Flavonoids and Phenolics E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Fachliteratur
- Serie: Medicinal Chemistry Lessons From Nature
- Sprache: Englisch
- Veröffentlichungsjahr: 2003
Medicinal chemists around the world have been inspired by nature and have successfully extracted chemicals from plants. Research on enzymatic modifications of naturally occurring compounds has played a critical role in the search for biologically active molecules to treat diseases.
This book explores compounds of interest to researchers and clinicians. It presents a comprehensive analysis about the medicinal chemistry (drug design, structure-activity relationships, permeability data, cytotoxicity, appropriate statistical procedures, molecular modelling studies) of different compounds. Each chapter brings contributions from known scientists explaining experimental results which can be translated into clinical practice.
Each chapter follows a specific format for a phytochemical agent with common chemical features:
• General background on the (phyto)chemistry of the scaffold
• General background on the pharmacological profile of the scaffold
• A Description of the proposed derivatives and their advantages with respect to the parent compounds (emphasizing the synthetic approaches and structure-activity relationships)
• In silico analysis of the crucial interactions with the biological target
• Clinical studies and patent survey (if available) on the new and proposed structures
The objective of this book set is to fulfil gaps in currently acquired knowledge with information from the recent years. It serves as a guide for academic and professional researchers and clinicians.
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FOREWORD
The intention of this volume is to give an overview of the latest discoveries in the research on natural products-derived compounds, through a medicinal chemistry approach of the most exciting topics on flavonoids and (poly)phenolics derivatives. It is structured with innovative book setting outlines and a clear distinction between experimental and clinical results, in order to aid the reader to know at which step of the pipeline each compound is. Due to the scarcity of information of the other competing books, the Guest Editor wants to fulfil the gap of the acquired knowledge in the last few years with the aim to provide a guide for academic and professional researchers and clinicians.
The exploration of the chemical space ranges from flavonoids to phenolic compounds, covering all the aspects relevant for medicinal chemistry (drug design, structure-activity relationships, permeability data, cytotoxicity, appropriate statistical procedures, molecular modelling studies and technological formulations). Each chapter reviews on agents with common chemical features, considering them as scaffolds to obtain various derivatives aiming at the biological activity. The chemical modifications of these agents could increase their intrinsic properties, overcome limitations as drug candidates and introduce new properties. Thus, the volume is intended to be useful to researchers for more concrete applications in the natural product field. As far as I know, this is the first time these data are organized focusing on the synthetic methods and their strategies comprehending the last years.
KEY FEATURES
Updated information on synthetic/natural compounds;In-depth analysis of novel findings and promising translational applications;Use of organic reactions as a powerful tool in drug discovery to improve the biological activity or give new chemical and biological properties to the parent molecules;Molecular mechanisms with innovative approaches for the readers to improve their own research investigations.PREFACE
Natural products are often used in drug development due to their ability to provide unique and chemically diverse structures unmatched by any synthetic chemical collection. Medicinal Chemists have always been inspired by nature because natural products are often perceived as safer and for their capability to interact with biological targets. Indeed, in recent years, there has been emerging research on traditional herbal medicines based on their efficacy in the treatment of diseases for which they have been traditionally applied.
Conversely, natural compounds suffer from several issues such as scarce availability and seasonality, high differences in the production/extraction/isolation, low purity in commercial products from worldwide suppliers, and side effects. Moreover, due to their chemical complexity and the optional presence of different chiral centers, the total synthesis of a natural compound can be also challenging and expensive.
This book series would propose the latest discoveries in the field of compounds inspired by nature and obtained by chemical/enzymatic modification of a natural compound in the search for biologically active molecules for the treatment of human/animal ailments and permit the disposal of a wider arsenal for clinicians. The natural compounds are grouped into three clusters. The chapters are built in the following format: • General background on the (phyto)chemistry of the scaffold; • General background on the pharmacological profile of the scaffold; • Description of the proposed derivatives and their potentialities with respect to the parent compounds (with a particular emphasis on the synthetic approaches and structure-activity relationships); • In silico analysis of the crucial interactions with the biological target, when available; • Clinical studies and patent surveys (if available) on the new and proposed structures.
The readership of this book is represented primarily by Academies, Researchers, Specialists in the pharmaceutical field, Industry sector, Contract Research Organizations and hospitals dealing with clinical research.
List of Contributors
Polyphenols and Flavonoids: Chemical, Pharmacological and Therapeutic Aspects
Stefania Cesa1,*,Francesco Cairone1,Celeste De Monte1Abstract
Polyphenols and flavonoids represent a group of compounds characterized by a large assortment of phenolic structures, which can be naturally found in vegetables, roots, stems, flowers, grains, and fruits. Thanks to their biological activities, molecules belonging to these classes of compounds, besides their nutritional role, have found applications in several fields such as pharmaceutical, cosmetic, and nutraceutical. In fact, like many natural derivatives from plants, they possess several therapeutic properties, including antitumor, anti-oxidative, anti-neurodegenerative, antimicrobial and anti-inflammatory effects. Nowadays, the growing interest in polyphenolics and flavonoids translates into constant research to better define their pharmacological mechanism of action. Extraction studies in order to obtain pure compounds with a more defined biological activity, as well as pharmacokinetic studies to understand the bioavailability, the involved metabolic pathways and the related active metabolites, are carried out. Molecular docking studies are also continuously in progress to expand the field of application. Moreover, toxicity experiments to clarify their safety and studies about the interaction with other compounds to understand their selectivity of action are continuously forwarded and deepened. Consequently, many recent studies are aimed at introducing polyphenols, more specifically flavonoids, and their semi-synthetic derivatives, in the prevention, management and treatment of several diseases.
INTRODUCTION
Polyphenols are the plant’s secondary metabolites, contained in specialized cells in small quantities and not necessary for cell viability that vegetal organisms produce to perform different functions. They include several classes of chemical molecules characterized by the presence of aromatic rings bearing more than a hydroxylic function, up to complex polycyclic and polymeric compounds.
All molecules presenting a simple phenolic group are theoretically able to act as anti-radical species since they could react with endogen radicals to undergo new and more stable radical residues, which tend to react by neutralizing rather than attacking macromolecules such as DNA or proteins causing damages up to mutagenesis or unfolding. Moreover, the presence of ortho-diphenol groups also allows the metals chelation, improving the antioxidant properties. Polyphenols are frequently classified in relation to their chemical structure into four principal molecules, represented by phenolic acids, flavonoids, stilbenes and lignans. Natural products have always been the subject of great interest thanks to their biological activities and pharmacological properties. The attention towards bioactive compounds is growing more and more over the years because they found applications in pharmaceutical, nutraceutical, cosmetic and medical fields [1]. By virtue of appropriate pharmacodynamic, pharmacokinetic, bioavailability and toxicity studies, they can potentially be included among dietary supplements and therapeutic tools for the prevention and treatment of many human diseases with important applications in phytotherapy and herbal medicine [2]. A recent review [3] reports the protection exerted by a polyphenol-rich diet, highlighting the potential ability of pure polyphenols and of phytocomplex to reverse oxidative stress-relative diseases and a “promising chemopreventive efficacy” through modulation of apoptosis and cellular growth, inhibition of DNA synthesis and modulation of signal transduction. Among the bioactive natural products, a preeminent position is occupied by flavonoids, plants and fungi secondary metabolites, of supreme interest both for their ubiquitous distribution in nature and for the wide structural diversification, to which is often correlated a specific bioactivity. They can be found in many parts of plants, including leaves, flowers (where flavonoids constitute the colored pigments of petals), roots, fruits, stems, seeds, rhizome, bark, gum and shell [4]. The reason for their ubiquitous location in many plant organs may be due to the flavonoids' important role in protecting them against oxidative stress and ultraviolet radiation, as well as in attracting pollinating animals [5].
Flavonoids have gained a special prominence among natural compounds of pharmaceutical and therapeutic interest, thanks to the wide range of chemical subclasses and their wide variety of pharmacological properties, such as the modulation of enzymatic activities by inhibiting lipid peroxidation and cyclo-oxygenase and lipoxygenase activity, anti-inflammatory, anti-mutagenic, antioxidative and antitumor effects [6].
In a recent review [7], authors underline the high interest, not only for polyphenols contained in several by-products of agroindustry processes but also for bound polyphenols, which could need hydrolytic treatment of the containing matrices, to make efficient their extraction yields. Analogously, Jablonsky et al. [8] studied the bioactivity potential of phenolics extracted by softwood bark, emphasizing the extract complexity and the wide applications in the pharmacologic field as cytotoxic, antioxidant, fungicidal and antibacterial substances. Moreover, Cotas et al. [9] evaluated the potential applications of polyphenols, tannins and many others extracted from seaweed (Chlorophyta, Rhodophyta and Phaeophyceae). As polyphenols are one of the most represented classes of seaweed phytochemicals and seaweeds are one of the most available organic matrices in nature, they could be more exploited for large-scale production of polyphenolic compounds.
Healthy food containing polyphenols found also application in the prevention of skin aging and skin cancer, as functional foods or as sources of nutraceuticals to be used both in food supplements, cosmetic products or a topical formulation for dermatologic applications. This application field was also recently reviewed, but authors concluded that ingredients used with this aim, are often poorly characterised or represent part of complex mixtures by which it is difficult to establish the relationship between a single molecule and its biological effect [10].
The antioxidant and free radical scavenging activities were shown for many compounds of this class, as well as the cardioprotective, antidiabetic and antiviral potential. Most researchers are actually involved in the deepening of the mechanisms underlying the anti-cancer activity and the apoptosis induction, focusing the attention on the key enzyme involved in cellular proliferation, angiogenesis progression, and metastatic processes [11]. Anyway, most parts of these results were obtained by experiments performed in vitro or ex vivo, but the real potential of a molecule or of a class of compounds needs to be evaluated on the basis of its ability to be absorbed and metabolized while maintaining its biological effect, and finally its capability to reach the active site. As flavonoids generally display low water solubility and consequently low bioavailability, a topic of greatest interest for the scientific community is the application of several strategies aiming to solve this problem. So, a valent strategy for the polyphenolic fraction valorization is represented by the possibility of enhancing their efficacy, bioavailability and release to the action site, mediated by the exploitation of nanotechnologies capable of solving many and different problems [12] related to the specific structures, which could undergo low intestinal absorption, rapid metabolism and excretion, low plasmatic contents. This research field found application in the formulation based on many different systems, differently organized, such as liposomes, solid lipid nanoparticles, nanostructured lipid carriers, nanosuspensions, and nanoemulsions. These could be able to enhance the solubility of single nutraceuticals rather than solving problems inherent with the more or less complex nature of organic extracts obtained by food, non-edible
by-products or waste of agricultural practices and, more generally, by biological resources [13, 14].
A number of different solutions were proposed, ranging among the use of adsorption enhancers, semisynthetic strategies, use of pro-drugs, transformation in more hydrophilic molecules by glycosylation, and the use of carrier systems as, among others, the just mentioned nanotechnologies. In a recent review by Zhao et al. [15], the advantages and limitations of the different applied solutions, together with an evaluation of their influence on dissolution rate, mucosal permeation and degradation during the passage throw the gastrointestinal tract are reported, and authors highlight a generally achieved great improvement of the pharmacokinetic behaviour of poorly absorbed flavonoids, significantly represented in nature and foods.
CHEMICAL STRUCTURES, CLASSIFICATION AND PROPERTIES OF FLAVONOIDS
Flavonoids are a wide group of polyphenolic natural, synthetic and semi-synthetic products with low molecular weight, whose name is due to their flavan nucleus that derives from 3,4-dihydro-2-phenyl-2H-1-benzopyran skeleton. They have a generic structure with C6-C3-C6 units, with a skeleton consisting of 15 carbons, organized in three rings, as reported in (Fig. 1). two phenyl rings (A and B rings) with a six-carbons chain, and a pyran nucleus (C ring,) with a three-carbons chain [16].
Fig. (1)) Chemical skeleton of flavonoids consisting of C6-C3-C6 units.The classification into which flavonoids are cataloged depends on the degree of oxidation and unsaturation of the C ring and also according to the position (the second, the third, or the fourth carbon atom) on which the B ring is attached to this heterocyclic ring. More frequently, the phenyl ring B is linked to the second position of the C ring and flavonoids are subdivided into many classes, such as flavones, flavonols, anthocyanidins, flavanones, flavanonols, flavanols or catechins. When the B ring is connected to the third position of the C ring, they are named isoflavonoids, as well as when the connection occurs at the fourth position, they are called neoflavonoids [17].Other kinds of phenolic compounds related to flavonoids are chalcones because they can be considered open-chain flavonoids [16]. According to Silva et al. [18], flavonoids represent about two thirds of the ingested polyphenols in the human diet and could be classified into seven classes, as shown by (Fig. 2).
Fig. (2)) Classification of polyphenols and flavonoids. For each class some molecules are mentioned.Classes of Flavonoids with the B Ring on C2
Flavones are characterized by a 2-phenyl-1-benzopyran-4-one skeleton, without substitution at the C3 position and a ketonic function at the C4 (Fig. 3). Flavones derived from plants are generally conjugated with glycosides and can be O-glycosides or C-glycosides. Examples of O-glycosides are the 7-O-glycosides diosmin or the 7-O-rhamnosylglucoside diosmetin, as well as examples of C-glucosides, are the 6-C-glucosides vitexin or the 8-C-glucosides apigenin [19]. Flavonols, such as kaempferol, quercetin and myricetin, also possess a ketone moiety in C4 and can be glycosylated, but they also present a hydroxyl group at the C3 of the core structure [20]. Flavanols, also known as catechins when there is a hydroxyl group at C3 (flavan-3-ol structure), present the saturation of the double bond between C2 and C3, and there is not a carbonyl group at the C4 position, so they have two chiral carbons (C2 and C3) and four diastereomers [21]: (+)-catechin (2R,3S), (−)-catechin (2S,3R), (+)-epicatechin (2R,3R), and (−)-epicatechin (2S,3S). Moreover, when they are esterified with gallic acid, they give rise to gallate conjugates [22]. In addition, in the class of flavanols can be found: flavan-3,4-diols, which are called leucoanthocyanins, because by heating with aqueous acidic conditions, anthocyanidins can be obtained from them [23]; proanthocyanidins, that consist of dimers, trimers, or oligomers of flavan-3-ols [24]; and condensed tannins produced by the polymerization of flavan-3-ols [25]. Flavanones, or dihydroflavones, possess a ketone group at C4 and, such as flavanols, and they have no double bond between C2 and C3 of the C ring. Among this class, there is naringenin, which shows the 4’-hydroxy moiety on the B ring, and its 7-O-glycoside, naringin, and hesperetin, with 3’-OH and 4’-methoxy groups, from which derives its glycoside hesperidin [26]. Flavanonols are 3-hydroxyflavanone derivatives such as taxifolin or dihydroquercetin [27]. Conversely, anthocyanidins are characterized by a positive charge at the oxygen atom (forming oxonium ion) of the C-ring that, for this reason, is called flavylium (2-phenylchromenylium) ion. As anthocyanidins represent the aglycone form, anthocyanins are the glycoside forms representing the plants' blue, red and purple pigments. Anthocyanidins do not possess the ketone moiety at C4 of the heterocyclic core and, according to the hydroxy and methoxy groups at the B ring, they are named cyanidin, delphinidin, pelargonidin, peonidin, malvidin, and petunidin [28].
Fig. (3)) Flavonoids with the B ring on C2.Classes of Flavonoids with the B Ring on C3
Isoflavonoids have the 3-phenylchromen-4-one nucleus and are involved in the phytoestrogens pathway in mammals (Fig. 4). They can be classified in isoflavones, such as genistein, found almost exclusively in Leguminosae and possess the 3-phenylchromen-4-one skeleton without the hydroxyl group at C2 in isoflavans, such as the nonsteroidal estrogen equol, when the skeleton is the 3-phenylchroman [29].
Fig. (4)) Flavonoids with the B ring on C3.Classes of Flavonoids Where the B Ring is Connected to the C Ring Through the 4th Position
Neoflavonoids possess the 4-phenylchromen skeleton without the hydroxyl moiety at C2. Among the 4-arylcoumarin derivatives, called neoflavones, calophyllolide was isolated first [6], as well as dalbergichromene, and belongs to the 4-phenyl-2H-1-benzopyran derivatives called neoflavenes [30].
Classes of Open-Chain Flavonoids
Chalcones (Fig. 5) or chalconoids, represent a subclass in which the C ring is absent [11]. Among chalconoids found in fruits and vegetables, there is phlorizin, that is, the glucosylated dihydrochalcone derived from the aglycone caphloretin [31], arbutin, glycosylated hydroquinone [32], chalconaringenin, from which spontaneous cyclization naringenin is obtained [33] and ellagic acid [34].
Fig. (5)) Chalcones and chemical skeleton of ellagic acid.PHARMACOLOGICAL ASPECTS OF POLYPHENOLS AND FLAVONOIDS
As described in the introduction section, the appropriate intake through the diet of polyphenolic compounds contained in fruits and vegetables could represent a valid approach in the prevention of the onset of chronic diseases, such as overweight and obesity, correlated inflammation, insulin resistance and diabetes, neurodegenerative and cardiovascular diseases and even various types of cancer [35-42]. These effects could be in part correlated with their inhibitive action on radical species of oxygen and nitrogen and with the ability to activate antioxidant enzymes, but it is also clear that each molecule could exerts more specific actions, not only attributable to its generic phenolic portion. A meta-analysis recently published [35] reports the correlation between flavonoid intake and life expectancy, with an evaluation of the associated total, cardiovascular and cancer risk. A lot of different factors were evaluated in the considered literature, among which age, sex and education level, body mass index and diabetes, habits such as smoking or physical activity, blood pressure, intake of healthy and non-healthy nutrients, such as vitamins and fibers or fatty acids, cholesterols and coffee, cardiovascular, cancer and other familiarities, and so on. The results of the meta-analysis indicated that flavonoid intake was inversely and significantly associated with total and cardiovascular risk but not with cancer mortality risk, a study whose reports are often contradictory. The conclusions support the potential protective role of polyphenols, also underlying the importance of the diet variety, including different flavonoid sources. But they also show the heterogenicity and different quality of the reviewed results. Effectively, many different aspects had to be evaluated with the aim of acquiring an overview of this complex issue. The specific character of every single molecule, as well as its capability to be absorbed and metabolized and give interaction with other nutrients or drugs, could also confer a specific activity connected to the interaction with complex biochemical pathways. The generical anti-inflammatory properties of flavonoids are probably connected to the inhibited formation of nitric oxide (NO) radicals, prostaglandin E2, tumor necrosis factor alpha and specifically proinflammatory cytokines, such as the interleukins 1β and 6. A more specific action is shown by flavonone glucosides, acting as cyclooxygenase-2 (COX-2) inhibitors, or by quercetin in reducing cancer cells proliferation, inducing apoptosis and the expression of genes related to cyclin D1, involved in the cellular cycle, or finally by catechins supporting the immune system increasing antibody production [36]. The same authors reported that “the biologic potential of phenolic acids is as wide as their structural diversity”, being able to act as antidepressant and neuroprotective as well as anticancer or antihyperglycemic, and even as enhancers of microbiota diversity with beneficium of cardiovascular and liver functionality. They conclude by asserting that clinical studies are needed to explore bioavailability, safety, and beneficial effects. One might also add that structure-activity relationship (SAR) studies, metabolism and toxicity need to be deepened to determine effective use of polyphenols as pharmaceutical compounds. Rolt & Cox [37] analysed polyphenols such as stilbenoids, flavonoids, and chalcones to support the molecular basis of inflammation and chronic disease prevention to provide a response to this strategy. Behind the scavenging action, these classes of compounds interact with transcription factors that regulate the oxidative status of cells. Resveratrol (Fig. 6). and flavonoids represent molecules of particular interest due to their capacity to undergo highly stabilised radicals after reaction with superoxide anion or other oxygen radicals. The study of resveratrol isomers showed that molecules bearing ortho hydroxy groups were able to scavenge radicals at lower concentrations and molecules bearing hydroxy groups on both rings are more efficacious. Different derivatives, still retaining the resveratrol key features, provide or enhance the antiradical activity. The study conducted on flavonoids furtherly shows a more modulated activity connected with the different scaffolds. Flavones, isoflavones, flavanones, flavonols, and hydroxy flavanones were evaluated, showing that flavonols, having the best character of electron donors and the highest stabilization by resonance, represented the most potent antioxidant structures [37]. This is well shown by the comparison of the higher antiradical activity of quercetin with respect to luteolin, which only differs for the flavon (luteolin) and flavonols (quercetin) scaffold. Another important regulation key is represented by the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), whose levels rise in the oxidized cells with the function of enhancing the antioxidant cellular mechanism. Consequently, the activation of Nrf2 signalling, induced by stilbenoids, flavonoids such as fisetin and its dihydroxy analogue in C7, or chalcones, as well as other small molecules interfering by dithiol groups, ameliorates the age-related diseases. SAR studies have elucidated that the α,β-unsaturated carbonyl group is needed for this indirect antioxidant activity. In conclusion, the authors highlighted different polyvalent scaffolds, evidencing that compounds which simultaneously target multiple therapeutic pathways are more efficient in modulating and delaying age-related diseases, preventing oxidative stress, inflammation and cellular senescence. The ability of flavonoids to scavenge reactive species of oxygen and nitrogen, regardless of their sources, was also evaluated in another recent review as an effect on mitochondria activity, these organelles representing the main source of intracellular radical species [38]. Baicalein, silibinin, quercetin and catechins were found to protect several organs by the inhibition of the Fenton reaction and nitrosative stress, by increasing ATP levels and scavenging superoxide anion. Hippocampal, neuronal, heart, kidney and liver cells were protected against oxidative injuries through activation of antioxidant enzymes transcription in the nuclear factor erythroid 2-related factor 2 with a delay of all the relative associated chronic diseases, such as diabetes, cardiovascular and coronary heart, vasoconstriction, high blood pressure and stroke, Alzheimer’s, Parkinson’s and other chronic neurodegenerative and cardiac diseases [38]. Regarding dysmetabolic syndrome and obesity-related diseases, in the review by Sandoval et al. [39], the effect of the different groups of flavonoids on the liver and white and brown adipose tissue was evaluated, highlighting the involved molecular mechanism. According to the reported results, anthocyanins, catechins and proanthocyanidins activity in the liver is joined to the activation of adenosine monophosphate-activated protein kinase. This enzyme is involved in the metabolism of glucose and lipid oxidation, with the upregulation of glycolysis and fatty acid oxidation and downregulation of gluconeogenic and lipogenic genes. Flavonoids activity as SIRT1 (sirtuin 1) activators was discussed in the review by Sayed et al. [40]. SIRT1 is a member of the sirtuin protein implicated in the maintenance of health status and longevity, which in response to external and stress stimuli, regulates the gene expression and cell survival, decreasing the transcription nuclear factor kappa B (NF-kB) and cytokine release, through the inhibition of cyclooxygenase-2 and of inducible nitric oxide synthase enzymes. As counteract, downregulation of SIRT1 is correlated with increased acetylated NF-kB and the onset of the inflammatory cascade. Accordingly, many recent studies were reviewed about the potential of polyphenols, and particularly of flavonoids, alone or in combination, in the complex battle against cancer [34, 41-43]. Cancer onset, proliferation, migration and tumour cell dissemination, highly correlated with other diseases, such as obesity and inflammation, could be hindered by the appropriate use of a plethora of molecules, highly represented in vegetal, microbial and marine matrices, which could find application alone, or as phytocomplex, or better in combination with drugs usually adopted in cancer therapy. Many different polyphenolic molecules (some of which are reported in Fig. 6. were evaluated in the four cited reviews [34, 41-43], e.g., the stilbene resveratrol, curcumin, coumaric acid, lignans as arctigenin, magnolol, honokiol; flavones, flavonols and flavonones as apigenin, luteolin and chrisin, quercetin, kaempferol, myricetin, taxifolin and fisetin, naringenin and hesperetin, as well as the corresponding glycosylated naringin, hesperidin and rutin; flavanols such as epigallocatechin gallate and catechin, isoflavones daidzein and genistein, the chalcone ellagic acid and the anthocyanin delphinidin.
Fig. (6)) Non-flavonoid polyphenols of pharmacological interest.Many different action mechanisms, among which stand out the activity towards ROS and the inhibition of nitric oxide synthase, the activation of caspases, the inhibition of the transcription nuclear factor NF-kB and of the tumour nuclear factor TNF-α, the interleukins inhibition, the downregulation of COX-2, the modulation of p53 protein and of P-glycoprotein were evidenced. All these actions and many others explained in their role on specific signalling pathways, result, in turn, in DNA protection, inhibition of proliferation and promotion of apoptosis of cancer cells, inhibition of angiogenesis and metastatic processes, interference with other toxic drugs with an increase of cellular uptake, side effects reduction and amelioration of multidrug resistance. Highly correlated with the anti-inflammatory potential of these molecules are also the effects studied on rheumatoid arthritis [44] and neuropathic pain [45], the prevention of urinary tract infections [46], hyperuricemia [47], as well as the health effects shown on the prevention of chronic cardiovascular and neurodegenerative diseases [48-50]. In the review by Singh et al. [44], a series of 33 medicinal plants used against biomarkers of inflammation onset and progression was evaluated with the aim to show their efficacy against rheumatoid arthritis, an autoimmune disease characterized by rheumatic organ disease, as well as systemic implications. Conclusions indicate that polyphenols and flavonoids, such as gallic, vanillic and syringic acids, proanthocyanidins and tannins are active towards TNF-α, NF-kB and the correlated interleukins 1, 1a, 1b, 4, 6 and 17, as well as against COX-1 and -2, lipoperoxidase (LOX-1 and -2), inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), mitogen-activated protein (MAP) kinase, preventing the joint damage, inflammation and pain, by targeting the inflammation mediators as a whole. Proinflammatory cytokines such as TNF-α and COX-2 are also produced in the case of nerve damage associated with neuropathic pain. Peripheral nerve injury causing sensitization and hyperexcitability, which lead to spontaneous, superficial, paroxysmal and finally neuropathic pain, is naturally counteracted by the γ-aminobutyric acid (GABA) activity. Two types of GABA receptors exist, GABA A and GABA B. GABA A receptor modulators, such as benzodiazepine drugs, were largely used in therapy. Flavonoids, with particular attention to 6-methoxy-flavones and flavanones, were widely studied for their activity as GABA A positive allosteric modulators [45]. Parkinson’s disease and other neurodegenerative diseases are strictly associated with oxidative stress [49] because the degeneration of dopaminergic neurons of the substantia nigra could be induced by neurotoxic events highly correlated with the oxidative status, both in terms of radical oxygen species (ROS) production and of mitochondrial dysfunction, which could trigger downregulation of neurotrophic factors and promote protein aggregation. Besides Parkinson’s, Alzheimer’s and Huntington’s diseases as well as multiple and amyotrophic lateral sclerosis, are induced by biochemical alterations strictly joined to cellular oxidative stress and ROS production. The phosphorylation of the nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) and its translocation in the cell nucleus is individuated as the key mechanism of flavonoid control of secondary oxidative stress. Many flavonoid structures such as flavanols catechin and epigallocatechin gallate, flavanones naringin, hesperetin and pinocembrin, flavononols as ampelopsin, flavones chrisin, baicalein, apigenin, luteolin, flavonols as quercetin, myricetin, fisetin, rutin, kaempferol, anthocyanidins as cyanidins and pelargonidins, isoflavones as genistein were all reported as anti-Parkinson bioactive molecules and deepened in their action mechanism [49]. As regards the isoflavones, well focused papers were also published in relation to their protective role as phytoestrogens in pregnancy, premenopausal and postmenopausal stages of a woman's life [51]. Besides isoflavones, dietary phytoestrogens were also recognized in coumestan and lignans structures, and relative studies indicate them as protective agents against osteoporosis progression, cardiometabolic and cognitive dysfunctions, breast and prostate cancer progression and menopausal symptoms. Among the modulating effects of the endocrine system, to these classes is recognized an action of thyroid stimulation, insulin-resistance amelioration and adiponectin activation, with overall positive effects on the prevention of the dysmetabolic syndrome [51]. Phytoestrogens can directly bind the estrogenic receptors, with different affinity for the α and β classes, which result in a modulated protection activity. Finally, in the last year, many studies were carried on in relation to the antiviral [52] and anti-COVID 19 (Coronavirus Disease 2019) potential [53]. Different flavonoids were already recognized for their antiviral activity. For example, apigenin, luteolin, quercetin, naringenin, and diosmetin were studied for their anti-HCV activity, epigallocatechin-3-gallate for anti-HCV (Hepatitis C virus), anti-HBV (Hepatitis B virus) and anti-HIV (Human immunodeficiency virus) activity, vitexin for anti-H1N1 (Hemagglutinin Type 1 and Neuraminidase Type 1 influenza virus) and anti-HAV (Hepatitis A virus) activity, myricetin for anti-HIV activity, vitexin and quercetin-3-rhamnoside against influenza virus, baicalin against Dengue virus, pinocembrin against Zika virus. The main antiviral mechanism is recognized in the inhibition of different enzymes essential for viral survival and replication, such as DNA/RNA polymerase, neuraminidase or proteases. Among flavonoids, a synergistic effect or absorption increase could be modulated by the same chemical compounds or by other metabolites present in the different phytocomplex [51]. In the last year, data were also collected to evaluate the potential capacity of specific known and less known flavonoid molecules to counteract COVID-19. Forty-seven molecules were well-known or very few widespread chalcones, flavans, flavanols, flavanons, flavanonols, flavones, isoflavones and procyanidins, were identified as lead compounds, mainly focusing the attention on their interference with 3-CL (3-chymotrypsin-like) and PL (papain-like) viral proteases. These represent, in fact, key targets involved in the genomic RNA replication and transcription within host cells. Deepened studies on the structure activity relations are reported, which seems to be very relevant for designing new potent drugs in this new challenge involving the scientific community.
BIOAVAILABILITY AND METABOLISM OF FLAVONOIDS, TOXICOLOGICAL ACTIVITY AND SEMI-SYNTHETIC STRATEGIES
The pharmacological effects of flavonoids are influenced not only by the great variety in the chemical structures but also by the natural source from which they are obtained, the concentration in the food intake, and the interaction with other dietary molecules. Therapeutic efficacy depends on the bioavailability and on metabolism of these bioactive phenolic derivatives [54, 55]. Furthermore, regarding their pharmacokinetic properties, absorption, distribution, metabolism and elimination (ADME), it is also important to consider the inter-individual differences and variability in the population [56, 57]. The bioavailability of flavonoids may actually differ from one individual to another and their metabolic pathway is influenced also by the microbiome in the intestinal tract. In fact, flavonoids are exposed to resident microorganisms reacting with them and converting these compounds into smaller aromatic and phenolic derivatives [58]. Another factor to be taken into consideration for the use of flavonoids as therapeutic tools, besides the bioavailability and metabolism, is represented by the problems related to their extraction. In fact, as flavonoids possess very similar chemical structures, the isolation and purification processes could be long and labor intensive. Therefore, research in recent years is also focusing on the study of new strategies in order to more easily obtain specific products. In particular, semi-synthetic natural flavonoids can be investigated from the point of view of the structure-activity relationship (SAR) to obtain specific information on the pharmacodynamics of these bioactive compounds [59].
ADME of Flavonoids
As reported in Fig. 7, flavonoids taken within the diet are mostly not absorbed in the small intestine and when they arrive in the colon tract, they interact with the local microbiota and are subjected to the action of hydrolysis. Flavonoids are metabolized in the epithelium and the derivatives are carried to the liver, metabolized again with phase I and phase II mechanisms and can return in the intestine through the biliary tract or reach systemic circulation and target tissues. Finally, flavonoid metabolites are excreted by kidneys and by the fecal route [55, 60].
Fig. (7)) Example scheme of flavonoids metabolism.In detail, with the exception of anthocyanins, which arrive in plasma and urinary tract in the form of glycosides [43], flavonoid glycosides are generally hydrolyzed before absorption in two different ways. In the small intestine, they are subjected to the action of the transmembrane enzymatic protein lactase phlorizin hydrolase (LPH, lactase) and transformed into aglycones endowed with greater lipophilicity that allows them to enter the epithelium by passive diffusion. Otherwise, the glycoside forms are carried in the epithelial cells by sodium-dependent glucose transporter and then they are converted into aglycone thanks to intracellular β-glucosidases [61, 63]. The transcellular transport of flavonoids takes place thanks to membrane bound ATP binding cassette (ABC) transport proteins, such as P-glycoprotein and multidrug resistance proteins, that influence flavonoids' bioavailability by acting in two different ways. They could enhance bioavailability by allowing the flux of flavonoids from the intestinal cells into the portal bloodstream or, otherwise, decrease bioavailability by carrying the molecules back into the intestinal lumen [63]. In the liver, flavonoids are converted in the oxidated or O-demethylated forms, thanks to the activity of cytochrome P450 (CYP) monooxygenases such as CYP3A4 and CYP2C9 isoforms [64]. Flavonoids, not absorbed by the small intestine, undergo the action of the enzymes produced by microflora in the large intestine, resulting in the formation of aglycones and phenolic acid derivatives [65]. All these aglycone metabolites are available for conjugation by phase II metabolism with the reactions of methylation, glucuronidation, or sulphation by catechol-O-methyltransferases (COMTs, whose reaction concerns the catecholic flavonoids like catechins, epicatechins, and epigallocatechins), urine-5’-diphosphate glucuronosyltransferases (UGTs), and sulphotransferases. By these reactions occurring in the intestine or in the liver, the resulting conjugated derivatives reach the bloodstream and are distributed in tissues [66]. Finally, a portion of metabolites is collected in the renal tubules through organic anion transporters and eliminated through the urine [67], whereas some of the metabolites are excreted through the biliary duct, and, thanks to enterohepatic recirculation, they come back in the intestine and undergo fecal elimination [68]. On the other hand, as regards the polymeric forms such as proanthocyanidins, only a few pharmacokinetic data are available and the in vitro and in vivo experiments show that their absorption is much lower than flavonoid monomers. Proanthocyanidins were detected in plasma with a degree of polymerization not higher than the dimer forms [65], maybe because they are hydrolyzed in the stomach by acid [69], even if the buffering effect of a specific food may differ from another and this could influence the stomach acidity necessary for their hydrolysis. So, as a result, there is also evidence of oligomer stability during gastric transit [70].
Factors that Could Affect ADME of Flavonoids
Flavonoids are taken within the diet together with other components present in foods such as proteins, carbohydrates, fats and ethanol. Even if the consumption of milk has no significant impact on the absorption of flavanols [69], quercetin and kaempferol present in green or black tea [72] reduce the bioavailability of epicatechin from dark chocolate [73]. Several studies have shown that bread and sugar can improve the bioavailability of catechin and epicatechin thanks to the enhanced secretions and motility in the stomach. Furthermore, it has been detected that ethanol in red wine considerably improves the elimination of catechins in urine, probably in relation to its diuretic action [65, 74]. An example of how fats taken with diet can affect the permanence in the gastrointestinal tract is given by a study on human patients in which it was proven that, when strawberries are eaten with cream, the excretion of anthocyanin metabolites is delayed even if without changes in AUC [75]. Another clinical experiment explained that the AUC of quercetin in plasma was elevated when a fat-rich breakfast was eaten in comparison to its bioavailability in subjects who consumed a fat-free breakfast [76]. With regard to the influence of sex and age on the human metabolism and bioavailability of flavonoids, there are restricted studies that demonstrate a correlation between age or sex with the activity of phase I and phase II enzymes on these molecules. Some experiments have shown that in females, the expression and activity of glucuronidation enzymes are higher than those of the male population [77], and some data claim that the metabolism of these bioactive compounds is reduced with age because of age-related metabolic ability [78]. However, at the moment, it is not possible to draw appropriate conclusions and more studies are needed. Other experiments are also required to investigate the impact of the genetic variability on phase I and phase II enzymes on the inter-individual differences in flavonoid pharmacokinetics [56]. Regarding the drug-drug interactions with natural compounds, the impact of the assumption of drugs in flavonoid metabolism and activity is plausible, even if more studies are necessary. For example, some data have been collected on silymarin, an extract from milk thistle which contains flavonolignan silibinin (Fig. 6). which possesses anti-inflammatory, anti-oxidant, anti-diabetic and anti-neurodegenerative effects. In fact, silymarin finds applications against diseases affecting the liver, pancreas, central nervous system, kidney, and heart. The experiments show that the administration of silymarin enhances the bioavailability of the β-blocker talinolol and of the anti-sickness drug domperidone, whereas it reduces the bioavailability of the antimicrobic metronidazole and of the antiviral indinavir. Silybin also influences the pharmacokinetics of antihypertensive losartan, reducing its conversion into the active form [79]. Among the many factors that can influence the bioavailability of flavonoids, their interaction with the efflux transporter P-glycoprotein must be taken into consideration, although the specific mechanisms of interaction are still unknown. P-glycoprotein is an ATP-dependent efflux pump [80], which affects the flux of drugs and could reduce the access of flavonoids into the systemic circulation and into the blood-brain barrier (BBB), limiting the bioavailability and the neuroprotection explained by some flavonoids. Indeed, depending on the type of binding/interactions of the flavonoids with the steroid-interacting regions and ATP binding sites in the P-glycoprotein nucleotide binding domains, the permeability of flavonoids or their circulating metabolites, and so their efficacy, could be reduced or enhanced. Regarding the ability of some flavonoids and their metabolites to cross the blood-brain barrier, recent findings have shown that hesperetin, naringenin, their glucuronidated metabolites, cyanidin-3-rutinoside and pelargonidin-3-glucoside, permeated in vitro the blood-brain barrier, according to their lipophilicity [81]. In a comparison of the permeability of sucrose, which is a marker of paracellular transport, the permeability of the more lipophilic hesperetin and naringenin was higher than that of the sucrose, suggesting their transcellular flux, while the permeabilities of the glucuronide conjugates of hesperetin and naringenin and the permeability of the anthocyanins were poorer than that of sucrose. However, further investigations are needed to better understand the pharmacokinetic profiles of flavonoids, which also means better identifying their localization in the central nervous system and the correlation with neuroprotective effects and pharmacological activity against neurodegenerative diseases [82].
Toxicity of Flavonoids
Due to the beneficial effects of flavonoids for human health, it is worth taking into account their possible toxic effects, which may be due to their chemical properties, the interactions with other molecules and the concentration after consumption. In fact, evidence has demonstrated that when administered at high doses, phenolic bioactive compounds could produce reactive oxygen species (pro-oxidant activity); moreover, they could be involved in negatively regulating the expression of chaperones and antioxidant enzymes, they could have hepatotoxic and nephrotoxic effects and worsen some pathologies of the digestive system like colitis and colon tumor [83]. Conversely, in an experiment carried out on rats, Cladis et al. [84] observed that the oral administration of blueberry polyphenols, at a range of concentration from 0 to 1000 mg total polyphenols/kg bw/day for three months, did not cause changes in behavior, body weight, consumption of food, development of pathologies at the maximum concentration given. The toxic action of flavonoids is influenced also by way of assumption and the resulting biotransformation that occurs in the body. In fact, Zheleva-Dimitrova et al. [85] established that the toxicity levels of Clinopodium vulgare L. aqueous extract, which is a source of flavonoids, in mice and rats depended on the route of administration because the LD50 (Lethal Dose 50) in acute intraperitoneal administration reached at lower concentrations (675 mg/kg for mice and 500 mg/kg for rats, with central nervous system toxicity) than those of the oral administration for which LD50 was major than 2000 mg/kg. This evidence demonstrates the importance of in vivo experiments and clinical trials to assess the safe doses of flavonoids taken with a daily diet [36].
Strategies for a Better Investigation of Flavonoids Properties
Since it is not easy to obtain information from a specific natural compound due to the difficulty of separation and purification from complex matrices during the extraction processes, in order to evaluate the structure-activity relationship and the mechanism of action of the natural products, in recent years, research has also focused on the study of new semisynthetic strategies regarding natural bioactive compounds [86]. Moreover, the use of polymers such as cyclodextrins and nanoparticles could modify the solubility and could regulate the metabolism of natural molecules, and more specifically flavonoids, with the aim of improving their bioavailability and the therapeutic efficacy for the prevention and the management of several human diseases [87]. Among the semi-synthetic approaches, reactions of acetylation, methylation, hydrogenation, and cyclization of several flavonoids isolated from Eriosema genus have been described and the resulting synthetic derivatives have shown to possess interesting pharmacological properties such as antifungal, antimicrobial, antioxidant effects and also activity against erectile dysfunctions [88]. Another example of a semi-synthetic approach is given by the use of xanthohumol, which is a prenylated chalconoid found in the female inflorescences of Humulus lupulus [89], for the production of the isomers 8-prenylnaringenin and 6-prenylnaringenin, which are two secondary metabolites in hops, endowed with estrogen-like, cytotoxic, and neuro-regenerative effects. Because of the poor yield reached from their extraction, these two flavonoids can be formed from the demethylation, with lithium chloride and dimethylformamide, of xanthohumol, obtainable from the extraction industry of lupulus. The problem of this traditional method is given by the presence of by-products and the low amount of the desirable products. To avoid this inconvenience, microwave-assisted demethylation of xanthohumol could be used to optimize the temperature and the time of the reaction, using lithium chloride and dimethylformamide as not-expensive reagents, in order to achieve a final yield of 76% of 8-prenylnaringenin and 6-prenylnaringenin without by-products [90]. These new methodologies to produce natural derivatives demonstrate the importance of obtaining pure bioactive compounds of therapeutic interest that can thus be better investigated from the pharmaceutical point of view as potential drugs for the prevention and treatment of several pathologies.
CONCLUSION
Phenolic compounds extracted from plants have been shown to possess a wide spectrum of biological activities and focusing on the many classes and subclasses of polyphenols and flavonoids, there is evidence of their pharmacological properties that make them of interest to the development of new drugs. In this context, several experiments and molecular docking studies have been carried out in order to better understand their mechanism of action. Many research works have been designed to obtain information on the pharmacokinetic, safety and toxicity profiles. Moreover, different extraction techniques and semisynthetic strategies are investigated with the aim of improving the purity profile of these molecules in order to better analyze them from the pharmaceutical point of view. In conclusion, the deepening of these studies together with the implementation of preclinical and clinical trials, allows defining polyphenols and flavonoids as potential therapeutic tools with applications in preventing and treating many human pathologies.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
ACKNOWLEDGEMENTS
Declared none.
