Chemical composition and bioactivity of different botanical parts of
Aesculus hippocastanum L. fruits
Asma Dridi
Dissertation made under the agreement of Double Diploma between Escola Superior
Agrária de Bragança | IPB and Tunisia Private University ULT, Tunisia to obtain the Master in Chemical Engineering
Supervisors:
João Carlos Martins Barreira Khalil Zaghdoudi
Isabel Cristina Fernandes Rodrigues Ferreira
This Dissertation does not include the criticisms and suggestions made by the Jury.
This work was funded by programme FEDER-INterreg Spain-Portugal through Project 0377_Iberphenol_6_E.
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Acknowledgements
I would like to show my sincere appreciation to those who have contributed to this dissertation and supported me in one way or the other during this amazing journey for without the generous support of them, this work would not have been possible.
First of all, I would like to express my pride to be supervised by widely known and highly experienced scientific researchers
Professor Isabel C. F. R. Ferreira, thank you for giving me the opportunity to be part
of a highly skilled team and carry out my thesis in the forefront of scientific engineering research in a very good working environment. It has been an honour to have someone with your immense knowledge and scientific experience as my co-supervisor.
Professor M. Khalil Zaghdoudi and Professor Doctor João C.M. Barreira, I am
very thankful to have such a wonderful, professional and perfectionist persons as my thesis supervisors. I am deeply thankful for your continuous support and patience in this learning path, for your persistent help, and for the constant motivation to do more and better. You offered continuous advices and encouragement and I benefitted from your knowledge and scientific experience. I appreciate your effort and I want to thank you for your wise guidance, kindness and continuous availability.
Doctor Filipa Sofia Dinis Reis my ultimate support and mentor. I am so grateful for
everything that I have learned from you during our lab work time, your irreplaceable guidance and great help in laboratory procedures; you are a kind person with a warm loving heart. Your enthusiasm and energy that I admire made the lab a wonderful place to work in and created the best environment ever to make up for the distance. Your positive outlook and your ability to smile despite the situation is your special super power. You are a wonderful and beautiful person both inside and out; I would like to
express my gratitude for all your support and encouragement. Thank you for always
finding the time to guide me through all the difficult moments of research and while writing this thesis, your support and words of encouragement gave me the power to commit myself to this work.
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Thank you for always being so friendly and supportive with your smile and generous words of encouragement. You were there by my side on every step of my path and for everything you’ve done for me I’m so glad to consider you as a FRIEND.
I would like to express my sincere appreciation to Doctor Lillian Barros, thank you for being such a wonderful and generous person. I appreciate your encouragement, good humour and your kindness.
I also want to thank all researchers of the Mountain Research Centre (CIMO) and the BioChemCore research group for their support and generosity. Thank you all for always being so helpful and friendly, as well as for all your efforts and support during the realization of this work.
Also, I’m grateful to FEDER-Interreg España-Portugal programme for financial support through the project.
I am deeply grateful to all members of the jury who kindly honoured me by their presence to participate in the defence of this thesis.
My big appreciation to my special and wonderful friends, Ahmed, Ghada and Raed who helped me in every possible way to achieve my dreams and successfully complete this research work. I will never forget all their support and effort. You’re the best.
Keeping the best for last, I would like to thank my parents and my sisters whose help made this journey possible from the beginning they believe in me and supported my studies abroad both morally and financially. They have been my inspiration and my unfailing source of passion and energy. Thank you for your irreplaceable support, for all your love, patience and kindness, for always encouraging me to pursuit my education, for believing in me and for everything that you have done to make me the person I am now. Without you none of this would have been possible. I love you!!
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Abstract
Since ancient times, natural products, especially those originated from plants have been an important source of therapeutic agents. Currently, several drugs are derived from natural products (plants, animals, bacteria and fungi). Although the focus the synthetic chemistry advances, research on natural pharmaceutical uses has been knowing an important increase in number. In addition, recent data from the pharmaceutical industry show that, for some complex diseases, natural products still represent an extremely valuable source for prospecting new chemical formulations, as they represent unique structures refined by evolutionary mechanisms throughout millions of years. The importance and contribution of natural substances in medicine treatments is especially evident in some Asian and African countries, where 80% of the population depends on traditional medicine, including herbal treatments like in our case of study the Aesculus hippocastanum L.
A. hippocastanum (also known as horse chestnut fruits) is an important source of
bioactive natural molecules. The biological activity of A. hippocastanum is mainly provided by its secondary metabolites, a class of molecules especially involved in the plant defence system against different threats. Thus, these secondary metabolites play an important role in the adaptation of plants to the environment, effectively participating in their tolerance to various stress factors (pathogen attacks, drought, UV light, among others). The evaluation of the therapeutic value of these metabolites is the subject of many researches, leading to the identification of the main bioactive compounds in this plant.
Saponins (triterpenes or steroid glycosides) and phenolic compounds are among the main classes of secondary metabolites. These compounds are of great interest because of their wide range of biological activities, having already been increasingly applied in pharmaceutical related formulation. In order to achieve effective valorisation strategies for these bioactive compounds, it is necessary to optimize separation processes to obtain them from natural sources. In addition to the former extraction procedure, obtaining bioactive compounds also requires other techniques (sometimes slow and expensive) such as purification and identification, which might be a limitation for their industrial application.
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Accordingly, it is mandatory to develop analytical techniques to improve the extraction process, and achieve rapid separation, miniaturization and coupling methodologies, continuously performed following green chemistry principles. The aim of this work was to demonstrate the antioxidant potential (free radical scavenging effect, reducing power and inhibition of lipid peroxidation), antimicrobial (antibacterial using Gram-positive and Gram-negative strains and antifungal activity) and the absence of toxicity (porcine liver primary cells) of the ethanol/methanol extracts, obtained from the previously mentioned plant and to contribute with updated information on natural sources of bioactive compounds, thorough chemical characterization and possible applications.
The extract of A. hippocastanum fruit presented antioxidant activity, without toxicity up to the maximal tested dose and contained different classes of various bioactive molecules, besides carbohydrates lipids, proteins, and minerals. Overall, the information collected from the different analyses realized on the extract samples reveal their potential use in developing new forms of biopharmaceuticals or could be replacing other chemical substances in cosmetic products without interfering with the product composition or affecting its characteristics.
Keywords: Bioactive compounds; Extraction; Aesculus hippocastanum; Analytical
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Resumo
Desde tempos ancestrais, os produtos naturais, especialmente aqueles originários de plantas, têm sido uma importante fonte de agentes terapêuticos. Atualmente, vários medicamentos são derivados de produtos naturais (plantas, animais, bactérias e fungos). Apesar da focalização na química sintética, a pesquisa sobre as aplicações de farmacêuticos naturais tem conhecido um aumento importante. Além disso, dados recentes da indústria farmacêutica mostram que, para algumas doenças complexas, os produtos naturais ainda representam uma fonte extremamente valiosa para a prospeção de novas formulações químicas, pois representam estruturas únicas refinadas por mecanismos evolutivos ao longo de milhões de anos. A importância e contribuição de substâncias naturais nos tratamentos medicamentosos é especialmente evidente em alguns países asiáticos e africanos, onde 80% da população depende da medicina tradicional, incluindo tratamentos à base de plantas, como no nosso caso de estudo, o
Aesculus hippocastanum L.
O A. hippocastanum (também conhecido como castanha-da-Índia) é uma fonte importante de moléculas naturais bioativas. A atividade biológica de A. hippocastanum é fornecida principalmente pelos seus metabolitos secundários, uma classe de moléculas especialmente envolvidas no sistema de defesa das plantas contra diferentes ameaças. Assim, estes metabolitos secundários desempenham um papel importante na adaptação das plantas ao meio ambiente, participando efetivamente na sua tolerância a diversos fatores de stress (ataques de patógenos, secas, luz UV, entre outros). A avaliação do valor terapêutico desses metabolitos é objeto de muitas pesquisas, levando à identificação dos principais compostos bioativos desta planta.
As saponinas (triterpenos ou esteróides glicosilados) e os compostos fenólicos estão entre as principais classes de metabolitos secundários. Estes compostos são de grande interesse devido à sua ampla gama de atividades biológicas, tendo sido cada vez mais aplicados em formulações relacionadas com produtos farmacêuticos
Para alcançar estratégias eficazes de valorização destes compostos bioativos, é necessário otimizar o processo de separação desses compostos das suas fontes naturais.
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Além do procedimento de extração anterior, a obtenção destes compostos também requer outras técnicas (às vezes lentas e caras), como purificação e identificação, que podem ser uma limitação para sua aplicação industrial. Portanto, é obrigatório o desenvolvimento de técnicas analíticas para melhorar o processo de extração e alcançar metodologias rápidas de separação, miniaturização e acoplamento, realizadas continuamente seguindo os princípios da química verde. O objetivo deste trabalho foi demonstrar o potencial antioxidante (efeito de eliminação de radicais livres, poder redutor e inibição da peroxidação lipídica), antimicrobiano (antibacteriano usando estirpes Gram-positivas e Gram-negativas e atividade antifúngica) e a ausência de toxicidade (fígado suíno células primárias) dos extratos etanol/metanol, obtidos da planta mencionada anteriormente e contribuir com informações atualizadas sobre fontes naturais de compostos bioativos, caracterização química completa e possíveis aplicações.
No presente caso, o extrato de A. hippocastanum apresentou atividade antioxidante, sem toxicidade até a dose máxima testada e apresentando diferentes classes de várias moléculas bioativas, além de hidratos de carbono, lípidos, proteínas e sais minerais.
No geral, as informações recolhidas das diferentes análises realizadas nas amostras de extrato revelam potencial no desenvolvimento de novas formas de produtos biofarmacêuticos como alternativas a outras substâncias químicas em produtos cosméticos sem interferir na composição do produto ou afetar suas características.
Palavras-chave: Compostos bioativos; Aesculus hippocastanum; Técnicas analíticas;
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Index
Acknowledgements ... i Abstract ... iii Resumo ... v Index of tables ... x List of abbreviations ... xi I. Introduction ... 151.1. Medicinal plant derived nutracerruticals ... 17
1.2. Botanical characterization of Aesculus hippocastanum L. ... 18
1.3. Natural bioactive compounds ... 20
1.3.1. Saponins ... 20
1.3.2. Organic acids ... 29
1.3.3. Phenolic compounds acids ... 30
1.3.4. Vitamins ... 34
1.3.5. Fatty acids ... 39
1.4. Bioactivity of A. hippocastanum and related health effects ... 40
1.4.1. Antioxidant activity ... 41
1.4.2. Antimicrobial activity ... 42
1.5. Potential industrial applications of A. hippocastanum ... 44
II. Objectives ... 46
III. Material and Methods ... 47
3.1. Sampling of A. hippocastanum fruits ... 47
3.2. Standards and reagents ... 47
3.3. Chemical characterization ... 48
3.3.1. Nutritional value ... 48
3.3.2. Determination of soluble sugars ... 51
3.3.3. Fatty acids profile ... 52
3.3.4. Organic acids profile ... 53
3.3.5. Phenolic compounds profile ... 53
4.1 Bioactivity evaluation ... 55
1.1.1. Antioxidant activity ... 56
1.1.1.1. TBARS formation inhibition ... 56
1.1.1.2. Oxidative haemolysis inhibition assay (OxHlIA) ... 57
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1.1.3. Cytotoxicity ... 59
1.1.3.1. Tumour cell lines ... 59
1.1.3.2. Non-tumour cell line: porcine liver cell line PLP2 ... 60
1.1.4. Anti-inflammatory activity ... 60
4.2 Statistical analysis ... 61
IV. Results and discussion ... 62
4.1 Nutritional value of A. hippocastanum fruit ... 62
4.2 Soluble sugars of A. hippocastanum fruit ... 62
4.3 Fatty acids profile of A. hippocastanum fruit ... 62
4.4 Phenolic profile of different parts of A. hippocastanum fruit ... 63
4.5 Organic acids ... 70
4.6 Antioxidant activity ... 71
4.7 Antimicrobial activity ... 72
4.8 Cytotoxicity and anti-inflammatory activity ... 74
V. Conclusion ... 75
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Index of figures
Figure 1. Chemical structures of spirostane (a) and furostane (b).... 22
Figure 2. Chemical structures of oleanane (a), ursane (b) and dammarane (c).... 22
Figure 3. Chemical structures of ursolic acid (a), oleanolic acid (b), diosgenin (c), glycyrrhizin (d), soyasapogenol A (e), soyasapogenol B (f), soyasaponin A (g), soyasaponin B (h).... 24
Figure 4. Chemical structures of escin Ia (a), escin Ib (b), escin IIa (c), escin Ib (d). ... 26
Figure 5. Chemical structure of some organic acids present in foods. Oxalic acid (2C), malic acid (4C), and fumaric acid (4C), quinic acid (7C), (based on Yildiz, 2009). ... 30
Figure 6. Basic structure of flavonoids. ... Erreur ! Signet non défini. Figure 7. General chemical structure of vitamin E isoforms. A- Tocopherols (α-tocopherol: R1 and R2 = Me; β-tocopherol: R1 = Me and R2 = H; γ-tocopherol: R1 = H and R2 = Me; δ-tocopherol: R1 and R2 = H). B - Tocotrienols (α-tocotrienol: R1 and R2 = Me; β-tocotrienol: R1 = Me and R2=H; γ- tocotrienol: R1 =H and R2 = Me; δ- tocotrienol: R1 and R2 = H) (Stpanuk and Caudill 2013). ... 38
Figure 8. A. hippocastanum fruit and peeled kernel. ... 47
Figure 9 - Muffle used in ash determination. ... 49
Figure 10 - Soxhlet equipment used in the determination of fat content. ... 50
Figure 11 - Equipment used in the determination of protein content. A - digestor. B - Kjeldahl equipment. ... 51
Figure 12 - Equipment (HPLC-RI) used in soluble sugars identification. ... 52
Figure 13 - Equipment (HPLC-MS) used in the determination of the phenolic compounds. .... 55
Figure 14. Methanolic/ethanolic fruit extract of A. hippocastanum.... 55
Figure 15. Example of different levels of MDA-TBA formation (samples on the left are the most antioxidant). ... 56
Figure 16. Microplate used in cytotoxicity evaluation. ... 60
Figure 17. Phenolic profile of pulp of the Aesculus hippocastanum recorded at 280 nm (A),
x
Index of tables
Table 1 - Basic structures of some common phenolic compounds... 31 Table 2 - Retention time (Rt), wavelenghts of maximum absorption in the visible region (λmax),
mass spectral data, tentative identification and quantification (mg/g extract) of phenolic compounds in pulp (peeled kernel), bark and skin, of Aesculus hippocastanum. ... 64
Table 3 - Organic acids profile (g/100 g dried weight) of different parts of Aesculus
hippocastanum. ... 70
Table 4 - Antioxidant activity of different parts of Aesculus hippocastanum (values are
presented EC50 values in g/ml of extract). ... 71
Table 5 – Antimicrobial activity of the ethanolic extracts of different parts of Aesculus
hippocastanum fruits. ... 72
Table 6 - Cytotoxicity of different parts of Aesculus hippocastanum (values are presented GI50
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List of abbreviations
AAPH ADHD ADI AOAC ATCC BC BHA BHT BOD BrdU CAC CEC CCCD CFU DAD DMEM DMSO DPPH EC EDTA EEC EGF ESI FAME FAO FBS FDA FID HAE HBSS 2,2’-Azobis(2-amidinopropane) dihydrochloride Attention deficit hyperactivity disorderAcceptable daily intake
Association of analytical communities American Type Culture Collection Before Christ
Butylated hydroxyanisole Butylated hydroxytoluene Biological Oxygen Demand
Bromodeoxyuridine (5-bromo-2’-deoxyuridine) The International Codex Alimentarius Commission European Commission Scientific Committee Circumscribed central composite design Colony forming units
Diode Array Detector
Dulbecco’s Modified Eagle Medium Dimethylsulfoxide
2,2-diphenil-1-picrilhydrazil European Commission Ethylenediaminetetraacetic European Economic Community Epidermal growth factor
Electrospray ionization Fatty acid methyl ester
The Food and Agriculture Organization Fetal bovine serum
Food and Drug Administration Flame ionization detector Heat Assisted Extraction Hank’s balanced salt solution
xii HCA HHP HPLC INT IS LPS MBC MDA MFC MH MIC NCI NED NO MS NCTC OXHLIA PBS PDA PEF PFA RI RNA RNS ROS RPMI RSS SE SRB TBA TBARS TCA TFA
Hierarchical Cluster Analysis High Hydrostatic Pressure
High-Performance Liquid Chromatography Iodonitrotetrazolium
Internal standard Lipopolysaccharide
Minimal Bactericial Concentration Malodialdehyde
Minimum Fungicidal Concentration Mueller-Hinton agar
Minimal Inhibitory Concentration National Cancer Institute
N- (1-naphthyl) ethylenediamine hydrochloride Nitric oxide
Mass Spectrometry
National Collection of Type Cultures Oxidative hemolysis inhibition assay Phosphate-buffered saline
Photo-diode array detector Pulsed Electric Field Paraformaldehyde Refraction Index Ribonucleic acid
Reactive nitrogen species Reactive oxygen species
Roswell park memorial institute medium Reactive sulphur species
Soxhlet Extraction Sulforhodamine B Thiobarbituric Acid
Thiobarbituric Acid Reactive Species Trichloroacetic Acid
xiii TSB UAE UE ULFC/UHPLC USA UV WHO Tryptic Soy Broth
Ultrasonic Assisted Extraction Ultrasonic Extraction
Ultra-fast liquid chromatography The United States of America Ultra violet
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I. Introduction
Thrоughоut the ages, humans have invoked nature for their basic needs because nature has always been a golden partner in the prominent phenomena of coexistence. Natural products from plants, animals, and minerals have been the basis for treating human diseases (Jamshidi et al, 2018).
Indeed, it was at the beginning of human life that the use of medicinal plants for the treatment of various diseases has appeared especially since man began his research for tools to recover from a disease at the beginning of this discovery, the manufacture of fire, the manufacture of fire, the cover of a shelter, the manufacture of clothing or the provision of nutrition are the main axes to which the use of plants has been limited and this is due to the lack of knowledge of its therapeutic conveniences. Today, research has changed the worldview and this change allows medicinal plants to take an increasingly preponderant place to the point of becoming the main concern in terms of treatments and has made us take more and more aware of its important role in the fight against disease and management of human race (Ahmed et al., 2015).
There is no doubt that plants play a major role in the provision of essential ecosystem services; whether for humans or other living beings, plants play a decisive role in a proper life on earth. In addition to its vital role, plants, especially the medicinal ones, have consistently served as a global indicator of ecosystem health (Pierangeli and al. 2009). It was in countries like China, Greece, Egypt, and India that medicinal plants were transformed into one of the oldest sciences and medicine that has been perpetuated and enriched over time. In ancient Persia, plants are grown and commonly used as medicines, antimicrobial agents used for disinfection as well as aromatic agents (Hayta et al., 2014). The demand for medicinal plants and their admission among other treatments is increasing rapidly in many countries. The treatment of diseases in more than 85% of the population in the Middle East, Latin America, Africa, and Asia is based mainly on traditional medicine, especially medicinal plants, (Dos Santos Reinaldo et al., 2015). Around 100 million people in the European Union and 90% of the population in other countries still use traditional medicines whose active ingredients are exclusively planted drugs. Medicinal plants and their use in several applications and various fields
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have led researchers to focus on evaluating their contribution to health maintenance and disease prevention (Murray, 2004). Recently, pharmacopeia has used between 50,000 and 70,000 plant species known for the development of modern medicines. Plants and their products have been integrated into the therapeutic process, humans from ancient civilizations around the world have exploited them in different forms such as infusions, creams, and drops to cure diseases (Hayta et al., 2014).
More than one-tenth of the plant species (more than 50,000 species) have been incorporated as different formulations in the pharmaceutical, cosmetic and food industries, and have been used as auxiliaries or even as promoters because of their composition in bioactive compounds. (Newman and Cragg, 2012). All parts of the plant such as seeds, roots, leaves, fruits, skin, flowers or even the whole plant can be used in herbal medicine. These different parts of plants are rich in active compounds that could also be used as medicinal agents through their direct or indirect therapeutic effects (Rates, 2001). To maintain health and to prevent, diagnose, treat and cure diseases, humans mainly use plant raw materials (Balunas and Kinghorn, 2005). Medicinal plants have healing properties based on a synergistic mode of action for which they are used for treatment. (Pal and Shukla, 2003). This synergy is ensured by either a benefit interaction for the different components of the plant or unfavorable to either of them or eliminates the harmful effects of both (WHO, 2004).
Medicinal plants can help relieve difficult-to-treat diseases, such as cancer. They are also characterized by their ability to prevent, relieve or cure more than a single disease (Mukherjee, 2002; Bodeker et al., 2005; Bandaranayake, 2006).
Research and evaluation of the field of medicinal plants are becoming increasingly important. Some medicinal plants are experiencing significant growth among people around the world because of their ability not only to be a source of adjuvant treatment in prevention and maintenance health systems but also anti-inflammatory, antimicrobial, anti-mutagenic, anti-cancer and/or antioxidant agents with satisfactory results (Wojdylo et al., 2007). For this reason, more and more species are being explored for their bioactive components, to identify the most active chemical compounds, to establish adequate amounts for their incorporation into drugs or nutraceuticals and to know their side effects (Raynor et al., 2011). Phenolic compounds and saponins are among the classes of bioactive compounds that are strongly reported in the treated plant in this work. Scientific studies and the identification of biologically
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active compounds and their properties may lead to the emergence of new therapeutic benefits and the future production of nature-based products despite the enhancement of plants and their use in traditional medicine (Pal and Shukla, 2003).
1.1. Medicinal plant derived nutracerruticals
Humans consider plants to be an essential source of food and medicine. The rapid increase in knowledge about nutrition, medicine, and plant biotechnology has radically changed the concepts of food, health, and agriculture. Plants play an important role as a food and/or therapeutic purposes for humans. As well as the increasing level of knowledge, medicine, phytotherapy, and nutrition that is becoming rapidly has radically changed the concepts of food, health, and agriculture. Plants contain a very large amount of active ingredients that are traditionally converted into primary and secondary metabolites and form the basis of the formulas of certain medicines and skincare products (Hans, 2007).
Primary metabolites are types of a metabolite that are directly involved in photosynthesis, respiration, growth, development and normal reproduction of the plant. These include phytosterols, acylated lipids, nucleotides, amino acids, and organic acids. In contrast, other phytochemicals that are not directly involved in plant growth but are involved in their adaptation to different environments such as UV protection, defense, the attraction of insects useful for pollination, are considered secondary metabolites. They accumulate at unexpectedly high concentrations in some species of the vegetal
kingdom under various and numerous structures (Frasher, 2006).
Indeed, humans can exploit these secondary metabolites of plants as raw materials in the food industry used as food additives, flavors, and dyes, in agriculture as insecticides as well as in cosmetics and pharmaceutical industries for the synthesis of medicines, perfumes, and other fine chemicals.
These secondary plant metabolites have been categorized into several large groups such as terpenoids, flavonoids, and alkaloids that have a wide variety of chemical structures and biological activities and exist widely in different vegetal rules. Hydroxytyrosol is a powerful antioxidant found mainly in olives and olive oil. Nuts and red wine have good sources of antioxidants, antithrombotic, inflammatory and anti-cancer thanks to their resveratrol content (Inti and Faoro., 2006). Lycopene, an oxidant
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that is part of the carotenoid family, is recognized as a contributor to cancer prevention and inhibits tumor cell growth in animals. The phenolic compounds and saponins contained in Aesculus hyppocastanum have multifunctional benefits for human health. Isothiocyanates from cruciferous vegetables and monoterpenes from citrus fruits, cherries, and herbs have biological effects on immunity included anti-carcinogenic and cardioprotective effects. Grapefruit contains Naringen (4 ', 5', 7-hydroxyflavanone) which can prevent rejection of transplanted organs as well as slow the liver detoxification of prescription drugs such as cyclosporin (Berger and Shenkin., 2006).
All of these examples of components represent only a small portion of scientific recommendations based on phytonutrients and nutritional therapies. In recent years, nutritional therapy and herbal medicine have become the new systems of complementary and alternative therapies in vogue and a new concept to use in health. Plant-based nutraceuticals remain increasingly popular and highly recommended to strengthen prevention strategies for treatment and disease control. Indeed, the term "nutraceutical" associating "nutrition" and "pharmaceutical" in 1989 by Dr. Stephen DeFelice, is defined as a food or part of a food offering medical or health benefits (Ramaa et al., 2006). Dietary supplements are concentrated sources of nutrients, which provide bioactive compounds essential to our body and beneficial to health thanks to it's preventive or medicinal properties. These nutraceuticals combine vitamins, proteins, fats, carbohydrates, minerals or other necessary nutrients, depending on their energy intake and importance (Pandey et al., 2010).
1.2. Botanical characterization of Aesculus hippocastanum L.
Horse chestnut (Aesculus hippocastanum L.), is native to a small area in the mountains of the Balkans in southeast Europe, where it grows in moist broadleaf forests up to 1000-1200 m in altitude (Tomanek 1994). It can be also found in small areas in northern Greece, Albania, the Republic of Macedonia, Serbia, and Bulgaria. Horse chestnut was introduced in other parts of Europe for a long time, with the exception of the northern area, which may suggest that the tree requires higher temperatures then those in this area. Today it is widely cultivated worldwide, in temperate zones, because of its large and beautiful flower clusters and its excellent resistance to environmental conditions. (Seneta and Dolatowski 2005).
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A. hippocastanum is a large deciduous tree up to 30 m high and 1 m thick, usually
with a short stem and dense rounded crown (Brus, 2004) that belongs to the genus
Aesculus, the most widespread genus of the Hippocastanaceae family. The genus Aesculus, comprises twelve species recognized in four subgenera: Hippocastanum, Pavia, Calothyrsus, and Macrothyrsus (Anwar et al., 2007).
Based on the bud viscidity, fruit exocarp ornamentation, flower colour, and petal morphology, the 12 species are grouped into five sections: section Aesculus in Europe and Japan (A. hippocastanum L. and A. turbinata Blume); section Calothyrsus (Spach) K. Koch in south-eastern Asia and western United States (A. assamica Griffith, A.
californica (Spach) Nutall, A. chinensis Bunge var. chinensis); section Pavia (Mill.)
Persoon in the south-eastern United States; section Macrothyrsus (Spach) K. Koch in the south-eastern United States (A. parviflora Walter), and Section Parryanae Wiggins in Baja California, Mexico (A. parryi A. Gray) (Fugile, 2000; Anwar et al., 2007).
The genus Aesculus prefers deep, nutritious and humid sandy-clay soil with air pockets. The tree is best grown individually in full sunlight, although it also grows well in the shade. This genus can survive winter temperatures below -20 ºC, although it is not a winter hardy tree species (Roloff et al., 2009; Wilczynski and Podlaski, 2007). The leaves of this genus are opposite and palmately compound with 5-7 leaflets. The sessile leaflets are green beneath with obtusely double-serrate. Its flowers are usually white with a small red spot. The fruit is a green, spiky capsule containing one (rarely two or three) brown seed, called chestnut or conker. (Zhang et al., 2010) The common name “horse chestnut” may have come from the uses of seeds for horses to treat overexertion or coughs by Turks and Greeks (Zhang et al., 2010).
Horse chestnut has historically been used as animal feed for farm animals; it is a valuable source of nutrients for deer, and some Native American people have included it in their diet. Today, A. hippocastanum has been investigated in several randomised clinical studies that provide evidence that the tree has therapeutic properties, presenting itself as a species of medical/clinical interest (Ramachandran et al., 1980). In Europe, the bark and leaves of A. hippocastanum have been employed as an astringent to treat diarrhoea and haemorrhoids and the seed extracts have traditionally been used as a therapy for chronic venous insufficiency such as feeling of pain, heaviness, and tension in the legs and are processed by the pharmaceutical industry (Wichtl, 2004). Currently,
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seeds of A. hippocastanum are used for ornamental purposes, but most of them end up in landfills. Most reports related to seeds of A. hippocastanum are focused on their biochemical composition and morphoanatomical features.
1.3. Natural bioactive compounds
Horse-chestnut seeds, like all other seeds, are natural products whose chemical composition is a very complex matrix. They contain many molecules and analytes, the majority of which are polysaccharides (both starches and starches), proteins, lipids, mineral salts and many minor components among others, with homogeneous or localized distributions in different districts, all of which interact strongly with each other in a synergistic way to form very complex structures. On the other hand, seeds are the promise to propagate plant biodiversity and to ensure its continuity. It is probably for these reasons that these complex structures are far from being detailed at the microscopic level, even though many researchers engaged in different fields (biologists and biotechnologists, bioengineers, chemists, pharmacists, etc.) (Baraldi et al, 2007).
Aesculus seeds are plant materials often used in medicine Populations in Eurasia and North America have often used vegetal materials such as Aesculus seeds to maintain health as well as to prevent, diagnose, treat and cure diseases. In recent decades, chemical and natural investigations have focused primarily on the active ingredients of seeds and fruits. To date, several researchers and scientists have isolated and characterized more than 210 compounds from Aesculus. These compounds include triterpenoids, triterpenoid glycosides (saponins), flavonoids, coumarins, carotenoids, long-chain fatty compounds and certain other classes of compounds. In Europe, they have used in a traditional application for the therapy of some diseases such as chronic venous insufficiency, traumatic edema, hemorrhoids, etc (Zhang et al, 2011).
1.3.1. Saponins
Saponins represent a class of chemical compounds that may be found in considerable abundance in several plant species. Actually, saponins are considered as ubiquitous phytochemicals, widely reported in many species of plants and some marine animals (Sparg et al., 2004; Van Dyck et al., 2010; Vincken et al., 2007). Saponins constitute a large group of glycoside derivatives, widely distributed in higher plants. Their surface-active properties distinguish them from other natural glycosides.
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These molecules are amphipathic glycosides that express the general feature of producing soap-like foam when shaken in aqueous solutions. Despite having been traditionally considered as anti-nutrients, there is an increasing interest in saponins, owing to recent findings that indicate a good range of bioactive properties, most likely due to the structural variability of their aglycones. In general, saponins have been related to immunostimulatory, hypocholesterolaemic, antitumor, anti-inflammatory, antibacterial, antiviral, antifungal, and antiparasitic activities (Francis et al., 2002, Rao and Gurfinkel, 2000, Sparg et al., 2004). Therefore, a wide range of applications in the pharmaceutical, food and cosmetic industry (e.g., as hypoglycaemic or surfactant agent) might be anticipated.
Saponins are high molecular weight amphiphilic compounds consisting of a hydrophobic aglycone (triterpenoid or steroid), designated as sapogenin, linked to one or more hydrophilic sugar moieties through an ether or ester glycosidic linkage, at one or two glycosylation sites (Güçlü-Üstündağ & Mazza, 2007).
The structural complexity of saponins arises from the variability in sapogenin structure, the nature of attached side chains, and the attachment positions of sugar moieties to the sapogenin. According to the chemical structure of the sapogenin, saponins can be classified into steroidal or triterpenoid saponins. Steroidal saponins consist mainly of a C27 spirostane skeleton (Figure 1a), generally comprising a six-ring structure, or a furostane skeleton (Figure 1b), which is pentacyclic (Sparg et al., 2004).
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Figure 1. Chemical structures of spirostane (a) and furostane (b).
Triterpenoid saponins (Figure 2) consist mainly of a C30 pentacyclic skeleton, commonly as oleanane and ursane structures, or C30 tetracyclic skeleton, as dammarane structures (Sparg et al., 2004), and are mainly found in the dicotyledonous (e.g., families Hippocastanaceae, Araliaceae, or Primulaceae) (Garai, 2016).
Figure 2. Chemical structures of oleanane (a), ursane (b) and dammarane (c).
Furthermore, there are two main types of triterpenoid saponins: neutral, when a typical sugar is attached to sapogenin, and acidic, when the sugar moiety contains uronic acid or one or more carboxylic groups attached to the sapogenin (Lásztity et al., 1998). Some typical examples of saponins and sapogenins are given in Figure 3.
Additionally, saponins can be categorized according to the number of sugar chains present as monodesmosidic, bidesmosidic, or tridesmosidic (Güçlü-Üstündağ and Mazza, 2007), according to the number of osidic chains (linear or branched) at different positions of the aglycone. Steroidal as well as triterpenic saponins are often monodesmosidic, generally containing the sugars unit attached through an acetal bond
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at C3. Bidesmosidic saponins, in turn, are found among the triterpene saponins of the oleanane type (with bonds at C-3 and C-28) and steroidal saponins of the furostane type (with bonds at C-3 and C-26) (Yabe et al., 2003). The most common monosaccharides that can be found attached are hexoses (glucose, galactose, fructose), uronic acids (glucuronic acid, galacturonic acid), 6-deoxyhexoses (rhamnose), and pentoses (arabinose, xylose) (Güçlü-Üstündağ and Mazza, 2007, Kharkwal, 2012).
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Figure 3. Chemical structures of ursolic acid (a), oleanolic acid (b), diosgenin (c), glycyrrhizin (d),
soyasapogenol A (e), soyasapogenol B (f), soyasaponin A (g), soyasaponin B (h).
The presence of saponins has been reported in more than 100 families of plants, and in a few marine sources such as starfish and sea cucumber. In what regards plant species, saponins were isolated from different parts of the plants, including roots, rhizomes, stems, bark, leaves, seeds and fruits. These compounds have been identified in more than 100 families of both wild and cultivated plants, belonging to the division of Magnoliophyta, in which two major classes can be found: Liliopsida and
Magnoliopsida, where the majority of saponins producing species are found (Vincken et
al., 2007). The steroidal saponins are mainly found in monocotyledon families (such as
Agavaceae, Dioscoreaceae and Liliaceae), while triterpene saponins are predominantly
present in dicotyledons (Güçlü-Ustündağ and Mazza, 2007).
Likewise, triterpenoid saponins are mostly found in cultivated crops, while steroid saponins are common in medicinal plants. Thus, triterpenoid saponins can be found in many legumes (alfalfa, mung beans, soybean, chickpeas, beans, peanuts, broad beans, kidney beans and lentils), which represent their main dietary sources. Saponins are also present in ginseng roots, sunflower seeds, horse chestnut, liquorice roots, spinach leaves, tea leaves, quillaja bark, quinoa seeds, sugar beet or allium species (leek, garlic). Examples of steroidal saponins are those found in oats, tomato seeds, yam, fenugreek seeds, ginseng roots, asparagus, aubergine or capsicum peppers (Güçlü-Ustündağ and Mazza, 2007; Moses et al., 2014).
The main saponins in Aesculus genus are called escins, presenting escin Ia (24%), escin Ib (17%), escin IIa (13%) and escin IIb (6%) as the major froms (Figure 4). These structures are distinguished by their melting point, specific rotation, haemolytic index and solubility in water, thus having potential to be used in different pharmaceutical applications (Helmi et al., 2015). Escins are mainly located in the seeds of A.
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hippocastanum L., specifically in the endosperm, where around 30 individual saponins
were isolated and identified (Sirtori 2001).
The extraction process is based on the solubility differences among the compounds of a mixture in a solvent. Several methods have been developed to improve the isolation and production of NBCs which include various extraction techniques and ME. The current tendency is dividing extraction techniques in conventional (used for a long time) and innovative (involve more recent techniques). Conventional techniques include steam distillation (hydrodistillation), Soxhlet extraction, batch extraction and sonication assisted extraction, among others.
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In the “innovative techniques” category, we can mention microwave assisted extraction, accelerated solvent extraction, or extraction with supercritical fluids (Cheok et al., 2014). The major advantages and drawbacks of each technique and the main compounds obtained according to current literature information are discussed in the next pages.
a) Soxhlet extraction: Soxhlet is a classic method for solid-liquid extraction. Its
main advantage is probably the fact that the sample contacts continuously with a fresh portion of solvent, which helps move the balance of transfer to the solvent. In addition, this method does not require filtration after extraction and is independent of the vegetable matrix. The most important drawbacks of this method, compared to other methods, are the long duration and the large amount of solvent consumed, which leads not only to economic losses but also to environmental problems. As samples are heated at high temperature for a long period, the risk of thermal degradation of certain compounds is not negligible if the plant material contains thermolabile compounds. Given the large amount of solvent used, the subsequent evaporation/concentration step becomes limiting. Furthermore, this technique is limited in what concerns the selectivity of the solvent and is not easily automatable (Sarvin et al., 2018).
b) Ultrasound-assisted extraction: extraction by sonication, is a simple, effective
and inexpensive method. Its most significant benefits are related to the increase in extraction efficiency and an acceleration of kinetics compared to a conventional extraction. It allows to work at relatively low temperatures and to avoid thermal degradation of compounds. The technique is generally easy to implement. Like Soxhlet extraction, extraction by sonication allows to use a wide range of solvents to obtain different natural compounds. However, the effect of ultrasound extraction on extraction efficiency and kinetics is related to the nature of the plant matrix. The presence of a dispersed phase leads to the attenuation of the ultrasonic waves and the active zones in the extractor remain close to the ultrasound emitter. This method does not allow the solvent to be renewed during the process. The limiting step is filtration and rinsing after extraction (Cheok et al., 2014).
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c) Extraction with supercritical fluids: extraction with a supercritical fluid such as
carbon dioxide is an alternative to the use of chemical solvents. In fact, supercritical CO2 has been used for extracting and separating natural products
with high added value. In the vicinity of the critical point, the solvent power is sensitive to variations in temperature and pressure. The separation of the extracts and the solvent is very easy and is achieved simply by reducing the pressure. In addition, the transport properties of supercritical fluids (viscosity, diffusivity, among others) allow a deeper penetration into the solid matrix of plants and thus in general a more efficient and faster extraction (Cheok et al., 2014; Sarvin et al., 2018).
Once the plant extracts have been obtained, identification and characterisation of bioactive compounds becomes a big challenge, because most plant extracts occur as a combination of various types of bioactive compounds or phytochemicals with different polarities. Phytochemical screening assay is a simple, quick, and inexpensive procedure that gives the researcher a quick answer to the various types of phytochemicals or secondary metabolites found in plants (Sasidharan et al. 2011).
In the isolation of these bioactive compounds, different chromatographic separation techniques, such as thin layer chromatography (TLC), column chromatography, flash chromatography, Sephadex chromatography and high-performance liquid chromatography (HPLC), may be used to obtain pure compounds. TLC is a favourite method of most researchers because it gives a quick answer as to how many components are there in a mixture. TLC is also used to support the identity of a compound in a mixture when the retention factor (Rf) of a compound is compared with the Rf of a known compound (Sasidharanet al. 2011).
The pure compounds are then used for the determination of structure and biological activity. Numerous analytical methods have been developed, which may facilitate structural determination of the bioactive compound, including TLC, HPLC,
LC/electrospray ionisation tandem mass spectrometry (MS/MS), capillary
electrophoresis, ion spray mass spectrometry (MS), gas chromatography/MS (GC/MS), and nuclear magnetic resonance (Jeong et al. 2012).
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MS provides highly specific information that is directly related to the chemical structure, such as accurate mass, isotope distribution patterns for elemental formula determination and characteristic fragment ions for structural elucidation or identification through spectral matching to authentic compound data. Moreover, the high sensitivity of MS allows detection and measurement of picomole to femtomole levels of many primary and secondary metabolites (Lei et al. 2011; Sumner et al. 2003). Non-chromatographic techniques such as immunoassay, which use monoclonal antibodies, phytochemical screening assay or Fourier-transform infrared spectroscopy, can also be used to obtain and facilitate the identification of the bioactive compounds (Sasidharan et al. 2011).
1.3.2. Organic acids
Organic compounds with acidic properties belong to OAs, such as formic and acetic acids (Figure 5). Their low dissociation in water and water solution makes them weak acids. In general, the carboxyle group (COOH) is the characteristic group of the acid character of an AO that is determined primarily by the relative stability of the combined base of molecules (Nollet, 2004). They are described as low molecular weight carbohydrates characterized by the possession of one or more carboxyl groups such as water-soluble formic and acetic acids. The high molecular weight compound is insoluble due to the increase in alkyle groups present. However, most organic acids are highly soluble in solvents (Theron and Lues, 2010; Lianou et al., 2012). They are categorized according to their chemical structure, namely the type of carbon chain, the degree of saturation and substitution, and the number of carboxyle groups (Nollet, 2004; Yildiz, 2009). HO OH O O OH OH HO OH OH OH O
Malic acid Quinic acid
OH
OH O
O
Oxalic acid Fumaric acid
OH
OH
O O
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Figure 5. Chemical structure of some organic acids presents in foods. Oxalic acid (2C), malic acid (4C),
and fumaric acid (4C), quinic acid (7C), (based on Yildiz, 2009).
Organic acids preserve the properties of raw materials and it is because of their acidity that they can greatly eliminate the risk of food deterioration and contamination. Following a transformation by the metabolic activity of a microorganism or as a result of hydrolysis and biochemical metabolism they were considered as the main constituents of fermented foods and products. Even in this case, their properties do not allow them to be nutrients. However, they are responsible for colonizing the food, changing its taste, texture, color and above all allow its preservation. For example, the food additive E-330 (critical acid) can be added in a very wide range of industrial foods and standardized as a preservative and antioxidant, acetic and lactic acids, are used as a biocide respectful of the environment, less dangerous to users and is increasingly becoming a first-rate need in industries to reduce pathogens that can contaminate food. These organic acids have bactericidal properties that allow them to be considered active agents to be used to reduce levels of bacterial pathogens on food surfaces (Therons and Lues, 2011; Lianou et al., 2012).
1.3.3. Phenolic compounds acids
As plant compounds, phenolic compounds belong to the secondary metabolites group. The term phenolic compounds include several thousand molecules that hardly meet a simple definition. From a chemical point of view, they are defined by their benzene cycle carrying at least one free or engaged hydroxyl grouping in another function (ether, ester...). The classification on the chemical structure alone is insufficient and it is important to take into account the biosynthetic origin of the molecules. The structures of phenolic compounds are distinguished by their basic carbon skeleton (number of carbon), then their degree of modification (oxidation, hydration, methylation...) and finally the bonds that may exist with other molecules such as sugars or other phenolic compounds. There are a dozen large classes of compounds (Table 1) comprising simple forms (simple phenols, hydroxybenzoic acids, hydroxycinnamic acids, coumarins, naphthoquinones, stilbene, flavonoids, lignanes...) and phenols condensed from the polymerization of lignans (lignins) or certain flavonoids (tannins) (Carocho and Ferreira, 2013a; Vermerris and Nicholson, 2007). Phenolic compounds are known to have various functions in plants in terms of plant physiology and
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relationships with its environment especially at the level of reproduction and plant defense mechanisms against agents’ fungal pathogens. Conversely, they can also have a positive role as a pollinator attractor. Moreover, as phenolic compounds, they all absorb in the spectral domain of ultraviolet (UV) (Solovchenko and Merzlyak, 2003). Their presence in the outermost tissues of plants (skin of leaves, fruits, etc.) would limit the penetration of these radiations and protect sensitive plant structures (Macheix, Fleuriet et al., 2005).
Table 1 - Basic structures of some common phenolic compounds.
Class Basic
skeleton Basic structure
Simple phenols C6 Benzoquinones C6 Phenolic acids C6–C1 Acetophenones C6–C2 Phenylacetic acids C6–C2 Hydroxycinnamic acids C6–C3 Phenylpropenes C6–C3
32 Coumarins C6–C3 Chromones C6–C3 Naphtoquinones C6–C4 Xanthones C6–C1–C6 Stilbenes C6–C2–C6 Anthraquinones C6–C2–C6 Flavonoids C6–C3–C6 Lignans (C6–C3)2 Ellagitannins (C6–C3)n
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Various studies have identified phenolic compounds in their antioxidant, anti-inflammatory, antitumor, and antimicrobial properties (Adeboye et al., 2014). Generally, when phenolic compounds are referred to in plant foods, flavonoids are the predominant class described, as they account for about two-thirds of dietary phenols (Lattanzio, 2013), but the concentration of these compounds in the seeds of A.hyppocastanum is low. Flavonoids have a basic C6-C3-C6 skeleton, consisting of two aromatic cycles (A and B) and a heterocycle dihydropyran (C) (Figure 6).
O A B C 1 2 3 4 5 6 7 8 1' 2' 3' 4' 5' 6' Flavonoids
Figure 6. Basic structure of flavonoids.
This group can be divided into a variety of classes that differ in the level of oxidation and substitution pattern of the C nucleus, such as flavones (e.g., flavone,
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apigenin, and luteolin), flavonols (e.g., quercetin, kaempferol, myricetin, and fisetin), flavanones (e.g., flavanone, hesperetin and naringin) and others (Min et al., 2015).
The term "phenolic acids" in general describes phenols that have a carboxylic acid functionality. However, in the description of plant metabolites, it is a distinct group of organic acids. These natural phenolic acids contain two distinctive carbon structures: hydroxycinnamic and hydroxybenzoic structures.
Although the basic skeleton remains the same, the number and position of the hydroxyl groups on the aromatic cycle create variety (Soto et al., 2015).
Hydroxybenzoic acids consist of a seven-carbon skeleton (C6-C1) and derive from benzoic acid. These acids are 26 of the simplest phenolic forms, prevalent in both gymnosperms and angiosperms, they are characterized by the presence of a substituecarboxyl group on phenol and are obtained under the action of shikimate (Vermerris and Nicholson, 2007). While hydroxycinnamic acids derive from cinnamic acid and have a carbon skeleton-type C6-C3. They are mainly found in transform, but cis isomers also exist. Because of their potential toxicity to plant cells, hydroxycinnamic acids are often complex to various molecules. Hydroxycinnamic acids generally exist in the plant in the form of glucosides, or in the form of esters (Soto et al., 2015).
1.3.4. Vitamins
Vitamins are essential nutrients that are required for various biochemical and physiological processes in the body. It is well known that most of the vitamins are unable to be synthesized in the body, so it is essential to obtain them through the diet primarily from fruits and vegetables.
In the plant and the animal kingdom, the endogenous production of vitamin C (or ascorbic acid) is widespread, only a few organisms do not synthesize it. This is the case for some fish (carp, rainbow trout), frugivorous bats, guinea pigs, humans, and some primates. They cannot convert glucose into ascorbic acid, through multiple chemical and enzymatic reactions, because they do not possess L-guluno- γ -lactone dehydrogenase, an enzyme contained in the liver. As a result of this deficiency, which is from a mutation that occurred about 40 million years ago, these organisms and therefore humans find themselves in the need to obtain these vitamins through the diet. It was Haworth in 1932 the first who established the chemical structure of ascorbic acid.
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It can be oxidized in dehydroascorbic acid (C-H-O) preferentially in alkaline environments (Gropper and Smith, 2012; Hickey and Saul, 2008). These two active molecules will coexist physiologically in the body's fluids at different proportions. Dehydroascorbic acid accounts for only 5-20% of the circulating vitamin C in the blood, compared to 80-95% for ascorbic acid. The absorption of ascorbic acid in the intestine is mostly regulated by a transport protein, SVCT1, which also regulates its reabsorption in the kidneys (190) (Malo C and Wilson JX, 2000; Takanga H, Mackenzie B and Hediger MA). The absorption of dehydroascorbic acid, brought by food or formed in the intestine as a result of the oxidation of ascorbic acid, is regulated by PROTEINs GLUT (mainly GLUT1), responsible for the glucose's transport (). After being absorbed, dehydroascorbic acid is quickly reduced to ascorbic acid by an enzyme. Thus, in a healthy individual, plasma concentrations of dehydroascorbic acid are very low (Smirnoff et al., 2001). The proportion of plasma ascorbic acid that results from this conversion is not known. Ascorbic acid can be absorbed beyond the saturation threshold of the body's cells but exerts back control on SVCT1, which helps limit the amount of excess ascorbic acid absorbed. Vitamin C has several physiological roles in our body, it is a powerful antioxidant to fight against cellular aging. Besides, it affects the regeneration of vitamin E, the main antioxidant in our body (Chambial and Dwivedi, 2013). It is essential for the synthesis of collagen as well as carnitine (an amino acid). It is involved in the synthesis of stress hormones (adrenaline and norepinephrine) and steroid hormones. It also helps strengthen our immune system by stimulating our defenses during microbial attacks. Besides, it promotes digestive absorption and the use of iron, which helps to accelerate the formation of red blood cells and thus reduce the risk of anemia (Gropper & Smith, 2012; Medeiros & Wildman, 2013). It appears to prevent the release of histamine, a compound involved in allergies and inflammatory reactions. It plays a protective role against various diseases related to oxidative stress (Ferreira et al., 2009).
Vitamin E is a commonly used term for two major groups of molecules: tocopherols and tocotrienols, each with 4 vitamers (Jensen and Lauridsen, 2007. Their chemical structures consist of a single-, di-, or tri-methylated chromanol cycle to which is attached a lateral chain of 16 carbon atoms, of isoprene structure.
This chain defines the two large families: tocopherols, saturated lateral chain, and tocotrienols, with a lateral chain with three unsaturations (Figure 7). The designation of
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the number and position of methyl groups on the aromatic cycle depends on the number and position of methyl groups (Aggarwal et al., 2010; Brigelius Flohe, 2009). The most frequently found forms are alpha- and gamma-tocopherols. It is also those with the highest vitamin activity, determining their content and composition depends on the source (Rizvi et al., 2014). The concentration of alpha tocopherols in the blood is higher than that of gamma-tocopherols while beta and delta-tocopherols are found in plasma but with a low concentration.
CH3 CH3 OH R2 CH3 CH3 CH3 R1 A CH3 CH3 OH R2 CH3 CH3 CH3 R1 CH3 B
Figure 7. General chemical structure of vitamin E isoforms. A- Tocopherols (α-tocopherol: R1 and R2 = Me; β-tocopherol: R1 = Me and R2 = H; γ-tocopherol: R1 = H and R2 = Me; δ-tocopherol: R1 and R2 = H). B - Tocotrienols (α-tocotrienol: R1 and R2 = Me; β-tocotrienol: R1 = Me and R2=H; γ- tocotrienol:
R1 =H and R2 = Me; δ- tocotrienol: R1 and R2 = H) (Stpanuk and Caudill 2013).
Most of the plant and animal species have the ability to synthesize vitamin C from glucose and galactose through uronic acid pathway but man and other primates cannot do so because of deficiency of enzyme gulonolactone oxidase [EC 1.1.3.8] required for its biosynthesis. Deficiency of this enzyme is a result of a mutation which occurred approximately 40 million years ago (Smirnoff et al., 2001). They are classified on the basis of their solubility as water soluble (C and B complexes) and fat-soluble vitamins (A, D, E, K).
Vitamin C or ascorbic acid (AA) was first isolated in 1923 by Hungarian biochemist and Nobel laureate Szent-Gyorgyi and synthesized by Howarth and Hirst.
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However, we can find vitamin C in its reduced forms as ascorbate and in its oxidized form as dehydroascorbic acid (Gropper & Smith, 2012; Hickey & Saul, 2008) which are easily inter-convertible and biologically active thus it acts as important antioxidant. Vitamin C is easily oxidized acid and destroyed by oxygen, alkali and high temperature. Vitamin C, consumed either with diet or dietary supplements, is absorbed by the epithelial cells of the small intestine by SVCT1 or, subsequently diffuses into the surrounding capillaries and then the circulatory system (Malo and Wilson, 2000; Takanga et al., 2003). The body requires vitamin C for normal physiological functions. It helps in the synthesis and metabolism of tyrosine, folic acid and tryptophan, hydroxylation of glycine, proline, lysine carnitine and catecholamine. C plays an important role in a number of metabolic functions including the activation of the B vitamin, folic acid, the conversion of cholesterol to bile acids and the conversion of the amino acid such as tyrosine, and neurotransmitters such as serotonin (Gropper & Smith, 2012; Medeiros & Wildman, 2013). As an antioxidant, it protects the body from various deleterious effects of free radicals, pollutants and toxins. The therapeutic effect of vitamin C was explored by Linus Pauling however his work on therapeutic role of vitamin C in his later years generated much controversy yet he was the first to introduce the concept of high doses of vitamin C for the treatment of various conditions from common cold to cancer (Chambial and Dwivedi, 2013).
In addition, ascorbic acid is thought to exert a protective role against various oxidative stress-related diseases such as heart disease, stroke, cancer, several neurodegenerative diseases and cataractogenesis (Ferreira et al., 2009).
Vitamin E is a term frequently used to designate a family of chemically related compounds, namely tocopherols and tocotrienols, which share a common structure with a chromanol head and isoprenic chain. Vitamin E is composed of eight chemical compounds namely, the alpha, beta, gamma and delta classes of tocopherol and tocotrienol, which are synthesised by plants from homogentisic acid. Alpha-and gamma-tocopherols are the two major forms of the vitamin, with the relative proportions of these depending on the source (Rizvi et al., 2014).
Among the tocopherols, the alpha- and gamma-tocopherols are found in the serum and the red blood cells, with alpha-tocopherol present in the highest concentration. Beta- and delta-tocopherols are found in the plasma in minute concentrations only. Tocopherols and tocotrienols are monophenols and derivatives of a 6-chromanol ring
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which differ between them only in the saturation or the unsaturation of their side chain
(Figure 7). CH3 CH3 OH R2 CH3 CH3 CH3 R1 A CH3 CH3 OH R2 CH3 CH3 CH3 R1 CH3 B
Figure 8. General chemical structure of vitamin E isoforms. A- Tocopherols (α-tocopherol: R1 and R2 = Me; β-tocopherol: R1 = Me and R2 = H; γ-tocopherol: R1 = H and R2 = Me; δ-tocopherol: R1 and R2 = H). B - Tocotrienols (α-tocotrienol: R1 and R2 = Me; β-tocotrienol: R1 = Me and R2=H; γ- tocotrienol:
R1 =H and R2 = Me; δ- tocotrienol: R1 and R2 = H) (Stpanuk and Caudill 2013).
Tocopherols and tocotrienols are found with a yellow coloration at an ambient temperature. They are compounds that are virtually insoluble in water but miscible in aprotic solvents. In the diet, the main sources of vitamin E are vegetable oils and products derived from these oils. Of the oils, those derived from wheat and sunflower germs are the richest in the tocopherol (Grilo et al., 2014). Oilseed fruits such as hazelnuts and almonds, and cereals are also sources of vitamin E. Tocopherols and tocotrienols are also found in the lipid fractions of certain animal products such as liver, eggs and milk fat, but to a lesser extent. Green vegetables contain small amounts of them (Franzen et al., 1991; Bartoli et al., 1997; Grusak and DellaPenna, 1999).
Like other fat-soluble vitamins, vitamin E is absorbed in the presence of bile salts and fats. From the intestine, it is transported to the liver but also to the adipose tissue, heart, muscles, adrenal glands and pituitary gland, where it is stored. The physiological roles of vitamin E has many physiological properties, it is widely recognized in particular by its antioxidant activity which prevents many diseases related to oxidative stress (Barros et al., 2008). It is a powerful antioxidant that neutralizes free radicals, protecting our body's cell membranes and slowing their premature aging. It helps
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protect red blood cells and thus avoids their hemolysis. It also acts at the platelet level by avoiding their excessive aggregation (risk of thrombosis) and on our immune system by intervening on the formation of antibodies.
In addition, e vitamins are widely used by the food industry in food preservation, which improves the nutritional value of food and helps overcome losses when processing food. Tocopherols are allowed as food additives. They are used as antioxidants in foods, individually or in combination, and are classified in the "E" classification as naturally occurring extract rich in tocopherol (E306), synthetic tocopherol (All-rac-α-tocopherol; DL-α-tocopherol; E307), synthetic γ-tocopherol (DL-γ-tocopherol; E308) and synthetic δ-tocopherol (E309) (Tomassi & Silano, 1986).
1.3.5. Fatty acids
Fatty acids are part of the large lipid family (Fahy et al., 2005), they are formed of a hydrocarbon chain with a carboxyl group (COOH) at one extremity and a methyl grouping (CH3) at the other extremity (Guesnet et al., 2005). They are unsaturated or saturated depending on whether they contain or not dual links. They can also have cis or trans configurations when they have dual links. In its natural state, the majority of fatty acids have a cis configuration. Different nomenclatures exist, nutritionists use the nomenclature that indicates the length of their carbon chain, the absence or presence of double bonds that reflects biochemical reactivity, and family belonging (position of the first double link to the terminal methyl cluster) (Calder, 2015; Wall et al., 2010).
The unsaturated fatty acids are classified into monounsaturated fatty acids that have only one double bond and polyunsaturated fatty acids which have several dual bonds. Among the polyunsaturated fatty acids, there are two fatty acids families called the Omega-3 or W3 family and the 6 or w6 family. The precursors of the omega-3 and omega-6 family are linolenic acid (18: omega-3womega-3) and linoleic acid (18: 2w6), respectively. These precursors can be elongated and desaturated in their long-chain derivatives in particular: arachidonic acid (20: 4w6), eicosapentaenoic acid (20: 5w3), docosapentaenoic acid (22: 5w3) and docosahexaenoic acid (22: 6w3). Linoleic acid and linolenic acid are called essential polyunsaturated fatty acids because our bodies are unable to synthesize them by themselves (McGuire - Beerman, 2012).
Only plants have the necessary enzymes for their synthesis. Therefore they must be provided with food. Polyunsaturated fatty acids and their properties are the subjects
