Resveratrol
Phytochemicals

non solo bene

RESVERATROL AND POLYPHENOLS IN CHEMOPREVENTION

INTRODUCTION

Cancer is a dynamic process that involves many complex factors, which may explain why a “magic bullet” cure has not been found. The lack of such a cure has led to increased interest in chemoprevention as an alternative approach to the control of cancer. Chemoprevention is defined as a pharmacological approach used to arrest or reverse the process of cancer development before invasion and metastasis occur, by the use of nontoxic substances, including many food factors, to interfere with carcinogenesis, in which oxidative stress plays a key role.

Chemopreventive agents

Polyphenols, which are among the most common anti-oxidants in fruits and vegetables, have been proposed as primary chemopreventive agents. However, it is very difficult to predict from these results the effects of polyphenol intake on disease prevention in humans. One of the reasons is that these studies have often been conducted at doses or concentrations far beyond those documented in humans. The development of polyphenols as direct inhibitors against target proteins is regarded as a rational approach for chemoprevention. Among polyphenols, resveratrol is one of the most promising chemopreventive molecule, which naturally occurs in many plants.

Polyphenols as small molecular inhibitors of signaling cascades in carcinogenesis Kang et al.

POLYPHENOLS

Structural diversity

Polyphenols (PPs) are characterised structurally by the presence of one or more six-carbon aromatic rings and two or more phenolic hydroxyl groups. Strictly speaking, mono-phenols such as p-coumaric acid are not PPs, but they share many of their properties and characteristics and are most usefully considered as functional PPs. Polyphenols can be divided into several classes according to the number of phenol rings that they contain and the structural elements that bind these rings to one another.
There are five major classes of PPs:

• flavonoids: quercetin, cathechins, cyanidin, procyanidins

• chalcons: curcumin

• stilbenes: resveratrol

• phenolic acids: caffeic acid, ferulic acid, chlorogenic acid

• lignans

Polyphenolic phytochemicals – just antioxidants or much more? Stevenson et al.

The major biosynthetic pathway starts with phenylalanine, which is converted into cinnamic acids and then elaborated into the various other classes of compounds, ending with the anthocyanins. There are numerous smaller classes of compounds arising from other biosynthetic pathways.

Dietary intake, absorption and metabolism

Several hundreds of different polyphenols have been identified in foods. They are found in berries, grapes/wine, tea, chocolate, coffee, soybeans, and other fruits and vegetables. The total dietary intake is about 1 g/d. It is much higher than that of all other known dietary antioxidants, about 10 times higher than that of vitamin C and 100 times higher than those of vitamin E and carotenoids.

Biological properties of polyphenols depend on their bioavailability, which in turn is largely influenced by their structure. The chemical structure of polyphenols determines their rate and extent of intestinal absorption, and nature of the metabolites circulating in the plasma. Phenolic acids like caffeic acid are easily absorbed through the gut barrier, whereas large-molecular-weight polyphenols such as proanthocyanidins are very poorly absorbed. Once absorbed, polyphenols are conjugated to glucuronide, sulphate and methyl groups in the gut mucosa and inner tissues. Non-conjugated polyphenols are virtually absent in plasma. Such reactions facilitate their excretion and limit their potential toxicity. The polyphenols reaching the colon are extensively metabolised by the microflora into a wide array of low-molecular-weight phenolic acids. These, in turn, can be absorbed via the colon and augment the phenolic acids directly absorbed from the diet.
Both the liver and intestine contain high levels of phase I and II metabolic enzymes which hydroxylate
and conjugate xenobiotic compounds, respectively.

Absorption and metabolism of polyphenols in the gut and impact on health Scalbert et al.

Functions

In plants - Polyphenolic phytochemicals function in various protective roles: they inhibit the development of pathogens and decay microorganisms, and they provide protection against UV radiation and oxidative stress.

In humans - Polyphenols are the most abundant antioxidants in our diets. Polyphenols are reducing agents, and together with other dietary reducing agents protect the body’s tissues against oxidative stress and associated pathologies such as cancers, cardiovascular diseases, diabetes, neurodegenerative diseases and osteoporosis. These degenerative diseases, are associated with aging. Oxidative damage to cell components, DNA, proteins, and lipids accumulates with age and contributes to the degeneration of the somatic cells and to the pathogenesis of these diseases. Anti-oxidants present in food can help limit this damage by acting directly on reactive oxygen species or by stimulating endogenous defence systems. The phenolic groups in polyphenols can accept an electron to form relatively stable phenoxyl radicals, thereby disrupting chain oxidation reactions in cellular components.

Polyphenols: do they play a role in the prevention of human pathologies? Tapiero et al.

Regulation of inflammation and redox signaling by dietary polyphenols Rahman et al.

Anticarcinogenic effects - Carcinogenesis is a multistage process that comprises three major steps, initiation, promotion and progression, and takes many years to develop into complete malignancy:

• initiation is a relatively short and irreversible process that involves a chain of extracellular and intracellular events. Polyphenols may act as blocking agents at this stage. They influence the metabolism of procarcinogens by modulating the expression of cytochrome P450 enzymes involved in their activation to carcinogens. They may also facilitate their excretion by increasing the expression of phase II conjugating enzymes. This induction of phase II enzymes may have its origin in the toxicity of polyphenols. Polyphenols can form potentially toxic quinones in the body that are, themselves, substrates of these enzymes. The intake of polyphenols could then activate these enzymes for their own detoxication and, thus, induce a general boosting of our defenses against toxic xenobiotics. Polyphenols may also limit the formation of initiated cells by stimulating DNA repair.

• promotion is a relatively lengthy and reversible process in which actively proliferating preneoplastic cells accumulate. Polyphenols can act as suppressing agents, and inhibit the formation and growth of tumors from initiated cells.

• progression, the final stage of neoplastic transformation, involves the growth of a tumor with invasive and metastatic potential.

Dietary Polyphenols and the Prevention of Diseases Scalbert et al.

RESVERATROL

Origin of the name:

res: might be an abbreviation of the class of molecules (resveratrol belongs to the resorcinols)
veratr: abbreviation of the plant name, Veratrum grandiflorum (the poisonous but medical plant from which it was first isolated by Michio Takaoka in Japan, 1939)
ol: indicating that it contains "alcohol" chemical groups

Discovery of resveratrol

Description

Resveratrol (3,4’,5 trihydroxystilbene) is the most well-known member of a class of substances called stilbenes.
It has been detected in more than 70 plant species, including grapes, peanuts, berries, and pines.
It is a phytoalexin, a low-molecular-weight secondary metabolite, that is involved in both structural and biochemical mechanisms which provide plants with resistance to pathogens.
Synthesis is induced in response to environmental stressors such as water deprivation, ultraviolet irradiation and fungal infection. Resveratrol and other phytoalexins act as defense mechanisms by biochemically deterring invaders, making the plant a less desirable target.
It is produced in huge amount in grapevine skin in response to infection by Bothrytis cinerea. This production of resveratrol blocks the proliferation of the pathogen, thereby acting as a natural antibiotic. Fresh grape skin contains about 50 to 100 μg of resveratrol per gram wet weight, which contributes to a relatively high concentration of resveratrol in red wine and grape juice.

Absorption

Resveratrol exists in both cis and trans isomeric forms. In plants, it mostly exists in glycosylated piceid forms.

Glycosylation is known to protect resveratrol from oxidative degradation, and glycosylated resveratrol is more stable and more soluble and readily absorbed in the human gastrointestinal tract.
Numerous studies exist that have utilized a wide range of concentrations of resveratrol, suggesting that its biological effects may vary depending on cell and tissue types.

Biosynthetic pathway

The resveratrol precursor is phenylalanine and the key cell enzyme is stilbene synthase which orientates the synthesis pathway toward resveratrol, instead of toward flavonoids through chalcone synthase.
Therefore, resveratrol can be classified either as a stilbene or as a polyphenol.

Functions

Numerous studies have reported interesting properties of trans-resveratrol as a preventive agent against important pathologies i.e. vascular diseases, cancers, viral infection or neurodegenerative processes.
Resveratrol is known to have potent anti-inflammatory and antioxidant effects and to inhibit platelet aggregation and the growth of a variety of cancer cells.

Resveratrol: A review of preclinical studies for human cancer prevention Athar et al.

Chemopreventive and chemotherapeutic activities have been demonstrated in all three stages of carcinogenesis (initiation, promotion, and progression). Extensive data in human cell cultures indicate that resveratrol can modulate multiple pathways involved in cell growth, apoptosis, and inflammation. The anti-carcinogenic effects of resveratrol appear to be closely associated with its antioxidant activity. Resveratrol has been shown to inhibit cyclooxygenase, hydroperoxidase, protein kinase C, Bcl-2 phosphorylation, Akt, focal adhesion kinase, NFκB, matrix metalloprotease-9, and cell cycle regulators.

Tumor initiation - the initiation phase consists of the DNA alteration (mutation) of a normal cell, which is an irreversible and fast change. The initiated cell is capable to autonomous growth. The initiating event can consist of a single exposure to a carcinogenic agent or in some cases, it may be an inherited genetic defect.
The anti-initiation activity of resveratrol is exerted through:

• inhibition of phase I enzymes: resveratrol can prevent metabolic activation of procarcinogens by a competitive inhibition of the aryl hydrocarbon receptor (AhR) which binds to polycyclic aromatic hydrocarbons. AhR is involved in various processes such as cell proliferation, differentiation and P450 1A1 induction after xenobiotics exposure. This phase I enzyme, P450 1A1, which is well known as an aryl hydrocarbon hydroxylase, is often considered to be one of the most important enzymes involved in tumor initiation.

• induction of phase II enzymes: phase II enzyme induction generally protects tissues and cells from endogen and/or exogen intermediate carcinogens. Phase II conjugation reactions lead to the formation of a covalent linkage between a functional group on the parent compound and glucuronic acid, sulfate, glutathione, amino acids, or acetate. Resveratrol also increases glutathione levels and the activity of glutathione-S transferase (GST) as well as the activity of glutathione peroxidase (GPX) and glutathione reductase (GR) in various cells and subsequently reduces DNA damage.

• ROS: resveratrol is an effective scavenger of hydroxyl, superoxide, and metal-induced radicals. Resveratrol exhibits a protective effect against lipid peroxidation in cell membranes and DNA damage caused by ROS. These antioxidant actions of resveratrol contribute to prevent oxidative DNA damage which plays a pivotal role in the carcinogenic activity of many genotoxic agents.

• stimulation of DNA repair: many drugs and ultraviolet irradiation cause DNA damage and failure to repair this damage results in carcinogenesis. This DNA alteration presents an obstacle to DNA polymerase. In order to protect against the effects of mutational rates, several genes such as p53 have to survey the genome damage and / or to repair these damages. Resveratrol is able to stimulate DNA repair by increasing the activity of p53 in various cell lines.

Resveratrol suppresses cell transformation and induces apoptosis through a p53-dependent pathway Huang et al.

Tumor promotion - The initiated cell may remain dormant for months or years and unless a promoting event occurs, it may never develop into a clinical cancer case. The promotion phase is the second major step in the carcinogenesis process in which specific agents (referred to as promoters) trigger the further development of the initiated cells. Promoters often, but not always, interact with the cellular DNA and influence the further expression of the mutated DNA so that the initiated cell proliferates and progresses further through the carcinogenesis process.
Anti-promotion activity of resveratrol is exerted through:

• inhibition of lipid mediators: lipid mediators such as prostaglandins have been shown to be involved in promoting cell proliferation, suppressing immune surveillance, and stimulating tumorigenesis. COX-1 and COX-2 are respectively constitutive and inducible enzymes that catalyze the production of pro-inflammatory prostaglandins from arachidonic acid. Various reports show that resveratrol inhibits COX-1 and COX-2 activity.

The active site of COX1 in complex with resveratrol is shown in this macromolecular X-ray crystallography

• cell cycle: Resveratrol is able to interfere with the molecular machinery of the cell cycle arrest which involves various key regulators. Resveratrol can block the G1/S transition of the cell cycle. Resveratrol is able to act at this point by decreasing the protein expression of cyclin D1, D2, E and the protein expression of cdk 2, 4, 6 and the activities of kinases examined in various cancer cell lines. A loss of cyclin D/cdks or cyclinE/cdks kinase activities before the restriction point prevents cells from entering S phase and so resveratrol increased the number of cells in G1 phase.

• induction of cell death: resveratrol is able to activate cell death by either the mitochondrial pathway or the death receptor pathway.

Tumor progression - the progression phase is associated with the evolution of the initiated cells into a biologically malignant cell population. In this stage, a portion of the benign tumor cells may be converted into malignant forms leading to a true cancer. Tumor progression is certainly too advanced for chemopreventive intervention but not for a chemotherapeutic intervention. During tumor progression, resveratrol can act as an antiproliferative agent by:

• blocking cell cycle progression

• inducing apoptosis of cancer cells

Resveratrol as a Chemopreventive Agent: A Promising Molecule for Fighting Cancer Delmas et al.

CONCLUSION

The attractiveness of naturally occurring compounds for cancer chemoprevention has escalated in recent years. An ideal chemopreventive/therapeutic agent would restore normal growth control to preneoplastic or cancerous cells by modulating aberrant signaling pathways and/or inducing apoptosis. It should target the multiple biochemical and physiological pathways involved in tumor development, while minimizing
toxicity in normal tissues. Resveratrol has been shown to be an effective chemopreventive agent in multiple
murine models of human cancers. It has the capacity to interact with multiple molecular targets and appears to be relatively nontoxic at least at the doses tested in these models.
Several phase-I clinical trials are currently underway to evaluate the pharmacokinetics and safety of resveratrol, utilizing various dosages of resveratrol.
Thanks to all these properties, resveratrol seems to be a potential weapon for new therapeutic strategies.

Il resveratrolo (trans-3,5,4’-triidrossistilbene) è un fenolo non flavonide rinvenuto nella buccia degli acini dell’uva, nella grappa, nelle nocciole e in oltre 70 tipi di piante diverse, in particolare nel ‘polygonum cuspidatum’, pianta erbaceo-arbustiva tipica dei luoghi acquitrinosi e paludosi.
Polygonum cuspidatum è il nome di una pianta erbacea perenne, appartenente alla stessa famiglia del grano saraceno (Polygonaceae). Di origine asiatica, questa specie cresce spontaneamente in zone aride degli Stati Uniti e nel sud del Canada. P. cuspidatum è una pianta nota sin dall’antichità, impiegata dalla medicina popolare come erba lassativa, e occasionalmente utilizzata come cibo. Oggi se ne conoscono diverse applicazioni terapeutiche per varie patologie, ma attualmente l’importanza di questa pianta è in relazione all’elevata concentrazione di resveratrol in essa presente, una fitoalexina dotata di potente attività antiossidante, antitumorale e cardioprotettiva. Il resveratrol si trova in quantità apprezzabili in varie piante (uva, arachidi, ecc.), ma grande interesse ha suscitato il fatto che si ritrovi nell’uva e nei suoi derivati. Alla luce di questo, negli ultimi anni è stato molto incentivato il consumo di vini, in particolare quello rosso, poiché è stato possibile associare all’uso quotidiano, una minor incidenza di malattie cardiovascolari. Tale fenomeno è conosciuto come “paradosso francese”, per uno studio compiuto sulle popolazioni del sud della Francia, le quali, pur consumando cibi ricchi di grassi, sono meno soggette alle malattie cardiovascolari di altre che non consumano vino rosso. La peculiarità della P. cuspidatum è quella di contenere, nell’estratto secco della sua radice, resveratrol in una quantità 400 volte superiore rispetto all’uva e ai suoi derivati, rappresentando così la maggior sorgente naturale di questo composto, nella forma biologicamente più attiva. Infatti, per assumerne quantitativi efficaci, bevendo vino rosso, molto probabilmente s’incorrerebbe nell’insorgenza di altre malattie (es. cirrosi) prima di poter riscontrarne i benefici. Inoltre, utilizzando un estratto naturale e non un derivato di sintesi, si possono sfruttare non solo le proprietà terapeutiche del fitocomponente, ma anche quelle di altre sostanze bioattive presenti, che ne completano l’attività e ne favoriscono la biodisponibilità.

Nel 1997 uno studio ha evidenziato l’effetto chemiopreventivo del resveratrolo nei vari stadi della carcinogenesi.Quest’osservazione ha dato il via ad una serie di studi volti a caratterizzarne meglio le attività, tali studi hanno poi evidenziato oltre cento targets differenti che mediano diverse attività, le principali sono:
-Attività antineoplastica (antiangiogenetica, antiproliferativa)
-Antinvecchiamento
-Antiossidante (attivo, anche se dimostrato per ora solo in vitro, contro alcuni radicali liberi e impedisce l’ossidazione del colesterolo LDL.)
-Cardioprotettiva: (vasodilatante, antiaggregante piastrinica, limitazione dell’infiammazionne aterosclerotica e nelle deposizioni trombocitiche vasali correlate alla sua somministrazione)
-Antiinfiammatoria (capacita’ di inibire es la ciclo-ossigenasi, effetto osservato solo in vitro.)
-Estrogeno simile
-Ipoglicemizzante

Le ragioni che mettono in dubbio l’utilità del resveratrolo sono il dosaggio(esperimenti condotti con dosi elevate per avere una concentrazione nel sangue consigliata di almeno 10mg/l, per ottenere tale concentrazione nel nostro corpo dovrebbero circolarne 50 mg. La buccia dell’acino d’uva rossa contiene circa 50-100 microgrammi di resveratrolo/grammo di peso secco e la sua concentrazione nel vino rosso è dell’ordine di 0,3-0,5 milligrammi/litro; onde per cui un individuo di 70 Kg per assumere le stesse dosi testate in laboratorio dovrebbe mangiare 3-6 Kg di buccia di acini di uva al giorno.) e la biodisponibilità che nel resveratrolo è bassissima in quanto viene metabolizzato molto velocemente.( una dose di 25 mg lascia tracce di concentrazione nel plasma trascurabili e dopo 30-60 minuti circa i valori di picco sono 10-100 volte inferiori alle dosi utilizzate in laboratorio).