Dr Algirdas Žemaitaitis

Dr. Zigmuntas Gražina Juodeikienė

Dr Jonas Beresnevičius

Dr Isabelle Fourasté

Sigitas Audrius

Dr Ruta Maruška

Galoburda D'

Professeur Petras

Rimantas Venskutonis

REMERCIEMENTS

Tout d'abord j'adresse mes remerciements aux différents organismes qui ont mis les moyens financiers pour l'aboutissement de cette thèse: aux gouvernements de la Lituanie et la France, ainsi que Campus France avec la bourse d'Excellence Égide Eiffel.

RESUME

Par ailleurs, une autre tâche importante pour la valorisation est l'utilisation rationnelle des plantes dans toute sa totalité, c'est-à-dire, la valorisation de tout le produit avec le moins de déchets. Les déchets ou sous-produits obtenus lorsque la transformation ou modification de transformer des matières premières des plantes peuvent représenter une ressource potentielle des antioxydants et d'autres substances importantes. Par exemple, les déchets produits pendant la distillation des plantes aromatiques pour la récupération de l'huile essentielle peuvent constituer jusqu'à 99,5 % des matières premières lesquels sont utilisés inefficacement ou ne sont pas utilisés. En effet, il y a des preuves croissantes que les composés non volatils présents dans ces résidus peuvent posséder diverses activités, qui pourraient être appliquées dans les aliments comme antioxydants et des additifs antimicrobiens pour la prévention de diverses maladies telles que le cancer, l'athérosclérose et des difficultés neurologiques. De plus, en considérant les études récentes [1][2][3] sur la fraction volatile de plantes, traditionnellement appelées huiles essentielles, qui considèrent leurs propriétés antioxydantes comme des substituts potentiels pour les antioxydants synthétiques [4]. Il est possible d'utiliser (ou revaloriser) les sous-produits, qui sont obtenus après l'élimination de l'huile volatile par diverses techniques de distillation, afin d'améliorer la faisabilité commerciale de la production de composés bioactifs de différentes plantes.

Dans notre recherche nous sommes intéressés par des espèces qui sont considérées comme de « plantes oubliées médiévales » et dans lesquelles les sous-produits ne sont pas encore analysés dans les études récentes. Ainsi, Calamintha grandiflora, Myrrhis odorata et Tussilago farfara, qui sont des plantes de type et qui poussent en Lituanie et au sud de la France, sont considérées comme candidat à réaliser une étude afin d'identifier les composant leurs propriétés et ses activités (antioxydants ou odorants) et définir la variation des composés bioactifs entre les différentes origines, une tâche considérée importante tant du point de vue chimique et pour l'optimisation de la production d'ingrédients fonctionnels bioactifs.

Objectif et les tâches du travail

Le but de ces travaux de thèse était d'évaluer les possibilités de la transformation des plantes Calamintha grandiflora, Myrrhis odorata et Tussilago farfara en arômes et ingrédients antioxydants en utilisant le concept d'agro-raffinerie.

Les tâches suivantes ont été définies pour atteindre l'objectif: 3. Évaluer les propriétés antioxydantes des sous-produits d'hydrodistillation au travers des différents essais in vitro avec différents solvants. 4. Évaluer les propriétés antioxydantes des sous-produits d'hydrodistillation in situ (huile de tournesol) en le comparant avec l'antioxydant synthétique commercial. 5. Déterminer les composés phytochimiques des sous-produits d'hydrodistillation et évaluer les propriétés antioxydantes de composants individuels en utilisant des méthodes chromatographiques et spectrométriques.

Originalité de la thèse

La nouveauté scientifique suite à ces travaux de thèse a été réalisée par l'accomplissement des tâches énumérées ci-dessus:

1. Les profils chimiques des composés volatils dans les plantes sélectionnées qui poussent en Lituanie et au sud de la France ont été compares par première fois. Les différences entre les plantes de Lituanie et de France ont été observées, ainsi qu'avec des travaux précédents rapportés dans la littérature.

Deux nouvelles méthodes analytiques (Artificial Crushing Finger device et Design of

Flash Aroma Dispenser) pour l'évaluation rapide de native huile essentielle de plantes aromatiques ont été développées et testées.

L'évaluation systématique et complète des composés phytochimiques dans les sousproduits d'hydrodistillation, particulièrement les composés phénoliques, dans

Calamintha grandiflora, Myrrhis odorata et Tussilago farfara a été réalisée pour la première fois. 4. Résidus désodorisés de Calamintha grandiflora, Myrrhis odorata et Tussilago farfara ont été valorisés à partir des bioressources d'un antioxydant pour la première fois. viii 5. La comparaison de profil chimique et des propriétés antioxydants de Myrrhis odorata et Tussilago farfara de Lituanie et la France a été réalisée pour la première fois et certains composés n'ont pas été identifiés dans les études précédemment publiées.

Apports de la thèse

Les sous-produits après hydrodistillation sont des ressources les plus prometteuses d'antioxydants naturels et qui permettent une utilisation rationnelle des matières premières. L'information obtenue dans cette étude peut être utilisée pour la sélection des plantes avec le plus grand contenue des composants actifs en fonction de leur origine géographique. L'information obtenue sur la composition des plantes sélectionnées et leurs propriétés biologiques élargir nos connaissances scientifiques sur la biodiversité des produits naturels.

En effet, cette information peut être utile pour les scientifiques dans le domaine concerné et aux spécialistes de l'alimentation, des produits pharmaceutiques et cosmétiques, en termes d'applications pratiques. Enfin, les méthodes développées pour l'évaluation rapide des composés volatils pourraient trouver application dans la reproduction de diverses espèces de plantes aromatiques et d'épices.

Les points clés de la thèse

1. Des arômes et ingrédients avec des antioxydants peuvent être obtenus des sousproduits après hydrodistillation de Calamintha grandiflora, Myrrhis odorata et Tussilago farfara en utilisant le processus de l'agro-raffinerie.

Résultats Analyse chimique des plantes aromatiques de deux différentes zones géographiques

La composition chimique des plantes aromatiques et médicinales varie selon la région de culture, les conditions climatiques, la phase végétative, la modification génétique pour n'en citer que quelques-uns. Par conséquent, la caractérisation de la diversité de la flore dans les différentes zones géographiques et pays est une tâche importante. Cette caractérisation est possible au travers de l'analyse des huiles essentielles des plantes aromatiques, car ils sont une grande source des composés chimiques. Les facteurs qui déterminent la composition chimique et sa quantité dans les huiles essentielles sont nombreuses et, dans certains cas, il est difficile de différencier entre eux, car ils peuvent être interdépendants et d'influencer réciproquement [START_REF] Anwar | Changes in composition and antioxidant and antimicrobial activities of essential oil of fennel (Foeniculum vulgare Mill.) fruit at different stages of maturity[END_REF]. Par exemple, dans ces facteurs où variables, on peut considérer les différentes variations: saisonnière, l'origine géographique, génétique, les stades de croissance, manipulation post-récolte et le stockage [START_REF] Burt | Essential oils: their antibacterial properties and potential applications in foods-a review[END_REF][START_REF] Anwar | Changes in composition and antioxidant and antimicrobial activities of essential oil of fennel (Foeniculum vulgare Mill.) fruit at different stages of maturity[END_REF]. Ainsi, l'objectif de ce manuscrit a été d'examiner les compositions chimiques des huiles essentielles de trois plantes aromatiques (Myrrhis odorata, Tussilago farfara et Calamintha grandiflora) de deux différentes origines afin de déterminer les facteurs plus importants qui influencent la composition et la quantité chimique de ces plantes et les comparer avec des résultats publiés antérieurement à ce manuscrit. Pour ce faire, les huiles essentielles de ces plantes ont été obtenues par hydrodistillation. Puis, pour l'identification des composées volatiles, les huiles sont analysées avec la méthode de chromatographie en phase gazeuse (GC) et détecteur à ionisation de flamme et avec la méthode chromatographie en phase gazeuse et de spectrométrie de masse (GC-MS). Les résultats de ces analyses sont détaillés ci-après.

Myrrhis odorata

La quantité d'huile essentielle extrait a été de 0.40±0.02% (Lituanie) et 0.50±0.01% (France) (w/w). vingt et vingt-deux composants volatiles ont été détectés dans les huiles essentielles de Cette variation dans la quantité de composant peut paraître étonnante, mais pas impossible parce que le polymorphisme chimique est une caractéristique significative pour plusieurs plantes de la famille des ombellifères. Ainsi, cette thèse fournit une information actualisée sur la composition chimique de l'huile essentielle de Myrrhis odorata de deux Mais, si bien que les princips de base sont similaires, la méthode ABTS est préférable pour sa capacité à piéger les radicaux libres dès les antioxydants lipophiles et hydrophiles.

Évaluation de l'activité antioxydante des résidus d'hydrodistillation de C. grandiflora

Pour l'évaluation de la capacité à piéger les radicaux libres des extraits de C. grandiflora, trois méthodes ont été utilisées: DPPH • , ABTS •+ et FRAP. Ces méthodes présentent des avantages, comme leur simplicité, leur utilisation généralisée pour la présentation rapide de résultats dans l'analyse des propriétés antioxydantes des plantes et des informations préliminaires fiables de la présence des composés antioxydantes actives dans les extraits.

La concentration nécessaire pour réduire la concentration initiale de DPPH • à 50% (IC 50 ) a été choisi pour comparer la capacité à piéger les radicaux libres des différentes extraits (Fig. Les effets des résidus de l'hydrodistillation évalués pour différentes concentrations de cellesci (0,05% ; 0,1% et 0,2%) sont présentés dans le tableau R.4.

LIST OF NOMENCLATURES

Abbreviations

GENERAL INTRODUCTION

Relevance of the work

Aromatic, medicinal and spice plants are immense and sustainable resources of natural compounds with various beneficial properties; they have been used for food flavouring, preservation, nearly all-medicinal therapies, and other applications since prehistoric times. Some of these plants are now grown commercially to produce a variety of valuable ingredients. However, some medicinal and aromatic herbs, Calamintha grandiflora, Myrrhis odorata and Tussilago farfara among them, for various reasons have not found wider application and sometimes are referred to as the "forgotten plants". Search of new natural bioactive compounds for nutraceuticals, pharmaceuticals, cosmetics and other products has led to the increase in bio-prospecting, i.e. collection, investigation and utilization of diverse biological resources; therefore, it is reasonable to revisit the "forgotten plants" in order to assess their applicability more systematically. Many medicinal and aromatic plants contain large amounts of antioxidants, such as polyphenols, which may play an important role in foods and human body in absorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing hazardous peroxides.

The occurrence of medicinal and aromatic plants and their composition depends on many factors, particularly on the climatic conditions and the type of soil. The biogenesis of biologically active compounds also depends on several factors: origin of the plant, geographical growing zone, its genotype, phenotype, and chemotype as well as annual climatic fluctuations. Most of natural compounds possess several biological activities: antioxidant, antimicrobial, anti-inflammatory, and others. Therefore, the comparison of the selected plant species grown in different geographical sites in order to assess their biological properties is an interesting and important task, particularly for the valorization of such plants for their commercial cultivation.

Another important task for the valorization of less widely used plants is their rational processing following the rule "more valuable products and less waste". The residues and/or by-products obtained during processing of raw plant materials may represent potential natural resources for antioxidants and other valuable substances. For instance, during distillation of aromatic plants for essential oil recovery the wastes may constitute up to 99.5% of the raw material, which currently are used rather inefficiently. There is increasing evidence that the nonvolatile compounds present in such residues may possess various activities, which might be applied in foods as antioxidant and antimicrobial additives and for preventing various diseases such as cancer, atherosclerosis and neurological disorders. Volatile fractions of plants, traditionally called essential oils, were also studied for their antioxidative properties [1][2][3] and were proposed as potential substitutes for synthetic antioxidants [4]. Considering that the production of essential oils of aromatic plants is very expensive, the rational use of by-products remaining after volatile oil removal by various distillation techniques could remarkably improve commercial feasibility of the production of bioactive compounds from such plants.

The by-products of Calamintha grandiflora, Myrrhis odorata and Tussilago farfara from different origins, as it may be judged from the available literature, have not been studied until now. Furthermore, the variation of bioactive compounds from different origins is an important task both from the chemical point of view and for optimization of the production of bioactive functional ingredients. Therefore, these species, which sometimes are referred as 'Medieval forgotten plants' growing in Lithuania and south of France, were selected in this study as candidates for the production of valuable ingredients and characterisation of their composition and properties.

For this work, coltsfoot (Tussilago farfara) has been chosen because it's valuable functionality as antioxidant. Although there exits some complains about this plant because it contains pyrrolizidine alkaloids which have toxic properties, It is known that these toxics compounds are mainly accumulated in the coltsfoot buds. Thus, in this work, we selected different parts of the plant where these alkaloids can be found in very small quantities that were too difficult to identify. These parts are flowers, stems, young leaves and roots. However, even though the amounts of alkaloids could be insignificant, it is not recommendable to consume long time and in large quantities to prevent any damage. Additionally, to eliminate the pyrrolizidine alkaloids, in this work the aqueous fractions were not used considering that these alkaloids dissolve in water. Nevertheless, there is a new coltsfoot new genotype, created in Austria, which does not contain the toxic pyrrolizidine alkaloids. Therefore, this new Tussilago farfara genotype may be used and then the aqueous fraction which remains after hydrodistillation can also be used.

Aim and tasks of the work

The aim of the work was to evaluate the possibilities of the processing of Calamintha grandiflora, Myrrhis odorata and Tussilago farfara grown in Lithuania and south of France into aroma and antioxidant ingredients by applying the so-called agro-refinery concept.

The following tasks were raised to achieve the aim: 1. To determine the composition of essential oils isolated from aromatic plants collected in Lithuania (Kaunas) and France (Midi-Pyrénées).

To develop new analytical techniques for rapid extraction of volatile compounds and

to compare the volatile compounds of Calamintha grandiflora leaves obtained by hydrodistillation and novel analytical methods (Artificial Crushing Finger device (CFD) and Design of Flash Aroma Dispenser (FAD). 3. To evaluate the antioxidant properties of hydrodistillation by-products prepared by using different solvents in different in vitro assays (DPPH • , ABTS •+ , FRAP and Oxipres). 4. To assess antioxidant properties of hydrodistillation by-products in situ (sunflower oil) by comparing with commercial synthetic antioxidant (BHT). 5. To determine phytochemical composition of hydrodistillation by-products and to evaluate antioxidant properties of individual constituents by using chromatographic and spectrometric methods. 6. To adapt the technological scheme in the valorization of aromatic plants to obtain high-added value substances.

Scientific novelty

The following scientific novelty was achieved by fulfilling the above-listed tasks: 2. Two new analytical methods for the rapid evaluation of volatile oils from aromatic plants were developed and tested. 3. Deodorized residues of Calamintha grandiflora, Tussilago farfara and Myrrhis odorata were valorized as an antioxidant's bioresource for the first time; some compounds (rosmarinic acid, salvianolic acid C, kaempferol-7-O-glucoside, cynarin, quercetin-pentoside and kaempferol-glucoside) of these plants were not identified in the previously reported studies.

Practical significance

The by-products after hydrodistillation are promising resources of natural antioxidants. For example, in our study, we identified some compounds with an antioxidant activity such as rosmarinic acid, salvianolic acid C, kaempferol-7-O-glucoside, cynarin, quinic acid, chlorogenic acid, dicaffeoylquinic acid, quercetin-3-rutinoside (rutin), quercetin-pentoside and kaempferol-3-O-glucoside.

Additionally, the results obtained are important in the selection and evaluation of cultivation area of medicinal plants. These allow us to standardize and ensures the quality of raw materials to obtain a product with the desired compounds.

Finally, two new analytical methods for the rapid evaluation of volatile compounds were developed. They are useful for quick assessment of different types of aromatic and culinary plants.

Key points of the thesis

1. Valuable aroma and antioxidant ingredients are obtained from Calamintha grandiflora, Myrrhis odorata and Tussilago farfara whole plants and hydrodistillation residues by applying agrorefinery concept to their processing.

Rapid aroma profiling methods, namely, Artificial Crushing Finger device (CFD) and

Design of Flash Aroma Dispenser (FAD) are used for the assessment of plant volatile compounds as alternative to the traditional extraction techniques.

Approbation of Results

The results of the research have been published in 3 scientific articles in the journals indexed by ISI Thomson Reuters (WOS) and 2 international conferences proceedings books. The results of the research have also been presented in the 8 international scientific conferences.

Structure and content of the dissertation

The thesis is written in English. It consists of the list of abbreviations; introduction; literature review; materials and methods; results and discussion; conclusions; references; and list of publications relevant to the subject of the dissertation. The dissertation has 106 pages, 18 tables and 27 figures. The list of references includes 208 bibliographic sources.

Medicinal and aromatic plants are very important sources of natural food additives, functional food products, dietary supplements, cosmetics, toiletries, domestic chemistry and herbal medicinal products for pharmacy [1][2][3][4]. Humankind has valued medicinal and aromatic plants since ancient times particulalry for their pleasant odours and healing properties. In addition, they had a knowledge how to use the scent of such plants in order to relax and stimulate their body, improve wellbeing and make skin look beautiful.

In the Middle Ages the herbs were used not only to flavour the food but for the medical treatment too. Herbal treatment originated from Ancient Greek medicine. For instance, Greek physicians used pine extracts to cure tuberculosis [5], the skills of Arab doctors, who preserved original Greek sources of treating different diseases with plants, assietd in preventing contagious diseases. These scientists passed their knowledge to the Europeans in the early Middle Ages. The influx of medical texts preserved in Arabic revived the Latin medical tradition from the tenth century on. Famous medicine centres were established in Italy; one of them was called Salemo where the famous tract Tractatus de Herbis was written in Latin. The mission of the tractate was to inform the townspeople about the plants. Those people used local herbs not only for treatment but also to flavour the food, as rich nobility in the Middle Ages used to do by various spices such as cinnamon and pepper, brought from the East that ordinary townspeople could not afford [6]. Moreover, medieval scientists taught the townspeople how to make ointments by themselves in order to cure wounds and burns. Also, they taught the people how to make various herbal elixirs and charms against incantations and ailments.

Plants which widely used in the medieval period recently have become more popular in the modern world too. Chefs of the modern ages have been using herbs which were known in the medieval ages not only to season fish, meat and vegetable dishes but also as additives enhancing nutritional value of the dishes. Plants used in the past return back to the modern world also due to highly trending vegetarianism. More and more humankind explores the properties of plants that may serve not only as spices but also as an individual food as well. Table 1.1 provides several medieval plants that have been widely used in modern cuisine because they contain active compounds (antioxidants, antimicrobials and flavourings) that both enhance the taste of food and demonstrate the value of medical effectiveness (e.g. ease coughs, help to relax and sleep well, etc.).

DIVERSITY OF AROMATIC AND MEDICINAL PLANTS Table 1.1. Some aromatic and medicinal plants widely used in medieval ages with therapeutic properties

Plant name Family

Achillea millefolium Apiaceae

Vitex agnus-castus Lamiaceae

Angelica archangelica Apiaceae

Calamintha grandiflora Lamiaceae

Chamaemelum nobile Asteraceae

Crataegus monogyna Apiaceae

Foeniculum vulgare Apiaceae

Heracleum sphondylium Apiaceae

Lavandula officinalis Lamiaceae

Levisticum officinale Apiaceae

Melissa officinalis Lamiaceae

Mentha piperita Lamiaceae

Myrrhis odorata Apiaceae

Ocimum basilicum Lamiaceae

Origanum majorana Lamiaceae

Origanum vulgare Lamiaceae

Tanacetum parthenium Asteraceae

Thymus serpyllum Lamiaceae

Tussilago farfara Asteraceae

It is well known that plants are the richest source of bioactive phytochemicals, aromatic compounds and antioxidant nutrients [2]. Around 500,000 different species of plants are deemed to exist on Earth, however, people use only up to 10% of these species. Most of the aromatic plants and essential oil commodities in terms of world trade belong to the families of Lamiaceae, Umbelliferae and Compositae [7].

Essential oils are complex mixtures of volatile compounds characterized by strong odour and diverse biological properties. They are formed as secondary plant metabolites [7]. Biosynthesis of essential oils in plants proceeds in three basic pathways -the mevalonate pathway, the methyl-erithrytol-pathway and the shikimic acid pathway [7][8]. Commonly, the key components of essential oils are monoterpenes, diterpenes and sesquiterpenes. These terpenoids in plant are synthesized via mevalonate and deoxyxylulose phosphate pathways (Fig. 1.1).

Essential oils are isolated from different aromatic plants generally distributed in Mediterranean and tropical countries. Aromatic extracts may be isolated from different parts

GENERAL CHARACTERIZATION OF ESSENTIAL OILS

Sources of essential oils

of a plant, for instance, flowers, buds, stems, leaves, fruits, seeds, bark, fruits rinds, and roots [7][8] (Table 1.2), by using different methods such as water and steam distillation, supercritical fluid extraction, macerating distillation, extraction by solvents, cold pressing, destructive distillation [7,[9][START_REF] Celiktas | Antimicrobial activities of methanol extracts and essential oils of> Rosmarinus officinalis, depending on location and seasonal variations[END_REF][START_REF] Anwar | Antioxidant and antimicrobial activities of essential oil and extracts of fennel (Foeniculum vulgare Mill.) seeds from Pakistan[END_REF][START_REF] Dang | Antioxidant activity of essential oils from various spices[END_REF][START_REF] Sood | Physiological and biochemical studies during flower development in two rose species[END_REF].

Fig. 1.1. Biosynthetic pathways leading to terpenoids in plant cell -compartmentation of the biosynthesis between cytosol (mevalonate pathway, MVA) and plastid (deoxyxylulose/methylerythritol phosphate pathway, MEP) [7-8].

The essential oils are used for food, animal feed as well as in pharmacy, perfumery or chemistry industry [START_REF] Moerman | An analysis of the food plants and drug plants of native North America[END_REF]. Due to the bactericidal and fungicidal properties of essential oils, their pharmaceutical and food uses are becoming increasing important as alternatives to synthetic chemical products to protect the ecological equilibrium [START_REF] Burt | Essential oils: their antibacterial properties and potential applications in foods-a review[END_REF]. Moerman analysed the use of different species of plants among the groups of Indians in America and found that up to 2564 different species of plants were used mostly for medical purposes, whereas for food it was used four times less [9,[START_REF] Moerman | An analysis of the food plants and drug plants of native North America[END_REF]. Presently, essential oils are produced from more than 2000 species of plants; however, only about 100 species have economical significance in the world of essential oils [9][START_REF] Celiktas | Antimicrobial activities of methanol extracts and essential oils of> Rosmarinus officinalis, depending on location and seasonal variations[END_REF][START_REF] Anwar | Antioxidant and antimicrobial activities of essential oil and extracts of fennel (Foeniculum vulgare Mill.) seeds from Pakistan[END_REF][START_REF] Moerman | An analysis of the food plants and drug plants of native North America[END_REF][START_REF] Burt | Essential oils: their antibacterial properties and potential applications in foods-a review[END_REF][START_REF] Novak | An α-Pinene Chemotype in Salvia offcinalis L. (Lamiaceae)[END_REF][START_REF] Naghdi Badi | Effects of spacing and harvesting time on herbage yield and quality/quantity of oil in thyme, Thymus vulgaris[END_REF][START_REF] Harrewijn | Natural terpenoids as messengers: a multidisciplinary study of their production, biological functions, and practical applications[END_REF][START_REF] Hussain | Chemical composition, antioxidant and antimicrobial activities of basil (Ocimum basilicum) essential oils depends on seasonal variations[END_REF][START_REF] Teixeira Da Silva | Mining the essential oils of the Anthemideae[END_REF][START_REF] Hammer | A review of the toxicity of Melaleuca alternifolia (tea tree) oil[END_REF]. Some essential oils or their bioactive components such as limonene from peel of lemon or orange, geranyl acetate, from leaves of lemongrass and carvone from seeds of caraway are also used as an important ingredients in tooth pastes and other hygienic products. They also act as antimicrobial agents in foods, and also have been employed for the treatment of different ailments in the folk medicine systems. Many species of the Lamiaceae have been used as culinary spices. For example, the leaves of oregano, rosemary, sage and thyme are typical seasonings in the Mediterranean region; oregano is consumed in particulalry large quantities all around the world as a part of pizza seasoning mix [START_REF] Celiktas | Antimicrobial activities of methanol extracts and essential oils of> Rosmarinus officinalis, depending on location and seasonal variations[END_REF][START_REF] Novak | An α-Pinene Chemotype in Salvia offcinalis L. (Lamiaceae)[END_REF][START_REF] Naghdi Badi | Effects of spacing and harvesting time on herbage yield and quality/quantity of oil in thyme, Thymus vulgaris[END_REF][START_REF] Hussain | Chemical composition, antioxidant and antimicrobial activities of basil (Ocimum basilicum) essential oils depends on seasonal variations[END_REF].

The sage (Salvia officinalis), one of the common species of this family is now widely cultivated in various parts of the world and is used as a culinary herb for flavoring and seasoning. Essential oil of sage was characterized by high concentrations of camphor, 1,8cineole and thujone [START_REF] Novak | An α-Pinene Chemotype in Salvia offcinalis L. (Lamiaceae)[END_REF].

Essential oils of some spices and herbs such as sage, oregano, thyme, savory and others have shown their antioxidant potential [START_REF] Dang | Antioxidant activity of essential oils from various spices[END_REF] and thus can be used as natural antioxidants for the protection of fats/oils and related products.

The extracted oils or natural antioxidants can vary in quality, quantity and in the chemical composition depending upon the agroclimate, plant organ, age and vegetative cycle stage [7,[START_REF] Novak | An α-Pinene Chemotype in Salvia offcinalis L. (Lamiaceae)[END_REF] Essential oils ussually conntain of a great number of chemical components mainly consisting of hydrogen, carbon and oxygen [7]. They can be essentially classified into two groups: Volatile fraction: Essential oil constituting of 90-95% of the oil in weight, containing the monoterpene and sesquiterpene hydrocarbons, as well as their oxygenated derivatives along with aliphatic aldehydes, alcohols, and esters. Nonvolatile residue: This comprises 1-10% of the oil, containing fatty acids, sterols, carotenoids, waxes and oxygen heterocyclic compounds [7].

However the composition of essentila oils isolated from the same species may vary in a wide range depending on different factors. For instance, Bonnardeaux investigated the oil composition and yield of different plant parts of basil (Ocimum basilicum) and found that lower oil yields were obtained from the distillation of basil with shorter flower spikes than from plants with long flower spikes [START_REF] Bonnardeaux | The effect of different harvesting methods on the yield and quality of basil oil in the Ord River irrigation area[END_REF]. Typical peppermint oil consists of about 50% of menthol, 20% of menthone and 8% of ester [START_REF] Burbott | Effects of light and temperature on the monoterpenes of peppermint[END_REF][START_REF] Clark | The importance of harvest date and plant density on the yield and quality of Tasmanian peppermint oil[END_REF]; however, flower oil contains much higher amounts of menthofuran and pulegone (> 50%) than leaf oil, with remarkablylower amounts of menthol and menthone [START_REF] Burbott | Effects of light and temperature on the monoterpenes of peppermint[END_REF][START_REF] Clark | The importance of harvest date and plant density on the yield and quality of Tasmanian peppermint oil[END_REF].

Chemical composition of essential oils also depends on such factors as plant chemotype (polymorphism), climatic conditions, geographical location, the type of soil, othre environmental and technological conditions [START_REF] Novak | An α-Pinene Chemotype in Salvia offcinalis L. (Lamiaceae)[END_REF][START_REF] Cava | Antimicrobial activity of clove and cinnamon essential oils against Listeria monocytogenes in pasteurized milk[END_REF]. In addition, quality and quantity of the essential oils most commonly are influenced by several factors that are closely related together. It is difficult to segregate these factors from each other [START_REF] Terblanche | The characterization, utilization and manufacture of products recovered from Lippia scaberrima Sond[END_REF], therefore, in this chapter four main interdependent factors will be briefly reviewed.

Environmental and botanical (taxonomic) factors.

Plants accumulate essential oil in different anatomical parts depending on their taxonomy. Polymorphism is characteristic for plants as well; consequently, plants of the same species can have different chemical composition which defines the quality of essential oils. Often producers of essential oils consider that the plants of the same species have the same main volatile compounds. However, essential oil composition highly depends on the plant's taxonomy. For example, main components of Mentha (species) plants are monoterpenes, however, Mentha × Piperita and Mentha arvensis ssp. piperascens contain high amounts of menthol which is characteristic only for these two species of mint. On the other hand, eugenol, the compound which is characteristic to cloves plants (Syzygium aromaticum) is also found in large amounts in cinamon (Cinnamomum zeylanicum) and basil (Ocimum basilicum). Therefore, the producers should carefully of essential oils to take into account the chemotaxonomy of the plants through consider chemical polymorphism of various plant cultivars in order to guarantee the consistent quality of essential oil. In many cases plant species posses typical quantitatively major or in some cases even minor compounds which may be considered as their fingerprints. For instance, δ-3-carene mybe used to distinguish Citrus grandis from other Citrus species [START_REF] Gonzalez | Chemotaxonomic value of essential oil compounds in citrus species[END_REF].

Factors influencing the production and the quality of essential oil

Climatic and environmental factors such as the length of the day, the activeness of the sun and radiation, the amount of water, the type of soil, the amount of emissions, the effect of vermins, etc. influence biosynthesis of essential oils in plants. However, in most cases the climate has the greatest influence on the quality of essential oils, as the temperature impacts the harvest of plants, while optimal interactions between humidity and temperature may increase the yield of essential oil. Humidity coupled with elevated temperatures produces conditions favorable to the proliferation of insect parasites and, most importantly, microorganisms. This sometimes causes plants to increase the production of essential oil for their own protection [START_REF] Letchamo | The selection of Thymus vulgaris cultivars to grow in Canada[END_REF].

Seasonal and geographical origin factors. They are closely related and may have important influence on the quality of essential oils. Literary sources provide large amount of information about differences of essential oils of the same plant species harvested in different months and seasons [START_REF] Celiktas | Antimicrobial activities of methanol extracts and essential oils of> Rosmarinus officinalis, depending on location and seasonal variations[END_REF][START_REF] Hammer | A review of the toxicity of Melaleuca alternifolia (tea tree) oil[END_REF][START_REF] Letchamo | The selection of Thymus vulgaris cultivars to grow in Canada[END_REF][START_REF] Kofidis | Seasonal variation of essential oils in a linalool-rich chemotype of Mentha spicata grown wild in Greece[END_REF][START_REF] Van Vuuren | Seasonal and geographical variation of Heteropyxis natalensis essential oil and the effect thereof on the antimicrobial activity[END_REF][START_REF] Juliani | Chemical diversity of Lippia multiflora essential oils from West Africa[END_REF]. For instance, Badi et al., concluded that spring is the most favourable time to collect thyme because its essential oil is richer in oxygenated compounds than collected in the summer, autumn or winter [START_REF] Naghdi Badi | Effects of spacing and harvesting time on herbage yield and quality/quantity of oil in thyme, Thymus vulgaris[END_REF][START_REF] Atti-Santos | Seasonal variation of essential oil yield and composition of Thymus vulgaris L. (Lamiaceae) from South Brazil[END_REF]. On the other hand, thyme collected in the beginning of winter was higher in phenolic compounds [START_REF] Mcgimpsey | Seasonal variation in essential oil yield and composition from naturalized Thymus vulgaris L. in New Zealand[END_REF]. The quality of essential oil may also greatly depend on their geographic location [START_REF] Marotti | Effects of variety and ontogenic stage on the essential oil composition and biological activity of fennel (Foeniculum vulgare Mill.)[END_REF][START_REF] Skoula | The taxonomy and chemistry[END_REF][START_REF] Yildirim | The variation of antioxidant activities and chemical composition of essential oils of Teucrium orientale L. var. orientale during harvesting stages[END_REF][START_REF] Anwar | Changes in composition and antioxidant and antimicrobial activities of essential oil of fennel (Foeniculum vulgare Mill.) fruit at different stages of maturity[END_REF][START_REF] Chalchat | Essential oil of Tagetes minuta from Rwanda and France: chemical composition according to harvesting location, growth stage and part of plant extracted[END_REF]. It was proved that chemical composition of Mentha longifolia (L.) and Tagetes minuta collected in various geographical areas differed significantly [START_REF] Chalchat | Essential oil of Tagetes minuta from Rwanda and France: chemical composition according to harvesting location, growth stage and part of plant extracted[END_REF][START_REF] Uribe-Hernández | The essential oil of Lippia graveolens HBK from Jalisco, Mexico[END_REF][START_REF] Souto-Bachiller | Terpenoid composition of Lippia dulcis[END_REF][START_REF] Viljoen | The Chemo-Geographical Variation in Essential Oil Composition and the Antimicrobial Properties of' Wild Mint'-Mentha longifolia subsp. polyadena (Lamiaceae) in Southern Africa[END_REF]. Some researchers hypothesized that chemical composition of plants varied due to different type of soil [START_REF] Chalchat | Essential oil of Tagetes minuta from Rwanda and France: chemical composition according to harvesting location, growth stage and part of plant extracted[END_REF][START_REF] Uribe-Hernández | The essential oil of Lippia graveolens HBK from Jalisco, Mexico[END_REF][START_REF] Souto-Bachiller | Terpenoid composition of Lippia dulcis[END_REF][START_REF] Viljoen | The Chemo-Geographical Variation in Essential Oil Composition and the Antimicrobial Properties of' Wild Mint'-Mentha longifolia subsp. polyadena (Lamiaceae) in Southern Africa[END_REF]. Besides, due to different growing site altitude above the sea level, the plants need to adapt and this also influenced the processes of biosynthesis in the plant. The same plant species collected in different regions with the same type of soil, yet often droughty or floody, showed different profiles of chemical composition. Therefore, it was stated that climate conditions due to which plant changes genetically and adapts have greater impact on plants biosynthesising essential oils [START_REF] Terblanche | The characterization, utilization and manufacture of products recovered from Lippia scaberrima Sond[END_REF][START_REF] Milos | Localities and seasonal variations in the chemical composition of essential oils of Satureja montana L. and S. cuneifolia Ten[END_REF][START_REF] Santos-Gomes | Organ-and season-dependent variation in the essential oil composition of Salvia officinalis L. cultivated at two different sites[END_REF][START_REF] Graven | The development of Artemisia afra (Jacq.) as a new essential oil crop[END_REF].

Other factors affecting yield and composition of essential oil.

Composition and quantity of essential oils are as well influenced by the following factors: raw material used for distillation (dried or fresh), technique of postharvest drying (availability of sun and water), timing of plant sowing, plant resistance to fungous diseases, conditions of harvesting, distillation method, etc. [START_REF] Burbott | Effects of light and temperature on the monoterpenes of peppermint[END_REF][START_REF] Baritaux | Effects of drying and storage of herbs and spices on the essential oil. Part I. Basil, Ocimum basilicum[END_REF].

The essential oils are produced from plant material by using different methods, such as hydrodistillation, water and steam distillation, direct steam distillation, solvent extraction, maceration, cold pressing, effleurage, super critical CO 2 extraction, etc. [7][8]. The extraction process generally depends on the rate of diffusion of the oil through the plant tissues to an exposed surface. The production of essentials oils for commercial use is based in three basic methods. Expression is used for the production of Citrus oils. The second method, hydrodistillation or steam distillation is the most commonly used one of the three methods and dry distillation is used very rarely in special cases [START_REF]Extraction of essential oil[END_REF].

Extraction of essential oils using hydrodistillation and steam distillation

Hydrodistillation is one of the simplest and oldest processes of essential oil isolation from the plants, which has been used mainly by small scale producers. Fresh or dried, whole or ground plant material may be hydrodistilled by boiling it in water at 100°C. During boiling the plant material soaks up the water and oil present within the plant cell diffuses via osmosis [7]; volatile oil is carried over to the condenser along with the steam. The condenser cools the vaporized oil and steam and the condensates trickle down into the graduated tube where the oil is collected. In steam distillation the plant material is placed in the chamber and steam is allowed to pass through the material; it breaks the essential oil containing cells and carries the oil into the condenser similarly like in hydrodistillation, producing two-phases, essential oil and hydrosol [7][8][START_REF] Charles | Comparison of extraction methods for the rapid determination of essential oil content and composition of basil[END_REF]. A number of factors determine the final quality of distilled essential oils. Apart from the plant material itself, most important factors are time, temperature and distillation equipment. The advantages of distillation are good oil yield, relatively cheap process; in case of steam distillation the compounds are better protected against oxidation. However, these methods have several disadvantages; the plant material becomes wet, which slows down distillation as the steam has to vaporize the condensed water. Hydrodistillation can thermally degrade and hydrolyze some of the oil components, which in some cases can lead to significant distortion of the native composition of the oils present in the ungtreted plants. The quality of oils is very variable and varied with each distillation. Monoterpenes are vulnerable to chemical changes under steam distillation conditions [7][8].

Extraction Methods for Recovery of Volatile Organic Compounds

Solvent-extraction

Aromatic extracts can be extracted by using solvents such as dimethyl ether, petroleum ether, or hexane. All the extractable components from the plant are dissolved in the solvent [7][8][START_REF] Charles | Comparison of extraction methods for the rapid determination of essential oil content and composition of basil[END_REF] and include volatile aroma molecules as well as non-volatile waxes, pigments and other constituents. The concentrates are usually further processed to remove the waxy materials which dilute the pure essential oil.

Maceration

Using maceration process plant material is soaked in vegetable oil thus obtaining the "infused oil" rather than the essential oil. This method is rarely used because product composition is largely different from natural oil.

Cold pressing

This method is used to extract the essential oils from citrus rinds. The rinds are separated from the fruit, are ground or chopped and then pressed, resulting in a watery mixture of essential oil and liquid [7][8]. Pressed oils retain high quality natural aroma, however their shelf life is rather short. This mechanical method, also called cold expression, is used in the industrial production of cold-pressed citrus oils. It has the advantage to protect the compounds as aliphatic and aldehydes from the thermal degradation as the formation of malodorous carboxylic acids. At the beginning, this process was an extremely laborious and characterized by substantial oil losses. Late, some improvements were development as the "scodella" method which was an apparatus with a metallic hemisphere lined inside with small spikes, with a tube attached at its center [START_REF] Arnodou | The taste of nature; industrial methods of natural products extraction[END_REF]. For extract the oils, the fruit is placed inside hemisphere which start to rotate and the fruit is squeezed against the spikes breaking the oils cells. The oil emulsion containing some wastes flowed into the central tube, thus the oils is separated by centrifugation. Nowadays, new machines are implemented to extract the oil: sfumatrice method and the FMC extractor which use high pressures, the Pellatrice and Brown process machine which use centrifugal forces [START_REF] Omidbaigi | Production and processing of medicinal plants, In Processing, Analysis and Application of Essential Oils[END_REF][START_REF] Reeve | [END_REF].

Dry distillation-enfleurage

This process is applicable to plants, that have low content of essential oil and so delicate that heating would destroy the blossoms before releasing the essential oils. The principle of the enfleurage method is that fats dissolve essential oils and thereby absorb their aromas. Every day or every few hours after the vegetable or fat has absorbed as much essential oil as possible; the depleted petals are removed and replaced with fresh ones. Thereafter, the oil is extracted from the fat with alcohol and then isolated. This method is expensive and complicated. Enfleurage has the advantage that even the most delicate components of the flower oils are preserved.

Supercritical CO 2 extraction

Supercritical CO 2 extraction method is attractive because essential oils are being isolated at low temperatures. Supercritical CO 2 extraction involves carbon dioxide heated to 31 °C and pumped through the plant material at around 75 bar under these conditions; the CO 2 is likened to a 'dense fog' or vapor. Supercritical CO 2 extraction method has several advantages: low pressure and low temperature processing, there are no solvent residues left behind, and essential oil is pure also, there is no heat applied to the plant material or essential oil to alter it in any way [7][8][START_REF] Charles | Comparison of extraction methods for the rapid determination of essential oil content and composition of basil[END_REF]. The essential oil is very accurate with respect to the original state of the plant. Despite these advantages, this method is expensive and this method cannot be used for dissolving polar molecules.

Modern methods of extraction of essential oils

Modern extraction methods have been developed using the latest inventions However, these methods are not designed for industrial use. These methods have been developed in order to minimize change the volatile compounds during the extraction process and these techniques are: solid phase micro-extraction, supercritical fluid extraction, microwave distillation, membrane extraction.

Synthetic antioxidants

Antioxidants are compounds that can retard the process of lipid oxidation; they can also block the propagation step of the radical chain reaction by destroying or binding free radicals, inhibition of catalysts or stabilization of hydroperoxides. Most common is to classify antioxidants to primary and secondary ones and to describe origin of antioxidants such as natural or synthetic. Synthetic antioxidants containing phenolic groups such as butylated hydroxylanisole (BHA), butylated hydroxyltoluene (BHT), gallic acid esters (propyl, octyl, dodecyl) and tert-butylhydroquinone (TBHQ) have been widely used as antioxidants in foods. TBHQ is the most effective antioxidant used by the food industry [START_REF] Yanishlieva | Stabilisation of edible oils with natural antioxidants[END_REF]. On the other hand, synthetic antioxidants are acceptable for their effectiveness and relatively low cost, but they may have negative side effects related to their toxicity. Because of negative aspects of synthetic antioxidants the interest in healthier and safer antioxidants from natural sources have increased. Therefore, the importance of the search for the exploitation of natural antioxidants, especially of plant origin, has greatly increased in recent years and natural antioxidants have gained popularity. Traditionally herbs and spices are added to food for improving organoleptic properties and they also may act as antioxidants and preservatives.

Natural antioxidants

ANTIOXIDANTS

Natural plant origin antioxidants are mainly phenolic or polyphenolic compounds. The structure of plant phenolics and polyphenols varies from simple molecules to polymerized compounds, such as proanthocyanidins; several thousands (among them over 8,150 flavonoids) different compounds have been identified so far. Several classes of phenolics have been categorized on the basis of their basic molecular skeleton (Table 1.3) [START_REF] Gil | Antioxidant capacities, phenolic compounds, carotenoids, and vitamin C contents of nectarine, peach, and plum cultivars from California[END_REF][START_REF] Chang | Low-density lipoprotein antioxidant activity of phenolic compounds and polyphenol oxidase activity in selected clingstone peach cultivars[END_REF][START_REF] Someya | Antioxidant compounds from bananas (Musa Cavendish)[END_REF]. Phenolic compounds are traditionally believed to play an important role in plant herbivore interactions and in disease resistance of plants. Flavonoids are one of the major subgroups of phenolic compounds wich mostly occur as their glycosylated derivatives in plants. Chemical structure of flavonoids are subdivided into flavones, isoflavones, anthocyanidins, flavan-3ols, flavan-4-ols, flavan-3,4-diols, proanthocyanidins [START_REF] Balasundram | Phenolic compounds in plants and agri-industrial byproducts: Antioxidant activity, occurrence, and potential uses[END_REF][START_REF] Augustyniak | Natural and synthetic antioxidants: an updated overview[END_REF]. Tocopherols are other important antioxidants, which may be divided into tocols and trocotrienols. Tocopherols are considered as safe natural antioxidants; however they do not always provide effective protection against oxidation in vitro. Therefore, research of other natural antioxidants is highly desirable.

Carotenoids. Carotenoids are also considered as secondary antioxidants. They are yellow or orange-red pigments. The carotenoids are molecules with limited solubility properties and very sensitive to oxygen and light. The antioxidant activity of carotenoids is based on their singlet oxygen quenching properties and their ability to trap peroxyl radicals [START_REF] Augustyniak | Natural and synthetic antioxidants: an updated overview[END_REF].

Recent studies have demonstrated that dietary plants are rich source of antioxidants and can contribute to the protection from age-related diseases Hinneburg et al. investigated antioxidant activities of extracts from selected culinary herbs and spices (basil, laurel, parsley, juniper, aniseed, fennel, cumin, cardamom, and ginger) [START_REF] Hinneburg | Antioxidant activities of extracts from selected culinary herbs and spices[END_REF]. The extracts from basil and laurel possessed the highest antioxidant activities and both extracts are promising alternatives to synthetic antioxidant. Yanishlieva et al. [START_REF] Yanishlieva | Stabilisation of edible oils with natural antioxidants[END_REF] in a review paper presented information about the antioxidative effects of rosemary, sage, oregano, thyme, ginger, summer savory, black pepper, red pepper, clove, marjoram, basil, peppermint, spearmint, common balm, fennel, parsley, cinnamon, cumin, nutmeg, garlic, coriander, etc. Sage and oregano, which belong to the same family, have gained the interest of many research groups as potential antioxidants.

In the last decade the popularity of a healthy lifestyle has increased, therefore the consumers more often give preference to natural products. This tendency also increases the demand for essential oils. On the other hand, for enterprises producing essential oils distillation waste disposal becomes an increasing problem. Currently, the manufacturers of essential oils are using solid waste mainly for composting whereas fluid wastes are simply discarded. So far as essential oil content of the raw material is low, usually less than 5%, the waste consists of more than 95%; therefore, recently, the interest in the effective use of waste has been increasing However, this topic of research is relatively new, there is little information about active compounds in both liquid and solid residues of essential oil distillation; therefore, in this section brief survey on by-products formed from the processing of fruits and vegetables will be provided.

Processing of fruits and vegetables almost always results in some by-products, which are called waste in the case they are not utilized for further processing. The quantities of the waste are usually quite large (Table 1.4). [START_REF] Schieber | By-products of plant food processing as a source of functional compounds-recent developments[END_REF][START_REF] Moure | Natural antioxidants from residual sources[END_REF][START_REF] Goñi | By-Products from Plant Foods are Sources of Dietary Fibre and Antioxidants. Phytochemicals -Bioactivities and Impact on Health[END_REF][START_REF] Aaby | Phenolic composition and antioxidant activities in flesh and achenes of strawberries (Fragaria ananassa)[END_REF][START_REF] Sójka | Composition of industrial seedless black currant pomace[END_REF] Production process % of wastes or by-products

Table 1.4. Amount of agro-industrial residues

White wine production 20-30

Red wine production 20-30

Fruit and vegetable juice production 30-50

Fruit and vegetable processing and preservation 5-50

Vegetable oil production 40-70

Essential oil production 85-99

Moure et al. suggested various ways of waste uses, one of these opportunities to recycle and use it as a source of phenol compounds, fiber and protein [START_REF] Moure | Natural antioxidants from residual sources[END_REF]. Other authors who studied the by-products such as the hulls of rice, buckwheat, and almond estimated that there is a large amount of minerals and active ingredients, possessing antioxidant properties; they have offered these active components to extract from the waste, thus increasing the complete recycling of primary raw material [START_REF] Ramarathnam | Chemical studies on novel rice hull antioxidants. 2. Identification of isovitexin, a C-glycosyl flavonoid[END_REF][START_REF] Takeoka | Antioxidant constituents of almond Prunus dulcis (Mill.) DA Webb hulls[END_REF][START_REF] Bryngelsson | Lipids and antioxidants in groats and hulls of Swedish oats (Avena sativa L)[END_REF]. On the other hand, in many developed countries by-products of vegetables are burned as a fuel, instead of being discarded; however, there is the opinion, that such utilistion of processing of by-products is not suficiently rational. Therefore, purposeful and rational ways of waste recycling remains the challenge both for the scientists and industry.

The quantities of solid and liquid waste formed in food industry mainly depend on the technological processes and the origin of raw material. However, the total formed waste in plants pose huge problems for their recovery, because there are complex technological and legal restrictions in waste recovery processes. Four recycling processes resulting in the formation of by-products in large quantities which could be further treated for isolating active

Plant residues 1.3.2 Valorization of plant processing by-products

components will be shortly discussed [START_REF] Goñi | By-Products from Plant Foods are Sources of Dietary Fibre and Antioxidants. Phytochemicals -Bioactivities and Impact on Health[END_REF][START_REF] Bryngelsson | Lipids and antioxidants in groats and hulls of Swedish oats (Avena sativa L)[END_REF]. Large amounts of residues obtained after extraction of oil from the seeds of soybean, cotton, sunflower etc. are produced in the edible oil industry. Oilseed meals come from oil processing after the recovery of the oil by mechanical pressing or solvent extraction. They are significant protein sources due to high protein content, low price, and high availability.

Molasses and sugar beet pulp in the sugar industry are other abundant by-products. Sugar cane bagasse is one of the largest cellulosic agro-industrial by-products. Several processes have been used for sugarcane bagasse. These include electricity generation, pulp and paper production, and products based on fermentation. Various products, which have been obtained from the processes involving bagasse include chemicals and metabolites such as alcohol and alkaloids, mushrooms, protein enriched animal feed (single cell protein), enzymes, etc. One of the significant applications of the bagasse is the production of protein-enriched cattle feed and enzymes. Bagasse could also been used for the production of industrially important enzymes and biofuel.

By-products from fruit processing include citrus and pineapple pulps, reject apples, etc. can be processed for the production of alcohol or other products. Pectin is also produced from the peel by acid extraction, dietary fibers by mechanical processing, while the recovery of flavonoids and carotenoids are new potential applications. Citrus seeds can be used for oil extraction and recovery of terpenoids, while the meal remaining from the extraction is a good source of proteins.

The solid residue (leaves, flowers, stems, roots) and water residue obtained after hydrodistillation of essential oil in the essential oil industry can be processed into a powder with high content of phenolic compounds and strong antioxidant capacity; the product could be used as a natural additive or healthy food ingredient in foods and nutraceuticals.

The recycling processes of isolating organic compounds or natural molecules are usually much simpler than using the raw material, mostly because the waste is left of certain components which impede to isolate active compounds, such as oil materials and resin, which complicate the extraction of polyphenols. As an example, fruits, vegetables, oilseeds and hydrodistillation of the by-products processing schemes are shown in Fig. 1.2. Using simple technological processes can be distinguished valuable components such as antioxidants, protein, fiber, carotenoids and pectin, which later would be used for food, cosmetic or pharmaceutical products [START_REF] Goñi | By-Products from Plant Foods are Sources of Dietary Fibre and Antioxidants. Phytochemicals -Bioactivities and Impact on Health[END_REF][START_REF] Bryngelsson | Lipids and antioxidants in groats and hulls of Swedish oats (Avena sativa L)[END_REF]. The solid residue remaining after second recycling phase could be used for composting or for more specific applications, e.g. for producing biodegradable cups for plants.

Growing interest is pointing towards natural antioxidants obtained using "green" processes. Natural antioxidants represented 38% of the market of antioxidants in 2004 and every year this market grows by 6-7% [START_REF] Meyer-Aurich | Impact of tillage and rotation on yield and economic performance in corn-based cropping systems[END_REF]. These products have a promising business perspective, for instance, in the formulation of functional foods when antioxidants can provide an important benefit for human health [START_REF] Pokornỳ | Preparation of natural antioxidants[END_REF]. It is also important to emphasise that the amount of raw materials and energy is limited therefore they should be used as efficiently as possible [START_REF] Garcia-Serna | New trends for design towards sustainability in chemical engineering: Green engineering[END_REF]. The extraction of antioxidants from agricultural waste is thus an opportunity to obtain profit in a sustainable way from low-cost raw material. Studies related to the antioxidants recovery from residues remaining after extractions have achieved positive results regarding their potential use [START_REF] Schieber | By-products of plant food processing as a source of functional compounds-recent developments[END_REF][START_REF] Balasundram | Phenolic compounds in plants and agri-industrial byproducts: Antioxidant activity, occurrence, and potential uses[END_REF].

Fig. 1.2. Flow diagram for the recovery of active componets from by-products

While in the developed countries of the world synthetic drugs replaced the majority of natural plant preparations used for treatment of various diseases a long time ago, the plants remain as the main source of treatment of various diseases in many developing countries. However, modern science have once again turned to the analysis of the plants for searching new active molecules, since the plants are the renewable resources; also a wide variety of plants enables finding a number of new previously unknown molecules possessing different functional properties [START_REF] Augustyniak | Natural and synthetic antioxidants: an updated overview[END_REF]. It has long been known that the plants accumulate phytocomponents which can absorb and neutralize free radicals, inhibit the oxidation processes and slow down the formation of peroxides [START_REF] Davidson | The Oxford companion to food[END_REF][START_REF] Hacskaylo | The National Herb Garden Guidebook[END_REF][START_REF] Smith | The National Herb Garden Guidebook[END_REF][START_REF] Kähkönen | Antioxidant activity of plant extracts containing phenolic compounds[END_REF][START_REF] Velioglu | Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products[END_REF][START_REF] Anderson | Walnut polyphenolics inhibit in vitro human plasma and LDL oxidation[END_REF][START_REF] Schwarz | Investigation of plant extracts for the protection of processed foods against lipid oxidation. Comparison of antioxidant assays based on radical scavenging, lipid oxidation and analysis of the principal antioxidant compounds[END_REF][START_REF] Chipault | The antioxidant properties of natural spices[END_REF][START_REF] Abdalla | Effect of plant extracts on the oxidative stability on sunflower oil and emulsion[END_REF]. Increased consumption of plantbased products due to the pervasive vegetarianism significantly reduced some of the diseases and mortality, resulting in a greater interest in natural additives which might replace synthetic ones [START_REF] Sharopov | Essential oil composition of Mentha longifolia from Wild Populations Growing in Tajikistan[END_REF]. Natural ingredients made of by-products are also gaining popularity due to the developments in recycling procedures applied for the isolating active components. For instance, during last years pomegranate and cranberry wastes have been widely analyzed because they contain components that inhibit inflammatory processes. Pomegranate peel was found to contain a large amount of phenolic compounds including anthocyanins (derivatives of delphinidin, cyanidin and pelargonidin), hydrolysable tannins (catechin, epicatechin, punicalin, pedunculagin, punicalagin, gallic and ellagic acid esters of glucose) [START_REF] Cuccioloni | Pomegranate fruit components modulate human thrombin[END_REF][START_REF] Gil | Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing[END_REF], and several lignans (isolariciresinol, medioresinol, matairesinol, pinoresinol, syringaresinol, and secoisolariciresinol). These phenolic compounds confer antioxidant, anti-mutagenic, antiinflammatory and anticancer activities [START_REF] Gil | Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing[END_REF][START_REF] Zheng | Solid-state bioconversion of phenolics from cranberry pomace and role of Lentinus edodes β-glucosidase[END_REF][START_REF] Naveena | Comparative efficacy of pomegranate juice, pomegranate rind powder extract and BHT as antioxidants in cooked chicken patties[END_REF]. In recent studies husks of pomegranate were successfully used in the production of ellagic acid [START_REF] Abdalla | Effect of plant extracts on the oxidative stability on sunflower oil and emulsion[END_REF][START_REF] Negi | Antioxidant and antimutagenic activities of pomegranate peel extracts[END_REF]. The pomace of cranberry, a byproduct of the cranberry juice, is a good source of ellagic acid and other phenol compounds as well [START_REF] Negi | Antioxidant and antimutagenic activities of pomegranate peel extracts[END_REF][START_REF] Hernández | Kinetic production of the antioxidant ellagic acid by fungal solid state culture[END_REF]. Many literature sources reported that antioxidants isolated from by-products may possess higher activity than synthetic compounds; for example, polyphenols in the skin of pomegranate better inhibited lipid oxidation occurring in meat during cooking process than BHT [START_REF] Cuccioloni | Pomegranate fruit components modulate human thrombin[END_REF][START_REF] Mccarthy | Evaluation of the antioxidant potential of natural food/plant extracts as compared with synthetic antioxidants and vitamin E in raw and cooked pork patties[END_REF].

Natural bioactive compounds from crop by-products

Povilaityte et al., reported the antioxidant effects of acetone and deodorized acetone extracts of Roman chamomile, purple peril and Moldavian dragonhead and compared it with synthetic antioxidant BHT [START_REF] Povilaitytė | Antioxidative activity of purple peril (Perilla frutescens L.), moldavian dragonhead (Dracocephalum moldavica L.), and roman chamomile (Anthemis nobilis L.) extracts in rapeseed oil[END_REF]. Chen et al., studied extracts from rosemary leaves and determined that hexane and acetone extracts showed higher antioxidative activity than BHA and BHT [START_REF] Chen | Effects of rosemary extracts and major constituents on lipid oxidation and soybean lipoxygenase activity[END_REF], while methanol extracts had lower activity. By-products, like skins and seeds of grapes [START_REF] Castilho | Production and extraction of pectinases obtained by solid state fermentation of agroindustrial residues with Aspergillus niger[END_REF][START_REF] Louli | Recovery of phenolic antioxidants from wine industry by-products[END_REF], peel and pulp of citrus [START_REF] Benavente-García | Uses and properties of citrus flavonoids[END_REF], pomace of apples [START_REF] Lu | Antioxidant and radical scavenging activities of polyphenols from apple pomace[END_REF][START_REF] Schieber | A new process for the combined recovery of pectin and phenolic compounds from apple pomace[END_REF], waste of potatoes peel [START_REF] Sotillo | Potato peel extract a nonmutagenic antioxidant with potential antimicrobial activity[END_REF][START_REF] Sotillo | Phenolics in aqueous potato peel extract: extraction, identification and degradation[END_REF], by-products of onions [START_REF] Chu | Flavonoid content of several vegetables and their antioxidant activity[END_REF], hulls of beans [START_REF] Onyeneho | Effect of navy bean hull extract on the oxidative stability of soy and sunflower oils[END_REF], plant residues after hydrodistillation [START_REF] Povilaitytė | Antioxidative activity of purple peril (Perilla frutescens L.), moldavian dragonhead (Dracocephalum moldavica L.), and roman chamomile (Anthemis nobilis L.) extracts in rapeseed oil[END_REF] were reported to be rich in various classes of compounds such as coumarins, cinnamic acid derivatives and chalcones, phenolic diterpenes and phenolic acids, with promising nutritional and therapeutic applications [START_REF] Li | Antioxidant properties in vitro and total phenolic contents in methanol extracts from medicinal plants[END_REF][START_REF] Trouillas | Antioxidant, anti-inflammatory and antiproliferative properties of sixteen water plant extracts used in the Limousin countryside as herbal teas[END_REF].

By-products of fruit processing as it was shown for skin and seeds of citrus fruits consitute up 50% of the whole fruit mass; in addition, many studies demonstrated that the largest amount of phenolic compounds are being accumulated specifically in the not edible part of fruit part, which compose the waste [START_REF] Bocco | Antioxidant activity and phenolic composition of citrus peel and seed extracts[END_REF][100]. The same patterns were found in processing apples, nectarines, peaches, pears, and pomegranate. Consequently it may be noted that the skin of the fruit should not be discarded because it contain at least two times more phenolic compounds than the peeled fruit. Therefore, the enterprises involved in fruits processing should also focus on recycling by-products, thus reducing the amount of waste and optimizing the effectiveness of the use of raw materials [START_REF] Gil | Antioxidant capacities, phenolic compounds, carotenoids, and vitamin C contents of nectarine, peach, and plum cultivars from California[END_REF][START_REF] Chang | Low-density lipoprotein antioxidant activity of phenolic compounds and polyphenol oxidase activity in selected clingstone peach cultivars[END_REF][START_REF] Someya | Antioxidant compounds from bananas (Musa Cavendish)[END_REF][101][102]. The studies on byproducts recycling also showed that the quantity of recovered phenolic compounds can depend on the process parameters. Jeong et al. found that exposure of lemon rinds at 150 °C for 40 min results in the increase of total phenolics from 71.8 to 171.0 μM [103]. On the other hand, Garrote et al. found that exposure of corn cobs at a higher temperature increased the amount phenolics compounds, however their activity decreased, leading to the hypothesis that heat treatment may change the structure of phenolic compounds, which determines the activity of compound [104]. The ability of phenolic compounds from by-products to act as antioxidants in vitro depends on the number and the arrangement of the hydroxyl groups and the extent of structural conjugation. Many studies established very important relationships between structures and activities of polyphenols [103-104].

The changes of phenol compounds structure can significantly alter the selected method of extraction or solvent, so the recycling of waste should evaluate these factors.

Phenolic compounds may demonstrate both primary and secondary antioxidant activities as chain breaking antioxidants able to donate hydrogen atoms to lipid radicals (radical scavenging) or by preventing initiation reactions by such means as chelating catalyst metals [105-107]. Moreover, the fact that a molecule of phenolic compound is capable to donate a hydrogen atom to a reactive molecule thus transforming it into a non-radical compound with lower activity demonstrates it being a typical radical scavenger [108]. Cai et al. investigated the relationship between the structure and radical scavenging activity of phenolic compounds and confirmed that the position and degree of hydroxylation and the related glycosylation, methoxylation and other substitution determine the antioxidant activity of phenolic compounds [109]. The effectiveness of phenolic antioxidants depends on several factors: The objective in extracting phenolic compounds from by-products of plant sources is to liberate these compounds from the vacuolar structures where they are found, either through rupturing plant tissue or through a process of diffusion; they are commonly extracted by alcoholic or other organic solvents. Usually before extraction plant samples are treated by milling, grinding and homogenization [112].

Structure-activity of phenolic compounds

Many methods are described in the literature for extracting phenolic compounds such as Soxhlet extraction, solid-liquid, liquid-liquid, supercritical fluid, accelerated solvent, ultrasound-assisted, microwave-assisted and other methods; however, a single extraction protocol which can be considered optimal for all types of residues does not exist. By-products of plant materials may be composed of variable amounts of phenolic acids, phenylpropanoids, anthocyanins and tannins, among others. This interaction might cause the formation of hardly soluble complexes, which has an effect on the extraction procedure and its efficiency [113][114][115]. Consequently, in many cases it is not easy to develop an universal method for all plant phenolics. In this chapter some widely used solvent extraction methods for phenolic compounds will be briefly reviewed.

The most popular extraction method is solid-liquid extraction, which efficiency depends on many factors, first of all plant origin and polarity of extraction solvent; however, temperature, solvent to materials ratio, number of extraction cycles, properties of antioxidant active components present in the by-products may also play an important role [116][117]. Nonpolar solvents (hexane, petroleum ether) can be used for the recovery of tocopherols and

Polyphenol extraction from by-products

certain phenolic terpenes. Ethyl ether and ethyl acetate are very efficient for the recovery of flavonoid aglycons, low-molecular-weight phenols, and phenolic acids. Solvents of higher polarity (ethanol or ethanol-water mixtures) additionally can extract flavonoid glycosides and higher molecular weight phenols, resulting in higher yields of total extracted polyphenols. In particular, methanol has been generally found to be more efficient in extraction of lower molecular weight polyphenols while the higher molecular weight flavanols are better extracted with aqueous acetone [118][119][120][121]. Ethanol is another good solvent for polyphenol extraction and is safe for food applications [122]. In preparing anthocyanin-rich phenolic extracts from by-products of plant materials, an acidified organic solvent, most commonly methanol or ethanol is used. Methanol was also used to extract phenolic acids from Lamiaceae family plants [123][124]. However, a dittany water extract was found to be richer in polar phenolic compounds and therefore more effective in retarding lipid oxidation and in scavenging free radicals than acetone, ethanol and methanol extracts of the same plant material [125]. It the study of Moller et al. [126] also has been shown that the aqueous extract of dittany is more effective than the methanol, ethanol and acetone extracts in scavenging hydroxyl radicals as generated by the Fenton reaction and in reducing oxygen consumption when initiated by metmyoglobin. Chen et al. analysed the amount of carnosol and carnosic acid from rosemary extracted by different solvents [START_REF] Chen | Effects of rosemary extracts and major constituents on lipid oxidation and soybean lipoxygenase activity[END_REF]. In contrast to the aforementioned results, they observed lower antioxidant activity for methanol extracts than for either hexane or acetone extracts. They concluded that acetone was the most efficient solvent for the antioxidant substances of rosemary [START_REF] Chen | Effects of rosemary extracts and major constituents on lipid oxidation and soybean lipoxygenase activity[END_REF]. The efficiency of phenolic compounds extraction greatly depends on origin of plant material but also on the extraction time and temperature [127].

An increase in the extraction temperature can promote higher analyte solubility by increasing both solubility and mass transfer rate. In addition, the viscosity and the surface tension of the solvents are decreased at higher temperature, which helps the solvents to reach the sample matrices, improving the extraction rate. However, increasing the temperature is limited by the nature of compound of interest: high temperatures can cause denaturation of these compounds; therefore the optimum temperature should be carefully selected taking into account several factors [START_REF] Schieber | By-products of plant food processing as a source of functional compounds-recent developments[END_REF]122] The soil and climatic conditions is good for growing of a lot of aromatic and medical herbs in Lithuania and France. However, nowadays many herbs, which were widely used in medieval ages, are underinvestigated and therefore being used in small amounts, if any. This chapter is focused on 3 such aromatic plants, namely Calamintha grandiflora, Myrrhis odorata and Tussilago farfara, which may be referred to some extent as "forgotten medieval plants" (Fig. 1.4). However, the authors of the study did not analyse the antioxidant composition of the extracts obtained. Furthermore, considering many factors influencing the composition and the concentrations of bioactive constituents in C. grandiflora as well as the remarkable importance of these plants to their health-promoting potential, studies should be continued by focusing on the identification of active compounds and comprehensive characterization.

REVIEW ON SELECTED AROMATIC PLANTS

Calamintha grandiflora (L.) Moench

M. odorata (L.) Scop. is an underrated plant deserving more attention as a culinary herb and as a sugar saver. Common names of M. odorata are sweet cicely, anise cicely, Spanish chervil, sweet chervil, anise chervil, garden myrrh, sweet scented myrrh. Sweet cicely is widely spread in whole Europe. It grows in very moist forests and mountainous regions. Sweet cicely is being cultivated in Lithuania too. Also, feral types of this species might be found. As it is related to chervil, it may also be called by this name. M. odorata is a perennial plant and it might reach the height of 90 cm. It blooms early in the spring up to the middle of summer. Blossoms are tiny and white; leaves resemble the fern, though the smell of anise might be felt after grinding it. A sweet cicely is very easy cultivated plant because it grows almost everywhere: both in sunny or shadowy places. This plant may be sowed either in autumn or in the end of winter. The leaves of sweet cicely produce essential oil which has the aroma of anise [137].

Previously this plant was widely used in cuisine; however, as a potherb, its cultivation dercreased. Fresh, sometimes frozen or dried leaves are usually used to season the food. Green or dried seeds and roots that are collected in autumn may be used too. It is worth mentioning that dried sweet cicely's aroma is noticeably weaker than that of fresh herb. Young stems and leaves can be cut all the time. They are especially soft. Green sweet cicely has the strongest taste; therefore, the leaves may be frozen. The taste of the fruits is best while they are still green and unripe. The roots can be dug out in autumn and used as vegetables. In cuisine the leaves of M. odorata may be used as a replacement for sugar in the sweet dishes as using this plant for desert decreases the use of sugar twice. Fresh leaves are used to season the salad, soup, sauce or omelette because the leaves of sweet cicely make the taste more delicate. Only a little of the leaf is enough to give a dish a certain taste of sweet anise. On the other hand, the stems are also used as the replacement for celery. Roots are useful to make wine or salad may also been consumed boiled. The seeds are used as a fragrant material which is also usable for making cream, ice cream or candy as it improves the texture too. In addition, the seeds may be used as a replacement for cloves while baking cakes. Children eat it as a snack. Blossoms are edible or used for decorations. In cuisine, this plant is also used because it has a lot of nutrients and is fat free. It has a sweet taste; therefore, diabetics might use it in their diet for replacing saccharose. Sweet cicely is not very popular in nowadays cuisine. But it is still used in the Scandinavian countries as the people make liquor or spice out of it to make meat, fish and vegetables taste better.

Previously, the oaken furniture used to be polished by the leaves of sweet cicely. The stalk of the plant was used to make the orange-brown paint for the textile. The seeds were used to make the varnish for the furniture to make the textile better and redolent.

A few centuries ago, all parts of M. odorata were used in the medicine. This plant was thought to be harmless, thus, a lot of scientists recommended it. Sweet cicely revs up the digestive system. It reduces the caught, tones the organism and acts as an antidepressant. M. odorata presents expectorant, diuretic and was proposed for malaria treatment [138]. Essential oils and flavonoids that are present in the plant act as anticeptic agents. It stabilizes the appetite and cleans the blood too. The essential oil gathered from sweet cicely is used in perfumery. odorata is 190 days. The quality of raw material was characterised analysing the bioactive compounds. The differences in phytochemical composition of M. odorata plant's secondary metabolites depend on plant's geographical location, chemotype, meteorological factors, raw material preparation time and conditions. Therefore, it is very important to evaluate those predisposing factors. On the other hand, no information is available on the antioxidant activity and chemical constituents of M. odorata essential oils from the plants grown in France and Lithuania T. farfara L., commonly called coltsfoot, is an Asteraceae family plant, which traditionally has been used as a medicinal herb. Coltsfoot which is native from Europe to Asia is also a common plant in North and South America where it has been introduced, most likely by settlers as a medicinal item. Early coltsfoot is not big herbaceous perennial plant with the creep branched underground rhizomes. It blooms of bright yellow blossoms before issuing leaves -in March-April. In the beginning of spring from rootstock grows scaly, leafless, 10-25 cm tall stem of blossom with one bud at the top. Before flowering and blooming the buds are pendulous. After flowering grow large, round heart-shaped, angular leaves. The upper surface of leaf is dark green, the bottom -white gray, because abundantly covered with white nap. The Latin name of coltsfoot "Tussilago" is derived from the words "tussis -cough" and "Agere -budge". This medicinal plant is found throughout Lithuania. Only one kind of coltsfoot -early grows in Lithuania. It prefers loamy and sandy soils; the plant also grows very well in wet soil areas. Most often it is found on the fields, on coasts of water's bodies, near ditches, on meadows. It can be cultivated and grown artificially.

In Europe early coltsfoots are some of the most popular medicinal plants used in folk medicine for treatment of various diseases; the dried leaves and already made preparations can be purchased in pharmacies. The flowers have been wider used in European countries, while in the Eastern countries the buds, containing pyrrolizidine alkaloids prevail in the Very small quantities of pyrrolizidine alkaloids, which severely damage the liver, are being detected in the early coltsfoots; therefore the preparations of this plant are dangerous when used in large quantities. It was found that very small quantities of pyrrolizidine alkaloids harmful to health, therefore, it was proposed to completely abandon the use of early coltsfoots for therapeutic purposes. Pyrrolizidine alkaloids break during decoction preparation. Consequently, the preparations of these plants can be used just under medical advice, not exceeding the treatment more than 4-6 weeks. The fresh squash leaves can place on sores, bruises, rose and other painful areas of the body. In folk medicine with dry shredded leaves fumigated the sick persons, suffering from shortness of breath, difficulty in breathing. With the juice of coltsfoot's fresh leaves can treat runny nose, wounds. Was thought to be sufficient to include the juice of coltsfoot into the nostrils and runny nose will pass. The leaves of coltsfoot contain 2.6% bitter, up to 10% of tanning materials, up to 8% mucilage, essential oil, organic acids, vitamin C and carotene [145][146][147].

The buds of early coltsfoots before blossoming or at the beginning of blossoming may be consumed fresh or stewed; also they can be added to salads. These plants have barely perceptible smell of anise. Very young leaves can be used in vinaigrettes, salads and soups. Fragrant tea can be prepared from fresh or dried leaves; wheras young shoots may be cooked and consumed as sweets. For food these plants are mostly used in the Scandinavian countries. The study reported by Gao et al. showed the methanolic extract from flower buds of T. farfara possessed the highest maltase inhibitory activity using maltose as a substrate [153]. 3,4-dicaffeoylquinic, 3,5-dicaffeoylquinic and 4,5-dicaffeoylquinic acids showed comparative maltase inhibitory activities with the IC 50 values of 0.91 mM, 0.90 mM and 0.89 mM, respectively. In the same study it was also suggested that the number of caffeoyl groups attached to a quinic acid core were important for the activities of dicaffeoylquinic, chlorogenic, quinic and caffeic acids T. farfara is a good source of proteins, lipids, essential oils and a wide range of polyphenolic phytochemicals. The amounts of these compounds in T. farfara from Kosovo reported by Faikul et al., varied of 2.3-2.7% (proteins), 1.6-2% (lipids) and 0.1-0.2% (essential oil).

T. farfara is also considered as a source iron, calcium, potassium, magnesium, coper, sodium and zinc. The biological activity of this plant depends on the minerals and on the composition of the essential oil [156].

The literature survey shows that plant biodiversity is very high; however due to increasing consumer's demand it is important to look for the new bioactive components and their sources. Despite several published reports dealing with bioactivity of isolated compounds from flower or buds of T. farfara, very little information was found about antioxidant activity of extracts from Calamintha grandiflora, Myrrhis odorata and Tussilago farfara leaves. On the other hand, by-products obtained during distillation of aromatic plants for essential oil were not studied. Considering the high content of wastes, it is reasonable to search for ways of a most effective use of the wastes with the aim to process the whole plant material of flavours and antioxidants.

The reference compounds for β-myrcene (≥ 95.0%), estragole (≥ 95.0%), E-anethole (≥ 98.0%), methyleugenol (≥ 98.0%), E-nerolidol (≥ 98.0%) (Fluka, Steinheim, Switzerland), nalkanes C 8 -C 28 (Sigma Chemical, USA) were used in the GC analysis. Acetone (analytical grade) was from Stanchem (Lublin, Poland), ethanol (rectified spirit 95%, Stumbras, Lithuania) and methanol (analytical grade) were from Fluka Chemie (Buchs, Switzerland) were used isolated non-volatile extracts from hydrodistillation residue.

Acetonitrile (HPLC grade), methanol (HPLC grade), acetic acid (HPLC grade), gallic acid, rutin, 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH • , 95%), 2,6-di-tert-butyl-4-methylphenol (BHT), anhydrous sodium sulphate and phosphate buffered saline (PBS) were from Sigma-Aldrich (Steinheim, Germany). Trolox ® (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), (97 %) was from Acros Organics (Geel, Belgium). Folin-Ciocalteu's phenol reagent, methanol (analytical grade), 2,4,6-tripyridyl-s-triazine (TPTZ), 2,2'-azinobis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS •+ ) were from Fluka Chemie (Buchs, Switzerland). Commercial refined sunflower oil "Vilnius" (Vilniaus AB "Aliejus", Lithuania). KCl, NaCl, NaH 2 PO 4 , K 2 S 2 O 8 (Merck, Darmstadt, Germany). KH 2 PO 4 (Jansen Chimica, Beerse, Belgium) were used to evaluate antioxidant properties in the residues extracts. The leaves and roots of coltsfoot (Tussilago farfara L.) were collected in July, 2011 and August, 2010 after flowering vegetation phase.

SOLVENTS AND REAGENTS

PLANT MATERIAL

Volatile extracts

The collected and separated from non-used parts plants were dried at 302C in a ventilated oven "Vasara" (Utena, Lithuania). Dried plant leaves, roots, flowers and stems were ground in a Moulinex D53 grinder (Moulinex, France).

The essential oils were obtained by hydrodistillation from 40 to 80 g grounded plants in a Clevenger type European Pharmacopoeia apparatus over a 3 h period. The obtained oil was separated from the water and dried over anhydrous sodium sulphate. Isolation of oil was performed in duplicate and the samples were stored in a freezer prior to further analysis. The yields of M. odorata, T. farfara and C. grandiflora essential oils were expressed in % (w/w) of dry weight (Fig. 2.1).

Non-volatile extracts for isolating antioxidants

For the preparation of deodorized acetone (AE) and methanol (ME) or ethanol extracts (EE) the essentials oils were removed (Fig. 2.2). The residues of hydrodistillation were separated into the solid and liquid fractions. The solid residue was dried at 30°C in a flake "abc Bio Dörrer" (Gengenbach, Germany). 12 g of dry residue were extracted with 120 mL of acetone, methanol or ethanol with automatic extractor IKAWERKE RET control visc (Staufen, Germany). The solvent was evaporated in a R114 vacuum rotavapor equipped with a B480 water bath (60C) and B169 vacuum pump (Büchi, Switzerland). The liquid fraction (water extract) was divided in two parts. One of it was spray-dried (SD) in a Büchi Mini Spray Dryer 190 (Donau, Switzerland) and the other one freeze-dried (FD) using Maxi-Dry Lyo (Heta, Germany) for C. grandiflora, and evaporated in a vacuum rotavapor (W) for M. odorata. The extractions were performed in triplicate, and the dry extracts were stored in a freezer below -18°C prior to further analysis. Acetone (AE), methanol (ME) or ethanol (EE) of T. farfara leaves and roots extracts were obtained by extracting 15 g of dry crushed leaves and roots with 200 mL of acetone, methanol or ethanol during 3 h in a Soxhlet apparatus. The extracts were concentrated in a R114 vacuum rotavapor equipped with a B480 water bath (60C) and B169 vacuum pump (Büchi, Switzerland). The oil was analyzed by GC using a Hewlett-Packard 6890 apparatus (Hewlett-Packard, Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and electronic pressure control (EPC) injector. DB-5 column (30 m × 0.25 mm, 0.25 µm film thickness) was used. The flow of the carrier gas (N 2 ) was 1.0 mL/min at a column pressure of 42 kPa. The analysis was performed using the following temperature program: oven temperature isothermal at 50°C for 5 min, increase at a rate of 5°C/min and isothermal at 300°C for 5 min (for M. odorata samples), for C. grandiflora samples GC oven temperature was programmed from 30°C (5 min hold) to 200°C (10 min hold) at 5°C/min, and for T. farfara samples the column temperature was programmed from 50°C (5 min hold) to 280°C (10 min hold) at a rate of 3°C/min Injector's and detector's temperatures were held at 280°C and 300°C, respectively.

PREPARATION OF PLANT EXTRACTS AND ISOLATION OF ESSENTIAL OILS

The GC-MS analyses were performed on a gas chromatograph HP 5890 (II) interfaced with an HP 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) with

ANALYSIS OF ESSENTIAL OILS (VOLATILE EXTRACTS)

GC-FID and GC-MS analysis of oil constituents

electron ionization (EI) 70 eV. An HP-5MS capillary column (60 m × 0.25 mm, 0.25 μm film thickness) was used. The column temperature was programmed the same as the GC-FID method. The carrier gas was He with a flow rate of 1.2 mL/min. Scan time and mass range were 1 s and 50-550 m/z, respectively. The injected volume was 1 μL, and the total run time was approximately 63 min. Identification was based on a comparison of peak retention indices relative to (C 8 -C 22 (Sigma Chemical, St. Louis, USA)) n-alkanes with literature data or authentic compounds available in our laboratory. Further identification was confirmed by matching recorded spectra with Wiley/NBS mass spectral library and other published mass spectra [157]. Quantitative data were obtained from the electronic integration of the FID peak areas. Some entire fresh leaves of C. grandiflora weighting about 500 mg were placed in the cell of the FAD, thermostated from 8 to 25°C. N 2 O and CO 2 gas cartridges were used for the explosion of the vesicles of storage of essential oil and the release of the main volatile compounds of the plant in atmosphere (Fig. 2.3). Volatiles emitted after explosion were absorbed during 10 min. using a SPME syringe equipped with a Carboxen/PDMS/DVB fibre (Supelco, Bellefonte, USA). In order, the temperature and pressure were controlled. For each sample three replicates were performed.

Figure 2.3. Instrumental Flash Aroma Dispenser system

Some entire fresh leaves of C. grandiflora weighting about 400 mg were placed in the cell of the CFD; piston was used for the explosion of the vesicles storage of essential oil, and the release the main volatile compounds of the plant in atmosphere (Fig. 2.4). Power changes and number of crushing were controlled for the optimization. Volatiles emitted after crushing were absorbed during 10 min. using a SPME syringe equipped with a Carboxen/PDMS/DVB fibre. For each sample three replicates were performed. A single-factor-at-a-time method studies the phenomenon by varying one factor while keeping all other conditions constant. However, the effect of each factor is not necessarily additive. It is, therefore, necessary to take into account the influence of each factor and the interaction between these factors which may be synergistic or antagonistic [158]. The most influential factors on the characteristics of volatiles compounds are temperature (U 1 ) and pressure (U 2 ) in FAD method, and power changes (U 1 ) and number of crushing (U 2 ) in CFD method, respectively. The statistical significance of the model was determined by the application of Fisher's F test. A Doehlert matrix was used to represent the responses studied in all experimental regions of these factors (temperature, pressure, power changes and number of crushing) [159]. The factors are given in the form of coded variables (X) with no units in order to permit comparison of factors of different natures. The transformation of natural variables (U) into coded corresponding variables (X) is made on the basis of the following equation:

DIRECT EVALUATION OF VOLATILE COMPOUNDS IN FRESH PLANTS LEAVES

Design of Flash Aroma Dispenser (FAD) method 2.5.2 Crushing Finger Device (CFD) method

; ) ( 0 j j ij ij U U U X    (1)
where: X ij is the value of coded variable j in experiment i; U ij is the value of natural variable j in experiment i; U j is the value of natural variable j in the center of the domain of interest; it corresponds to X j =0; ∆U j is the variation of the natural variable j corresponding to a variation of the coded variable j equal to +1.

Doehlert's experimental matrix is given in Table 2.2.

This combination is chosen in such a way that the factors are not correlated with each other and that modelling can be done with uniform accuracy over the whole parameter space [160]. This analysis was achieved by response surface methodology which gave satisfactory results. Each response (Y) can be described by a second order model adequate for predicting the responses in all experimental regions:

2 1 12 2 2 22 2 2 11 2 2 1 1 0 X X a X a X a X a X a a Y       ; (2)
where: X 1, X 2 are the coded variables related to the natural variables U 1 , U 2, ; a 0 is the intercept term, a constant that corresponds to the response when X i is zero for each factor; a 1 determines the influence of X 1 ; a 2 determines the influence of X 2 ; a 12 is the interaction effect between the X 1 and X 2 ; a 11 , and a 22 can be regarded as a curve 'shape' parameter.

Methodology of experimental design for FAD and CFD techniques

For each response (Y ), the coefficients (a i , a ij ) of the postulated model were calculated on the basis of the experimental responses by least squares regression using the NEMROD software (New Efficient Methodology for Research using Optimal Design) [161]. To confirm the presence of essential oil on leaves, flower, and stem, anatomical study has been used (free hand sections). This method allowed examining the samples in a few min. It is also suitable for a variety of plant materials, such as soft herbaceous stems and small woody twigs [162]. Fresh tissues of leaves, stem, and flower were harvested, and free finest hand section was prefomed, and placed for one min in the Gazet du Châtelier reagent [163].

Then the sections were mounted on clean glass slide with a help of glycerin-water (50/50, v/v), and covered by a glass cover slip. Then the slides were observed directly on a light microscope (Leica DM-LB, magnification 100-400; Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) and photographed using a digital camera (Canon Power Shot S40; Canon, New York, U.S.A).

Radical scavenging capacity (RSC) of plant extracts against stable 2.2-diphenyl-2picrylhydrazyl hydrate (DPPH • ) was determined spectrophotometrically by a slightly modified method of Brand-Williams and co-workers [164]. The solution of DPPH • in methanol (6.510 -5 M) was prepared daily before measurements on a UV/visible light spectrophotometer Spectronic Genesys 8 (Thermo Spectronic, Rochester, NY) at 515 nm. Extract solutions were prepared by dissolving dry extract in methanol. Two mL of this solution were mixed with 50 μL of different concentration (3, 2.5, 2, 1.5 and 1 mg/mL) extract solution in 1 cm path length disposable microcuvette. The samples were kept in the dark for 30 min at room temperature and then the decrease in absorbance was measured. The absorbance of a blank sample containing the same amount of methanol and DPPH • solution was prepared and measured daily. The experiment was carried out in triplicate. The RSC was calculated by the following formula:

SAMPLE PREPARATION (FREE HAND SECTIONING METHOD)

ASSESSMENT OF NON-VOLATILE EXTRACTS AND ANTIOXIDANT ACTIVITY

DPPH • scavenging assay

% 100     B A B A A A (3)
where: I is DPPH • inhibition, %; A B is the absorbance of a blank sample (t=0 min); A A is the absorbance of extract solution (t=30 min).

The amount of extract required to decrease the initial DPPH • concentration in the reaction by 50% is referred as an effective concentration; the lower the IC 50 the higher radical scavenging effect.

The RSC of extracts was also measured by ABTS •+ assay [165]. Stock solution of (2 mM) ABTS was prepared by dissolving in 50 mL of phosphate buffered saline (PBS) obtained by dissolving 8.18 g NaCl, 0.27 g KH 2 PO 4 , 1.42 g Na 2 HPO 4 and 0.15 g KCl in 1 L of ultra-pure water. If the pH was lower than 7.4, it was adjusted with NaOH. Ultra-pure water was used to prepare 70 mM solution of K 2 S 2 O 8 . ABTS •+ was produced by reacting 50 mL of ABTS stock solution with 200 μL of K 2 S 2 O 8 solution and allowing the mixture to stand in the dark at room temperature for 15-16 h before use. The radical was stable in this form for more than 2 days when stored in the dark at room temperature. For the assessment of extracts, the ABTS •+ solution was diluted with PBS to obtain the absorbance of 0.800±0.030 at 734 nm. Three mL of ABTS •+ solution were mixed with 30 μL ethanol solution of leaf extract in 1 cm path length microcuvette. The absorbance was read at ambient temperature after 1, 4, 6 and 10 min. PBS solution was used as a blank sample. All determinations were performed in triplicate. The percentage decrease of the absorbance at 734 nm was calculated by formula:

% 100 I    C C D A A A ( 4
)
where: I is ABTS •+ inhibition, %; A D is the absorption of blank sample (t=0 min); A C is the absorption of extract solution (t=10 min).

The RSC was expressed as a percentage of inhibition of Trolox (6-hydroxy-2.5.7.8tetramethylchroman-2-carboxylic acid), mM equivalent per 1000 g using prepared calibration curve. The extent of quenching of ABTS •+ by extract was compared with standard amounts of Trolox. The concentrations of Trolox standard used for calibration curve were 0.001, 0.01, 0.1, 0.5, 1, 1.5 mM.

The ability of plant extracts to reduce ferric ion to the ferrous one, (FRAP assay) is another indicator frequently used for assessing antioxidant power [166]. Ferrous ion (Fe 2+ ) produced in this assay forms a blue complex (Fe 2+ /TPTZ) absorbing at 593 nm. Briefly, the reagent was prepared by mixing acetate buffer (300 mM, pH 3.6), a solution of 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM HCl, and 20 mM FeCl 3 • 6H 2 O at 10:1:1 (v/v/v). Firstly 300 μL of freshly prepared FRAP reagent was heated to 37°C and an absorbance (A 0 ) of a blank reagent was read at 593 nm in a Biotek EL808 microplate reader (Vermont, USA). Then 10 μL of 0.1% extract solution in water and 30 μL H 2 O were added (final dilution of

ABTS •+ decolourisation assay 2.7.3 Ferric reducing/antioxidant power (FRAP) assay

samples in the reaction mixture was 1:34) and the absorbance (A) was recorded every 1 min during the whole monitoring period which lasted up to 30 min. The change in the absorbance (ΔA593 nm) between the final reading and A 0 was calculated for each sample and related to the ΔA593 nm of a Fe 2+ reference solution which was measured simultaneously.

The content of TPC in extracts was determined with Folin-Ciocalteu reagent [167]. Calibration curve was prepared by using 1 mL reference gallic acid solutions in ethanol (0.025, 0.075, 0.100, 0.175 and 0.350 mg/mL), which were mixed with 5 mL of a standard Folin-Ciocalteu reagent and diluted with distilled water (1:10) and 4 mL of 7.5% sodium carbonate solution in distilled water. The absorption was read after 30 min at 765 nm. The concentration of TPC was expressed in mg of gallic acid equivalents (GAE) per g of plant extract. The TPC was calculated by the following formula:

; m V c C   (5)
where: C is concentration of the total phenolics, in gallic equivalents (GAE); c is the concentration of gallic acid, determined form the calibration curve (mg/mL); V is the volume of plant extract (100 mL); m is the weight of pure plant extract (g).

The content of flavonoids was measured using rutin as a reference [168]. One mL of plant extract solution in 95% ethanol (10 g/L) and 1 mL of aluminium trichloride solution in 95% ethanol (20 g/L) were pipetted into a 25 mL volumetric flask and made up with 95% ethanol. The absorbance was read at 415 nm after 40 min at 20°C. Blank samples were prepared from the mixture of 1 mL of plant extract and 1 drop of diluted acetic acid. The absorbance of a reference solution, which was prepared by using 1 mL of rutin solution instead of plant extract, was read simultaneously. Rutin solution was prepared from 0.05 g of dried at 130-150°C for 3 h rutin, which was diluted in 100 mL of 95% ethanol. All determinations were performed in triplicate. The concentration of flavonoids in plant extracts was calculated by formula:

; 10 0 0 m A m A X     (6)
where: X is concentration of the total flavonoids in rutin equivalents (RE); A is the absorbance of extract; A 0 is the absorbance of rutin; m is the weight of pure extract (g); m 0 is the weight of rutin (g).

The content of flavonols was determined by the previously reported method [168]. Series of reference rutin solutions containing 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, and 0.5 mg/mL of rutin were prepared. Two milliliters of such reference were mixed with 2 mL of aluminum trichloride solution (20 g/L) in 95% ethanol and 6 mL of sodium acetate solution in ethanol (50 g/L) were added. The absorbance was read at 440 nm after 2.5 h at 20°C and the calibration curve on the dependence of the absorbency on the concentration of rutin was drawn. Plant extract samples were prepared under the same conditions by using 2 mL of 2.7.4 Analysis of total phenolic compounds (TPC) 2.7.5 Determination of flavonoids 2.7.6 Determination of flavonols extract (10 g/L) in 95% ethanol instead of rutin. All determinations were performed in triplicate. The concentration of flavonols was calculated by the formula:

; m V C Y   (7)
where: Y is concentration of the total flavonols in rutin equivalents (RE); C is the concentration of rutin, determined form the calibration curve (mg/mL); V is the volume of plant extract (mL); m is the weight of pure plant extract (g).

The samples were prepared by mixing commercial refined sunflower oil "Vilnius" with 0.05, 0.1 and 0.2% of extracts. Five g of oil were placed in a reaction vessel and thermostated at 110°C under oxygen atmosphere (5 bars) in Oxipres apparatus (Mikrolab, Aarhus, Denmark). Pressure changes which occur due to the absorption of oxygen consumed for oil oxidation were recorded. The protection factor (PF) value of sunflower oil in case of using plants extracts and their antioxidant activity (AA) were calculated by the following formulas:

; K X IP IP PF  (8) . K BHT K X IP IP IP IP AA    ; (9)
where: IP X is induction period of sample with additive (h); IP K is induction period of sample without additive (h); IP BHT is induction period of sample with synthetic antioxidant BHT (h).

The extracts were analysed on a HPLC/UV system supplemented with DPPH • scavenging detector. In this system two chromatograms were recorded simultaneously, one of which representing UV absorbance of effluent at 265 nm prior to the reaction, while a mirror chromatogram was obtained recording absorbance at 517 nm after reaction of effluent with DPPH • solution in the reaction coil. Mobile phase was supplied to the column by a model 9012 HPLC gradient pump (Varian. USA) at flow rate 0.75 mL/min. The samples of 20 μL were injected into the HPLC system by means of Cheminert C1 injector (Valco Instruments, USA). Reverse-phase LiChroSpher RP-18e 5 μm 12.5 × 0.4 cm column and 0.5 × 0.4 cm precolumn (Merck, Germany) were used for separation. The DPPH • reagent was prepared by dissolving 0.01 M DPPH • in 0.1 M sodium citrate buffer (pH=7.6), methanol and acetonitrile (50:25:25 v/v). It was continuously supplied into a reaction coil (3 m of 0.25 mm ID fusedsilica capillary) by a model 2200 HPLC pump (Bischoff, Germany) at a flow rate of 0.75 mL/min. Signals were acquired at 265 and 517 nm wavelengths by means of Linear 206 PHD and Linear UVIS 200 UV-VIS detectors, respectively. Solution A (bidistilled water with 0.05% TFA) and B (methanol with 0.05% TFA) were used as the mobile phase components for gradient elution. Extracts were separated using the following gradient: 10% of B at 0 min, 25% of B at 5 min, 40% of B at 25 min, 95% of B at 40 min, 95% of B at 43 min and 10% of B at 44 min. Clarity chromatography software (DataApex. Czech Republic) was used for data acquisition.

Measurement of the inhibition effect of plant extracts on oil oxidation in

Oxipres apparatus

On-line HPLC-DPPH assay

LC system consisted of a Thermo-Fisher Spectra System (TFSP, San Jose, CA) P1000XR pump, a TFSP 6000LP Photodiode Array Detector and a TFSP AS 3000 autosampler. Separation of compounds was performed on a Varian Pursuit XRs 5 C18 column (250 mm × 4.6 mm ID, 5 μm) using a linear gradient of 0-30 min, 3%-97% of acetonitrile (A) to bidistilled water with 0.1% TFA (v/v) (B), 30-35 min, 20%-80% of A to B, 35-45 min, 3%-97% of A to B. Flow rate was 1 mL/min and 10 μL of the sample was injected.

Mass spectra were acquired using a Thermo-Fisher LCQ mass spectrometer (Finnigan MAT, San Jose, CA) equipped with an atmospheric pressure chemical ionization source (APCI) using both positive and negative ion mode. The APCI source operating parameters were as follows: the capillary and APCI vaporizer temperatures were set to 250°C and 450°C, respectively, and the spray was stabilized with nitrogen sheath and auxiliary gas (80 and 25 arbitrary units, respectively). Discharge current was 5 μA and capillary voltage was +15 V and -15 V in the positive and negative ion mode, respectively. The mass spectra were acquired in the data dependent mode with wideband activation (i.e. the most intense ion obtained for each scan in the full mass spectrum is further submitted to 38% collision energy for MS/MS).

The values are expressed as a means of 3 replicates with standard deviations (SD). RSC tests and content of TPC, flavonoids and flavonols were calculated using MS Excel software (CORREL statistical function). Statistical analysis of the data was performed by using one-way analysis of the variance (ANOVA), followed by the Duncans' post hoc test to compare the means that showed significant variation (p < 0.05). All the data were subjected to variance analysis using the GLM procedure of SAS [169]. Analyses were performed using MSStat (ANALYT MTC, Muehlheim, Germany, MS Statistical Software Version 3.02u); the software was looking for differences in the plant origins.

HPLC and LC-MS analysis

STATISTICAL ANALYSIS

RESULTS AND DISCUSSION

CHAPITRE

Chemical composition of aromatic and medicinal plants depends on cultivation area, climatic conditions, vegetation phase, genetic modifications and other factors; therefore characterization of flora present in different growing sites, countries and geographical zones is an important task. Factors that determine the composition and the yield of essential oil are numerous and in some instances it is difficult to segregate these factors from each other, since many are interdependent and influence one another [START_REF] Anwar | Changes in composition and antioxidant and antimicrobial activities of essential oil of fennel (Foeniculum vulgare Mill.) fruit at different stages of maturity[END_REF]. These variables may include seasonal and maturity variation, geographical origin, genetic variation, growth stages, part of plant and post-harvest drying, and storage [START_REF] Burt | Essential oils: their antibacterial properties and potential applications in foods-a review[END_REF][START_REF] Anwar | Changes in composition and antioxidant and antimicrobial activities of essential oil of fennel (Foeniculum vulgare Mill.) fruit at different stages of maturity[END_REF]. There are many reports in the literature showing the variation in the yield and chemical composition of the essential oil with respect to geographical regions. For instance, variations in the yield and chemical profile of essential oils were reported for Rosmarinus officinalis and Tagetes minuta populations, collected from different geographical locations [START_REF] Celiktas | Antimicrobial activities of methanol extracts and essential oils of> Rosmarinus officinalis, depending on location and seasonal variations[END_REF][START_REF] Uribe-Hernández | The essential oil of Lippia graveolens HBK from Jalisco, Mexico[END_REF][START_REF] Souto-Bachiller | Terpenoid composition of Lippia dulcis[END_REF]. Such differences could be linked to the soil textures and possible adaption response of different populations, resulting in different chemical products being formed, without morphological differences being observed in the plants [START_REF] Souto-Bachiller | Terpenoid composition of Lippia dulcis[END_REF].

The aims of this part of study were to examine the composition of M. odorata, T. farfara and C. grandiflora essential oils of two different origins and to compare to previously published results on these species.

The yields of the oils were 0.40±0.02% (Lithuania) and 0.50±0.01% (France) (w/w) which was in accordance with literature [140,142]. Chemical composition of the oils of two origins is presented in Table 3.1. Twenty and twenty-six components were detected in Lithuanian and French oils, respectively. Six volatile compounds, namely camphor, iso-menthone, cisthujone, pinocarveol, carvacrol and p-anisaldehyde which were identified in M. odorata French oil, were not detected in Lithuanian oil. Volatile constituents identified in the sweet cicely belong to the following classes of organic compounds: phenylpropanoids, terpenoids, alcohols and aldehydes.

INVESTIGATION OF AROMATIC PLANTS COLLECTED FROM TWO GEOGRAPHICAL LOCATIONS

Essential oil composition of Myrrhis odorata (L.) Scop. leaves grown in Lithuania and France

Phenylpropanoids were the most abundant components in M. odorata leaves constituting 63.3±0.7%-68.2±0.6% of total oil, followed by terpenes (33.3±0.2%-29.3±0.1%). Hydrocarbon monoterpenes constituted 3.7±0.1%-4.1±0.1%; while oxygenated monoterpenes (1.2±0.1%) were detected only in French origin plant. Higher percentage of hydrocarbon sesquiterpenes was determined in M. odorata from Lithuania (16.9±0.1%), than in French origin oil (11.7±0.1%), while the content of oxygenated sesquiterpenes was almost similar, 12.3±0.4% and 12.7±0.3%, respectively. The essential oil of sweet cicely contained higher content of hydrocarbon sesquiterpenes than hydrocarbon monoterpenes. It is known that the parent sesquiterpenes are biosynthesized from geranyl diphosphate by its condensation with isopentenyl diphosphate. It can be suggested that monoterpenes are consumed by the plants faster at late vegetation phases as compared to sesquiterpenes. The major components in both M. odorata oils were E-anethole, methyleugenol, E-nerolidol, and the differences in their percentages were not remarkable. The odor of these compounds were preliminary assessed and is described as sweet, green, anethole herbal anise, licorice (E-anethole), clove, spicy (methyleugenol) and floral, green, waxy, citrus, woody (E-nerolidol), and they are found in such important spices as Illicium verum, Foeniculum vulgare, Piper claussenianum [170][171][172]. From this point of view M. odorata from France and Lithuania may be preliminary assigned to the same E-anethole, methyleugenol and E-nerolidol chemotype. However, some qualitative and significant quantitative differences in oil composition of two remote origins may be observed (Table 3.1). Cis-thujone, camphor, iso-menthone, pinocarveol, carvacrol and p-anisaldehyde were detected only in M. odorata oil of French origin; the content of estragol in it was more than 4 times higher than in the oil of Lithuanian origin plant material. The odor of estragol is described as anise, licorice so the French oil had a stronger anise, licorice flavor than Lithuanian essential oil. On the other hand, the percentage of βcaryophyllene, germacrene-D, spathulenol and limonene was significantly higher in the oil from Lithuania and this oil had fresh, sweet, woody, spice, clove dry, earthy, herbal, fruity odor. Comparison of the percentages of the main components in M. odorata oils analyzed in our study and previously reported in literature are summarized in Table 3.2. E-Anethole was dominating constituent (82-85%) in M. odorata oils from Finland [137], Russia [139] and North USA [140], and it may be assumed that the plants from these countries were purely Eanethole chemotype. It should be noted that the plants from North USA contained reasonable amount of methyleugenol, which was not found in Finnish and Russian origin sweet cicely, while E-nerolidol was not detected in Russian and Serbian chemotypes. Completely different M. odorata chemotype was reported in Serbia (Prokletije region) with p-cymene as the major compound constituting 62.0% in the total oil [141]. This variation in component level it is surprising, but not impossible because chemical polymorphism is significantly characteristic of many Umbelliferae family species [173]. The oil reported from Serbia may be would not have the typical sweet, anise, licorice odor of M. odorata because p-cymene had a stronger fresh, citrus, terpene, woody, spice smell. These differences may be dependent on climatic conditions prevailing during plant development and their genetic peculiarities. Indeed, the composition of essential oils as well as other chemical molecules of plants may vary considerably depending on annual climatic changes and genotype [146,174]. These parameters influence the total content of essential oils and their composition. Unfortunately, weather conditions were not reported in studies performed in Finland, Serbia and USA.

The present study provides new information about essential oil chemical composition of M. odorata from two geographical origins (Lithuania and France), not studied before. The percentage of the main oil compounds in the studied M. odorata collected in remote habitats was quite similar; however comparison of the obtained data with previously studied M. odorata oils revealed that there are different plant chemotypes. Therefore, further investigations are necessary to ascertain our results by studying a collection of accessions from different geographic regions cultivated under same climatic conditions. This could allow identifying the genetic potential which could help for improvement M. odorata breeding programs and diversification of it industrial uses.

The yield of T. farfara flower essential oil was 0.07±0.01% (Lithuania) and 0.09±0.00% (France) (w/w). A close essential oil yield 0.10% (w/w) from T. farfara buds grown in China was obtained by Liu et al. [148].

Essential oil composition of Tussilago farfara L) from Lithuania and France

However, in this study aerial biomass (flower and peduncle) was used. This result highlighted the possibility and the interest to used whole plant for essential oil extraction instead of buds. Moreover, European Food Safety Authority (EFSA) have addressed in 2009 an opinion to the scientific substantiation of health claims in relation to T. farfara and normal function of the upper respiratory tract and "immune health" [175]. The report of EFSA have provided information on the conditions uses of flower and leaf of T. farfara plant as infusion, powder, tincture and fluid extract. However, no information was provided on the conditions of use of stem part. In order to prove the potential interest of the peduncle as provider of essential oil, we performed anatomical study. The result emphasized clearly the presence of glandular trachoma in stem of T. farfara similar to those showed in flowers (Fig. 3.1) which can be a useful organ for essential oil supply. This result reinforces the fact of using the aerial part (flower and peduncle) of T. farfara plant for essential oil production. It should be noted that up to date, no sufficient scientific information on T. farfara specie was conducted in literature as noted in EFSA report [175]. Moreover, stem represented an important percentage of the aerial part (50±3.5%) of plant dry matter; while those of the bud and flower were two times lower (28.6±2.4% and 21.4±3.7%, respectively). Accordingly it could be suggested the use of peduncle as potential and additional part of T. farfara specie for essential oil supply. GC-MS analysis exhibited 27 and 22 components were detected in Lithuanian and French oil, respectively, accounting for 62.2±0.1% and 53±0.5% of the total intedgarted GC area. Chemical composition of T. farfara essential oil previously studied from different origin and reported in several works is presented in Table 3 The content of 1-nonene in French oil was four times higher than in the Lithuanian oil. On the other hand, the percentage of α-phellandrene, αpinene, p-cymene and 1-dodecene was higher in the French oil, than in Lithuanian oil (Table 3.3). It is important to note that the climatic conditions from two studied areas countries are clearly different; mean temperature and rainfall amount during the month of collection were higher in Midi-Pyrenées-France (10.5°C and 97.1 mm) than in Kaunas-Lithuania (6.9°C and 34.0 mm).

Comparison of the percentages of main components in T. farfara oils analyzed in our study and those previously reported in literature are summarized in Table 3.4. Comparing T. farfara oil of Lithuania origin analyzed in our study and that previously reported by Judzentienė and Budienė [149] it may be observed that it have roughly the same chemical profile with n-tricosane (22.7-21.7%) as a major compound. However, 1nonene (8.1±0.3%) was the second major compound in our study, while in previous studies this compound was absent. Also n-undecane and npentacosane were found at the concentrations of 7.8±0.2% and 1.3±0.0% respectively; however their percentage was different comparing to published data [149] reporting n-undecane and n-pentacosane 3.9% and 9.4%, respectively. In addition, the organs and the collection area of T. farfara plant investigated in our study (flower-stem from Kaunas area) were not the same as those studied by Judzentienė and Budienė (flower or stem from Vilnius and Kėdainiai area) [149]. On the other hand, significant differences were found between the main constituents of T. farfara oils studied in this work and reported by Liu et al.

[148] and Hãdãrugã et al. [176] from China and Romania origin, respectively. Chinese T. farfara buds oil contained reasonable amount of Ecycloundecene (8.5%), which was not found in studied oils from Lithuania and France. Moreover, β-bisabolene was the major constituent (13.9%) in Chinese essential oil, which does not exceed 0.7% and 1.2% in French and Lithuanian oil, respectively. Romanian T. farfara leaf oil contained two main compounds, α-bisabolol (20.7%) and chamazulene (7.2%), which were not found in Lithuanian and French oils. These significant differences can be related to climatic conditions or studied part of plant. Also, genotype and chemotype may be important factors responsible for this difference [146,174] Together with other factors such as climatic conditions, soil characteristics, harvesting time.

The present study provides new information about flower and stem essential oil chemical composition of T. farfara from two geographical origins. Microscopy analysis revealed that stem contain secretory pockets (glandular trichoma) which can be a useful organ for essential oil supply which reinforce the fact of using all the aerial part of T. farfara plant for essential oil production. Also, these new data can be used for the assessment of plant taxonomy. The percentage of the main oil compounds in the studied T. farfara collected in remote habitats was significantly different; however comparison of the obtained data with previously studied T. farfara oils revealed that there are different plant parts. Therefore, further investigations are necessary to advance our results by studying a collection of accessions from different geographic regions cultivated under same climatic conditions. This could allow identifying the genotypic difference which could help for improvement T. farfara breeding programs and diversification of it industrial uses. Also, for an optimal valorization of aerial parts of T. farfara plant, it is useful to investigate the chemical composition and the yield of essential oil of each plant part (flower, stem, bud, leaf) taken separately from the same origin.

Experimental design (Doehlert matrix) has been used to optimize the experimental conditions for the CFD and FAD analysis. Concerning the design analysis, two-factor model was selected for each response Y. The Doehlert matrix consists of N experiments with N=K 2 +K +1, where K is the number of variables studied. In this case K=2 and, therefore, the matrix was comprised of seven experiments (Table 3.5 and 3.6). These 7 points are scattered uniformly in the space of the coded variables (X i ) and are located in the corners of a hexagonal polyhedron. The responses studied were the number of volatile organic compounds (VOCs) using two new CFD and FAD methods (Y 1 , Y 2 ). Following the model

RESPONSE ANALYSIS AND INTERPRETATION OF CFD AND FAD DATA

established for response, we can represent graphically the surface of the corresponding responses. The values of the coefficients provide information on the influence of the factors and the curves obtained show the extreme, or at least privileged, directions (zones of interest of the factors). The main objective of the optimization was to determine the optimal conditions of power and number of crushing for the new CFD method and for FAD method was to determine temperature and pressure. So far as these two new methods are intended to be used in a rapid analysis of medicinal and aromatic plants, it was necessary to establish the domains of variation of response (the number of VOCs) in relation to the required characteristics.

From an economical point of view, the total number of VOCs (Y 1 , Y 2 ) is a positive parameter. The total VOCs measured using CFD and FAD methods is an important value, because the number of VOCs is very similar compared with essential oil obtained by hydrodistillation. The number of VOCs (Y 1 ) in CFD analysis is shown in Fig. 3.2. These results indicate that the number of VOCs is very low at low power (around 0.6 W) and high at high power (around 4.6 W) and high number of crushing. The effect of power on these characteristics is noticeable only at high number of crushing, especially in the case of number of VOCs. Thus, at power below 0.6 W the number of crushing did not affect the capacity of the VOCs, whereas at high number of crushing, a small increase in power brings about a major increase in a number of VOCs.

The optimal point using CFD indicated by the model corresponds to a power of 2.6 W and at number of crushing 10 (Fig. 3.2). The response values of number of VOCs calculated from the model for these experimental conditions are given in Table 3.6.

Fig. 3.2. Variation of the number of VOCs (Y 1 ) versus (X 1 , X 2 ) in CFD method

The optimum conditions are schematized as an ellipse. The center represents the optimum conditions in terms of power and number of crushing. The maximal efficiency is obtained for power and number of crushing at the 0 points in coded variable, i.e., 2.6 W and 10 number of crushing in real variable. The analysis of the isoresponse curves at the chosen experimental field delimited by a circle show that the maximum number of VOCs was obtained when the power and the number of crushing are increased. According to these obtained results, the coefficients of the polynomial model were calculated using the NEMROD Software [161].

Number of VOCs =22 + 9.17X 1 + 4.33X 2 -11X 2 1 -10.67X 2 2 + 0.58X 1 X 2 .

Fig. 3.3. Variation of the number of VOCs (Y 1 , Y 2 ) versus (X 1 , X 2 ) in FAD method. A) -using FAD with CO 2 , B) -using FAD with N 2 O

Two types of gases, N 2 O and CO 2 (Y 1 , Y 2 ) were used to obtain the responses for FAD method. Following the model established for response, we can represent graphically the surface of the corresponding responses. The values of the coefficients provide information on the influence of the factors and the curves obtained show the extreme or at least privileged directions (zones of interest of the factors). The number of VOCs (Y 1 ) and (Y 2 ) analysis of this response are presented in Fig. 3.3. These results indicate that the number of VOCs is very low at low temperatures (around 8 ºC) and high at high temperatures (around 26 ºC). The effect of pressure on these characteristics is noticeable only at high temperatures, especially in the case of number of VOCs. Thus, at temperatures below 8 ºC the pressure did not affect the capacity of the VOCs, whereas at high temperatures, a small increase in pressure brings about a major increase in number of VOCs.

The optimal point using FAD with N 2 O indicated by the model corresponds to a temperature of 19 to 26 °C and a pressure of 9 to 12 bar (Fig. 3.3). The response values of number of VOCs calculated from the model for these experimental conditions are given in Table 3.6. The center represents the optimum conditions in terms of temperature and pressure. The maximal efficiency is obtained for temperature 0.2 and pressure 0.5 in coded variable, i.e., 19 ºC and 10 bar in real variable. The analysis of the isoresponse curves at the chosen experimental field delimited by a circle show that the maximum of number of VOCs was obtained when the temperature and the pressure are increased.

The optimal conditions using FAD with CO 2 is obtained for temperature (-0.09-1) and pressure (-0.07-1) in coded variable, i.e., 16-26 ºC and 6-12 bar in real variable. Using FAD method with N 2 O gas we obtained more volatile compounds, than in case of CO 2 (Table 3.5); consequently the release of VOCs in a new FAD method would be more efficient when N 2 O gas is used, because the gas is aggressively attacking the cuticle of the multicellular headed glandular trichomes of the leaf.

A decade ago, several attempts were performed by the companies of aromatic industry sector both in South-America and West-Africa in order to investigate the aroma potentials of rare exotic plants as a possible source of novel flavors and fragrances formulations. As Midi-Pyrenees region (Southwest of France) has a rich and poorly valorized wild vegetal heritage, especially regarding aromatic and medicinal plants, the studies were carried out in order to collect and analyze various regional so-called 'medieval' aromatic plants, e.g. Achillea millefolium, Myrrhis odorata. So far as many of such plants are rather rare or protected (their harvesting is problematic), the sampling of such plants becomes a limiting factor in their phytochemical investigations [177]. One of such plants is C. grandiflora, which can be

Rapid evaluation of volatile compounds in fresh plants leaves by the two novel techniques Crushing Finger Device (CFD) and Flash Aroma Dispenser (FAD)

found in France, whereas in Lithuania it is very rare. The quantities of the available plants material are very important for the comprehensive analysis of their volatile compounds and further valorization. Therefore, new methods (techniques) for the release and collection of volatile compounds from small amounts of raw material are quite important. The essential oil components are being secreted by glandular trichomes present in the leaves. Therefore, before the analysis of aromatic extracts of plants, they have to be extracted and concentrated using a number of different methods for that purpose such as traditional hydrodistillation, steam distillation and solvent extraction techniques and some new methods. However, these extraction methods usualy require comparatively large amounts of raw materials. In addition, monoterpenes are known to be vulnerable to chemical changes during steam distillation conditions, and even conventional solvent extraction is likely to involve the losses of more volatile compounds during removal of the solvent [178].

During extraction process essential oil storage vesicles were exploded due to temperature and mechanical factors and consecutively aromatic compounds could be more easily extracted by water vapor. Florists and farmers are simulating this treatment when they crush between their fingers a couple of leaves or flowers followed by the sniffing of the generated odor in order to perform a rapid evaluation of the aromatic potential of such plants. Air dried leaves are usually subjected to hydrodistillation or steam distillation for about 3-8 hours. The losses of volatile compounds, long extraction times, degradation of unsaturated compounds and toxic solvent residue in the extract may be encountered with the current extraction methods. Hydrodistillation can thermally degrade and hydrolyze some of the oil components, which in some cases can lead to significant distortion of the composition of the oils naturally found in the intact plants.

In order to avoid degradation of volatile compounds a simple extraction technique, called Crushing Finger Device (CFD) method has been developed. The method developed is easy to perform using common laboratory equipment. In this method the sample is being crushed with an artificial finger. The advatages of CFD in comparison with the more traditional approaches are its high efficiency, low energy requirements and no use of solvent or boiling water. Another simple extraction method, so called Flash Aroma Dispenser (FAD) uses two types of gas, N 2 O and CO 2 . A strategic approach based either on the use of a specific/high effective sampling system with a classic detection (GC or GC-MS) was developed or tested in this study. CFD and FAD methods may become an important alternative to the traditional extraction methods, since the above mentioned limitations can be overcome using this new techniques.

Previously published results indicate that the isolated and naturally present aromatic extracts may vary depending on their isolation and preparation method [179-181]. Therefore, it was interesting to compare the composition of C. grandiflora by using hydrodistillation and two new techniques, namely CFD and FAD.

The yield of C. grandiflora essential oil was 0.25±0.07%. It was reported previously that the yield of oil obtained by steam distillation from dried leaves of C. grandiflora was approximately 0.35% [134]. Table 3.7 lists the composition of C. grandiflora oil obtained by different methods according to the results of GC-MS. Twenty eight compounds in the C. grandiflora aromatic extracts have been identified, pulegone, isomenthone and neo-isomenthol being the main volatile components of C. grandiflora (Table 3.7). In general, the qualitative composition of C. grandiflora oils extracted by different methods is quite similar, whereas relative concentrations of the identified compounds are apparently different. Volatile constituents identified in the C. grandiflora belong to the terpenoid class of organic compounds. Hydrocarbon monoterpenes constituted in essential oil 1.34%; while oxygenated monoterpenes were dominating (87.05%). Remarkably higher percentage of hydrocarbon monoterpenes was determined in C. grandiflora oil isolated by FAD (22.25-39.96%) and CFD (21.28%) techniques, than in the hydrodistilled essential oil (1.34%), whereas the content of oxygenated monoterpenes was higher in essential oil (87.05%) than in the volatiles isolated by FAD and CFD; 72.77%, and 38.78-23.29%, respectively. The essential oil of C. grandiflora contained higher content of oxygenated monoterpenes than hydrocarbon monoterpenes, while the volatiles isolated using new FAD method contained almost similar amount of oxygenated monoterpenes and hydrocarbon monoterpenes, 23.28, 38.78% and 22.25, 39.96% respectively. Some qualitative and quantitative differences between hydrodistilled oils and the products isolated by FAD and CFD were observed. Four volatile compounds, hexanal, α-thujene, α-phellandrene and α-terpinene which were identified in the volatiles isolated by CFD and FAD techniques were not detected in C. grandiflora hydrodistilled essential oil. On the other hand, 8 volatile compounds which were present in the hydrodistilled oil, namely linalool, p-menth-8-en-3-ol, piperitone, isomenthyl acetate, santalene, bicyclogermacrene, isoeugenyl acetate, α-cadinol, were not detected in the oils obtained by CFD and FAD methods. The differences might be due to the water and the thermal effect during hydrodistillation, because water is a polar solvent, which accelerates many reactions, especially reactions via carbocations as intermediates; hydrodistillation is also more aggressive method (Fig. 3.4). Another possible reason might be too little amounts of these compounds for GC/MS detection, which were released in CFD and FAD devices at the used parameters.

Fig. 3.4. Hydrolysis of isopulegyl acetate

In general, based on the composition of hydrodistilled and FAD-CFDisolated oils the chemotype of analyzed C. grandiflora may be characterized by the oxygenated p-menthane skeleton monoterpenes, namely menthone, isomenthone, isomenthol, neo-isomenthol and pulegone. Isomenthone and pulegone were the major constituents in C. grandiflora oil. The same structures were most abundant in Greek and previously studied French origin plants, however their percentages were different; pulegone in Greek C. grandiflora constituted 35.2%, menthone 20.2% and isomenthone 15.2% [134], while in the oil from France the contents of pulegone, isomenthone, neo-isomenthone and menthol were 27.6%, 24.7%, 23.7% and 10.8%, respectively [133]. Completely different C. grandiflora chemotype was reported in Kütahya Domaniç-Daritepe region of Turkey; isopinocamphone was the major compound constituting 52.6% in the total oil [132]. It is not surprising because chemical polymorphism is characteristic for many Lamiaceae family species. For instance, 5 chemotypes were identified for another Lamiaceae family species Thymus pulegioides previously; some of these chemotypes produced completely different terpene compounds, such as geraniol/geranial/neral, thymol and linalool [182]. In addition, the composition of essential oils varies significantly depending on annual climatic changes; therefore, even within the same chemotype, the remarkable variations in essential oil composition are possible. Each of two techniques developed (CFD and FAD) can be coupled with Fast-GC or GC in order to identify different chemotypes, to identify the key aromas compounds and to control the quality of plants.

The investigation of the essential oils by direct sampling from secretory glands is of fundamental importance in studying the true essential oil composition of unharvested aromatic plants, since the usual applied techniques such as hydrodistillation and extraction are known to produce in some cases several artifacts. Essential oil is deposited on the surface of the plant inside fragile glandular trichomes. The plants emit volatiles from all tissues, although certain volatiles are evolved only from particular anatomical structures. Most volatile compounds are not emitted by all plant parts. Volatile emission rates are influenced by the resistance within the diffusive pathway from biosynthetic sites to the atmosphere. The physical path probably involves diffusion through cells, intercellular air spaces and passage through the stomata [183]. The secretion and subsequent volatilization of essential oils, which consists largely of terpene hydrocarbons, is generally associated with specialized secretory structures such as glandular trichomes of leaves.

In distillation processes, the fresh or dried aromatic plant material is contacted with vapour or boiling water, a process that results in the disruption of the glandular trichomes and, consequently, in the release of the volatile substances contained therein [184].

Fig. 3.5. Microscopic analysis of C. grandiflora. glandular trichome. A and C before the experiment. B after affecting with CFD and D after affecting with FAD

The CFD and FAD procedures studied in this work involve contacting a bed of intact herbaceous material with artificial finger and pressure with CO 2 or N 2 O, thus disrupting the glandular trichomes in which the essential oils are encapsulated. As a result, the extraction of volatile compounds with CFD and FAD is often preceded by a mechanical treatment of the matrix in order to rupture these glandular trichomes. Glandular trichome before and after this fast chrushing and pressure showed evidence of disruption due to a bursting action from within the interior of the glands (Fig. 3.5). Two different extraction methods (CFD and FAD) produce distinguishable physical changes in the plant material. Fig. 3.5 compares scanning optical micrographs of C. grandiflora. The structures of the untreated plant material can be compared with those of the material treated by CFD or FAD. The untreated C. grandilfora leaf shows glandular trichomes which contain volatile oils; amongst glandular trichomes, two types are recognizedunicellular and multicellular glandular trichomes. The capitate trichomes were situated on the adaxial and abaxial leaf surface. They consisted of a basal cell, a short mono or bicellular stalk and a rather large one or two celled secretory head. After CFD and FAD, cuticle and cell walls have been affected and it can be observed damages of the oil-containing glands. Glandular trichomes (Fig. 3.5 B and D) seem to be destroyed. The changes observed for CFD and FAD extractions showing clearly that the cells are broken and damaged during crushing and decompression treatment, but the mechanical strain induced by the rapid decompression in the surface tension of the glandular wall, causing it to crumble or rupture more readily, than pressure with CO 2 or N 2 O.

Valorization of plant residues after essential oil distillation is an important task. Dry herbal products are mainly composed of nondigestable carbohydrates and their derivatives incorporated into the cell walls, which in their native form are of a little value for food and/or nutraceutical applications. However, non-volatile secondary metabolites may be of interest for such applications and therefore one of the main tasks of this study was to assess antioxidant potential and the amount of polyphenolics in C. grandiflora, M. odorata and T. farfara hydrodistillation residue, which actually may be considered as some kind of "herbal soup"consisting of solid and liquid fractions. It should be said that in some cases deodorized plant extracts produced from the essential oil free material were reported as possessing higher antioxidant power than the extracts isolated from the whole material [START_REF] Povilaitytė | Antioxidative activity of purple peril (Perilla frutescens L.), moldavian dragonhead (Dracocephalum moldavica L.), and roman chamomile (Anthemis nobilis L.) extracts in rapeseed oil[END_REF]108]. The composition of hydrodistillation fractions will depend on the solubility of plant constituents in boiling water, which is a high polarity solvent. The results of RSC of extracts in DPPH • and ABTS •+ reactions as well as their reducing power in FRAP assay are listed in chapter 3.3.1, 3.3.2 and 3.3.3. The RSC measurements in DPPH • and ABTS •+ assays are easy, rapid and sensitive methods and therefore most frequently applied for the preliminary assessment of antioxidant potential of various natural substances. Although the basic principles are similar, the ABTS •+ scavenging assay is preferable for its ability to evaluate RSC of both lipophilic and hydrophilic antioxidants.

ANTIOXIDANT ACTIVITY OF PLANT HYDRODISTILLATION RESIDUES

The model reaction systems containing stable DPPH • , ABTS •+ and reducing power in FRAP were used to assess radical scavenging capacity (RSC) of deodorized C. grandiflora extracts. These methods are simple and widely used for the fast screening of plant antioxidant properties and they provide quite reliable preliminary information on the presence of antioxidatively active constituents in the extracts.

The concentration necessary to decrease the initial concentration of DPPH • by 50% (IC 50 ) was chosen to compare RSA of different extracts (Fig.

3.6).

A low IC 50 is the result of high radical scavenging effect. IC 50 values of C. grandiflora extracts (solid residue) in DPPH • assay varied from 1.10 (AE) to 0.23 (ME) mg/mL (Fig. 3.6), while TEAC values in ABTS •+ reaction were from 0.11 (AE) to 0.66% (EE) (Fig. 3.7). It is evident that lower polarity acetone (AE) extract was several times weaker radical scavenger than polar alcoholic (ME, EE) extract. It is in agreement with many previously published results which showed that polar solvents extract more antioxidants from botanicals than lower polarity solvents [START_REF] Povilaitytė | Antioxidative activity of purple peril (Perilla frutescens L.), moldavian dragonhead (Dracocephalum moldavica L.), and roman chamomile (Anthemis nobilis L.) extracts in rapeseed oil[END_REF]108,185]. Methanol and ethanol as organic solvents are quite similar, however, the EE extract was stronger radical scavenger than ME in ABTS •+ reaction. Moreover, ethanol is a food grade solvent and from this point of view is preferable to the toxic methanol. It is also interesting noting that in DPPH • reaction the RSC of alcoholic extracts obtained from the solid residue were quite similar to the RSC of water extracts, 0.23 (ME), 0.22 (EE), 0.22 (FD) and 0.18 (SD) mg/mL, respectively. Meaning that, some antioxidativey active compounds do not dissolve in boiling water and still remain in the solid residue. FRAP assay is a versatile method and can be readily applied to both aqueous, alcohol and acetone extracts of different plants. In this assay, the antioxidant activity is determined on the basis of the ability to reduce ferric (III) iron to ferrous (II) iron and the results are expressed in mg ferrous iron equivalents per mL of sample. In FRAP assay a ferric salt (Fe III) is used as an antioxidant and its redox potential (0.70 V) is comparable to that of ABTS •+ (0.68 V), therefore there is not much difference between TEAC assay and FRAP assay [186]. However, the results obtained by these two assays for C. grandiflora extracts were rather different, which shows that the assay type plays a most important role in antioxidant activity measurements. The ferric reducing power of different extracts was in the range of 0.24-0.68 mg Fe (II)/mL (Fig. 3.6) and the most remarkable difference was observed for the AE, which was several times weaker antioxidant in TEAC assay (Fig. 3.7), while in FRAP assay AE was the strongest reducing agent. ME and AE produced from the solid hydrodistillation residue of plant were remarkably stronger antioxidants than dried water extracts. The observed differences between DPPH • /ABTS •+ and FRAP methods can be explained by different solvent polarities and pH in the reaction media [187].

Antioxidant properties of deodorized residues extracts of C. grandiflora in electron/hydrogen transfer based assays

Fig. 3.7. TEAC values in ABTS radical cation decolourisation assay of C. grandiflora

The soil, climate and altitude may be affected on the antioxidant activity; therefore the aim of this study was to determine the dependence of antioxidants properties and plant origin.

Antioxidant analysis shows that all extract plant residues samples were antioxidatively active; however, their RSC varied in a wide range (Fig. 3.8). The RSC of French (FR) origin extracts was from 0.12±0.00 to 0.83±0.08 mg/mL in DPPH • test and from 0.02±0.00 to 0.79±0.07 mg/mL in FRAP reaction system, while that of Lithuanian (LT) plant extracts varied from 0.11±0.00 to 0.98±0.08 mg/mL in DPPH • test and from 0.14±0.01 to 0.99±0.02 mg/mL in FRAP reaction system (Fig. 3.8).

In DPPH • test, M. odorata AE and ME extracts from FR grown plants shows little better antioxydant activity than those of the same botanical source but from different geographical origin (LT), while FRAP test show that LT ME, EE and WE extracts have better antioxidant activity compared to those from FR country. Based on the result obtained from DPPH • analysis, it is important to notice that there is not significant similitude between the same plant from different climate and country (LT and FR), but only important The previous data demonstrated that measurement of RSC in model system is a reliable method to assess antioxidant properties of plant extracts. However, it is known that antioxidants act very differently with various free radicals. In order to obtain more information about radical scavenging properties of M. odorata extracts, we introduced the second model system (ABTS •+ ) to test RSC. ABTS radical cation decolourisation assay, showed similar results compared to those obtained in DPPH • reaction. EE and ME extracts were stronger radical scavengers than AE extract in the ABTS •+ assay. The concentrations of extracts were equivalent to the activity of 1mmol of Trolox solution in ABTS •+ reaction are given in Fig. 3.9. Although some pure compounds such as a number of phenolics, mucopolysaccharides and water-soluble polysaccharides have been isolated from the flower buds of T. farfara [151], no flavonoids have been reported. So far, there are no studies performed on the T. farfara residues after plant hydrodistillation. However, these deodorized residues may contain a panel of bioactive molecules, previously not determined and therefore not used. Recent developments in green chemistry and speculation on plant material for industrial uses led to exploit plant residues after extraction and transformation process. Latest studies have proved that some species may contain a lot of bioactive molecules such as antioxidants [188]. There is no information on antioxidant activities in T. farfara deodorized residues and different anatomical parts of this plant. Therefore, it was interesting to characterize the antioxidant activities of T. farfara deodorized residues obtained after flowers-stems (FS) hydrodistillation and extracts of leaves (L) and roots (R) for inexpensive exploitation. The antioxidant activity in different anatomical parts, FS, L and R of T. farfara plants of two different origins were analyzed in our work.

The IC 50 values for DPPH radical scavenging activity of T. farfara anatomical parts were the lowest for FR R AE extract (0.88 mg/mL), and LT R AE extract (0.54 mg/mL), the highest antioxidant activity were in FR L AE extract (0.05 mg/g) (Fig. 3.10). [START_REF] Povilaitytė | Antioxidative activity of purple peril (Perilla frutescens L.), moldavian dragonhead (Dracocephalum moldavica L.), and roman chamomile (Anthemis nobilis L.) extracts in rapeseed oil[END_REF]108,185]. In contrast, another antioxidant activity screening method, applicable for both lipophilic and hydrophilic antioxidants ABTS •+ decolourisation assay, exhibited rather inverse range compared to those obtained in DPPH • reaction (Fig. 3.11). For TEAC values in ABTS •+ reaction AE (FS) and roots (R) extracts were the most effective ABTS radical scavengers, than FS ME, R ME and FS EE, but R EE extract showed higher activity, than R AE. The main difference is that DPPH • can be only dissolved in organic solvents (e.g., methanol), while ABTS •+ is soluble in both aqueous and organic media. Therefore, the ABTS •+ test can be performed in hydrophilic and lipophilic systems [165].

Antioxidant activity in different anatomic parts of T. farfara from two different geographical origins

The ferric reducing power (FRAP) assay of the different parts of the plant extracts was in the range of 0.18-0.77 mg Fe (II)/mL in LT extracts and 0.15-0.68 mg Fe (II)/mL in FRh extracts. Antioxidant activity of FS extracts measured by FRAP reaction showed the same relationships as did ABTS •+ . In FRAP assay a ferric salt (Fe III) is used as an antioxidant and its redox potential is comparable to that of ABTS •+ , therefore the results of TEAC and FRAP assays showed similar trends as previously reported [186]. FS AE extracts were remarkably stronger antioxidants than other (FS ME and FS EE) extracts in ABTS •+ and FRAP reaction, but for leaves extracts relationships between ABTS •+ and FRAP did not find, LT L EE showed higher activity in ABTS •+ and lowest in FRAP reaction, the observed difference can by explained by different pH in the reaction media (pH FRAP =3.6 and pH ABTS =7.4).

Fig. 3.11. TEAC values in ABTS radical cation decolourisation assay of different anatomical parts of T. farfara from Lithuania and France

Comparing antioxidant activity indicators we observed that the T. farfara extracts isolated from LT and FR origin were different. The differences in climatic conditions (the amount of rainfall during plant development was nearly three times less abundant in Lithuania (340 mm) than in France (971 mm), the average temperature was also lower in Lithuania and it might have the impact on these differences; however, other factors, such as soil, sunlight may also have the impact on plant properties and composition. Determination of the effects of these factors on T. farfara properties would require special experimental design which was beyond the scope of this study.

Phenolic components are very important plant constituents with radical scavenging ability due to their hydroxyl groups. It has been established that phenolic compounds are the major plant constituents with antioxidant activity that results from their redox properties. Phenolic compounds are a class of antioxidant agents which can adsorb and neutralize the free radicals [190]. Application of Folin-Ciocalteu reagent gained popularity and is commonly known as the total phenols assay, although the standard reagent used in this method measures a sample's reducing capacity [186]. The content of phenolic compounds was determined from regression equation of a calibration curve (y=8.3166x + 0.2207, R²=0.9985) and expressed in gallic acid equivalents (GAE) in C. grandiflora, M. odorata and T. farfara extracts. In C. grandiflora extracts it was from 28.0±0.12 in AE to 82.8±1.32 mg GAE/g in EE (Table 3.8). The concentration of TPC was lower in FD samples than in SD. Most likely, the reason for this finding is that the yield of FD (8.20±0.25% (w/w)) was higher than SD (5.15±0.10% (w/w)) and phenolic compounds in FD were more diluted. It is in agreement with previously obtained results for black currant buds [188]. It can be observed that the content of phenolics in the extracts correlates with their antiradical activity; strong correlation was observed between total phenolics and DPPH • (R 2 =0.8204) and ABTS •+ scavenging capacity (R 2 =0.8023), confirming that phenolic compounds are important contributors to the antioxidant properties of these extracts. However, the correlation between TPC and FRAP was very weak (R 2 =0.157), which suggests that in FRAP assay nonphenolic compounds may play more important role.

CONTENT OF TOTAL PHENOLIC COMPOUNDS (TPC), FLAVONOIDS AND FLAVONOLS

Consequently, M. odorata extracts isolated from the essential oil distilation by-products, particularly by such polar solvents as ethanol, methanol and water might be promising sources of natural antioxidants. The content of TPC extracted from M. odorata was solvent dependent and varied in the range of 10.78±0.02 to 85.25±1.02 mg GAE/g (Table 3.8). The content of polyphenols isolated by different solvents decreased in the following order: ME > EE > WE > AE for FR M. odorata and EE > ME > WE > AE for LT M. odorata. EE and ME are quite similar protonic solvents; however both of them were tested because EE is more acceptable for the isolation of food grade ingredients. The differences in the TPC content in EE and ME between LT and FR origin plants were not remarkable, however, the biggest sum of TPC in FR M. odorata (ME+WE+AE=179.47 mg GAE/g) was higher than in LT M. odorata (EE+WE+AE=126.93 mg GAE/g). It is interesting to note that lower polarity phenolics extracted with acetone were almost 4 times more abundant in FR M. odorata than in LT M. odorata.

The M. odorata water extracts have a strong hydroxyl radical inhibitory activity relative to the other investigated extracts, whereas its phenol content is lower, according to Table 3.8. This finding showed that the content of total phenolics in herbs is not a reliable indicator of their antioxidant activity. The structures of the individual constituents need to be elucidated and assessed in order to obtain more precise results and information. To support of this argument, we compared the TPC with RSC. Thus, the correlation between the RSC and TPC of the ten samples was studied using a linear regression analysis. The correlation coefficient between TPC and FRAP values (R²=0.283), DPPH • scavenging activity (R²=0.3078), and ABTS •+ assay (R²=0.7006). Usually the TPC are in a strong correlation with RSC, however in our study of M. odorata this correlation was weak, less than 0.8. For instance, the content of TPC in WE and AE of FR M. odorata was remarkably higher that in LT M. odorata, while the differences in the RSC between the extracts isolated from the two different origin plants with the same solvents were negligible (Fig. 3.8). EE and ME contained high amount of TPC and showed very good RSC, while the content of TPC in WE, which also possessed good RSC was remarkably lower than in EE and ME extracts.

The correlation between phenols content and these two DPPH • , FRAP assays was found to be very weak, less than 0.5. These low correlation values between total phenols and the antioxidative activity suggest that the major antioxidant compounds in studied extracts might be nonphenolics. TPC in different anatomical parts, L, FS and R were determined of T. farfara from two origins. Various solvents (ethanol, methanol and acetone) were used to achieve extraction of active substances with diversity in their polarity. The highest concentration of TPC in FS extracts of T. farfara were in AE followed by ME and EE except for ME isolated from T. farfara from LT; it contained similar amount of TPC to AE. It is interesting noting that AE of FR origin plants contained higher amount of TPC than AE of LT origin T. farfara, while the content of TPC in ME and EE was higher in LT origin plants. However, the sums of TPC in AE+ME and AE+EE were similar (189.4 mg GAE/g) or very close (161.2 and 171.0 mg GAE/g) for both origin plants.

The concentrations of TPC in L and R extracts were inverse than in FS extracts. The highest concentration of TPC in the LT T. farfara was L EE extract, but for the FR origin was L ME extract. The ME extracts of R have higher concentration of TPC, than EE and AE extracts. However, the sums (AE+ME+EE) of TPC in different anatomical part of T. farfara were very different, for LT and FR FS were (255.9 and 238.2 mg GAE/g), but for L the TPC were two time highest (403.6 and 487.8 mg GAE/g) respectively. Therefore, high amount of TPC in L extracts showed very good RSC, while the content of TPC in FS and R extracts, possessed lower RSC.

So far as acetone and alcohol were used as consecutive extraction solvents it may be suggested that individual phenolic compounds are present in the plants of two origins at different proportions. Results of the present study showed that acetone does not provide exhaustive extraction of phenolic compounds; polar solvent should be applied as a second solvent. According to our study, the high content of these phenolic compounds in AE FS extract of T. farfara could explain their high ABTS •+ scavenging capacity and FRAP. This reducing capacity of the extracts may serve as an indicator of potential antioxidant activities through the action of breaking the free radical chain by donating hydrogen atom [191].

It can be observed that the content of phenolics in the FS, L and R extracts correlates with their antiradical activity; strong correlation was observed between total phenolics and FRAP in FS and R extracts (R 2 =0.8341 and R 2 =0.7935 respectively), also we found correlation between TPC and DPPH • for R extract (R 2 =0.6969) and TPC with ABTS •+ assay in L extracts (R 2 =0.553), confirming that phenolic compounds are important contributors to the antioxidant properties of these extracts. However, the correlations between TPC, DPPH • , ABTS •+ in FS extracts and DPPH • , FRAP in L extracts were very weak, less than 0.5.

Flavonoids are important group of phenolic compounds; therefore, their concentration was assessed separately, by exploring the ability of flavonoids to form yellow colour chelate type internal complexes with Al 3+ , which may be measured spectrophotometrically.

The content of flavonoids, in rutin equivalents (RE), of C. grandiflora varied from 0.79 (in FD extract) to 4.30 mg (rutin)/g (in ME extract). The concentration of flavonoids was higher in alcoholic extracts than in AE. Relatively low amounts of flavonoids were determined in aqueous extracts of C. grandiflora, which contained high amount of TPC and the differences between FD and SD again may be explained by the differences in extract yield. There was no correlation between the amount of flavonoids and RSC.

The content of flavonols was also estimated spectrophotometrically and it was found that the concentration of this class of compounds varied from 0.03 to 1.18 mg RE/g of plant extract (regression equation of a calibration curve y=6.0727x + 0.0504, R²=0.9929). The highest amounts were measured in the AE extract, while in water extracts this class of compounds was present in trace concentrations. Flavonols are known as important compounds in terms of radical scavenging properties. It may be observed that the extract with higher content of flavonols showed lower IC 50 values in DPPH • assay.

The total flavonoids and the flavonol subclass of the M. odorata plant extracts were also measured. EE extracts had the highest flavonoid contents 4.00±0.31 and 4.15±0.71 mg RE/g dry weight plant extract, respectively, whereas WE extracts although demonstrating high RSC contained the lowest contents of flavonoids, phenolics and flavonols. It is noteworthy that, apart from the phenolic compounds, there may be other secondary metabolites in plants that could be responsible for antioxidant activities. The M. odorata water extracts have a strong hydroxyl radical inhibitory activity relative to the other investigated extracts, whereas its phenol content is lower, according to Table 3.8. This finding showed that the content of total phenolics in herbs is not always a reliable indicator of their antioxidant activity

The amounts flavonoids and flavonols extracted from different anatomical part and origins of T. farfara were analyzed. The highest amounts flavonoids and flavonols were found in LT origin plants for all their anatomical parts than in FR origin. It is important to note that the climatic conditions in the countries were very different; mean temperature and rainfall amount during the month of collection were higher in Midi-Pyrenées-France (10.5°C and 97.1 mm) than in Kaunas-Lithuania (6.9°C and 34.0 mm). These factors may be among the reasons of the differences in chemical composition.

High concentrations of flavonoids were determined in FS and L extracts, than R extract. The highest flavonoid content was found in the AE extract from the FS (9.20 and 9.18 mg RE/g) and in the ME extracts from the L (12.16 and 11.02 mg RE/g).

However, the content of total flavonoids and flavonols measured by using spectrophotometric method [192] cannot be directly used as a reliable indicator of the total antioxidant capacity. The structures of individual constituents need to be elucidated and assessed in order to obtain more comprehensive information.

Numerous plant extracts have been studied in various lipid systems. The results of these studies showed that there is further need for measurements of plant composition and properties; in many cases data obtained are still insufficient and sometimes contradictory, depending on the origin of primary plant material, extraction solvent, lipid substrate and method chosen for stability tests. Recently, many antioxidants were isolated from aromatic and medicinal plants [193][194][195][196]. The primary antioxidant activity analysis data obtained in sunflower oil are presented in this chapter. Sunflower oil is largely used in Europe. It is rather unstable because of the presence of a substantial amount of -linolenic acid (60-65%) and can be used for the assessment of antioxidant properties of plant extracts. The antioxidant activity of various plant extracts in sunflower oil has been studied previously. Rosemary, sage, nutmeg, clove and allspice extracts showed their capacities to stabilize sunflower oil [START_REF] Li | Antioxidant properties in vitro and total phenolic contents in methanol extracts from medicinal plants[END_REF][197][198]. Edible oils containing high percentage of polyunsaturated fatty acids are very sensitive to auto and photooxidation. So far as autooxidation proceeds via radical formation chain reaction, the compounds able to transfer electron and/or hydrogen may retard the process of lipid oxidation. Various methods exists to assess the effects of antioxidants on lipid oxidation; however, the Oxipres method, which was used to evaluate the antioxidant activity (AA) of extracts in our study, is a very convenient procedure as it is performed without using any chemicals. The change of oxygen pressure in the reaction vessel at the end of the induction period indicating rapid formation of hydroperoxides can be quite precisely measured. This is measured from the cross-section point of tangents of the first part and the subsequent part of

ANTIOXIDANT ACTIVITY OF EXTRACTS IN SUNFLOWER (HELIANTHUS ANNUUS) OIL

the curve recording the pressure changes. For edible oils analysis, the optimum conditions are: 5.0 g samples; initial pressure 0.5 MPa; temperature 100°C [198].

Antioxidant activity of C. grandiflora essential oil was reported previously and PF at the concentration of 0.2% was 1.63 [177]. In case of adding plant extracts at 0.05, 0.1 and 0.2% concentrations to sunflower oil, the pressure drop was delayed for some period of time, indicating the antioxidative effect of the added substance (Table 3.9). The effect of 0.2% EE extract on the stability of sunflower oil during accelerated oxidation was twice lower as compared with 0.02% butylated hydroxytoluene (BHT). Different concentrations of each extract were tested to receive more information about antioxidant properties of C. grandiflora extracts in sunflower oil. In general, all extracts showed oil stabilizing effect, which increased with increasing concentration of the extract in the oil. The effectiveness of the 0.2% concentration of plant extracts decreases in the following order: EE > ME > SD > AE > FD. However, the antioxidant activity of C. grandiflora EE was most effective and considerably exceeded the effect of other used herbal (M. odorata and T. farfara) extracts. However, all C. grandiflora and T. farfara extracts possessed slightly higher positive effect on such characteristics as IP, PF and AA as compared with M. odorata. For instance, 0.05 addition of AE, ME, EE and WE of LT and FR origin M. odorata, prolonged induction period by 2.5%, 0.5%, 1% 9.5% and 3.5%, 0%, 3.5%, 14.5% respectively as compared with the blank samples (Table 3.9). The changes of IP in sunflower oil after addition of M. odorata were not considerable; e. g. IP of the sample with 0.05% of M. dodrata ME was almost equal to IP of the sample without additives. It should be noted that so far as plant extracts are very complex mixtures consisting of various compounds the concentration of pure compound, BHT in oil was 2.5 times lower (0.02%) than the concentration of the extracts (0.05%), but synthetic (BHT) antioxidant was stronger, than plant extracts. Also from these data it may be observed that there are no significant differences between the same plants of two different origins, but the important difference are obtained in case of using different extraction solvents: WEs of M. odorata possessed stronger antioxidant activity, than AE, ME and EE. For LT T. farfara we observed that the highest IP was in AE extract from FS and L but for R was EE extract. In FR origin the EE extracts of FS and R showed the most effective effect, but for L AE was stronger. BHT was active antioxidant allowing stabilizing lipid for two times longer than T. farfara extracts. Comparing the influence of the same solvent and plant origin in Oxipres test it was found the same regularities between M. odorata and T. farfara, there were no significant differences between origins (LT and FR), the only important difference is obtained by the use of different solvents for extraction.

Summarizing the results obtained by oxipres method it can be stated that almost all herbal extracts obtained some AA by stabilizing sunflower oil during storage. In terms of effectiveness the extracts at 0.2 % concentration can be put into the following succession: C. grandiflora  T. farfara  M. odorata. It has also should be emphasized that no positive correlation was found between the TPC and AA. For instance, M. odorata extracts which were rich in the TPC possessed the lowest AA as compared with the roots of T. farfara (low amount of TPC and medium AA) and C. grandiflora (medium TPC and highest AA). It should be noted that different solvents were used for the isolation of phenolic compounds (ethanol or methanol) and (acetone). However, we suppose that the solvent in this case was an important factor, so far as the procedure of the extraction in Soxhlet apparatus is sufficiently efficient to isolate herb phenolics with ethanol or methanol, probably due to their higher compatibility with the medium or the more polar nature of the contained analytes. The absence of the correlation between the TPC and AA of the extracts can be better explained by the well-known fact that the AA of individual phenolic compounds can vary in a very wide range.

The on-line RP-HPLC-DPPH • method can be used for a rapid assessment of radical scavenging properties of separated in chromatographic column compounds which are present in complex mixtures such as plant extracts [199][200]. The HPLC/UV/DPPH • chromatograms of EE and FD extracts (Fig. 3.12) indicate that several separated compounds are able to scavenge radicals. This study was aimed at the identification of individual components in the extracts; the most abundant compound in EE was identified as rosmarinic acid, which is well known natural antioxidant. High amount of the radical scavenger eluted from the C. grandiflora FD at approximately 35.9 min: it was identified as salvianolic acid C. In LC-MS spectra it gave m/z 491 and characteristic fragmentation at m/z 311, and possessed a very high radical scavenging activity (30.4% of total area of the peaks).

Salvianolic acid was previously found in Salvia miltiorrhiza root; this polyphenolic acid protects against dopamine-induced apoptosis in SH-SY5Y cells and may be effective in treating neurodegenerative diseases associated with oxidative stress [203].

To the best of our knowledge rosmarinic acid and salvianolic acid C were not previously reported in C. grandiflora. Thus, these results showed that the residue of WEs from C. grandiflora possess strong antioxidant activity and therefore might be promissing for industrial applications, e.g. in foods and the cosmetics, as a means of protecting against negative effects of excessive free radicals.

The assessment of antioxidant properties of M. odorata by TPC and RSC-DPPH • assays suggests that there might be remarkable differences in the extract composition between the plants of FR and LT origin, particularly in case of WE and AE. It is known that the antioxidant activity of individual phenolic compounds can differ significantly depending on their molecular structure; therefore the content of TPC in plant extracts is not very informative indicator of their RSC. The structures of isolated constituents need to be elucidated and assessed in order to obtain more precise results. An on-line HPLC-DPPH • method is a convenient option for the preliminary screening of RSC of the compounds present in the extracts. Therefore, further studies were performed to evaluate RSC of chromatographically separated constituents. Kaempferol-7-O-glucoside and cynarin were identified in M. odorata and they were the major antioxidatively active compounds in the analyzed extracts, however the former compound was more abundant in all LT M. odorata extracts while the latter one was dominant in WE of FR M. odorata. To the best of our knowledge these compounds were not previously reported in M. odorata. Peak areas of kaempferol-7-O-glucoside and cynarin in EE and WE were in the range of 639.7-6038.2 and 0-849.1, while their input in the total RSC was 34.5-97.1 and 0-45.4%, respectively (Table 3.10). Kaempferol-7-O-glucoside was previously found in Smilax china; this flavonoid induced G2/M phase arrest and apoptosis on HeLa cells in a p53-independent manner [204]. Cynarin is an ester formed from quinic acid and two units of caffeic acid and was reported as biologically active chemical constituent of artichoke 58 years ago [205]; diuretic, choleretic and hepatoprotective activities of artichokes leaves extract were often related to the cynarin content [204][205]. In addition, cynarin is responsible for the sensation of sweetness that occurs after eating artichokes; it appears to suppress bitter tongue taste receptors while enhancing the sweet ones; consequently it may also be responsible for the sweet taste of M. odorata. Most recently it was reported that cynarin, which was abundant in sunflower (Helianthus annuus) sprouts possessed both antiglycative and antioxidant activities and was able to intervene against glycoxidation [206].

The chromatographic profiles of WE (Fig. 3.13) obtained by the on-line HPLC-DPPH • methods clearly show that FR M. odorata contain more radical scavenging constituents than LT M. odorata. Strong antioxidant cynarin was present in FR M. odorata in remarkably higher amounts than in LT M. odorata, however, the content of kaempferol-7-O-glucoside was more abundant in LTMO (Fig. 3.13, Table 3.10). Total peak area of radical scavenging peaks in W of LT M. odorata and FR M. odorata was 2814.6 and 1801.4, respectively, and 3.6.2 Phytochemical composition and RSC of M. odorata constituents these values are in agreement with RSC (IC 50 ) of these extracts, which were 0.12 and 0.13 mg/l, respectively.

Fig. 3.13. HPLC-DPPH • chromatograms of Myrrhis odorata extracts of French (FR) and Lithuanian (LT) origin and m/z and the structures of identified compounds

However, in case of EE direct comparison of on line and batch evaluation of RSC is not relevant. The IC 50 of EE of FR M. odorata was only slightly lower than that of LT M. odorata, while peak area of the main EE constituent kaempferol-7-O-glucoside was more than 4 times bigger in the LT. Possibly EE of FR contains other radical scavengers which were not detected at the applied HPLC analysis conditions. As it may be observed in the chromatograms the extracts contain more antioxidatively active compounds, however, their input in the total RSC was less remarkable and constituted from 0.8% (WE of LT M. odorata) to 20.5% (EE of FR M. odorata) of the total RSC (Table 3.10). Mass spectra and UV data was not sufficient to identify these compounds. Also the content of detected constituents in AE was remarkably lower than in EE and WE. Flavonoid glycosides, quercetin 3-O-β-L-arabinopyranoside and quercetin 3-O-β-Dglucopyranoside [152] as well as quinic, chlorogenic and dicaffeoylquinic acids were reported previously in T. farfara flower bud extract from China [153]. The on-line HPLC-DPPH • method was used for the detection of radical scavenging components. The T. farfara extract of the flowers-stems had clear differences from the extracts obtained from other parts of the plant. The R AE exhibited smaller peaks than those seen in the extracts of the L and FS (Fig. 3.14). The analysis revealed that FS, L and R extracts contain 5 similar active components: quinic acid, dicaffeoylquinic acid, quercetin-pentoside and kaempferol-glucoside, while other detected compounds were different. The ESI-MS of the AE, ME and EE of T. farfara from two origins were qualitatively different (Table3.11). Compounds 3, 4 and 5 showed [M-H] - signal at m/z 515 with fragmentation at m/z 353 and 179, from which a molecular formula of C 25 H 24 O 12 was assigned. The ion at m/z 353 indicated a chlorogenic acid fragment derived from the loss of a caffeoyl group. The ion at m/z 179 indicated fragments of caffeic acid moieties. These MS data indicated that the compounds 3-5 were dicaffeoylquinic acid isomers. As chromatographic separation was performed, it wasn't possible to determine which isomers of dicaffeoylquinic acid were present, however all isomers are common components of T. farfara and possess antioxidant activity [117]. Usually, the location of the caffeoyl groups in dicaffeoylquinic are at C-3'; C-4', C-3'; C-5' and C-1'; C-5' (fig. 3.14, A). RSC of T. farfara extracts isolated from LT origin plants showed slightly better antioxidant activity than those of FR origin; however, based on DPPH • assay (Fig. 3.10), no significant differences in IC 50 values were observed between the same plant extract from different countries. Chromatograms profiles of extracts in HPLC-DPPH • assay (Fig. 3.14) were not similar between the two origin plants. T. farfara FS extracts isolated from LT origin plants showed more big peaks in negative chromatogram comparing to FR origin plants. Dicaffeoylquinic acids and quercetin-pentoside were major active compounds with total RSC (39.6%), (10%), (17.1%) and (16.9%), (13.8%), (13.1%) respectively (Table 3.11) in LT and FR AE deodorized FS extracts.

Evaluation of active compounds from T. farfara

Major active compounds from LT AE L extracts was dicaffeoylquinic acids and quercetin-pentoside, but in FR origin was dicaffeoylquinic acid, quercetin-pentoside and tricaffeoylquinic acid and these compounds have RSC (47.7%), (9.2%), (11.5%) and (36.5%), (10.4%), (17.6%) respectively.

As it has already been noted, phytochemical composition of plant's secondary metabolites depends on several factors, such as cultivation area, climatic conditions, vegetation phase, genetic modifications, part of plants and others; therefore evaluation of plant properties from different geographical regions was in the focus of numerous studies [147,207].

So far as the yields of volatile compounds which might be isolated from the selected aromatic plants are relatively low, it would be more economical to focus future efforts on the processing of by-products into high-added value nonvolatile substances, such as antioxidants. The performed studies suggest that solid and liquid residues remaining after essential oil isolation from the whole plant material could have potential uses in food industry and some other applications. In addition to reducing the waste associated with by-products production, could provide additional economic value to the aromatic plant material. Phenolic compounds from by-products may be extracted and spray dried producing powder with high content of phenolic compounds and strong antioxidant capacity; the product could be used as a natural additive or healthy food ingredient in foods and nutraceuticals.

All experiments were performed with small amounts of plant material on laboratory scale. Therefore it would be very important to upscale the production quantities. First step in implementation of the results would be testing of technology in pilot plant equipment and afterwards it may be upscaled to the industrial level. Possible technological scheme for the valorization of aromatic plant by-products processing is presented in Fig. 3.15.

The procedures used in this work on a laboratory scale may be implemented by using existing commercial equipment, possibly by adding to the existing lines some new apparatus fitting selected plant material properties. As an example an existing technological scheme developed by the company Dig-Maz [208] with some modifications (Fig. 3.15) may be applied; it represents the full technology scheme for plant material processing: the production line is divided into six stages, which are briefly outlined further in this section (detailed description of the technological scheme is given in appendix).

First stage is conducted for checking of the primary raw materials for various impurities and grinding. This stage is recommended by Dig-Maz using their equipment i.e., the raw materials

THE POSSIBILITIES OF TECHNOLOGICAL IMPLEMENTATION

are filled into the feeding funnel (AT 06) of the conveyor belt FB-4 (segregation line). The conveyor belt (FB 4) is transferring the raw material through the metal detector (GLS) to the cutting mill (GSL 180/120). After the cutting mill the crushed material particles are filled in the feeding tank. The feeding tank is placed on a balance and filled as long until the adjusted weight is obtained. A lifting-tipping device (filling device) is loading the extraction plant (hydrodistillation).

Second stage is a hydrodistillation process, resulting in one of the products -essential oil and two-type of by-products (liquid and solid) -liquid residue containing water soluble plant constituents and solid residue, containing water insoluble plant substances. This stage was not included into the existing technological scheme developed by the company Dig-Maz; therefore some modifications were introduced by adding a hydrodistillation apparatus with condenser and spray dryer, which will be used in third stage. Hydrodistillation is a method of extraction, which is sometimes used instead of steam distillation. This process of extraction is one of the most frequently used traditional methods of extraction. In this process the plants are soaked in the water; then the water container is heated untill the steam comes out together with volatile oil and afterwards is cool for condensation. The oil is collected from the top of the hydrosol.

The remaining by-products in the third stage can be processed according to our proposed technology: the liquid residue is filtered, thus solid particles can be separated, and sprayed in spray dryer to obtain a second product -powder containing active water soluble components such as glycosidic polyphenol compounds, phenolic acids and others. Spray drying is a very fast method. The short drying time, and consequently fast stabilization of material at moderate temperatures, means spray drying is also suitable for heat-sensitive substances. As a technique, spray drying consists of four basic stages: atomization, drying, particle formation, and their recovery. A liquid stock is atomized into droplets by means of a nozzle or rotary atomizer. Nozzles use pressure or compressed gas to atomize the feed while rotary atomizers employ an atomizer wheel rotating at high speed. Hot process gas (air or nitrogen) is brought into contact with the atomized liquid guided by a gas disperser, and evaporation begins. The balance between temperatures, of rate and droplet size controls the drying process. As the liquid rapidly evaporates from the droplet surface, a solid particle forms and falls to the bottom of the drying chamber. The powder is recovered from the exhaust gas using a cyclone or a bag later. The whole process generally takes no more than a few seconds. In addition, agglomeration may be used as a final stage, which performed by rewetting spray dried particles by mixing them with a mist of water and vapor and finally by gentle impacting to form the right size of light and stable agglomerates.

The remaining solid residue after hydrodistillation is dried at a temperature of 30-40°C (fourth stage) and then is transported by conveyor belt to the extraction; this stage was also added to the Dig-Maz technological scheme.

Extraction of solid residue is carried out using selected solvent; later the resulting extract is concentrated and the solvent is evaporated, thus obtaining a third product -the extract containing water insoluble substances. In this stage commercial DIG -MAZ Extraction system (DIG-MAZ 500) may be used. The DIG-MAZ extraction system is designed as a multifunctional system which combines important methods of extraction, such as percolation, digestion, maceration as well as distillation in one compact plant. The extraction process can be performed under pressure up to 10 bar and under vacuum. DIG-MAZ extraction plants' special counter-current feature, causing the solvent to stream through the herbs bottom up, makes them a unique extraction system. The process starts with the filling the extractor with hackled solids. The lid opens the whole cross-section of the extraction vessel at the touch of a button whereby an optimum filling is ensured. Batch-extraction of fine-grained solids proceeds by leaching with solvents, such as water and ethanol. Thus, the extract filtration is executed by bottom and lid sieves then, the solids are squeezed using the sieve bottom, which is employed as movable press piston and controlled via hydraulics. The good extraction is then dehumidified reducing the solvent loss and extraction vessel is tipped over on a horizontal axis and completely evacuated with the help of the hydraulically shifted sieve bottom. Finally, in the Vacuum evaporation, the solid-content of the extract is increased by vacuum evaporation at short holding time. The solvent is condensed, accumulated and can be re-circulated into the extraction process [208]. A solid residue after extraction (mainly lignocelullosic substances) may be further processed, for instance by transporting it to the pressing line for producing biodegradable cups for plants or briquettes for fuel or to composting line (these processes were not studied in the present work; however such technologies recently have been developed). Industrial implementation of the technology would require additional optimization of process parameters, such as temperature, the ratio of solvent and plant material, particle size, process time and others. In fact, main processes could give three main products -essential oil, water soluble fraction and water insoluble fraction, which may be isolated with organic solvents. These fractions may be further processed in to finer and more specific products. For instance, rectification column may be used as an additional apparatus for fractionation of volatile oil into different fractions possessing special odour and antimicrobial properties, while nonvolatile fractions may be further fractionated by chromatographic methods into the targeted compound's groups possessing strong antioxidant and other biological activities.

In our study it was shown that phytochemical composition of the selected aromatic and medicinal plants was highly dependent on their origin of cultivation and vegetation phase. Therefore, another important recommendation for the industrial implementation is associated with plant material selection and cultivation as well as various harvesting issues. These issues should be carefully considered by selecting cultivation area, agrotechnological aspects, plant chemo-and biotype peculiarities. These issues are important for the standardization of the products, which may be obtained by using agrorefinery concept. They should be focused on the crop yield and the concentrations of the most valuable constituents in the plant material harvested for processing. It should be considered that phytochemical composition of plant's secondary metabolites depends on several factors, such as cultivation area, climatic conditions, vegetation phase, genetic modifications and others. Therefore, comprehensive assessment of all these factors could help identifying genetic potential, which could assist in improvements in breeding programs and diversification of its industrial uses. Also, additional future work should include further investigation of the bioactivity; bioavailability and toxicology of phytochemicals need to be carefully assessed by the in vitro and in vivo assays.

Undoubtedly, functional foods as representing an important, innovative and rapidly growing part of the overall food market may be considered as one of the main end user of the developed plant products. Other areas of application include cosmetics, pharmaceuticals, and fine chemicals for other uses. So far as the majority of such products are intended for human consumption, future work should also be focused on investigating their design, sensory properties, consumer acceptability and also careful assessment of all potential risks which might arise from the natural compounds recovered from by-products. Furthermore, investigations on stability and interactions of phytochemicals with other food ingredients during processing and storage need to be performed.

CONCLUSION

CONCLUSION Conclusion

1. Based on main essential oil components, M. odorata of French and Lithuanian origin may be preliminary assigned to the same E-anethole, methyleugenol and E-nerolidol chemotype. However, the percentage of estragol was more than 4 times higher in essential oil of French origin plant materialwhereas the percentage of β-caryophyllene, germacrene-D, spathulenol and limonene was significantly higher in the Lithuanian origin oil. The climatic conditions from two studied areas countries are different; mean temperature and rainfall amount during the month of collection were higher in France than in Lithuania; due to these differences the former oil had stronger anise and licorice-type flavor notes while the latter one possessed stronger fresh, sweet, woody, spice, clove dry, earthy, herbal, and fruity notes.

2. The main constituents of T. farfara volatile fraction of French and Lithuanian origin plants were 1-nonene (34.1±0.0%) and n-tricosane (21.7±0.1%), respectively. Chemical composition of T. farfara volatiles in flowers and stems were compared for the first time; microscopy analysis revealed that stems contain secretory hairs (glandular trichoma), the organs for essential oil supply suggesting that all the aerial parts of T. farfara might be useful for essential oil production.

3. Two new analytical methods for the analysis of volatile compounds, namely Crushing Finger Device (CFD) and Flash Aroma Dispenser (FAD) were developed and tested as alternatives to the traditional extraction methods; it was shown that these methods may avoid some disadvantages of traditionally used analysis techniques, such as the thermal degradation of volatile compounds or large amounts of raw materials.

4. All extracts isolated from plant hydrodistillation residues were antioxidatively active. M. odorata and C. grandiflora alcoholic and water extracts were remarkably stronger antioxidants than acetone (AE) extracts in DPPH • and ABTS •+ reaction systems. Radical scavenging capacity (RSC) of extracts obtained from the solid residue was quite similar to the RSC of water extracts, showing that some antioxidatively active compounds do not dissolve in boiling water and still remain in the solid residue. Some differences were determined in the antioxidant activity indicators of T. farfara and M. odorata extracts isolated from Lithuanian and French origin plants proving that such factors as cultivation area, climatic conditions, vegetation phase, genotype and others may be important in the accumulation of active constituents in the plants. 5. Plants extract additives retarded sunflower oil oxidation; C. grandiflora extract added at 0.2 % had the strongest effect followed by T. farfara and M. odorata. However, synthetic butylated hydroxytoluene (BHT) antioxidant in oil was 10 times lower (0.02%) than the concentration of the extracts (0.2%), but synthetic (BHT) antioxidant was stronger, than plant extracts. It has also should be emphasized that no positive correlation was found between the total amount of phenolic compounds (TPC) and antioxidant activity (AA); for instance, M. odorata extract possessing high TPC value was weaker antioxidant that T. farfara root extract containing lower TPC.

6. Several antioxidants were identified in the studied plants by using modern chromatographic and spectrometric analysis techniques such as on-line HPLC-DPPH • method. Rosmarinic acid was the major antioxidant in C. grandiflora ethanol extract, while salvianolic acid C was major active compound in freeze-dried extract. Kaempferol-7-O-glucoside and cynarin were identified as the major antioxidatively active compounds in M. odorata extracts; these compounds were not previously reported in M. odorata and C. grandiflora. Eight antioxidant compounds were identified in T. farfara two of them (quercetin-pentoside and kaempferol-glucoside) previously not reported in this plant.

7. C. grandiflora, M. odorata and T. farfara examined in the study contain valuable functional constituents -antioxidants and volatile aroma compounds. The results obtained in this study may be considered as a first step in valorizing the processing of these raw materials in order to obtain high-added value substances, present both in volatile and nonvolatile fractions, which can found wider practical application in different areas, particularly in formulation and production of food additives, functional food components and healthy supplements. Agrorefinery approach applied in this study may substantially increase the feasibility of practical implementation in terms of agro-technological, economical, processing and application issues.

GALAXIE Spray Dryer Model 2520

Oven  Temperature controller and adjustable  Motor control push buttons  Low pressure air circulation system Successful drying depends on heat, air dryness and air circulation.

  aromatiques, médicinales et d'épices sont une immense source de ressources naturelles et durables de composés avec des propriétés bénéfiques. Elles sont utilisées, depuis la préhistoire, comme aromatisants ou préservent des aliments ou comme traitement médicaux. Actuellement, certains d'entre eux sont déjà cultivés commercialement pour utiliser ses propriétés. Néanmoins, il existe encore certaines plantes médicinales et aromatiques qui ne sont pas encore valorisées et donc elles sont dénommées comme « plantes médiévales oubliées ». Par exemple, Calamintha grandiflora, Myrrhis odorata et Tussilago farfara correspondent à ce type de plantes et lesquelles seront considérées dans ce manuscrit. Afin de revaloriser ces plantes oubliées, la bioprospection est mise en place et laquelle permet la recherche, la collection de nouveaux composés bioactifs naturels et son utilisation dans les produits pharmaceutiques et cosmétiques, par exemple. De plus, la bioprospection permet d'analyser les profils chimiques de différentes plantes et définir une liste des composés phytochimiques associés à chaque plante. Ainsi, par exemple, au travers de l'analyse d'une plante spécifique, il est possible définir les différents types antioxydants existant ainsi que sa quantité. Donc, cette plante peut utiliser comme une plante médicinale ou aromatique pour la consommation humaine afin d'absorber et neutraliser les radicaux libres ou la décomposition des peroxydes dangereux. L'apparition des plantes médicinales et aromatiques et leur composition dépendent de nombreux facteurs, mais particulièrement, sur les conditions climatiques et le type de sol. La biogenèse des composés biologiquement actifs dépend de plusieurs facteurs: l'origine de la plante, la zone géographique, son génotype, phénotype et chémotype, ainsi que les fluctuations climatiques annuelles. La plupart des composés naturels possèdent plusieurs activités biologiques: antioxydants, antimicrobiens, anti-inflammatoires et autres. En conséquence, la comparaison des espèces végétales cultivées dans des sites géographiques différents afin d'évaluer leurs propriétés biologiques, il est une tâche intéressante et importante, en particulier, pour la valorisation de ces plantes pour leur culture commerciale.

vii 1 . 2 .

 12 Déterminer la composition d'huiles essentielles à partir de plantes aromatiques collectées en Lituanie et sud de la France et les comparer avec les données publiées antérieurement. Développer de nouvelles techniques d'extraction rapide des composés volatils et les comparer avec les méthodes traditionnelles. Pour ce faire, les composés volatils des huiles essentielles de Calamintha grandiflora obtenues par la méthode d'hydrodistillation sont comparés avec ceux obtenus avec les nouvelles méthodes Artificial Crushing Finger device (CFD) et Design of Flash Aroma Dispenser (FAD).

2 .

 2 Les méthodes de rapid arôme profilage, à savoir 'Artificial Crushing Finger device (CFD)' et 'Design of Flash Aroma Dispenser (FAD)' peuvent être utilisées pour l'évaluation des composés volatils des plantes comme des méthodes alternatives aux techniques d'extraction traditionnelle. Les résultats de cette recherche ont été publiés dans trois articles scientifiques dans les revues indexées par 'ISI Web of Science' et dans deux d'actes de colloque internationaux. De plus, les résultats de cette recherche ont été présentés également dans les huit conférences scientifiques internationales.

  Lituanie et de France, respectivement. De plus, six composants volatils qui ont été détectés dans l'huile d'origine française ne sont pas trouvés dans l'huile d'origine lituanienne: camphre, iso-menthone, cis-thujone, pinocarveol, carvacrol et p-anisaldéhyde. Par ailleurs, les deux huiles essentielles ont montré une plus grande quantité des hydrocarbures sesquiterpènes que d'hydrocarbures monoterpènes (tableau R.1). Les composants de major quantité ont été E-anéthol, methyleugenol et E-nerolidol, dans lequel aucune différence significative n'a pas été constatée entre les deux origines. Par rapport à ses arômes, ce trois composants ont été xi La comparaison entre les pourcentages des principales composantes des huiles analysées dans notre étude et précédemment rapporté dans la littérature sont résumées dans le tableau R.2. Ainsi, dans la littérature, E-anéthole a été le composant la plus significative (82-85%) dans les huiles d'origine Finlande [137], de la Russie [139] et des États-Unis [140] et on peut considérer que ces plantes ont été que de chémotype E-anéthole. En autre, les plantes des États-Unis ont eu une raisonnable quantité de méthyleugénol qui n'a pas été trouvé dans les plantes d'origine de la Russie ou de Finlande. Par ailleurs, dans les plantes d'origine de la Russie [139] et de la Serbie [141], le chémotype E-nerolidol n'est pas identifié. De plus, un chémotype complément différent a été identifié dans l'huile d'origine de la Serbie (région Prokletije) avec p-cymène comme le composant le plus représentatif constituant le 62.0% du total d'huile [141].

  Fig.R.1. Crushing Finger Device" (CFD)

Fig.R. 2 .

 2 Fig.R.2. Flash Aroma Dispenser (FAD)

R. 4 )

 4 . Ainsi, un faible IC 50 est le résultat d'une capacité à piéger les radicaux libres.

Fig. R. 4 .

 4 Fig. R.4. Activité antioxydante de C. grandiflora Les valeurs de IC 50 des extraits de C. grandiflora (résidus solides) dans DPPH • ont été variable de 1.10 (AE) à 0.23 (ME) mg/mL (Fig. R.4) quand les valeurs de TEAC dans ABTS •+ ont été de 0.11(AE) à 0.66% (EE) (Fig. R.5). Il est évident que dans la polarité inférieure de l'extrait de l'acétone (AE) était significativement inférieur la capacité à piéger

Fig. R. 5 .

 5 Fig. R.5. TEAC valeurs dans ABTS test de C. grandiflora

Fig. R.

  Fig. R.6. Activité antioxydant de M. odorata de la Lituanie et la France

  cation radicalaire a montré un résultat similaire comparé avec ceux obtenus par DPPH • . Les extraits EE et ME ont une capacité à piéger les radicaux libres plus forts quelle extraits AE dans l'ABTS •+ teste. La concentration des extraits ont été similaires à l'activité de 1 mmol d'une solution de Trolox dans une réaction ABTS •+ (Fig. R.7).

Fig. R. 7 .

 7 Fig. R.7. TEAC valeurs dans ABTS test de M. odorata de la Lituanie et France

Fig. R. 8 .

 8 Fig. R.8. Activité antioxydant de T. farfara de la Lituanie et France.

Fig. R. 9 .

 9 Fig. R.9. TEAC valeurs dans ABTS test de T. farfara de la Lituanie et France

  Le résidu de l'hydrodistillation de C. grandiflora montre une activité antioxydante plus efficace, en comparaison des autres résidus de l'hydrodistillation. Les résidus de l'hydrodistillation de C. grandiflora et de T. farfara ont été les plus efficaces pour la stabilisation de l'huile de tournesol. L'efficacité des résidus de l'hydrodistillation de plante diminue en fonction de l'ordre suivant: C. grandiflora  T. farfara  M. odorata, pour de concentration de 0,2%.Évaluation de l'activité antioxydante des radicaux libres (RSC) par une méthode HPLC-DPPH • L'activité antioxydante des radicaux libres dans les extraits de C. grandiflora La méthode HPLC-DPPH • peut être utilisée pour faire une analyse rapide des propriétés antioxydantes des radicaux libres des composées des extraits des plantes obtenues à travers une colonne chromatographique[199-200]. Les chromatogrammes HPLC/UV/DPPH • des extraits EE et FD (Fig. R.10) montrent que plusieurs composés séparés sont capables de piéger les radicaux libres. Prendre en compte que l'objectif de ce manuscrit est l'identification des différends composés dans les extraits des plantes, nous avons réalisé l'analyse de C. grandiflora. Les résultats ont montré que le composé le plus abondant identifié dans EE a été l'acide rosmarinique, qui est bien connu comme un antioxydant naturel. La spectrométrie de masse (LC-MS) de l'acide rosmarinique (RT=30.6 min.) en mode d'ionisation positive a observé à 359 m/z et m/z 161 de fragmentation, lequel possède une grande RSC (49.8% de la superficie totale de pics). Une grande activité d'antioxydants des radicaux libres élues de C. grandiflora FD à environ 35.9 min: Il était identifié comme acide Salvianolique C. La spectrométrie de masse (LC-MS) observé à 491 m/z et m/z 311 de fragmentation, lequel possède une grande RSC (30.4% de la superficie totale de pics).

Fig. R. 10 .

 10 Fig. R.10. HPLC-DPPH • chromatogrammes des extraits de C. grandiflora

Fig. R. 12

 12 Fig. R.12 HPLC-DPPH • chromatogrammes des (AE) extraits de T. farfara

:

  Abbreviations:

Fig. 1 .

 1 Fig. 1.3. Antioxidant The hydroxyl group in ortho or para positions of phenolic acid increases antioxidant activity while the second hydroxyl group in meta position change antioxidant capacity by decreasing the stability of radical. Glycosilation and esterification of phenolic compounds also decrease antioxidant activity if comparing with the corresponding aglycones [110]; for example, the esterification of phenolic acids by quinic acid decreases antioxidant activity of the compound [111].

  . Duration of extraction is another important factor for exhausting extraction. In general, when shorter extraction time is applied, more extraction cycles are used isolate phenolic compounds more completely. It should also be taken into account that long extraction time at high temperature increases the risk of oxidation of phenolics. According to Wettasinghe et al., to reduce the risks of damage of antioxidant compounds the temperatures lower than 60-70 °C are suggested for drying of the raw material and extraction of antioxidants from by-products for both extraction and [128-129].

Fig. 1 . 4 .

 14 Fig. 1.4. The selected plants used in the present study

Fig. 1 . 5 .

 15 Fig. 1.5. Chemical structure of major components of C. grandiflora essential oils isolted from plant leaves Literature data showed that the ethyl acetate fractions (EtOAc) possessed highest antioxidant activity, probably due to major content of polyphenols. The lowest radical scavenging activity was exhibited by petrol ether fraction (Fig.1.6).

Fig. 1 . 6 .

 16 Fig. 1.6. IC 50 values of antioxidant activities and total phenolics content of MeOH extract and fractions from Calamintha grandiflora: (A) DPPH (ascorbic acid, IC 50 =2 mg mL -1, was used as positive control); (B) βcarotene bleaching test after 30 min of incubation; (C) β-carotene bleaching test after 60 min of incubation (propyl gallate, IC 50 =1 mgmL -1, was used as positive control); (D) Total phenolics content using Folin-Ciocalteau method. Values expressed as milligram of chlorogenic acid/g of extract [136].

1. 4 . 2

 42 Myrrhis odorata (L.) Scop.In some literature sources is mentioned that M. odorata contains E-anethole up to 80% in the total oil[137,[139][140]. p-Cymene was dominating constituent (62.0%) in M. odorata oils from Serbia (Prokletije region) (Fig.1.7) [141]. Flavonoids, apigenin-and luteolin-7-O-β-glucopyranosides [142], some coumarins [143] and several phenolic acids [144] were reported previously in M. odorata from Poland, however the information about antioxidative activity of M. odorata extracts has not been found in the available literature sources.

Fig. 1 . 7 .

 17 Fig. 1.7. Chemical structure of major components of M. odorata essential oils M. odorata species has been introduced into the collection of medicinal plants at Kaunas Botanical Garden of Vytautas Magnus University. The average vegetation period of M.odorata is 190 days. The quality of raw material was characterised analysing the bioactive compounds. The differences in phytochemical composition of M. odorata plant's secondary metabolites depend on plant's geographical location, chemotype, meteorological factors, raw material preparation time and conditions. Therefore, it is very important to evaluate those predisposing factors. On the other hand, no information is available on the antioxidant activity and chemical constituents of M. odorata essential oils from the plants grown in France and Lithuania

1. 4 . 3

 43 Tussilago farfara L. market. Rhizomes accumulating various nutrients also have been used for disorders treatments. Coltsfoots have bneen mostly used for treatment of respiratory diseases: bronchitis, cough, and asthma [145]. Crushed flowers supposedly cured skin conditions. Coltsfoot is not commonly used as a food but it is listed by the Council of Europe as a source of natural food flavouring [146-147].

  The extraction and composition of T. farfara essential oils has been widely studied[148- 149]. The extracts of T. farfara was reported as exhibiting various activities, such as antioxidant effect, antimicrobial activities and inhibitory effects on NO synthesis in LPSactivated macrophage and diacylglycerol acyltransferase activity[147, 150]. Some pure compounds such as a number of phenolics, mucopolysaccharides and water-soluble polysaccharides were isolated from the flower buds of T. farfara [151]. Quercetin 3-O-β-Larabinopyranoside and quercetin 3-O-β-D-glucopyranoside were reported previously in Korean T. farfara [152] and 3,4-dicaffeoylquinic, 3,5-dicaffeoylquinic, 4,5-dicaffeoylquinic acids and rutin were isolated from Chinese T. farfara [153].Two sugar conjugates of quercetin, quercetin 3-O-β-L-arabinopyranoside and quercetin 3-O-β-D-glucopyranoside exhibited higher antioxidative activity than their aglycone quercetin in the NBT superoxide scavenging assay (Fig. 1.8) [152].

Fig. 1 . 8 .

 18 Fig. 1.8. Two glycosides of quercetin from Korean of T. farfara

  [153]. Kokoska et al. reported that ethanol extract of T. farfara aerial parts did not show antibacterial activity against E. coli [154], while in the study of Turker et al. water extract of T. farfara leaves inhibited the growth of these bacteria [155].

  The leaves of large flowered calamint (Calamintha grandiflora (L.) Moench) were collected in Midi-Pyrenées (south-west of France, 43° 36′ 16.2″ N, 1° 26′ 38.4″ E) in May, 2009 at flowering vegetation phase, which is considered as a preferable time in terms of phytochemical composition and functional properties of herbs.The leaves of sweet cicely (Myrrhis odorata (L.) Scop.) were collected after flowering in Saint-Cyr-en-Val, region of France (47° 49′ 9.09″ N/1° 56′ 51.3″ E) during July, and in Kaunas Botanical Garden of Vytautas Magnus University, Lithuania (54° 52′ 14″ N/ 23° 54′ 40″ E), during August, 2009.The flowers and peduncle of coltsfoot (Tussilago farfara L.) were collected in Midi-Pyrenées (south-west of France 43° 36′ 16.2″ N/ 1° 26′ 38.4″ E) in April, 2011 and in Kaunas Botanical Garden of Vytautas Magnus University, Lithuania (54° 52′ 14″ N/ 23° 54′ 40″ E), during April, 2010 at flowering vegetation phase.

Figure 2 . 1 .Figure 2 . 2 .

 2122 Figure 2.1. Yields of M. odorata, T. farfara and C. grandiflora essential oils

Figure 2 . 4 .

 24 Figure 2.4. Instrumental Crushing Finger Device system

Fig. 3 . 1 .

 31 Fig. 3.1. Microscopic photographs of glandular trachoma from two parts of (A) Tussilago farfara (L.) plant; (B) flower; (C1 and C2) peduncle. Magnification 400˟

. 4 .

 4 Up to now, no information is available on the chemical constituents of T. farfara essential oils from the plants grown in France or other Southern European countries. There is only one study on volatile oil of coltsfoot from Eastern European origin[149]. Qualitative and quantitative differences in oil composition of two origins (Lithuania and France) are presented in Table3.3. 1-Nonene (34.1±0.0%) is the major compound in French oil; however, n-tricosane (21.7±0.1%) is the major compound in Lithuanian oil. Moreover, 7 volatile compounds, namely tetracosane, n-tricosane, n-pentacosane, n-hexacosane, n-pentadecane, 1tetradecene and 1-decanol were detected only in Lithuanian T. farfara oil.

Fig. 3 . 6 .

 36 Fig. 3.6. Ferric reducing/antioxidant power (FRAP) and DPPH radical scavenging assay of C. grandiflora

3. 3 . 2

 32 Antioxidant properties of deodorized residues of M. odorata from two different geographical origins in electron/hydrogen transfer based assays difference is obtained by using different solvents extraction. On the other hand, the results obtained from the FRAP analyses, shows that the plants located in LT has more antioxidant activity than located in the Midi-Pyrénées zone (FR), exept AE extract.

Fig. 3 . 8 .

 38 Fig. 3.8. Ferric reducing/antioxidant power (FRAP) and DPPH radical scavenging assay of M. odorata from Lithuania and France

Fig. 3 . 9 .

 39 Fig. 3.9. TEAC values in ABTS radical cation decolourisation assay of M. odorata from Lithuania and FranceThe strongest antioxidant activity was demonstrated by EE (from 0.76 to 0.8%) and ME (from 0.54 to 0.91%) extracts. All extracts were remarkably stronger antioxidants than AE extracts in DPPH • , FRAP and ABTS •+ reaction systems. Comparing different origins, the RSC in ABTS •+ reaction systems we can see the FR origin extracts were stronger, than the extracts from LT plants materialexcept WE extract. But in DPPH • assay, the RSC of EE and WE extracts was slightly stronger than that of ME, while AEs were approximately 6-9 times weaker radical scavengers than polar extracts (Fig.3.8). So far as polar phenolic constituents are dominant antioxidants in the majority medicinal and aromatic plants, this result is in agreement with many previously performed studies[188]. Preliminary screening of antioxidant potential indicate that non-volatile fraction of M. odorata contain relatively high amount of phenolic compounds which might be of interest for the isolation of valuable food and or nutraceutical ingredients. It also showed that phytochemical composition of plant's secondary metabolites depends on several factors, such as cultivation area, climatic conditions, vegetation phase, genetic modifications and others; therefore, evaluation of plant properties from different geographical regions is an interesting task, which was in the focus of numerous studies [188-189].

Fig. 3 . 10 .

 310 Fig. 3.10. Ferric reducing/antioxidant power (FRAP) and DPPH radical scavenging assay of different anatomical parts of T. farfara from Lithuania and France.IC 50 values of T. farfara FS, L and R extracts in DPPH • assay varied from 0.09 (L ME) to 0.88 (R AE) mg/mL in Lithuanian extracts and from 0.05 (L AE) to 0.54 (R AE) mg/mL in FR extracts. As expected, low polarity AE extracts FS and R were weaker radical scavengers than polar alcoholic extracts in DPPH • reaction that is in agreement with many previous reports[START_REF] Povilaitytė | Antioxidative activity of purple peril (Perilla frutescens L.), moldavian dragonhead (Dracocephalum moldavica L.), and roman chamomile (Anthemis nobilis L.) extracts in rapeseed oil[END_REF] 108, 185]. In contrast, another antioxidant activity screening method, applicable for both lipophilic and hydrophilic antioxidants ABTS •+ decolourisation assay, exhibited rather inverse range compared to those obtained in DPPH • reaction (Fig.3.11). For TEAC values in ABTS •+ reaction AE (FS) and roots (R) extracts were the most effective ABTS radical scavengers, than FS ME, R ME and FS EE, but R EE extract showed higher activity, than R AE. The main difference is that DPPH • can be only dissolved in organic solvents (e.g., methanol), while ABTS •+ is soluble in both aqueous and organic media. Therefore, the ABTS •+ test can be performed in hydrophilic and lipophilic systems[165].

Fig. 3 .

 3 Fig. 3.12. HPLC-DPPH • chromatograms of C. grandiflora extracts and m/z of identified compounds In LC-MS spectra rosmarinic acid (RT=30.6 min.) in a positive ionization mode gave m/z at 359 and characteristic fragmentation at m/z 161, which possessed a very high RSC (49.8% of total area of the peaks). It was described by other authors that rosmarinic acid is one of the most common caffeic acid esters occurring in the Lamiaceae family plants. The production of this compound has been investigated both in numerous Lamiaceous plants and in tissue cultures of several species. Zgorka et al. described the possible role of rosmarinic acid as the chemotaxonomic marker of the Lamiaceae family [201]. Rosmarinic acid is known as an antiviral, antibacterial, antioxidant, anti-inflammatory and immunostimulating agent [202].

3. 6

 6 ASSESSMENT OF RADICAL SCAVENGERS (RSC) BY ON-LINE HPLC-DPPH • METHOD 3.6.1 Radical scavengers in C. grandiflora extracts

Fig. 3 .

 3 Fig. 3.14. A) Structure of dicaffeoylquinic acid derivatives. B) HPLC-DPPH chromatograms of acetone (AE) extracts of T. farfara from Lithuanian (LT) and French origin (FR) The ESI-MS of the LT T. farfara FS extracts presented ions corresponding to: 1-(m/z 191)-quinic acid, 2-(m/z 354)-chlorogenic acid, 3-5 (m/z 515)-dicaffeoylquinic acid, 6-(m/z 609)-quercetin-3-rutinoside (rutin), 7-(m/z 434)-quercetin-pentoside and 8-(m/z 447)kaempferol-glucoside. FS extract of FR origin plants was qualitatively different: in this

Fig. 3 . 15 .

 315 Fig. 3.15. Technological scheme for the implementation of agrorefinery concept for the efficient processing of aromatic and medicinal plant material into different products[205]

  Water tank  Cooling water out/into  Hot water out  Vacuum/gas inlet  Condenser  Heat control  Separator Hydrodistillation is one of the simplest, oldest and primitive process known to man for obtaining essential oils from plants. Mostly used by small scale producers of essential oils In water / hydrodistillation the plant material is almost entirely covered with water as suspension in the still which is placed on a furnace Water is made to boil and essential oil is carried over to the condenser along with the steam. Useful for distillation of powders of spices and comminuted herbs etc. Extractor-ALAMBIC PRO 2 Spray drying  Air intel  Spray cylinder  Spray nozzle  Aspiration filter  Cyclone chamber  Collecting vessel  PumpSpray drying is a very fast method of drying due to the very large surface area created by the atomization of the liquid feed and high heat transfer coefficients generated. The short drying time, and consequently fast stabilization of feed material at moderate temperatures, means spray drying is also suitable for heatsensitive materials. .

FiguresFig. 1 . 1 .Fig. 1 . 2 .Fig. 1 . 3 .Fig. 1 . 4 .Fig. 1 . 5 .Fig. 1 . 6 .

 111213141516 Figures

Fig. 1 . 7 .

 17 Fig. 1.7. Chemical structure of major components of M. odorata essential oils ..................

Fig. 1 . 8 .

 18 Fig. 1.8. Two glycosides of quercetin from Korean of T. farfara ......................................

Fig. 3 . 1 .Fig. 3 . 2 .Fig. 3 . 3 .Fig. 3 . 4 .Fig. 3 . 5 .Fig. 3 . 6 .

 313233343536 Fig. 3.1. Microscopic photographs of glandular trachoma from two parts of (A) Tussilago farfara (L.) plant; (B) flower; (C1 and C2) stem. Magnification 400˟ ....Fig. 3.2. Variation of the number of VOCs (Y 1 ) versus (X 1 , X 2 ) in CFD method ................ Fig. 3.3. Variation of the number of VOCs (Y 1 , Y 2 ) versus (X 1 , X 2 ) in FAD method. A) -using FAD with CO 2 , B) -using FAD with N 2 O ............................................. Fig. 3.4. Hydrolysis of isopulegyl acetate ...................................................................... Fig. 3.5. Microscopic analysis of C. grandiflora. glandular trichome. A and C before the experiment. B after affecting with CFD and D after affecting with FAD ..............Fig. 3.6. Ferric reducing/antioxidant power (FRAP) and DPPH radical scavenging assay of C. grandiflora ............................................................................................

Fig. 3 . 7 .

 37 Fig. 3.7. TEAC values in ABTS radical cation decolourisation assay of C. grandiflora .......

Fig. 3 . 8 .

 38 Fig. 3.8. Ferric reducing/antioxidant power (FRAP) and DPPH radical scavenging assay of M. odorata from Lithuania and France .................................................

Fig. 3 . 9 .

 39 Fig. 3.9. TEAC values in ABTS radical cation decolourisation assay of M. odorata from Lithuania and France ......................................................................................

Fig. 3 . 10 .FiguresFig. 3 . 11 .Fig. 3 . 12 .Fig. 3 . 13 .Fig. 3 . 14 .Fig. 3 . 15 .

 310311312313314315 Fig. 3.10. Ferric reducing/antioxidant power (FRAP) and DPPH radical scavenging assay of different anatomical parts of T. farfara from Lithuania and France. ........

Table 3 . 6 .Table 3 . 7 .Table 3 . 8 .Table 3 . 9 .Table 3 . 10 .Table 3 . 11 .

 36373839310311 conditions and experimental responses ............................................................. Experimental domain ................................................................................... Chemical composition of essential oil of C. grandiflora and volatile compounds isolated from the leaves by CFD and FAD methods ......................... Total amount of plant phenolic compounds, flavonoids and flavonols ............... Antioxidant activity (AA) the extracts of C. grandiflora, M. odorata and T. farfara as compared with the effect of BHT and their effect on the stability of sunflower oil, expressed in protection factor values (PF) .................................... Composition and radical scavenging capacity of Myrrhis odorata constituents evaluated by the on-line HPLC-UV-DPPH • method (ni=not identified) ..................................................................................................... Composition and radical scavenging capacity of Tussilago farfara constituents evaluated by the on-line HPLC-UV-DPPH • method ........................

  Tableau R.2. Comparaison des principaux constituants d'huiles essentielles de Myrrhis odorata (L.) Scop.

	Composés	Lituanie a	France a	Finlande b	Russie c	Serbie d	Etats-Unis e
		(%)	(%)	(%)	(%)	(%)	(%)
	-Myrcène	1.7	1.8	1.1	-	-	-
	p-Cymène	0.2	0.5	-	-	62.0	-
	Limonène	1.7	0.4	0.1		2.2	0.3
	Estragole	0.9	4.1	2.6	-	-	1.7
	E-Anéthole	48.1	50.7	83.1	82.0	-	85.5
	-Caryophyllène	9.7	6.1	1.0	1.7	0.7	-
	Methyleugenol	14.3	13.1	-	-	-	9.1
	Germacrène-D	5.0	4.3	-	2.9	-	-
	E-Nerolidol	10.2	12.0	1.0	-	-	-

a Notre étude; b Uusitalo et al. 1999 [137]; c Tkachenko et al. 1993 [139]; d Rančić et al. 2005 [141]; e Hussain et al. 1990 [140].

  ). Ces résultats sont en accord avec les résultats publiés précédemment lesquels montrent que les solvants polars extraient plus de composants antioxydants que les solvants de polarité faible[START_REF] Povilaitytė | Antioxidative activity of purple peril (Perilla frutescens L.), moldavian dragonhead (Dracocephalum moldavica L.), and roman chamomile (Anthemis nobilis L.) extracts in rapeseed oil[END_REF] 108, 185]. Le méthanol et l'éthanol, comme des solvants organiques sont relativement similaires, mais la capacité à piéger les radicaux libres dans l'extrait EE ont été plus élevés que ME dans la méthode ABTS •+ . Néanmoins, l'éthanol est un solvant de qualité alimentaire donc son utilisation est préférable que le méthanol qui est toxique. Il faut aussi noter qu'en DPPH • , la réaction de la capacité à piéger les radicaux libres des extraits alcoolisés obtenus des résidus solides a été assez similaire à celui de la capacité à piéger les radicaux libres des extraits de l'eau, 0.23 (ME), 0.22 (EE), 0.22 (FD) et 0.18 (SD) mg/mL, respectivement. Ce qui est scientifique que

certains composés actifs antioxydants ne se dissolvent pas dans l'eau bouillante et donc ils restent dans les résidus solides. Par ailleurs, au travers de la méthode FRAP, il est possible d'appliquer aux trois extraits aqueux, alcoolisé et de l'acétone de différentes plantes. Dans cette méthode, l'activité antioxydante est déterminée sur la base de la capacité à réduire de l'état ferrique (Fe 3+ ) à l'état ferreux (Fe 2+ ). Cette réduction est définie en mg de ferreux par mL d'échantillon. Dans la méthode FRAP, un sel ferrique (Fe III) est utilisé comme un antioxydant et son potentiel d'oxydoréduction (0.70 V) est comparable à celui de ABTS •+ (0.68 V), et dont, par conséquent, il n'y a pas beaucoup de différences entre les méthodes TEAC et FRAP [186]. Pourtant, les résultats obtenus pour les extraits C. grandiflora d'avec ces deux méthodes étaient assez différentes. Cela montre que le type de test joue un rôle plus important dans la mesure de l'activité antioxydante. Ainsi, le pouvoir de réduction des différends extraits était entre 0.24-0.68 mg Fe (II)/mL (Fig. R.4). La différence la plus remarquable a été observée pour l'AE, lequel était plus faible antioxydant dans la méthode TEAC (Fig. R.5) à lorsque la méthode FRAP, AE l'agent réducteur ls plus forte. ME et AE produits avec les résidus solides de l'hydrodistillation des plantes ont été remarquablement élevée qu'avec les extraits séchées. Les différences observées entre les méthodes DPPH/ABTS et FRAP peuvent être expliqués pour les solvants avec une différence en sa polarité et un pH dans du milieu réactionnel [187].

  antioxydante de M. odorata a été testée via la méthode DPPH • qui a montré que les AE et ME extraits des plantes d'origine FR ont une activité supérieure à celles de la même famille botanique, mais de différentes origines (LT). De plus, la méthode FRAP a montré que les extraits LT ME, EE et WE ont une activité antioxydante plus élevée en comparaison avec les extraits FR. Ainsi, en considérant les résultats de l'analyse DDPH • , il est important de mentionner que n'existe pas une similitude significative entre les mêmes plantes de différents climats et origine (FR et LT). En revanche, les résultats obtenus de l'analyse FRAP montrent que les plantes localisées en LT une activité antioxydante supérieure à ceux localisés dans le

	Midi-Pyrénées.

6. Activité antioxydant de M. odorata de la Lituanie et la France xix L'activité Les informations précédentes ont démontré que la mesure de la capacité à piéger les radicaux libres dans ce modèle est une méthode fiable pour évaluer les propriétés antioxydante des plantes. Pourtant, il est bien connu que les antioxydants agissent très différemment avec les différents radicaux libres. En conséquence, afin d'obtenir plus d'informations sur la capacité à piéger les radicaux libres, nous avons présenté un deuxième modèle (ABTS •+ ) pour tester cette capacité. Ainsi, un test avec l'ABTS •+ de décoloration de

  Le test FRAP des extraits des différentes parties des plantes ont été entre le 0.18-0.77 mg Fe (II)/mL dans l'extrait LT et entre 0.15-0.68 mg Fe (II)/mL dans l'extrait FR.

•+ peut être appliquée aux extraits lipophiles. Pour les valeurs TEAC dans la méthode ABTS •+ , les extraits AE (FS) et R ont été les radicaux libres les plus actives que dans les extraits FS ME, R ME et FS EE, mais l'extrait R EE a montré une activité supérieure à R AE. La principale différence ce que DPPH • peut être dissous seulement dans les solvants organiques (méthanol), alors qu'ABTS •+ est soluble en milieu aqueux et organique. En conséquence, le test ABTS •+ peut être réalisé dans systèmes hydrophiles et lipophiles [165]. xxii L'activité antioxydante des extraits de FS mesurés avec le test FRAP a montré la même correspondance que ABTS •+ . Dans le test FRAP le sel ferrique Fe (III) a été utilisé comme antioxydant et son potentiel redox est comparable à celui d'ABTS •+ . En conséquence, les résultats des tests TEAC et FRAP ont montré de tendances similaires à celles indiqués dans les travaux précédemment [186]. Les extraits FS AE les antioxydants ont été remarquablement actifs que dans les autres extraits (FR ME et FR EE), mais pour les extraits de L, aucune relation existait entre ABTS •+ et FRAP, LT L EE a montré une activité élevée en ABTS •+ et une faible activité in FRAP, la différence observée peut-être explique pour la différence entre le pH dans la réaction dans le milieu media (pH FRAP =3.6 et pH ABTS•+ =7.4).

  Tableau R.4. Activité antioxydante (AA) des résidus de l'hydrodistillation comparée à l'efficacité du BHT

	(Continuation du Tableau R.4)
						Origine Origine
	Plant Plant C. grandiflora T. farfara-FS M. odorata T. farfara-L T. farfara -R	Solvent Solvent Conc % Conc AE 0.20 % 0.10 0.20 2.72±0.02 2.54±0.01 1.30±0.01 1.22±0.00 Lituanie France Lituanie France IP PF Lituanie France Lituanie France -2.68±0.01 -1.34±0.00 IP PF -2.72±0.02 -1.36±0.01 0.05 -2.48±0.00 -1.24±0.00 AE 0.10 2.61±0.03 2.50±0.01 1.25±0.02 1.20±0.00 ME 0.20 -2.81±0.01 -1.41±0.01 0.05 2.56±0.02 2.34±0.03 1.22±0.01 1.12±0.02 0.10 -2.71±0.03 -1.36±0.02 0.05 -2.58±0.02 -1.29±0.01 EE 0.20 -2.98±0.01 -1.49±0.00 0.10 -2.93±0.03 -1.47±0.02 0.05 -2.78±0.00 -1.39±0.00 FD 0.20 -2.21±0.01 -1.31±0.00 ME 0.20 2.57±0.01 2.67±0.01 1.23±0.01 1.28±0.01 0.10 2.49±0.01 2.51±0.01 1.19±0.01 1.20±0.01 0.05 2.46±0.01 2.38±0.01 1.18±0.01 1.14±0.01 EE 0.20 2.59±0.01 2.75±0.01 1.24±0.01 1.32±0.01 0.10 2.55±0.01 2.64±0.01 1.22±0.01 1.26±0.01 0.05 2.48±0.00 2.53±0.01 1.19±0.00 1.21±0.00 0.10 -2.02±0.01 -1.27±0.00 0.20 2.80±0.03 2.82±0.01 1.40±0.03 1.41±0.01 0.05 -2.01±0.00 -1.15±0.00 AE 0.10 2.62±0.01 2.64±0.01 1.31±0.01 1.32±0.01 SD 0.20 -2.78±0.03 -1.39±0.02 0.10 -2.51±0.02 -1.26±0.01 0.05 -2.37±0.01 -1.19±0.00 AE 0.20 2.17±0.03 2.19±0.01 1.09±0.02 1.10±0.01 0.08±0.02 0.09±0.01 Lituanie France Lituanie France AA -AA 0.35±0.00 -0.32±0.01 0.23±0.00 0.33±0.00 -0.26±0.02 0.21±0.00 0.23±0.00 -0.24±0.01 0.13±0.02 0.39±0.00 -0.34±0.01 -0.28±0.00 -0.47±0.00 -0.45±0.01 -0.38±0.00 -0.24±0.00 0.29±0.00 0.20±0.00 0.21±0.00 0.19±0.00 0.15±0.00 0.25±0.00 0.33±0.00 0.23±0.00 0.28±0.00 0.20±0.00 0.22±0.00 0.29±0.00 -0.39±0.03 0.40±0.01 0.26±0.00 -0.30±0.01 0.31±0.01 0.14±0.00 -0.38±0.01 -0.25±0.00 -0.18±0.00 0.10 0.05 2.58±0.01 2.59±0.00 1.29±0.01 1.30±0.00 0.28±0.01 0.29±0.00 ME 0.20 2.32±0.01 2.29±0.01 1.16±0.01 1.15±0.01 0.15±0.01 0.14±0.01 0.10 2.30±0.01 2.21±0.01 1.15±0.01 1.11±0.01 0.14±0.01 0.10±0.01 0.05 2.28±0.02 2.18±0.02 1.14±0.02 1.09±0.02 0.14±0.02 0.09±0.02 0.20 2.46±0.01 2.39±0.01 1.23±0.01 1.20±0.01 0.22±0.01 0.19±0.01 2.09±0.02 2.11±0.01 1.05±0.01 1.06±0.01 0.04±0.01 0.05±0.01 0.05 EE 0.10 2.40±0.01 2.36±0.01 1.20±0.01 1.18±0.01 0.19±0.01 0.17±0.01 2.05±0.02 2.07±0.01 1.03±0.01 1.04±0.01 0.02±0.01 0.03±0.01 ME 0.20 0.05 2.30±0.01 2.28±0.01 1.15±0.01 1.14±0.01 0.14±0.01 0.14±0.01 2.19±0.01 2.10±0.01 1.10±0.00 1.05±0.01 0.09±0.00 0.05±0.01 0.10 0.20 2.62±0.00 2.53±0.01 1.31±0.00 1.27±0.01 0.30±0.00 0.26±0.01 2.07±0.00 2.02±0.01 1.04±0.00 1.01±0.01 0.03±0.00 0.01±0.01 0.05 AE 0.10 2.39±0.01 2.48±0.01 1.20±0.01 1.24±0.01 0.19±0.01 0.23±0.01 2.01±0.01 2.00±0.01 1.01±0.01 1.00±0.00 0.00±0.00 0.00±0.01 EE 0.20 2.30±0.00 2.30±0.01 1.15±0.01 1.15±0.00 0.14±0.01 0.14±0.01 0.10 2.10±0.01 2.20±0.01 1.05±0.01 1.10±0.01 0.05±0.01 0.10±0.01 0.05 2.02±0.01 2.07±0.01 1.01±0.01 1.04±0.01 0.01±0.01 0.03±0.01 WE 0.20 2.36±0.04 2.42±0.01 1.18±0.01 1.21±0.01 0.17±0.01 0.20±0.01 0.10 0.05 2.26±0.02 2.30±0.01 1.13±0.02 1.15±0.01 0.13±0.02 0.14±0.01 ME 0.20 2.41±0.01 2.45±0.01 1.21±0.01 1.23±0.01 0.20±0.01 0.22±0.01 0.10 2.38±0.01 2.40±0.00 1.19±0.01 1.20±0.00 0.18±0.01 0.19±0.00 0.05 2.31±0.01 2.33±0.01 1.12±0.01 1.17±0.01 0.15±0.01 0.16±0.01 0.20 2.58±0.03 2.60±0.01 1.29±0.03 1.30±0.01 0.28±0.03 0.29±0.01 2.21±0.01 2.34±0.01 1.11±0.01 1.17±0.01 0.10±0.01 0.16±0.01 0.05 EE 0.10 2.50±0.01 2.51±0.01 1.25±0.01 1.26±0.01 0.24±0.01 0.25±0.01 2.19±0.01 2.29±0.01 1.10±0.01 1.15±0.01 0.09±0.01 0.14±0.01 0.05 2.42±0.01 2.46±0.01 1.21±0.01 1.23±0.01 0.20±0.01 0.22±0.01
	BHT		0.02±0.00	4.07±0.00	2.04±0.00	1.00±0.00
	L'huile tournesol	de	0.00±0.00	2.00±0.00	1.00±0.00	-
							xxv

TABLE OF CONTENTS 1 LITERATURE REVIEW ................................................................................................... 1.1 Diversity of aromatic and medicinal plants ............................................. 1.2 General characterization of essential oils ............................................... 1

 OF .2.1 Sources of essential oils .............................................................................. 1.2.2 Factors influencing the production and the quality of essential oil ............... 1.2.3 Extraction Methods for Recovery of Volatile Organic Compounds ..............

1.3 Antioxidants ............................................................................................... 1

  .3.1 Plant residues ............................................................................................ 1.3.2 Valorization of plant processing by-products ............................................. 1.3.3 Natural bioactive compounds from crop by-products ................................. 1.3.4 Structure-activity of phenolic compounds .................................................. 1.3.5 Polyphenol extraction from by-products .....................................................

1.4 Review on selected aromatic plants ...................................................... 1

  .4.1 Calamintha grandiflora (L.) Moench ........................................................... 1.4.2 Myrrhis odorata (L.) Scop. ......................................................................... 1.4.3 Tussilago farfara L. ....................................................................................

2 MATERIALS AND METHODS ..................................................................................... 2.1 Solvents and Reagents ........................................................................... 2.2 Plant material ........................................................................................... 2.3 Preparation of plant extracts and isolation of essential oils ............... 2.4 Analysis of essential oils (volatile extracts) ..........................................

	2.4.1 GC-FID and GC-MS analysis of oil constituents ........................................
	2.5

Direct evaluation of volatile compounds in fresh plants leaves ..........

	2.5.1 Design of Flash Aroma Dispenser (FAD) method ......................................
	2.5.2 Crushing Finger Device (CFD) method ......................................................
	2.5.3 Methodology of experimental design for FAD and CFD techniques ..........
	2.6

Sample preparation (free hand sectioning method) ............................. 2.7 Assessment of non-volatile extracts and antioxidant activity ............. xxxvi 2

  .7.1 DPPH • scavenging assay .......................................................................... 2.7.2 ABTS •+ decolourisation assay .................................................................... 2.7.3 Ferric reducing/antioxidant power (FRAP) assay ....................................... 2.7.4 Analysis of total phenolic compounds (TPC) ............................................. 2.7.5 Determination of flavonoids ....................................................................... 2.7.6 Determination of flavonols ......................................................................... 2.7.7 Measurement of the inhibition effect of plant extracts on oil oxidation in Oxipres apparatus ..................................................................................... 2.7.8 On-line HPLC-DPPH assay ...................................................................... 2.7.9 HPLC and LC-MS analysis ........................................................................

2.8 Statistical analysis ................................................................................... 3 RESULTS AND DISCUSSION ....................................................................................... 3.1 Investigation of aromatic plants collected from two geographical locations ................................................................................................... 3

  .1.1 Essential oil composition of Myrrhis odorata (L.) Scop. leaves grown in Lithuania and France ................................................................................. 3.1.2 Essential oil composition of Tussilago farfara L) from Lithuania and France………….. .......................................................................................

3.2 Response analysis and interpretation of CFD and FAD data ..............

	3.2.1 Rapid evaluation of volatile compounds in fresh plants leaves by the two
	novel techniques Crushing Finger Device (CFD) and Flash Aroma
	Dispenser (FAD) ........................................................................................
	3.2.2 Microscopic analysis of samples after CFD and FAD extraction ................

3.3 Antioxidant activity of plant hydrodistillation residues .......................

  Antioxidant activity in different anatomic parts of T. farfara from two different geographical origins ...................................................................................

	3.3.1 Antioxidant properties of deodorized residues extracts of C. grandiflora in
	electron/hydrogen transfer based assays ..................................................
	3.3.2 Antioxidant properties of deodorized residues of M. odorata from two
	different geographical origins in electron/hydrogen transfer based assays
	3.3.3

3.4 Content of total phenolic compounds (TPC), flavonoids and flavonols …………………………………………………………………………………….66 3.5 Antioxidant activity of extracts in sunflower (Helianthus annuus) oil.70 3.6 Assessment of radical scavengers (RSC) by on-line HPLC-DPPH • method…………. ...................................................................................... 3

  .6.1 Radical scavengers in C. grandiflora extracts ............................................ 3.6.2 Phytochemical composition and RSC of M. odorata constituents .............. Evaluation of active compounds from T. farfara .........................................

	3.6.3
	xxxvii

3.7 The possibilities of technological implementation ............................... 4 CONCLUSION .................................................................................................................. REFERENCES .................................................................................................................................... TECHNICAL SPECIFICATION ....................................................................................................... FIGURES........................................................................................................................................... TABLES ............................................................................................................................................ PUBLICATIONS ET PARTICIPATION À DES CONGRÈS ......................................................

Table 1 . 2 .

 12 Different parts of a plant which can be used for the isolation essential oil

	Leaves	Flowers	Peel	Seeds		Wood
	Basil	Chamomile	Bergamot	Almond		Camphor
	Bay leaf	Clary sage	Grape fruit	Nutmeg oil	Cedar
	Cinnamon	Clove	Lemon			Rosewood
	Eucalyptus	Geranium	Lime			Sandalwood
	Lemon grass	Hyssop	Orange
	Melaleuca	Jasmine	Tangerine
	Oregano	Lavender
	Patchouli	Manuka
	Peppermint	Marjoram
	Pine	Orange
	Rosemary	Rose
	Spearmint	Ylang-ylang
	Tea tree
	Wintergreen
	Thyme
	Lovage
	Parsley
	Pimento
	Berries	Bark	Resins	Root	Rhizome
	Allspice	Cassia	Frankincense	Valerian	Ginger
	Anise	Cinnamon	Myrrh	Lovage
	Celery			Parsley
	Cumin
	Fennel
	Juniper
	Pimento

Table 1 . 3 .

 13 Classes of phenolic compounds in plants

	Class

Table 2 .1. Investigated plants

 2

	Plant name	Family	Anatomical	Hervesting	Origin
			part	stage
	Calamintha grandiflora Lamiaceae Leaves	Full flowering	France
	Myrrhis odorata	Apiaceae Leaves	After flowering France, Lithuania
	Tussilago farfara	Asteraceae Flowers,	Flowering, after	France, Lithuania
			stems, leaves,	flowering
			roots

Table 2 . 2 .

 22 Doehlert's experimental matrix and experimental domain

	No. exp.	X 1 1ij	X 2 2 ij
	1	1	0
	2	-1	0
	3	0.5	0.866
	4	-0.5	-0.866
	5	0.5	-0.866
	6	-0.5	0.866
	7	0	0
	8	0	0
	9	0	0

Table 3 . 1 .

 31 Essential oil composition (%) of Myrrhis odorata (L.) Scop. leaves from Lithuania and France.

	No Compounds	RI th RI  Content GC area	Odor perception c
			(%)
	1 α-Pinene 2 β-Myrcene * 3 p-Cymene	Lithuania France 939 936 0.1±0.0 a 0.1±0.0 a fresh, camphor, sweet pine, earthy, woody 992 986 1.7±0.1 a 1.8±0.1 a peppery, terpene, spicy, balsam, plastic 1027 1022 0.2±0.0 a 0.5±0.1 a fresh, citrus, terpene, woody, spice
	4 Limonene	1029 1030 1.7±0.0 a 0.4±0.0 b citrus, fresh, peppery
	5 E-β-Ocimene 6 γ-Terpinene	1038 1032 0.3±0.1 b 0.8±0.0 a sweet, herbal, green, celery spicy, floral 1074 1058 0.3±0.0 b 0.6±0.1 a oily, woody, terpene, lemon, tropical, herbal
	7 cis-Thujone 8 Nonanal	1102 1092 1104 1103 0.2±0.0 b 0.4±0.0 a aldehydic rose, fresh, orange peel, fatty peely -0.2±0.0 thujonic, cedarleaf
	9 Camphor	1139 1131	-	0.3±0.0 medicinal, camphoreous
	10 iso-Menthone	1162 1168	-	0.2±0.1	powerful, refreshing cleanminty, cool, peppermint
	11 Pinocarveol	1169 1169	-	0.5±0.0 herbal, camphor, woody, pine, balsam
	12 Carvacrol	1192 1192	-	0.4±0.0 spice, woody, camphor, thymol
	13 Estragole *	1200 1197 0.9±0.1 b 4.1±0.4 a sweet, licorice, anise spice, green herbal fennel
	14 p-Anisaldehyde 1263 1271 15 E-Anethole * 1285 1290 48.1±1.4 a 50.7±1.1 a sweet, green, anethole herbal -sweet, powdery, mimosa, floral, hawthorn, 0.3±0.0 balsam
	16 Methyleugenol * 1407 1401 14.3±0.5 a 13.1±0.4 a sweet, fresh, warm, spicy, clove, carnation, cinnamon
	17 β-Caryophyllene 1419 1409 9.7±0.3 a 6.1±0.3 b sweet, woody, spice, clove dry
	18 α-Bergamotene 1431 1430 0.9±0.1 a 0.5±0.0 b woody, warm, tea 19 α-Humulene 1467 1459 0.9±0.2 a 0.5±0.0 a woody, herbal 20 Germacrene-D 1487 1485 5.0±0.1 a 4.3±0.3 a woody, spice 21 β-Bisabolene 1498 1496 0.1±0.0 a 0.1±0.0 a balsamic, citrus, myrrh, opoponax, spicy
	22 α-Farnesene	1500 1504 0.3±0.0 a 0.2±0.0 b citrus, herbal, lavender, bergamot, myrrh, neroli, green
	23 E-Nerolidol *	1563 1559 10.2±0.8 a 12±0.3 a floral, green, waxy, citrus, woody
	24 Spathulenol 25 Hexadecanol	1619 1618 2.1±0.5 a 0.7±0.0 a earthy, herbal, fruity 1870 1864 1.2±0.2 a 0.7±0.0 a waxy, floral
	26 Phytol	2128 2125 1.4±0.1 a 0.4±0.0 b delicate, floral, balsam, powdery, waxy
	Total		99.6±0.2 99.9±0.1
	Phenylpropanoids (%) Monoterpene hydrocarbons (%) 4.1±0.1 a 3.7±0.1 a 63.3±0.7 a 68.2±0.6 a
	Oxygenated monoterpenes (%) Sesquiterpene hydrocarbons (%) 16.9±0.1 a 11.7±0.1 b -1.2±0.0 Oxygenated sesquiterpenes (%) 12.3±0.4 a 12.7±0.3 a Others (%) 3.0±0.1 a 2.4±0.1 b
	RI  : Retention indices relative to C8-C22 alkanes on AnHP-5MS column RI th : index presented from from database http://www.flavornet.org * authentic standards were co-injected for the compounds identification c Odour description adapted from www.thegoodscentscompany.com
	Values with different letters (a-b) are significantly different at P < 0.05

Table 3 . 2 .

 32 Comparison of main essential oil constituents in Myrrhis odorata (L.) Scop. leaves from different geographic origins

	Compound	Lithuania a	France a	Finland b	Russia c	Serbia d	North USA e
		(%)	(%)	(%)	(%)	(%)	(%)
	-Myrcene	1.7	1.8	1.1	-	-	-
	p-Cymene	0.2	0.5	-	-	62.0	-
	Limonene	1.7	0.4	0.1		2.2	0.3
	Estragole	0.9	4.1	2.6	-	-	1.7
	E-Anethole	48.1	50.7	83.1	82.0	-	85.5
	-Caryophyllene	9.7	6.1	1.0	1.7	0.7	-
	Methyleugenol	14.3	13.1	-	-	-	9.1
	Germacrene-D	5.0	4.3	-	2.9	-	-
	E-Nerolidol	10.2	12.0	1.0	-	-	-
	a Our study; b Uusitalo et al. 1999 [137]; c Tkachenko et al. 1993 [139]; d Rančić et al. 2005 [141];

e

Hussain et al. 1990 [140]

.

Table 3 . 3 .

 33 Chemical composition (%) of flower and peduncle essential oils of Tussilago farfara from two origins

	No Compounds	R.I. ap	R.I. p	Content (%)
	1 2	1-Nonene α-Pinene	890 935	922 1024	Lithuania 8.1±0.3 b 3.7±01 a	France 34.1±0.0 a 4.2±0.2 a
	3	Camphene	951	1068	-	tr
	4 5 6 7 8	1-Decene α-Phellandrene p-Cymene n-Undecane 1-Dodecene	988 1004 1029 1100 1191	1040 1163 1270 1100 1190	-0.8±0.1 a 0.1±0.0 b 7.8±0.2 a 0.2±0.0 b	2.1±0.0 1.4±0.3 a 0.8±0.0 a 4.8±0.1 b 0.5±0.0 a
	9 10 1-Tridecene 1-Decanol	1273 1291	1275 1365	0.1±0.0 0.1±0.0 b	-0.3±0.0 a
	11 α-Copaene 12 β-Cubebene	1376 1387	1489 1522	0.2±0.0 0.2±0.0 b	tr 0.3±0.0 a
	13 1-Tetradecene 14 β-Caryophyllene 15 γ-Elemene 16 α-Humulene 17 Germacrene D 18 β-Selinene	1389 1419 1439 1454 1485 1489	1414 1589 1470 1665 1689 1696	0.9±0.1 1.9±0.1 a 0.2±0.0 b 0.4±0.0 a 0.7±0.1 a 0.7±0.0 a	-0.3±0.0 b 0.4±0.0 a 0.2±0.0 b 0.6±0.0 a 0.6±0.1 a
	19 n-Pentadecane 20 β-Bisabolene 21 δ-Cadinene 22 Spathulenol 23 Caryophyllene oxide	1500 1509 1522 1576 1585	1500 1715 1737 2128 1918	0.5±0.1 1.2±0.1 a 0.4±0.0 a 2.1±0.1 a 1.3±0.1 a	-0.7±0.1 b 0.2±0.0 b 0.7±0.1 b 0.2±0.0 b
	24 Tetradecanol	1680	2177	-	0.2±0.0
	25 n-Nonadecane 26 Phytol	1900 2112	1900 2547	0.4±0.0 1.4±0.1 a	-0.4±0.0 b
	27 n-Tricosane	2300	2300	21.7±0.1	-
	28 n-Tetracosane	2400	2400	0.7±0.0	-
	29 n-Pentacosane	2500	2500	1.3±0.0	-
	30 n-Hexacosane Total	2600	2600	5.1±0.1 62.2±0.1 a	-53.0±0.5 b
	(ap) Retention indices relative to C8-C28 alkanes on DB5 column (p) Retention indices relative to C8-C28 alkanes on Carbowax 20M column
	Values with different letters (a-b) are significantly different at P <0.05
	tr-trace <0.05%

Table 3 . 4 .

 34 Comparison of main essential oil constituents of Tussilago farfara (L.) from different plant parts and different geographic origins

				Lithuania			France China	Romania
	Compound	Ka-FS	V-F	flower V-S	Ke-F	Ke-S	M-FS	buds Ku-B	leaves C-L
	1-Nonene	8.1	-	-	-	-	34.1	0.1	-
	α-Pinene	3.7	tr	-	-	1.4	4.2	-	-
	n-Undecane	7.8	-	1.8	-	3.9	4.8	4.8	-
	n-Tricosane	21.7	21.6	22.7	19.4	7.2	-	-	-
	β-Bisabolene	1.2	0.6	-	0.8	-	0.7	13.9	-
	n-Pentadecane	0.5	0.7	0.4	1.0	0.4	-	4.6	-
	Spathulenol	2.1	2.0	1.6	3.8	2.2	0.7	3.4	-
	n-Hexadecanoic acid	-	-	-	-	-	-	3.1	-
	E-Cycloundecene	-	-	-	-	-	-	8.5	-
	α-Bisabolol	-	-	-	-	-	-	-	20.7
	n-Hexacosane	5.1	4.4	5.8	4.5	4.9	-	-	-
	n-Pentacosane	1.3	9.4	2.0	9.0	4.0	-	1.0
	n-Tetracosane	0.7	2.1	-	1.8	-	-	0.16	-
	Chamazulene	-	-	-	-	-	-	-	7.2
	Phytol	1.4	1.8	2.1	2.5	2.4	0.4	0.3	-
	References	This study		[149]		This study	[148]	[176]
	Collection area: [Ka] Kaunas, [V] Vilnius, [Ke] Kedainiai, [M] Midi-pyrénées, [Ku] Kuandonghua, [C] Câmpeni.
	Plant part: [FS] Flower and stem, [F] Flower, [S] Stem, [B] Bud, [L] Leave.
	tr-trace <0.05%

Table 3 . 5 .

 35 Doehlert's experimental matrix and the corresponding experimental conditions and experimental responses

				CFD
	No. exp.	Number of crushing X 1 1	Power of piston 2 X 2	Number of crushing	W Power of piston,		VOCs Y 1 a
	1	1	0	20	2.6		22
	2	-1	0	0	2.6		0
	3	0.5	0.866	15	4.3		18
	4	-0.5	-0.866	5	0.9		5
	5	0.5	-0.866	15	0.9		10
	6	-0.5	0.866	5	4.3		12
	7	0	0	10	2.6		22
	8	0	0	10	2.6		22
	9	0	0	10	2.6		22
	No. exp.	Temperature X 1 1	Pressure X 2 2	FAD Temperature, °C	Pressure, bar	VOCs Y 1 c	VOCs Y 2 d
	1	1	0	26	6	19	12
	2	-1	0	8	6	5	5
	3	0.5	0.866	22	12	20	12
	4	-0.5	-0.866	13	0	0	0
	5	0.5	-0.866	22	0	0	0
	6	-0.5	0.866	13	12	17	8
	7	0	0	17	6	18	10
	8	0	0	17	6	18	10
	9	0	0	17	6	18	10

a Number of volatile organic compounds (VOCs) using CFD method.

1

Number of crushing in coded variable.

2

Power of piston in coded variable.

c Number of VOCs using FAD with N2O.

d Number of VOCs using FAD with CO2.

Table 3 .6. Experimental domain CFD

 3

		Factors	Units	Centre	Step
	U 1	Number of crushing	-	10	10
	U 2	Power of piston	W	2.6	2
			FAD
	U 1	Temperature	°C	17	9
	U 2	Pressure	bar	6	7

Table 3 . 7 .

 37 Chemical composition of essential oil of C. grandiflora and volatile compounds isolated from the leaves by CFD and FAD methods

					Peak area
	No Compounds
		RI a	RI th	Essential oil% CFD% FAD-N 2 O% FAD-CO 2 %
	Hexanal	802	802	-	1.48±0.00 0.69±0.00	-
	α-Thujene	930	931	-	4.99±0.08 10.50±1.00 16.78±0.75
	α-Pinene	934	939	0.03±0.00 0.94±0.00 0.63±0.00	1.14±0.00
	Sabinene	973	975	0.19±0.01 5.69±0.65 3.50±0.05 10.90±0.90
	β-Pinene	980	979	0.21±0.02 0.95±0.00 0.95±0.00	1.44±0.30
	Myrcene	986	991	0.09±0.00 3.33±0.03 0.61±0.00	-
	3-Octanol	993	991	0.25±0.00 0.35±0.00 0.07±0.00	0.80±0.00
	α-Phellandrene	1004 1005	-	0.07±0.00 3.26±0.00	4.95±0.07
	α-Terpinene	1018 1019	-	3.42±0.01 0.33±0.00	-
	Limonene	1029 1029	0.71±0.03 1.14±0.00 1.11±0.06	1.14±0.00
	cis-β-Ocimene	1033 1037	0.08±0.00	-	-	-
	Terpinolene	1070 1089	0.03±0.00 0.75±0.00 1.62±0.10	2.81±0.00
	Linalool	1099 1091	0.04±0.00	-	-	-
	p-Menth-8-en-3-ol	1150 1150	0.24±0.01	-	-	-
	Menthone	1158 1153	4.40±0.05 1.36±0.09 0.92±0.00	-
	Isomenthone	1169 1162 34.07±0.00 15.99±0.15 8.35±0.70	4.61±0.60
	Isomenthol	1181 1183	7.65±0.10 1.85±0.01 1.62±0.06	-
	Neo-isomenthol	1193 1187 19.83±0.09 1.82±0.00 15.82±0.85 11.05±0.09
	Pulegone	1243 1237 19.54±0.50 51.75±0.60 12.07±0.65 7.63±0.03
	Piperitone	1255 1253	1.28±0.01	-	-	-
	Isomenthyl acetate	1304 1305	0.03±0.00	-	-	-
	Copaene	1362 1377	0.04±0.00 0.13±0.00	-	-
	β-Bourbonene	1369 1388	0.13±0.00 0.24±0.00	-	-
	β-Caryophyllene	1397 1409	1.99±0.04 1.23±0.02 0.42±0.00	0.42±0.00
	Santalene	1443 1447	0.37±0.01	-	-	-
	Germacrene-D	1477 1485	3.55±0.15 0.32±0.03 0.31±0.00	-
	Bicyclogermacrene	1495 1500	1.78±0.03	-	-	-
	α-Farnesene	1501 1506	0.11±0.00 0.55±0.00	-	-
	γ-Cadinene	1518 1514	0.12±0.02 0.67±0.00 0.10±0.00	-
	Isoeugenyl acetate	1578 1568	0.93±0.00	-	-	-
	Caryophyllene oxide	1584 1583	0.43±0.00	-	0.20±0.00	-
	α-Cadinol	1655 1654	0.2±0.00	-	-	-
	Total			98.32±0.00 99.02±0.00 63.08±0.00 63.67±0.00
	Monoterpene hydrocarbons (%)		1.34	21.28	22.25	39.96
	Oxygenated monoterpenes (%)		87.05	72.77	38.78	23.28
	Sesquiterpene hydrocarbons (%)		8.09	3.14	0.83	0.42
	Oxygenated sesquiterpenes (%)		0.63	-	0.20	-
	Others (%)			1.21	1.83	1.02	-
	a -Retention indices relative to C8-C22 alkanes on polar DB-5column.
	th-relative retention indices (literature values [157]).

Table 3 . 8 .

 38 Total amount of plant phenolic compounds, flavonoids and flavonols Superscript letters (a-b) indicate if the values for the extracts isolated with the same solvent from Lithuanian and French origin plants are statistically different (P≤0.05); Values are mean ± SD of triplicate measurements.

	Plant	Part of the plant	Extraction solvent	Total phenolic compounds (TPC), mg GAE/g LT FR	Total flavonoids mg/g plant extract ( in RE) LT FR	Total flavonols mg/g plant extract (in RE) LT FR
			Acetone (AE)	-	28.01±0.12	-	1.80±0.03	-	1.18±0.01
			Methanol (ME)	-	64.80±0.22	-	4.30±0.09	-	0.88±0.02
	C. grandiflora	Leaves (L)	Ethanol (EE)	-	82.80±1.02	-	2.44±0.17	-	0.75±0.01
			Water (FD)	-	58.90±0.72	-	0.79±0.05	-	0.03±0.00
			Water (SD)	-	72.78±1.11	-	1.24±0.05	-	0.10±0.01
	M. odorata	Leaves (L)	Acetone (AE) Methanol (ME) Ethanol (EE) Water (WE)	10.78±0.02 a 62.32±0.08 a 80.60±0.97 b 35.55±0.06 a	39.19±0.32 b 85.25±1.32 b 68.97±0.30 a 55.03±0.04 b	1.87±0.08 a 1.63±0.02 a 4.00±0.31 a 0.36±0.02 b	2.07±0.04 b 9.61±0.78 b 4.15±0.71 a 0.23±0.06 a	0.27±0.02 a 1.36±0.01 b 0.98±0.02 b 0.34±0.01 b	0.87±0.09 b 0.61±0.04 a 0.75±0.08 a 0.08±0.00 a
		Flower and Stem (FS)	Acetone (AE) Methanol (ME) Ethanol (EE)	94.79±0.35 a 94.55±0.22 b 66.45±0.15 b	123.16±0.12 b 9.20±0.17 a 66. 22±0.30 a 5.95±0.03 a 48.79±0.22 a 3.25±0.09 b	9.18±0.03 a 5.12±0.09 a 2.20±0.07 a	0.70±0.01 b 0.53±0.02 b 0.39±0.01 b	0.49±0.01 a 0.27±0.02 a 0.19±0.01 a
	T. farfara	Leaves (L)	Acetone (AE) Methanol (ME) 144.33±1.20 a 204.57±1.54 b 12.16±0.15 b 11.02±0.30 a 102.00±0.02 a 151.36±1.87 b 7.91±0.30 a 8.21±0.06 a Ethanol (EE) 157.25±0.08 b 131.88±0.99 a 10.96±0.15 b 7.81±0.30 a	0.96±0.03 b 1.73±0.02 b 1.98±0.05 b	0.86±0.02 a 0.82±0.03 a 0.42±0.02 a
		Roots (R)	Acetone (AE) Methanol (ME) Ethanol (EE)	6.06±0.01 a 41.08±0.78 a 38.87±0.65 a	13.58±0.01 b 41.64±0.03 a 41.30±0.06 b	0.47±0.02 b 0.35±0.01 b 0.47±0.03 b	0.29±0.02 a 0. 22±0.00 a 0.22±0.02 a	0.25±0.01 b 0.17±0.01 b 0.19±0.02 b	0.01±0.00 a 0.14±0.00 a 0.12±0.00 a

Table 3 . 9 .

 39 Antioxidant activity (AA) of the extracts of C. grandiflora, M. odorata and T. farfara as compared with the effect of BHT and their effect on the stability of sunflower oil, expressed in protection factor values(PF)

						Plant origin
	Plant	Solvent	Conc	Lithuania France	Lithuania France	Lithuania France
			%	IP			PF		AA
			0.20	-	2.68±0.01	-	1.34±0.00	-	0.35±0.00
		AE	0.10	-	2.72±0.02	-	1.36±0.01	-	0.33±0.00
			0.05	-	2.48±0.00	-	1.24±0.00	-	0.23±0.00
			0.20	-	2.81±0.01	-	1.41±0.01	-	0.39±0.00
		ME	0.10	-	2.71±0.03	-	1.36±0.02	-	0.34±0.01
	C. grandiflora	EE	0.05 0.20 0.10 0.05 0.20	-----	2.58±0.02 2.98±0.01 2.93±0.03 2.78±0.00 2.21±0.01	-----	1.29±0.01 1.49±0.00 1.47±0.02 1.39±0.00 1.31±0.00	-----	0.28±0.00 0.47±0.00 0.45±0.01 0.38±0.00 0.29±0.00
		FD	0.10	-	2.02±0.01	-	1.27±0.00	-	0.26±0.00
			0.05	-	2.01±0.00	-	1.15±0.00	-	0.14±0.00
			0.20	-	2.78±0.03	-	1.39±0.02	-	0.38±0.01
		SD	0.10	-	2.51±0.02	-	1.26±0.01	-	0.25±0.00
			0.05	-	2.37±0.01	-	1.19±0.00	-	0.18±0.00
			0.20	2.17±0.03 2.19±0.01 1.09±0.02 1.10±0.01 0.08±0.02 0.09±0.01
		AE	0.10	2.09±0.02 2.11±0.01 1.05±0.01 1.06±0.01 0.04±0.01 0.05±0.01
			0.05	2.05±0.02 2.07±0.01 1.03±0.01 1.04±0.01 0.02±0.01 0.03±0.01
			0.20	2.19±0.01 2.10±0.01 1.10±0.00 1.05±0.01 0.09±0.00 0.05±0.01
	M. odorata	ME EE	0.10 0.05 0.20 0.10	2.07±0.00 2.02±0.01 1.04±0.00 1.01±0.01 0.03±0.00 0.01±0.01 2.01±0.01 2.00±0.01 1.01±0.01 1.00±0.00 0.00±0.00 0.00±0.01 2.30±0.00 2.30±0.01 1.15±0.01 1.15±0.00 0.14±0.01 0.14±0.01 2.10±0.01 2.20±0.01 1.05±0.01 1.10±0.01 0.05±0.01 0.10±0.01
			0.05	2.02±0.01 2.07±0.01 1.01±0.01 1.04±0.01 0.01±0.01 0.03±0.01
			0.20	2.36±0.04 2.42±0.01 1.18±0.01 1.21±0.01 0.17±0.01 0.20±0.01
		WE	0.10	2.21±0.01 2.34±0.01 1.11±0.01 1.17±0.01 0.10±0.01 0.16±0.01
			0.05	2.19±0.01 2.29±0.01 1.10±0.01 1.15±0.01 0.09±0.01 0.14±0.01

Table 3 . 10 .

 310 Composition and radical scavenging capacity of Myrrhis odorata constituents evaluated by the online HPLC-UV-DPPH • method (ni=not identified)

	Plant			Ethanol extract	Water extract	Acetone extract
	origin			EE	WE	AE
		Active	RT,	Peak area,	% of total	Peak area,	% of total	Peak area,	% of total
		compound	min	mV×s	RSC	mV×s	RSC	mV×s	RSC
		ni	8.5	-	-	51.4	3.4	-	-
		ni	13.2	-	-	201.2	10.8	-	-
		Cynarin	15.5	-	-	849.1	45.4	-	-
	FR	ni K-7-O-glc	26.7 29.7	-1436.6	-77.2	26.4 639.7	1.4 34.5	-375.1	-90.4
		ni	30.7	229.9	12.4	-	-	-	-
		ni	32.0	58.0	3.1	33.6	1.8	-	-
		ni	34.2	67.6	3.6	-	-	-	-
		ni	35.0	26.5	1.4	-	-	-	-
		ni	36.5	-	-	-	-	32.6	7.9
		ni	15.5	-	-	231.2	7.8	-
		K-7-O-glc	29.8	6038.2	97.1	2547.8	86.4	214.4	77.2
	LT	ni	35.5	55.5	0.9	22.3	0.5	-	-
		ni	36.4	115.9	1.9	-	-	43.4	15.6
		ni	37.9	-	-	13.3	0.3	-	-

DIG-MAZ 500 -Extractor Extract concentrator all

  Solvent vesselFilling the extractor with hackled solids. The lid opens the whole cross-section of the extraction vessel at the touch of a button whereby an optimum filling is ensured. Batch-extraction of fine-grained solids by leaching with solvents, such as water and ethanol. Free choice of solvent flow direction in the extractor: inflow from the bottom or from top, flow direction also changeable according to pressure. Both, extract and solvent can be re-circulated. Extract filtration: Executed by bottom and lid sieves, if necessary additionally by an external filter. Squeezing the solids: Executed by a solid-squeezer that is integrated into the extract vessel: the sieve bottom is employed as movable press piston and controlled via hydraulics. The extraction good is dehumidified and the solvent loss reduced.Evacuation of the extractor:The extraction vessel is tipped over on a horizontal axis and completely evacuated with the help of the hydraulically shifted sieve bottom -and this in one single working step. The handling of the leached residuals is thereby substantially simplified. Vacuum evaporation: The solid-content of the extract is increased by vacuum evaporation at short holding time. The solvent is condensed, accumulated and can be re-circulated into the extraction process.

	6	Hydraulic press  Hydraulic pump	Most hydraulic presses are driven by one or more cylinders that use fluid pressure.
		 Vane pump
		 Pump with axial
		pistons for the
		working-phase.
		 Calibration valve
		 Double acting
		cylinder

Extraction system  Extractor  Valve block-control  Hydraulic system  Heat exchanger  Evaporation plant  Extract tank  Filter  Extract concentrator 

Table 1 . 1 .

 11 Aromatic and medicinal plants widely used in medieval ages with therapeutic properties .......................................................................................

	Figures

Table 1 . 2 .

 12 Different parts of a plant which can be used for the isolation essential oil ............

Table 1 . 3 .

 13 Classes of phenolic compounds in plants .......................................................

Table 1 . 4 .

 14 Amount of agro-industrial residues ...............................................................

Table 2 . 1 .

 21 Investigated plants .......................................................................................

Table 2 . 2 .

 22 Doehlert's experimental matrix and experimental domain ................................

Table 3 . 1 .

 31 Essential oil composition (%) of Myrrhis odorata (L.) Scop. leaves from Lithuania and France. .....................................................................................

Table 3 . 2 .

 32 Comparison of main essential oil constituents in Myrrhis odorata (L.) Scop. leaves from different geographic origins ..........................................................

Table 3 . 3 .

 33 Chemical composition (%) of flower and stem essential oils of Tussilago farfara from two origins .................................................................................

Table 3 . 4 .

 34 Comparison of main essential oil constituents of Tussilago farfara (L.) from different plant parts and different geographic origins .........................................

Table 3 . 5 .

 35 Doehlert's experimental matrix and the corresponding experimental

LITERATURE REVIEWCHAPITRE

MATERIALS AND METHODSCHAPITRE

extract chlorogenic acid was absent. Quinic acid, chlorogenic acid, rutinoside and dicaffeoylquinic acid isomers were previously isolated from the flower buds of T. farfara [153] while, to the best of our knowledge, quercetin-pentoside and kaempferol-glucoside were not previously reported in this plant.