.6 ng mL-1) and LOQ value (between 0.8-125 ng mL-1), it was possible to determine and quantify taxanes even when their concentration was extremely low. Intra- and inter- assay precision 192 and accuracy were determined and relative standard deviation (RSD) was always lower than 7% and accuracy was between 98- 104%, thereby demonstrating the reliability and reproducibility of the method. Recovery was always higher than 80% and despite a certain level of matrix effect, the high efficiency of the methodology allowed us to detect and quantify our samples in a precise way. We were able to determine, for the first time, taxanes at the beginning of the culture, when the production was considerably lower, which allowed us to screen for highly productive cell suspension lines at an early stage. As mentioned before, only baccatin III and taxol were quantified in the first screening and in the scale-up experiments, but after the development of our HPLC-MS/MS methodology it will be possible to target five taxanes. Therefore, the post-elicitation production will be higher and the taxane profile may change, providing a new hypothesis, such as a new scale-up process to produce other related compounds that may be the predominant products of our cultures. After compiling all the data obtained thus far, as described in the three first chapters, we optimized the medium culture, type of elicitor, moment of elicitation, the scale-up process, and the methodology to analyze the taxanes released in our cultures. In future work, it will be mandatory to study all the optimized conditions in the optimum bioreactor, using the best media for growth (supplemented with NAA and sucrose) and elicitation with 1 µM Cor at 50% pcv. The HPLC-MS/MS methodology developed will be used for the correct and reliable quantification of taxanes.

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Finally, given the greater effect of taxol and the high variety of compounds in the taxane family, chapter four focuses on the study of the compounds present in C. avellana plant extracts with the aim of finding new taxol-like compounds or other compounds with potential antiproliferative activity. C. avellana leaves and stem extracts were evaluated in three different cell lines (HeLA, HepG2 and MCF-7) that were selected based on different, previously described assays with pure taxol (Liebmann et al., 1993; Gagandeep et al., 1999 and Brenes et al., 2007). It is important to mention that the screening and isolation of new compounds from plants is extremely complex because of the difficulties in selecting and developing an appropriate screening. Moreover, after assaying the developed methodology, the result may lack novelty, or a potentially interesting compound may be underestimated (Balunas and Kinghorn, 2005; McRae et al., 2007). Once the most active extract was identified (the MeOH extract from leaves using Zippertex® technology), as well as the most antiproliferative fractions (fractions 6 and 7), the isolation and characterization of the compounds allowed us to identify two main compounds: quercetin-3-O-rhamnoside (quercitrin) and (3R,5R)-3,5-dihydroxy- 1,7-bis(4-hydroxyphenyl)heptanes 3-O-β-D-glucopyranoside. Although we did not identify any compound with a taxol-like structure, other fractions with a lower antiproliferative activity remain to be studied, so the presence of taxol and other taxanes is not excluded. Also, it has already been described that taxol and taxane production is influenced by the genotype and the season (Hoffman et al., 1998), so production could be really low. 194

After compiling all the data published about these two identified compounds (Sak, 2014; Yokosuka et al., 2002; Oliveira et al., 2008; Quan et al., 2012 and Riethmüller et al., 2013), we estimated that we were testing concentrations lower than 30 nM of each one in the cancer cell lines. This was far from the IC50 of 200 µM described in the literature, and did not correspond with the huge antiproliferative effect observed. Therefore, we hypothesized that these compounds may be interacting with each other, resulting in the high effect that we determined. In the future, studies with the pure compounds will be mandatory in order to confirm the IC50 of the compounds in our cell lines and to assay our synergy hypothesis. Considering and analyzing all the data presented in this thesis on C. avellana as a new biotechnological source of anticancer agents, we are still far from an industrial-level production. Nevertheless, the continously expanding market for taxol and taxanes, the over- exploitation of Taxus spp. plants, the recalcitrant growth behaviour of Taxus spp. in in vitro cultures and the economical unfeasibility of chemical synthesis are all reasons why the study of new sources of taxane production is more than reasonable. Although in vitro cell cultures of Taxus spp. are currently used by different private companies (e.g. Phyton Biotech, Samyang Genex, Mitsui Petrochemical Ind.) producing enormous yields of taxol, the highly specialized techniques required keeps the price of the final product high. Analyzing the time-course of the different optimization approaches in Taxus spp. at an academic level, although far from the industrial scale, it can be seen that taxane production is being enhanced at every stage of the process. 195

At the very beginning, without an optimized media, taxol production in T. baccata cell suspension cultures was 0.11 mg/L for baccatin III and 0.13 mg/L for taxol (Cusido et al., 1999). Then, the establishment of a two-stage culture increased baccatin III and taxol production to 2 mg/L and 2.56 mg/L, respectively (Cusido et al., 2002). In C. avellana cell suspension cultures, the initial values that we obtained in control conditions were between 0.022-0.026 mg/L for baccatin III and 0.011 mg/L for taxol at mL-scale. Controversially, also in C. avellana, Rezaei et al. (2011 and 2013) reported 0.05-0.07 mg/L of taxol in control conditions, and Safari et al. (2012) reported 0.01 mg/L of taxol. Again in T.baccata cell suspension cultures, the combination of a two-stage culture with 100 µM MeJA gave a concentration of up to 10.7 mg/L of baccatin III and 10 mg/L of taxol, while the combination with 1 µM of Cor increased the production to 51 mg/L of baccatin III and 36 mg/L of taxol (Onrubia et al., 2013b). In our C. avellana cell suspensions cultured in the TubeSpin® Biorector 50, we obtained about 0.5 mg/L of both baccatin III and taxol after elicitation with 100 µM of MeJA and up to 8 mg/L of baccatin III and 0.2 mg/L of taxol after elicitation with 1 µM Cor. Although Bestoso et al. (2006) reported an increase in taxane production after MeJA elicitation, they showed the data as a total taxane content. Therefore, there are no studies in C. avellana about the individual increase of taxol and baccatin III production after elicitation with MeJA or Cor. The scale up of T. media cell cultures to a 5L stirred bioreactor achieved concentrations of 1 mg/L of both baccatin III and taxol, and after the elicitation with 1 µM MeJA the production 196 increased to 56 mg/L of baccatin III and 21 mg/L of taxol (Cusido et al., 2002). Also, a scale up of a T. baccata cell suspension culture to a 5L stirred bioreactor was described, but using cells immobilized with alginate, in which taxol production was increased to 43 mg/L and baccatin III production to 5.06 mg/L (Bentebibel et al., 2005). Our scale-up study, although we did not immobilize the cells, yielded concentrations of 6 mg/L baccatin III and approximately 0.2 mg/L taxol in the stirred UniVessel® SU bioreactor. Very recently, the combination of cyclodextrins, specifically randomly methylated-β-cyclodextrins, with MeJA in T. media cell cultures resulted in a baccatin III production of 23 mg/L and a taxol production of 65.01 mg/L (Sabater-Jara et al., 2014). Taxane production in cell suspension cultures of Taxus spp. Has been studied for over 20 years and is still being studied, but C. avellana cell cultures as a source of taxol and related taxane production is quite recent and the process optimization remains preliminary, therefore more work is required to obtain a higher production of these compounds. In our studies, baccatin III production was always higher than taxol production but, as mentioned before, this metabolite is also important for semisynthetic processes. Based on all the data discussed before regarding cell suspension cultures of Taxus spp. it will be interesting to assay a two-stage C. avellana culture once the production medium has been optimized. Also, taking into account the results obtained after the cell immobilization with alginate in the stirred bioreactor, it will be interesting to study this approach using C. avellana cells. Finally, given the high increase in the production 197 obtained by Sabater-Jara et al. (2014), the assay of two elicitors, such as MeJA plus cyclodextrins or Cor plus cyclodxtrins, may contribute to enhance taxane production in C. avellana cultures. Focusing on the positive aspects of C. avellana, obtaining in vitro cultures is easier and faster than Taxus spp. and the derived calli are white and friable, giving a fine cell suspension culture faster. Calli showed a high growth rate and the fast growth of the derived cell culture contributed to increasing its productivity. Also, the taxane quantification in the medium of the cell suspension culture and the dramatic positive effect of the elicitation encourage further study of this new source. In conclusion, the present work used empirical approaches to improve the production of taxanes in suspension cell cultures of C. avellana. After completing the optimization of the culture conditions, it will be necessary to proceed with a rational approach. This new approach has proved to be very effective, as demonstrated in a large number of publications related with Taxus spp. (Van der Fits and Memelink, 2000; Ho et al., 2005; Expósito et al., 2010; Zang et al., 2011; Li et al., 2012, 2013). It involves the elucidation of the biosynthetic pathway of the compounds of interest, modifying the gene expression by metabolic engineering techniques with the aim of avoiding flux-limiting steps or competitive pathways, and therefore obtaining more productive cultures. Unfortunately, no data about the taxane biosynthetical pathway in C. avellana is available, hence it is hypothesized that C. avellana may have a similar taxane pathway as Taxus spp. As soon as new key steps in the C. avellana biosynthetic pathway are elucidated, 198 since this plant is an angiosperm, it will be a good candidate to undergo transformation with Agrobacterium. All the results presented here contribute to increase the expectations of C. avellana as a new future source of anticancer agents.

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200

Conclusions

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Conclusions As a general conclusion of this thesis, we can say that Corylus avellana is a potential new biotechnological platform to produce taxol and related taxanes, although the level of production obtained until now is not enough to satisfy commercial requirements. Further studies are therefore required to establish this species as an alternative source of the target compounds.

The specific conclusions are:

1. The best taxane production in C. avellana cell suspension cultures was obtained with a cell density of 50% and 1 µM coronatine as the elicitor. Coronatine increased baccatin III production 26-fold and taxol production 5-fold in shake flask experiments, and baccatin III production more than 403-fold and taxol production 17-fold at pcv 50% in the TubeSpin® Bioreactor 50.

2. A scale-up process of taxane production in C. avellana was studied for the first time. The UniVessel®SU Bioreactor showed the highest growth of a C. avellana cell suspension culture, producing 12.5 mg of taxanes (baccatin III + taxol). This yield was similar to that of the TubeSpin® Bioreactor 50.

3. Seeing that the productivity of the culture could be improved with a higher biomass production, several factors connected with C. avellana cell growth were studied. We found that 1- 203

naphalene acetic acid and sucrose had a statistically significant positive effect on the growth of a C. avellana cell suspension culture. On the contrary, cytokinins and basal medium did not have a statistically significant effect.

4. To preserve C. avellana calli for further use, a suitable stock may be maintained at 4 ºC in half-strength MS for 5 months with good rates of growth in the subsequent subcultures.

5. Considering the need to quantify traces of taxanes in a C. avellana cell suspension medium, a new liquid chromatography-tandem mass spectrometry method has been described.

6. Regarding the antiproliferative activity of C. avellana plant extracts, MeOH extracts from leaves obtained by Zippertex® technology showed the highest reduction (up to 90%) in cell viability in the three cancer lines tested (HepG2, MCF-7 and HeLA).

7. Quercitrin and (3R,5R) -3,5-dihydroxy-1,7-bis (4 hydroxyphenyl) heptane 3-O-β-D-glucopyranoside were identified by NMR in the fraction with the highest antiproliferative activity.

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References

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Annex

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Annex Compounds described on the literature in C. avellana. Formula, CAS Nº and molecular weight (MW) are reported for each one. The compounds listed are grouped as following:

1. Organic acids 2. Triacylglycerols 3. Phytosterols 4. Tocopherols 5. Phenolic compounds 6. Terpenes 7. Taxanes 8. Diarylheptanoids

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1. Organic acids:

Malic acid Galacturonic acid C4H6O5 C6 H10 O7 CAS Nº: 6915-15-7 CAS Nº 14982-50-4 MW: 139.09 MW: 194.14

Levulinic acid Succinic acid C5 H8 O3 C4 H6 O4 CAS Nº: 123-76-2 CAS Nº: 623158-99-6 MW: 116.12 MW: 118.09

Citric acid Oxalic acid C6 H8 O7 C2 H2 O4 CAS Nº: 77-92-9 CAS Nº: 144-62-7 MW: 192.12 MW: 90.03

Acetic acid Butyric acid C H O 2 4 2 C4 H8 O2 CAS Nº: 64-19-7 CAS Nº: 107-92-6 MW: 60.05 MW: 88.11

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2. Triacylglycerols

Trilinoleoylglycerol Trioleoyl glycerol C57 H98 O6 C57 H104 O6 CAS Nº: 537-40-6 CAS Nº: 122-32-7 R1, R2, R3: Linoleic acid R1, R2, R3: Oleic acid MW: 879.38 MW: 885.43 Oleoyl-dilinoleoyl glycerol Dipalmitoyl-oleoyl glycerol C57H100 O6 C53 H100 O6 CAS Nº: 2190-21-8 CAS Nº: 28409-94-1 R1, R2: Linoleic acid, R3: Oleic acid R1: Oleic acid, R2, R3: Palmitic MW: 881.40 acid MW: 833.37 Palmitoyl-dilinoleoyl glycerol Tripalmitoyl glicerol C55 H98 O6 C51 H98 O6 CAS Nº: 2190-1-0 CAS Nº: 555-44-2 R1, R2: Linoleic acid, R3: Palmitic acid. R1, R2, R3: Palmitic acid MW: 855.36 MW: 807.32 Dioleoyl-linoleoylglycerol Stearoyl-dioleylglycerol C57 H102 O6 C57H106O6 CAS Nº: 2790-20-7 CAS Nº: 113829-10-0 R1, R2: Oleic acid, R3: Linoleic acid R1, R2: Oleic acid, R3: stearoyl MW: 883.42 MW: 887.45 Palmitoyl-dioleoyl glycerol Palmitoyl-stearoyl- C55 H102 O6 oleoylglycerol CAS Nº: 2190-30-9 C55 H104 O6 R1, R2: Oleic acid, R3: Linoleic acid CAS Nº: 5512-58-3 MW: 859.39 R1: Palmitic acid, R2: stearoyl, R3: Oleic acid MW: 861.41 Dipalmitoyl-linoleoyl glycerol CAS Nº: 2442-56-0 R1, R3: Palmitic acid, R3: Linoleic acid C53 H98 O6 MW: 831.34

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3. Phytosterols

Cholesterol Cholesterol C27 H46 O C27 H48 O CAS Nº: 57-88-5 CAS Nº: 80-97-7 MW: 386.65 MW: 388.67

Campestanol Campesterol C H O C H O 28 50 28 48 CAS Nº: 474-60-2 CAS Nº: 474-62-4 MW: 386.65 MW: 400.68

Δ7-Campesterol β-Sitosterol C28 H48 O C29 H50 O CAS Nº: 516-78-9 CAS Nº: 83-46-5 MW: 400.68 MW: 414.71

Stigmasterol Δ5,23-Stigmastendiol C29 H48 O C29 H48 O CAS Nº: 83-48-7 CAS Nº: 2364-23-0 MW: 412.69 MW: 412.69

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Δ5,24-Stigmastendiol Δ7-Stigmasterol C29 H52 O C29 H46 O CAS Nº: 83-45-4 CAS Nº: 481-19-6 MW: 412.69 MW: 410.67

Δ7-Avenasterol 5-Avenasterol C29 H48 O C29 H48 O CAS Nº: 23290-26-8 CAS Nº: 18472-36-1 MW: 412.69 MW: 412.69

Clerosterol C29 H48 O CAS Nº: 2364-23-0 MW: 412.69

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4. Tocopherols

α-Tocopherol C29 H50 O2 CAS Nº: 59-02-9 MW: 430.71

α-Tocotrienol C29 H44 O2 CAS Nº: 588864- 81-6 MW: 424.66

β-Tocopherol C28 H48 O2 CAS Nº: 490-23-3 MW: 416.68

β-Tocotrienol C28 H42 O2 CAS Nº: 490-23-3 MW: 410.63

δ-Tocopherol C27 H46 O2 CAS Nº: 119-13-1 MW: 402.65

δ-Tocotrienol C27 H40 O2 CAS Nº: 14101-61- 2 MW: 396.60 γ -Tocopherol C28 H48 O2 CAS Nº: 54-28-4 MW: 416.68

γ-Tocotrienol C48 H42 O2 CAS Nº: 14101-61- 2 MW: 410.63 232

5. Phenolic compounds

Gallic acid Caffeic acid p-coumaric acid C H O C7 H6 O5 C9 H8 O4 9 8 3 CAS Nº: 149-91-7 CAS Nº: 331-39-5 CAS Nº: 7400-08-0 MW: 170.12 MW: 180.16 MW: 164.16

Ferulic acid Sinapic acid p-hydroxibenzoic acid C7 H6 O3 C10 H10 O4 C11 H12 O5 CAS Nº: 1135-24-6 CAS Nº: 530-59-6 CAS Nº: 99-96-7 MW: 194.18 MW: 224.21 MW: 138.12

p-hydroxyphenylacetic acid Vanillic acid Protocatechuic acid C8 H8 O3 C8 H8 O4 C7 H6 O4 CAS Nº: 156-38-7 CAS Nº: 121-34-6 CAS Nº: 99-50-3 MW: 152.1 MW: 168.15 MW: 154.12

Syringic acid Catechin Epicatechin C15 H14 O6 C9 H10 O5 C15 H14 O6 CAS Nº: 530-57-4 CAS Nº: 154-23-4 CAS Nº: 490-46-0 MW: 198.17 MW: 290.27 MW: 290.27

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Quercetin Quercetin-o-rhamoside C15 H10 O7 C21H20O11 CAS Nº: 117-39-5 CAS Nº: 522-12-3 MW: 302.24 MW: 448.38

Quercetin: R1= hexose / pentose Caffeoyltartaric acid C22 H18 O12 CAS Nº: 70831-56-0 MW: 474.37

3-Caffeoylquinic 5-Caffeoylquinic C16 H18 O9 C16 H18 O9 CAS Nº: 327-97-9 CAS Nº: 906-33-2 MW: 354.31 MW: 354.31

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p-Coumaroyltartaric Kaempferol C13 H12 O8 C15 H10 O6 CAS Nº: 69222-59-9 CAS Nº: 520-18-3 MW: 296.23 MW: 286.24

Kaempferol-3-O-Rhamnoside Myricetin-3-O-Rhamnoside C21H20O10 C21H20O12 CAS Nº: 482-39-3 CAS Nº: 17912-87-7 MW: 432.38 MW: 464.38

Phloretyn-2’O-glucoside Rosmarinic acid C18 H16 O8 C21H24O10 CAS Nº: 60-81-1 CAS Nº: 20283-92-5 MW: 436.41 MW: 360.31

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Cyanidin C15H11O6 CAS Nº: 13306-05-3 Epicatechin-3-gallate MW: 287.24 C22 H18 O10 CAS Nº: 1257-08-5 MW: 442.37

Formonetin Epigallocatechin C16 H12 O4 C15 H14 O7 CAS Nº 485-72-3 CAS Nº: 970-74-1 MW: 268.26 MW: 306.27

Daidzein Genistein C15 H10 O4 C15 H10 O5 CAS Nº: 486-66-8 CAS Nº: 446-72-0 MW: 270.24 MW: 254.24

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Glycitein Matairesinol C16 H12 O5 C20 H22 O6 CAS Nº: 40957-83-3 CAS Nº: 580-72-3 MW: 284.26 MW: 358.39

Lariciresinol Pinoresinol C20 H24 O6 C20 H22 O6 CAS Nº: 27003-73-2 CAS Nº: 487-36-5 MW: 360.40 MW: 358.39

Secoisolariciresinol Coumestrol C20 H26 O6 C15 H8 O5 CAS Nº 487-36-5 CAS Nº 479-13-0 MW: 362.42 MW: 268.22

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Epigallocatechin-(2βO7, 4β8) Epigallocatechin-(2βO5, 4β6) C H O C30H24O13 30 24 13 MW: 592.12 MW: 592.12

6. Terpenes

Squalene C30 H50 CAS Nº: 111-02-4 MW: 410.72 Betulin C30 H50 O2 CAS Nº: 473-98-3 MW: 442.72

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7. Taxanes

10-Deacetylbaccatin III Baccatin III C29H36O10 C31H38O11 CAS Nº: 32981-86-5 CAS Nº: 27548-93-2 MW: 544,23 MW: 586,24

10-Deacetyltaxol Taxol C45H49NO13 C47H51NO14 CAS Nº: 78432-77-6 CAS Nº: 33069-62-4 MW: 811,31 MW: 853,33

10-Deacetyl-7- Cephalomannine xylosylcephalomannine C45H53NO14 C48 H59 N O17 CAS Nº: 71610-00-9 CAS Nº: 90332-64-2 MW: 831,3460 MW: 921.98

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10-Deacetyl-7-xylosyltaxol 7-Epi-10-Deacetyltaxol C50 H57 N O17 C45 H49 N O13 CAS Nº: 90332-63-1 CAS Nº: 78454-17-8 MW: 943.98 MW: 811.87

10-Deacetyl-7-xylosyltaxol C 7-Xylosyltaxol C49 H63 N O17 C52 H59 N O18 CAS Nº: 90332-65-3 CAS Nº: 90332-66-4 MW: 938.02 MW: 986.02

Taxinine M C35 H42 O14 CAS Nº: 135730-55-1 MW: 686.70

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8. Diarylheptanoids

Hirsutenone C19H20O5 CAS Nº: 41137-87-5 MW: 328.36

1,7-bis-(4- hydroxyphenyl)-4- hepten-3-one C25H32O9 CAS Nº: 640272-81- 7 MW: 476,20 5-hydroxy-1-(4- hydroxyphenyl)-7- (3,4- dihydroxyphenyl)- heptan-3-one C19H22O5 MW: 330,146 5-hydroxy-1-(3,4- hydroxyphenyl)-7- (4- dihydroxyphenyl)- heptan-3-one C19H22O5 MW: 330,146 1,7-bis-(4- hydroxyphenyl)-4,6- heptendien-3-one C25H30O9 MW: 474,18

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Appendix

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Appendix Related publications and meetings attendance:

Other publications:

Gallego A, Ramirez-Estrada K, Vidal-Limon HR, Hidalgo D, Lalaleo L, Khan Kayani W, Cusido RM and Palazon J. (2014) Biotechnological production of centellosides in cell cultures of Centella asiatica (L) Urban. Eng. Life Sci. 00:1–10.

Book chapters:

Cusidó Vidal RM. Vidal H, Gallego A, Abdoli M, Palazon J. Biotechnological production of taxanes: a molecular approach. In: Diego Muñoz Torrero, Amparo Cortés & Eduardo L. Mariño (Ed). Recent Advances in Pharmaceutical Sciences III. Lugar: Transworld Research Network; 2013. p. 91-107.

Meeting attendance

XIII Congresso Luso-Espanhol de Fisiologia Vegetal, Lisbon (Portugal); 2013. Gallego A, Lalaleo L, Pedreño MA, Cusidó RM, Moyano E, Bonfill M. Effect of the methyl jasmonate and cyclodextrins on taxane production in cell suspension cultures of C.avellana. Poster

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V Annual COST FA0804 Meeting: Molecular farming: plants as production plataform for high value proteins, Warsaw (Poland); 2012. Abdoli Nasab M, Jalali-Javaran M, Alizadeh H, Ana Gallego, Cusido RM and J Palazon. Transformation of Tobacco Chloroplasts with the truncated Human Tissue Plasminogen Activator (K2S) gene and obtainment of homoplasmic plants. Poster.

Natural anticancer drugs, Olomouc (Czech Republic); 2012. Gallego A, Lalaleo L, Palazón J, Cusidó RM, Moyano E, Bonfill M. Improving taxane production in cell suspension cultures of Corylus avellana L. Oral communication.

Exploitation et valorisation des plantes médicinales et aromatiques, Fès (Marroc); 2012. Bonfill M. Biotechnological production of phytochemicals. Attendance.

59th International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Product Research. Antalya (Turquia); 2011. Onrubia M, Gallego A, Ramirez K, Vidal-Limón HR, Cusidó RM, Bonfill M, Palazón J, Moyano E. Changes in taxane production and gene expression during the development of in vitro Taxus baccata plant cultures. Poster.

XIX Meeting of the Spanish society of plant biology, Castelló de la Plana (Spain); 2011. Gallego A, Fattahi M, Griffe L, Bonfill M, Moyano E. Improving taxane production in cell suspension cultures of Corylus avellana L. Poster. 246

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