Tag Archives: flavonoids

Flavonoid biosynthesis pathway: genes and enzymes

The biosynthesis of flavonoids, probably the best characterized pathway of plant secondary metabolism, is part of the phenylpropanoid pathway that, in addition to flavonoids, leads to the formation of a wide range of phenolic compounds, such as hydroxycinnamic acids, stilbenes, lignans and lignins.
Flavonoid biosynthesis is linked to primary metabolism through both mitochondria- and plastid-derived molecules. Since it seems that most of the involved enzymes characterized to date operate in protein complexes located in the cell cytosol, these molecules must be exported to the cytoplasm to be used.
The end products are transported to different intracellular or extracellular locations, with flavonoids involved in pigmentation usually transported into the vacuoles.
The biosynthesis of this group of polyphenols requires one p-coumaroyl-CoA and three malonyl-CoA molecules as initial substrates.

Flavonoid Biosynthesis
Fig. 1 – Biosynthesis of Flavonoids

CONTENTS

Biosynthesis of p-coumaroyl-CoA

p-Coumaroyl-CoA is the pivotal branch-point metabolite in the phenylpropanoid pathway, being the precursor of a wide variety of phenolic compounds, both flavonoid and non-flavonoid polyphenols.

Skeletal formula of p-Coumaroyl-CoA, the pivotal branch-point metabolite in the phenylpropanoid pathway
Fig. 2 – p-Cumaroyl-CoA

It is produced from phenylalanine via three reactions catalyzed by cytosolic enzymes collectively called group I or early-acting enzymes, in order of action:

  • phenylalanine ammonia lyase (EC 4.3.1.24);
  • cinnamate 4-hydroxylase or cinnamic acid 4-hydroxylase (EC 1.14.13.11);
  • 4-cumarato: CoA ligase or hydroxycinnamic: CoA ligase (EC 6.2.1.12).

They seems to be associated in a multienzyme complex anchored to the endoplasmic reticulum membrane. The anchoring is probably ensured by cinnamate 4-hydroxylase that inserts its N-terminal domain into the membrane of the endoplasmic reticulum itself. These complexes, referred to as “metabolons”, allow the product of a reaction to be channeled directly as substrate to the active site of the enzyme that catalyzes the consecutive reaction in the metabolic pathway.
With the exception of cinnamate 4-hydroxylase, the enzymes which act downstream of phenylalanine ammonia lyase are encoded by small gene families in all species analyzed so far.
The different isoenzymes show distinct temporal, tissue, and elicitor-induced patterns of expression. It seems, in fact, that each member of each family can be used mainly for the synthesis of a specific compound, thus acting as a control point for carbon flux among the metabolic pathways leading to lignan, lignin, and flavonoid biosynthesis.

Note: Phenylalanine is a product of the shikimic acid pathway, which converts simple precursors derived from carbohydrate metabolism, phosphoenolpyruvate and erythrose-4-phosphate, into the aromatic amino acids phenylalanine, tyrosine and tryptophan. Unlike plants and microorganisms, animals do not possess the shikimic acid pathway, and are not able to synthesize the three above-mentioned amino acids, which are therefore essential nutrients.

Phenylalanine ammonia lyase (PAL)

It is one of the most studied and best characterized enzymes of plant secondary metabolism. It requires no cofactors and catalyzes the reaction that links primary and secondary metabolism: the deamination of phenylalanine to trans-cinnamic acid, with the release of nitrogen as ammonia and introduction of a trans double bond between carbon atoms 7 and 8 of the side chain.

Flavonoid biosynthesis: the reaction catalyzed by phenylalanine ammonia lyase
Fig. 3 – Phenylalanine Ammonia Lyase Reaction

Therefore, it directs the flow of carbon from the shikimic acid pathway to the different branches of the phenylpropanoid pathway. The released ammonia is probably fixed in the reaction catalyzed by glutamine synthetase.
The enzyme from monocots is also able to act as tyrosine ammonia lyase (EC 4.3.1.25), converting tyrosine to p-coumaric acid directly, (therefore without the 4-hydroxylation step), but with a lower efficiency.
In all plant species investigated,  several copies of phenylalanine ammonia lyase gene are found, copies that probably respond differentially to internal and external stimuli. Indeed, gene transcription, and then enzyme activity, are under the control of both internal developmental and external environmental stimuli. Here are some examples that require increased enzyme activity.

  • The flowering.
  • The  production of lignin to strengthen the secondary wall of xylem cells.
  • The production of flower pigments that attract pollinators.
  • Pathogen infections, that require the production of phenylpropanoid phytoalexins, or exposure to UV rays.

Cinnamate 4-hydroxylase (C4H)

It belongs to the cytochrome P450 superfamily (EC 1.14.-.-), is a microsomal monooxygenase containing a heme cofactor, and dependent on both O2  and NADPH. It catalyzes the formation of p-coumaric acid  through the introduction of a hydroxyl group in 4-position of trans-cinnamic acid (this hydroxyl group is present in most flavonoids).

Flavonoid biosynthesis: the reaction catalyzed by 4-coumarate:CoA ligase
Fig. 5 – 4-Coumarate:CoA Ligase

This reaction is also part of the biosynthesis of hydroxycinnamic acids.
Increases in transcription rates and enzyme activity are observed in correlation with the synthesis of phytoalexins (in response to fungal infections), lignification as well as wounding.

4-Coumarate:CoA ligase (4CL)

With Mg2+ as a cofactor, it catalyzes the ATP-dependent activation of the carboxyl group of p-coumaric acid and other hydroxycinnamic acids, metabolically rather inert molecules, through the formation of the corresponding CoA-thioester.

Flavonoid biosynthesis: the reaction catalyzed by 4-coumarate:CoA ligase
Fig. 5 – 4-Coumarate:CoA Ligase

Generally, p-coumaric acid and caffeic acid are the preferred substrates, followed by ferulic acid and 5-hydroxyferulic acid, low activity against trans-cinnamic acid and none against sinapic acid. These CoA-thioesters are able to enter various reactions such as:

  • reduction to alcohol (monolignols) or aldehydes;
  • stilbene and flavonoid biosynthesis;
  • transfer to acceptor molecules.

It should finally be pointed out that the activation of the carboxyl group can also be obtained through an UDP-glucose-dependent transfer to glucose.

Biosynthesis of malonyl-CoA

Malonyl-CoA does not derived from the phenylpropanoid pathway, but from the reaction catalyzed by acetyl-CoA carboxylase (EC 6.4.1.2, the cytosolic form, see below). The enzyme, with biotin and Mg2+ as cofactors, catalyzes the ATP-dependent carboxylation of acetyl-CoA, using bicarbonate as a source of carbon dioxide (CO2).

Acetyl-CoA + HCO3 + ATP → Malonyl-CoA + ADP + Pi

It is found both in the plastids, where it participates in the synthesis of fatty acids, and the cytoplasm, and is the latter that catalyzes the formation of malonyl-CoA that is used in the biosynthesis of flavonoids and other compounds. Increases in the transcription rate of the gene and enzyme activity are induced in response to stimuli that increase the biosynthesis of these polyphenols, such as exposure to pathogenic fungi or UV-rays.
In turn, acetyl-CoA is produced in plastids, mitochondria, peroxisomes and cytosol through different metabolic pathways. The molecules used in the biosynthesis of malonyl-CoA, and therefore of the flavonoids, are  the cytosolic ones, produced in the reaction catalyzed by ATP-citrate lyase (EC 2.3.3.8) that cleaves citrate, in the presence of CoA and ATP, to form oxaloacetate and acetyl-CoA, plus ADP and inorganic phosphate.

First steps in flavonoid biosynthesis

The first step in flavonoid biosynthesis is catalyzed by chalcone synthase (EC 2.3.1.74), an enzyme anchored to the endoplasmic reticulum and with no known cofactors.
From one p-coumaroyl-CoA and three malonyl-CoA, it catalyzes sequential condensation and decarboxylation reactions in the course of which a polyketide intermediate is formed. The polyketide undergoes cyclizations and aromatizations leading to the formation of the A ring. The product of the reactions is naringenin chalcone (2′,4,4′,6′-tetrahydroxychalcone), a 6′-hydroxychalcone and the first flavonoid to be synthesized by plants.

Skeletal formula of naringenin chalcone, the first flavonoid to be synthesized by plants
Fig. 6 – Naringenin Chalcone

The reaction, cytosolic, is irreversible due to the release of three CO2 and 4 CoA.
The B ring and the three-carbon bridge of the molecule originate from p-coumaroyl-CoA (and therefore from phenylalanine), the A ring from the three malonyl-CoA units.

Flavonoid biosynthesis: the biosynthetic origin of the flavonoid skeleton
Fig. 7 – The Origin of the Flavonoid Skeleton

Also 6’-deoxychalcone can be produced; its synthesis is thought to involve an additional reduction step catalyzed by polyketide reductase (EC. 1.1.1.-).
Chalcone synthase from some plant species, such as barley (Hordeum vulgare), accepts as substrates also caffeoil-CoA, feruloil-CoA and cinnamoyl-CoA.
It is the most abundant enzyme of the phenylpropanoid pathway, probably because it has a low catalytic activity, and, in fact, is considered to be the rate-limiting enzyme in flavonoid biosynthesis.
As for phenylalanine ammonia lyase, chalcone synthase gene expression is under the control of both internal and external factors. In some plants, one or two isoenzymes are found, while in others up to 9.
Chalcone synthase belongs to polyketide synthase group, present in bacteria, fungi and plants. These enzymes are able to catalyze the production of polyketide chains through sequential condensations of acetate units provided by malonyl-CoA units. They also includes stilbene synthase (EC 2.3.1.146), which catalyzes the formation of resveratrol, a non flavonoid polyphenol compound that has attracted much interest because of its potential health benefits.
Generally, chalcones do not accumulate in plants because naringenin chalcone is converted to (2S)-naringenin, a flavanone, in the reaction catalyzed by chalcone isomerase (EC 5.5.1.6).

Skeletal formula of (2S)-naringenin, a flavanone
Fig. 8 – (2S)-Naringenin

The enzyme, the first of the flavonoid biosynthesis to be discovered, catalyzes a stereospecific isomerization and closes the C ring. Two types of chalcone isomerases are known, called type I and II. Type I enzymes use only 6′-hydroxychalcone substrates, like naringenin chalcone, while type II, prevalent in legumes, use both 6′-hydroxy- and 6′-deoxychalcone substrates.
It should be noted that with 6′-hydroxychalcones, isomerization can also occur nonenzymically to form a racemic mixture, both in vitro and in vivo, enough to allow a moderate synthesis of anthocyanins. On the contrary, under physiological conditions 6′-deoxychalcones are stable, and so the activity of type II chalcone isomerases is required to form flavanones.
The enzyme increases the rate of the reaction of 107 fold compared to the spontaneous reaction, but with a higher kinetics for the 6′-hydroxychalcones than 6′-deoxychalcones. Finally, it produces (2S)-flavanones, which are the biosynthetically required enantiomers.
As other enzymes in flavonoid biosynthesis, also chalcone isomerase gene expression is subject to strict control. And, as phenylalanine ammonia lyase and chalcone synthase, it is induced by elicitors.
In the reaction catalysed by flavanone-3β-hydroxylase (EC 1.14.11.9), (2S)-flavanones undergo a stereospecific isomerization that converts them into the respective (2R,3R)-dihydroflavonols. In particular, naringenin is converted into dihydrokaempferol.

Skeletal formula of dihydrokaempferol, a dihydroflavonol
Fig. 9 – Dihydrokaempferol

The enzyme is a cytosolic non-heme-dependent dioxygenase, dependent on Fe2+ and 2-oxoglutarate, and therefore belonging to the family of 2-oxoglutarate-dependent dioxygenase (which distinguishes them from the other hydroxylases of the flavonoid biosynthetic pathway which are cytochrome P450 enzymes).
Naringenin chalcone, (2S)-naringenin, and dihydrokaempferol are central intermediates in flavonoid biosynthesis, since they act as branch-point compounds from which the synthesis of distinct flavonoid subclasses can occur. For example, directly or indirectly:

Not all of these side metabolic pathways are present in every plant species, or are active within each tissue type of a given plant. Like enzymes previously seen, the activity of those involved in these “side-routes” is subject to strict control, resulting in a tissue-specific profile of flavonoid compounds. For example, grape seeds, flesh and skin have distinct anthocyanin, catechin, flavonol and condensed tannin profiles, whose synthesis and accumulation are strictly and temporally coordinated during the ripening process.

References

Andersen Ø.M., Markham K.R. Flavonoids: chemistry, biochemistry, and applications. CRC Press Taylor & Francis Group, 2006

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Heldt H-W. Plant biochemistry – 3th Edition. Elsevier Academic Press, 2005

Vogt T. Phenylpropanoid biosynthesis. Mol Plant 2010;3(1):2-20. doi:10.1093/mp/ssp106

Wink M. Biochemistry of plant secondary metabolism – 2nd Edition. Annual plant reviews (v. 40), Wiley J. & Sons, Inc., Publication, 2010

Polyphenols from grapes and wines: content, biological activities, and benefits

The consumption of grapes and grape-derived products, particularly red wine but only at meals, has been associated with numerous health benefits, which include, in addition to the antioxidant/antiradical effect, also anti-inflammatory, cardioprotective, anticancer, antimicrobial, and neuroprotective activities.
Grapes contain many nutrients such as sugars, vitamins, minerals, fiber and phytochemicals. Among the latter, polyphenols from grapes are the most important compounds in determining the health effects of the fruit and derived products.
Indeed, grapes are among the fruits with highest content in polyphenols, whose composition is strongly influenced by several factors such as:

  • cultivar;
  • climate;
  • exposure to disease;
  • processing

Nowadays, the main species of grapes cultivated worldwide are: European grapes, Vitis vinifera, North American grapes, Vitis rotundifolia and Vitis labrusca, and French hybrids.
Note: Grapes are not a fruit but an infructescence, that is, an ensemble of fruits (berries): the bunch of grapes. In turn, it consists of a peduncle, a rachis, cap stems or pedicels, and berries.

CONTENTS

What are polyphenols from grapes and wines?

Polyphenols from red grapes and wine are significantly higher, both in quantity and variety, than in white ones. This, according to many researchers, would be the basis of the more health benefits related to the consumption of red grapes and wine than white grapes and derived products.
Polyphenols from grapes and wine are a complex mixture of flavonoid compounds, the most abundant group, and non-flavonoid compounds.
Among flavonoids, they are found:

Among non-flavonoid polyphenols:

Most of the flavonoids present in wine derive from the epidermal layer of the berry skin, while 60-70% of the total polyphenols are present in the grape seeds. It should be noted that more than 70% of grape polyphenols are not extracted and remain in the pomace.
The complex chemical interactions that occur between these compounds, and between them and the other compounds of different nature present in grapes and wine, are probably essential in determining both the quality of the grapes and wine and the broad spectrum of therapeutic effects of these foods.
In wine, the mixture of polyphenols play important functions being able to influence:

  • bitterness;
  • astringency;
  • red color, of which they are among the main responsible;
  • sensitivity to oxidation, being molecules easily oxidizable by atmospheric oxygen.

Finally, they act as preservatives and are the basis of long aging.

Anthocyanins

They are flavonoids widely distributed in fruits and vegetables.
They are primarily located in the berry skin (in the outer layers of the hypodermal tissue), to which they confer color, having a hue that varies from red to blue. In some varieties, called “teinturier”, they also accumulate in the flesh of the berry.
There is a close relationship between berry development and the biosynthesis of anthocyanins. The synthesis starts at veraison (when the berry stops growing and changes its color), causes a color change of the berry that turns purple, and reaches the maximum levels at complete ripening.
Among wine flavonoids, they are one of the most potent antioxidants.
Each grape species and cultivars has a unique composition of anthocyanins. Moreover, in grapes of Vitis vinifera, due to a mutation in the gene coding for 5-O-glucosyltransferase, mutation that determines the synthesis of an inactive enzyme, only 3-monoglucoside derivatives are synthesized, while in other species  the glycosylation at position 5 also occurs. Interestingly, 3-monoglucoside derivatives are more intensely colored than 3,5-diglucoside derivatives.

Skeletal formula of malvidin-3-glucoside, an anthocyanin
Fig. 1 – Malvidin-3-glucoside

In red grapes and wine, the most abundant anthocyanins are the 3-monoglucosides of malvidin (the most abundant one both in grapes and wine), petunidin, delphinidin, peonidin, and cyanidin. In turn, the hydroxyl group at position 6 of the glucose can be acylated with an acetyl, caffeic or coumaric group, acylation that further enhances the stability.
Anthocyanidins, namely the non-conjugated molecules, are not present in grapes and in wine, except as traces.
Anthocyanins are scarcely present in white grapes and wine.
The composition of anthocyanins in wine is highly influenced both by the type of cultivar and by processing techniques, since they are present in wine as a result of extraction by maceration/fermentation processes. For this reason, wines deriving from similar varieties of grapes can have very different anthocyanin compositions.
Together with proanthocyanidins, they are the most important polyphenols in contributing to some organoleptic properties of red wine, as they are primarily responsible for astringency, bitterness, chemical stability against oxidation, as well as of the color of the young wine. In this regard, it should be underscored that with time their concentration decreases, while the color is due more and more to the formation of polymeric pigments produced by condensation of anthocyanins both among themselves and with other molecules.
During wine aging, proanthocyanidins and anthocyanins react to produce more complex molecules that can  partially precipitate.

Flavanols or catechins

They are, together with condensed tannins, the most abundant flavonoids, representing up to 50% of the total polyphenols in white grapes and between 13% and 30% in red ones.
Their levels in wine depend on the type of cultivar.

Polyphenols from grapes: skeletal formula of catechin, a flavanol
Fig. 2 – Catechin

Typically, the most abundant flavanol in wine is catechin, but epicatechin and epicatechin-3-gallate are also present.

Proanthocyanidins or condensed tannins

Composed of catechin monomers, they are present in the berry skin, seeds and rachis of the bunch of grapes as:

  • dimers: the most common are procyanidins B1-B4, but also procyanidins B5-B8 can be present;
  • trimers: procyanidin C1 is the most abundant;
  • tetramers;
  • polymers, containing up to 8 monomers.
Skeletal formula of procyanidin C1, a proanthocyanidin
Fig. 3 – Procyanidin C1

Their levels in wine depend on the type of grape varieties and wine-making technology, and, like anthocyanins, are much more abundant in red wines, in particular in aged wines, compared to white ones.
In addition, as previously said, together with anthocyanins, condensed tannins are important in determining some organoleptic properties of the wine.

Flavonols

They are present in a large variety of fruit and vegetables, even if in low concentrations.
They are the third most abundant group of flavonoids from grapes, after proanthocyanidins and catechins.
They are mainly present in the outer epidermis of the berry skin, where they play a role both in providing protection against UV-A and UV-B radiations and in copigmentation together with anthocyanins.
Flavanol synthesis begins in the sprout; the highest concentration is reached a few weeks after veraison, then it decreases as the berry increases in size.
Their total amount is very variable, with the red varieties often richer than the white ones.
In grapes, they are present as 3-glucosides and their composition depends on the type of grapes and cultivar:

  • the derivatives of quercetin, kaempferol and isorhamnetin are found in white grapes;
  • the derivatives of myricetin, laricitrin and syringetin are found, together with the previous ones, only in red grapes, due to the lack of expression in white grapes of the gene coding for flavonoid-3′,5′-hydroxylase.
Polyphenols from grapes: skeletal formula of quercetin-3-glucoside, a flavonol
Fig. 4 – Quercetin-3-glucoside

In general, the 3-glucosides and 3-glucuronides of quercetin are the major flavonols in most of the grape varieties. Conversely, quercetin-3-rhamnoside and quercetin aglycone are the major flavonols in muscadine grapes.
In wine and grape juice, unlike grapes, they are also found as aglycones, as a result of the acid hydrolysis that occurs during processing and storage. They are present in wine in a variable amount, and the major molecules are the glycosides of quercetin and myricetin, which alone represent 20-50% of the total flavonols in red wine.

Hydroxycinnamates

Hydroxycinnamic acids are the main class of non-flavonoid polyphenols from grapes and the major polyphenols in white wine.
The most important are p-coumaric, caffeic, sinapic, and ferulic acids, present in wine as esters with tartaric acid.
They have antioxidant activity and in some white varieties of Vitis vinifera, together with flavonols, are the polyphenols mainly responsible for absorbing UV radiation in the berry.

Stilbenes

They are phytoalexins which are produced in low concentrations only by a few edible species, including grapevine (on the contrary, flavonoids are present in all higher plants).
Together with the other polyphenols from grapes and wine, also stilbenes, particularly resveratrol, have been associated with health benefits resulting from the consumption of wine.

Polyphenols from grapes: skeletal formula of trans-resveratrol, a stilbene
Fig. 5 – trans-Resveratrol

Their content increases from the veraison to the ripening of the berry, and is influenced by the type of cultivar, climate, wine-making technology, and fungal pressure.
The main stilbenes present in grapes and wine are:

  • cis- and trans-resveratrol (3,5,4′-trihydroxystilbene);
  • piceid or resveratrol-3-glucopyranoside and astringin or 3′-hydroxy-trans-piceid;
  • piceatannol;
  • dimers and oligomers of resveratrol, called viniferins, of which the most important are:

α-viniferin, a trimer;
β-viniferin, a cyclic tetramer;
γ-viniferin, a highly polymerized oligomer;
ε-viniferin, a cyclic dimer.

In grapes, other glycosylated and isomeric forms of resveratrol and piceatannol, such as resveratroloside, hopeaphenol, or resveratrol di- and tri-glucoside derivatives, have been found in trace amounts.
Glycosylation of stilbenes is important for the modulation of antifungal activity, protection from oxidative degradation, and storage of the wine.
The synthesis of dimers and oligomers of resveratrol, both in grapes and wine, represents a defense mechanism against exogenous attacks or, on the contrary, the result of the action of extracellular enzymes released from pathogens in an attempt to eliminate undesirable compounds.

Hydroxybenzoates

The hydroxybenzoic acid derivatives are a minor component in grapes and wine.
In grapes, gentisic, gallic, p-hydroxybenzoic and protocatechuic acids are the main ones.

Skeletal formula of gallic acid, an hydroxybenzoic acid
Fig. 6 – Gallic Acid

Unlike hydroxycinnamates, which are present in wine as esters with tartaric acid, they are found in their free form.
Together with flavonols, proanthocyanidins, catechins, and hydroxycinnamates they are among the responsible of astringency of wine.

References

Andersen Ø.M., Markham K.R. Flavonoids: chemistry, biochemistry, and applications. CRC Press Taylor & Francis Group, 2006

Basli A, Soulet S., Chaher N., Mérillon J.M., Chibane M., Monti J.P.,1 and Richard T. Wine polyphenols: potential agents in neuroprotection. Oxid Med Cell Longev 2012. doi:10.1155/2012/805762

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Flamini R., Mattivi F.,  De Rosso M., Arapitsas P. and Bavaresco L. Advanced knowledge of three important classes of grape phenolics: anthocyanins, stilbenes and flavonols. Int J Mol Sci 2013;14:19651-19669. doi:10.3390/ijms141019651

Georgiev V., Ananga A. and Tsolova V. Recent advances and uses of grape flavonoids as nutraceuticals. Nutrients 2014;6: 391-415. doi:10.3390/nu6010391

Guilford J.M. and Pezzuto J.M. Wine and health: a review. Am J Enol Vitic 2011;62(4):471-486. doi:10.5344/ajev.2011.11013

He S., Sun C. and Pan Y. Red wine polyphenols for cancer prevention. Int J Mol Sci 2008;9:842-853. doi:10.3390/ijms9050842

Xia E-Q., Deng G-F., Guo Y-J. and Li H-B. Biological activities of polyphenols from grapes. Int J Mol Sci 2010;11-622-646. doi:10.3390/ijms11020622

Waterhouse A.L. Wine phenolics. Ann N Y Acad Sci 2002;957:21-36. doi:10.1111/j.1749-6632.2002.tb02903.x

Polyphenols in olive oil

Olive oil, which is obtained from the pressing of the olives, the fruits of olive tree (Olea europaea), is the main source of lipids in the Mediterranean diet, and a good source of polyphenols.
Polyphenols, natural antioxidants, are present in olive pulp and, following pressing, they pass into the oil.
Note: olives are also known as drupes or stone fruits.
The concentration of polyphenols in olive oil is the result of a complex interaction between various factors, both environmental and linked to the extraction process of the oil itself, such as:

  • the place of cultivation;
  • the cultivars (variety);
  • the level of ripeness of the olives at the time of harvesting.
    Their level usually decreases with over-ripening of the olives, although there are exceptions to this rule. For example, in warmer climates, olives produce oils richer in polyphenols, in spite of their faster maturation.
  • the climate;
  • the extraction process. In this regard, it is to underscore that the content of polyphenol in refined olive oil is not significant.

Any variation of the concentration of different polyphenols influence the taste, nutritional properties and stability of olive oil. For example, hydroxytyrosol and oleuropein (see below) give extra virgin olive oil a pungent and bitter taste.

CONTENTS

Key polyphenols in olive oil

Among polyphenols in olive oil, there are molecules with simple structure, such as phenolic acids and alcohols, and molecules with complex structure, such as flavonoids, secoiridoids, and lignans.

Flavonoids

Flavonoids include glycosides of flavonols (rutin, also known as quercetin-3-rutinoside), flavones (luteolin-7-glucoside), and anthocyanins (glycosides of delphinidin).

Phenolic acids and phenolic alcohols

Among phenolic acids, the first polyphenols with simple structure observed in olive oil, they are found:

  • hydroxybenzoic acids, such as, gallic, protocatechuic, and 4-hydroxybenzoic acids (all with C6-C1 structure).
  • hydroxycinnamic acids, such as caffeic, vanillin, syringic, p-coumaric, and o-coumaric acids (all with C6-C3 structure).

Among phenolic alcohols, the most abundant are hydroxytyrosol (also known as 3,4-dihydroxyphenyl-ethanol), and tyrosol [also known as 2-(4-hydroxyphenyl)-ethanol].

Hydroxytyrosol

Hydroxytyrosol can be present as:

  • simple phenol;
  • phenol esterified with elenolic acid, forming oleuropein and its aglycone;
  • part of the molecule verbascoside.
Hydroxytyrosol, a phenolic alcohol, and one of the polyphenols in olive oil
Gig. 1 – Hydroxytyrosol

It can also be present in different glycosidic forms, depending on the –OH group to which the glucoside, i.e. elenolic acid plus glucose, is bound.
It is one of the main polyphenols in olive oil, extra virgin olive oil, and olive vegetable water.
In nature, its concentration, such as that of tyrosol, increases during fruit ripening, in parallel with the hydrolysis of compounds with higher molecular weight, while the total content of phenolic molecules and alpha-tocopherol decreases. Therefore, it can be considered as an indicator of the degree of ripeness of the olives.
In fresh extra virgin olive oil, hydroxytyrosol is mostly present in esterified form, while in time, due to hydrolysis reactions, the non-esterified form becomes the predominant one.
Finally, the concentration of hydroxytyrosol is correlated with the stability of olive oil.

Secoiridoids

They are the polyphenols in olive oil with the more complex structure, and are produced from the secondary metabolism of terpenes.
They are glycosylated compounds and are characterized by the presence of elenolic acid in their structure (both in its aglyconic or glucosidic form). Elenolic acid is the molecule common to glycosidic secoiridoids of Oleaceae.
Unlike tocopherols, flavonoids, phenolic acids, and phenolic alcohols, that are found in many fruits and vegetables belonging to different botanical families, secoiridoids are present only in plants of the Oleaceae family.
Oleuropein, demethyloleuropein, ligstroside, and nuzenide are the main secoiridoids.
In particular, oleuropein and demethyloleuropein (as verbascoside) are abundant in the pulp, but they are also found in other parts of the fruit. Nuzenide is only present in the seeds.

Oleuropein

Oleuropein, the ester of hydroxytyrosol and elenolic acid, is the most important secoiridoid, and the main olive oil polyphenol.

Oleuropein, a secoiridoid, and one of the polyphenols in olive oil
Fig. 2 – Oleuropein

It is present in very high quantities in olive leaves, as also in all the constituent parts of the olive, including peel, pulp and kernel.
Oleuropein accumulates in olives during the growth phase, up to 14% of the net weight; when the fruit turns greener, its quantity reduces. Finally, when the olives turns dark brown, color due to the presence of anthocyanins, the reduction in its concentration becomes more evident.
It was also shown that its content is greater in green cultivars than in black ones.
During the reduction of oleuropein levels (and of the levels of other secoiridoids), an increase of compounds such as flavonoids, verbascosides, and simple phenols can be observed.
The reduction of its content is also accompanied by an increase in its secondary glycosylated products, that reach the highest values in black olives.

Lignans

Lignans, in particular (+)-1- acetoxypinoresinol and (+)-pinoresinol, are another group of polyphenols in olive oil.
(+)-pinoresinol is a common molecule in the lignin fraction of many plants, such as sesame (Sesamun indicum) and the seeds of the species Forsythia, belonging to the family Oleaceae. It has been also found in the olive kernel.
(+)-1- acetoxypinoresinol and (+)-1-hydroxypinoresinol, and their glycosides, have been found in the bark of the olive tree.

Examples of lignans, a class of pholyphenols, in olive oil
Fig. 3 – Lignans in Olive Oil

Lignans are not present in the pericarp of the olives, nor in leaves and sprigs that may accidentally be pressed with the olives.
Therefore, how they can pass into the olive oil becoming one of the main phenolic fractions is not yet known.
(+)-1- acetoxypinoresinol and (+)-pinoresinol are absent in seed oils, are virtually absent from refined virgin olive oil, while they may reach a concentration of 100 mg/kg in extra-virgin olive oil.
As seen for simple phenols and secoiridoids, there is considerable variation in their concentration among olive oils of various origin, variability probably related to differences between olive varieties, production areas, climate, and oil production techniques.

References

Cicerale S., Lucas L. and Keast R. Biological activities of phenolic compounds present in virgin olive oil. Int. J. Mol. Sci. 2010;11: 458-479. doi:10.3390/ijms11020458

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Manach C., Scalbert A., Morand C., Rémésy C., and Jime´nez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79(5):727-47 doi:10.1093/ajcn/79.5.727

Owen R.W., Mier W., Giacosa A., Hull W.E., Spiegelhalder B. and Bartsch H. Identification of lignans as major components in the phenolic fraction. Clin Chem 2000;46:976-988.

Tripoli E., Giammanco M., Tabacchi G., Di Majo D., Giammanco S. and La Guardia M. The phenolic compounds of olive oil: structure, biological activity and beneficial effects on human health. Nutr Res Rev 2005:18;98-112. doi:10.1079/NRR200495

Black tea: benefits and processing of tea leaves

Black tea, like the other types of tea, is an infusion of processed leaves of Camellia sinensis, the tea plant, a shrub that belongs to the Theaceae family.
Black tea, a type of fully fermented tea, is the most consumed tea worldwide, accounting for about 78% of the consumed tea. It is preferred by Western populations, while the favorite tea in Asia, particularly in Japan and China, is green tea.

Processing of black tea

The processing of the leaves of Camellia sinensis, that leads to the production of black tea, proceeds through three main steps:

  • withering or drying;
  • rolling;
  • oxidation

black-teaThe last step, oxidation, gives it the specific organoleptic characteristics and composition in polyphenols, that are extremely different from those of green tea (green tea undergoes very slight oxidative processes during processing).

Withering or drying

The withering or drying step is the first, and most basic process during processing. In this step, water in the leaves is removed (about 75% of the leaf’s weight is made up of water), thus determining the concentration of the sap of the leaf itself. The withering also makes the next step easier.
Withering can be achieved in three different ways:

  • exposing leaves to sunlight, that is, sun withering;
  • heating in an appropriate manner the rooms where the leaves are placed;
  • using machines that artificially ventilate the leaves.

Rolling

The rolling step follows the withering of the leaves. It breaks the leaf tissue, facilitating the outflow of lymph; thus, it promotes the subsequent enzymatic oxidation of polyphenols. This step is essential for the creation of the aroma, color and flavor of black tea.

Oxidation

The oxidation, also improperly called fermentation, is the last stage of its processing, and is crucial in determining the quality of the tea. In this step, the enzymatic oxidation of about 90–95 % of the polyphenols occurs, accompanied by other changes that make the green tea leaves into red color.
Temperature (typically 25°C), pH, relative humidity (95% or more), ventilation, and duration are crucial factors too.

Black tea polyphenols

During the oxidation step, the main compounds that undergo oxidation processes, both enzymatic, by polyphenol oxidase, and chemical, by the action of atmospheric oxygen, are:

  • monomeric catechins and gallate catechins;
  • to a lesser extent, the glycosides of catechins, especially myricetin;
  • but also not flavonoids compounds, such as teogallin (ester of gallic acid).

Therefore, throughout the process, a reduction in the concentration of monomeric catechins, characteristics of fresh leaves of Camellia sinensis and green tea, occurs, with the formation of complex polyphenols, such as:

  • thearubigins, red-brown or dark-brown in color;
  • theaflavins and theaflavic acids, red-orange in color.

Thearubigins, polymers of catechins not yet well characterized, are the major polyphenols in black tea, accounting for about 20% of extracted solids. In addition to the reddish color, thearubigins contribute the richness in taste, the so-called “body” to black tea.
Theaflavins, dimers of catechins much better characterized than thearubigins, account for about 3-5% of the solids in black tea infusion. Theaflavins contribute the astringent and brisk taste, as well as the red-orange color of the beverage.
The main theaflavins are:

  • theaflavin digallate;
  • theaflavin-3-gallate;
  • theaflavin-3′-gallate.

Black tea benefits and oxidized polyphenols

Although this type of tea is still able to improve health, oxidative processes suffered from the leaves during the processing attenuate its health benefits , which are instead reported after intake of green tea (particularly, the benefits of green tea are ascribed to its content of catechins, such as EGCG, epicatechin and epicatechin gallate).

Its caffeine content does not vary significantly.

References

Asil M.H., Rabiei B., Ansari R.H. Optimal fermentation time and temperature to improve biochemical composition and sensory characteristics of black tea. Aust J Crop Sci 2012;6(3):550-8.

Kuhnert N. Unraveling the structure of the black tea thearubigins. Arch Biochem Biophys 2010;501(1):37-51 doi:10.1016/j.abb.2010.04.013

Li S., Lo C-Y., Pan M-H., Lai C-S. and Ho C-T. Black tea: chemical analysis and stability. Food Funct 2013;4:10-18 doi:10.1039/C2FO30093A

Menet M-C., Sang S., Yang C.S., Ho C-T., and Rosen R.T. Analysis of theaflavins and thearubigins from black tea extract by MALDI-TOF mass spectrometry. J Agric Food Chem 2004;52:2455-61 doi:10.1021/jf035427e

Sharma V.K., Bhattacharya A., Kumar A. and Sharma H.K. Health benefits of tea consumption. Trop J Pharm Res 2007;6(3):785-792.

Processing, properties, and benefits of green tea

Green tea is an infusion of processed leaves of tea plant, Camellia sinensis, a member of the Theaceae family.
It is the most consumed beverages in Asia, particularly in China and Japan.
Western populations consume black tea more frequently than green tea. However, in recent years, thanks to its health benefits, it has been gaining their attention.
Currently, it accounts for 20% of the tea consumed worldwide.
As all other types of tea, it is produced from fresh leaves of Camellia sinensis.
The peculiar properties of the beverage depend on the type of processing that the leaves undergo. In fact, they are processed in such a way as to minimize both enzymatic and chemical oxidation processes of the substances contained in them, in particular catechins.
Therefore, among the different types of tea, it undergoes the lowest degree of oxidation during processing.
At the end of the processing, tea leaves retain their green color, thanks to the little chemical modifications/oxidations they have undergone. The infusion shows off a yellow-gold color.
Finally, the processing of tea leaves ensures that green tea flavor is more delicate and lighter than black tea.

CONTENTS

Processing of tea leaves

After harvesting, tea leaves are exposed to the sun for 2-3 hours and withered/dried; then, the real processing starts.
It consists of three main steps: heat treatment, rolling and drying.

Heat treatment

Heat treatment, short and gentle, is the crucial step for the quality and properties of the beverage.
It occurs with steam (the traditional Japanese method) or by dry cooking in hot pans (a large wok, the traditional Chinese method). Heat treatment has the purpose of:

  • inactivate the enzymes present in the tissues of the leaves, thus stopping enzymatic oxidation processes, particularly of polyphenols;
  • eliminate the grassy smell in order to stand out tea flavor;
  • evaporate part of the water present in the fresh leaf (water constitutes about 75% of the weight of the leaf), making it softer, so as to make the next step easier.

Rolling

The rolling step follows the heat treatment of the leaves; this step has the purpose of:

  • facilitate the next stage of drying;
  • destroy the tissues of the leaves in order to favor, later, the release of aromas, thus improving the quality of the product.

Drying

The drying is the last step, which also leads to the production of new compounds and improves the appearance of the product.

Benefits of green tea

All types of tea are rich in polyphenols, compounds that are also present in fruits, vegetables, extra virgin olive oil, and red wine.
Fresh tea leaves are rich in water-soluble polyphenols, especially catechins (or flavanols) and glycosylated catechins (both belonging to the class of flavonoids), molecules which are believed to be the responsibles of the benefits of green tea.
The major catechins in green tea are epigallocatechin-3-gallate (EGCG, see figure), epigallocatechin, epicatechin-3-gallate, epicatechin, epicatechin, but also catechin, gallocatechin, catechin gallate, and gallocatechin gallate are present, even if in lower amount.

Gree Tea

These polyphenols account for 30%-42% of the dry leaf weight (but only 3%–10% of the solid content of black tea).
Green tea caffeine accounts for 1,5-4,5% of the dry leaf weight.

Catechin absorption and lemon juice

In vitro studies have shown the high antioxidant power of catechins, greater than that of vitamin C and vitamin E. In vitro, EGCG is generally considered the most biologically active catechin.
In vivo studies and several epidemiologic studies have shown the possible preventive effects of green tea catechins, especially EGCG, in preventing the development of:

  • cardiovascular disease, such as hypertension and stroke;
  • some cancers, such as lung cancer (but not among smokers) and oral and digestive tract cancers.

For these reasons, it is essential to maximize the intestinal absorption of catechins.
Catechins are stable in acidic environment, but not in non-acidic environment, as in the small intestine; also for this reason, after digestion, less than 20% of the total remains.
Studies with models of the digestive tract of rat and man, that simulate digestion in stomach and small intestine, have shown that the addition of citrus juice or vitamin C to green tea significantly increases the absorption of catechins.
Among tested citrus juices, lemon juice is the best, followed by orange, lime and grapefruit juices. Citrus juices seem to have a stabilizing effect on catechins that goes beyond what would be predicted solely based on their ascorbic acid content.

References

Clifford M.N., van der Hooft J.J.J., and Crozier A. Human studies on the absorption, distribution, metabolism, and excretion of tea polyphenols. Am J Clin Nutr 2013;98:1619S-1630S. doi:10.3945/ajcn.113.058958

Dwyer J.T. and Peterson J. Tea and flavonoids: where we are, where to go next. Am J Clin Nutr 2013;98:1611S-1618S. doi:10.3945/ajcn.113.059584

Green R.J., Murphy A.S., Schulz B., Watkins B.A. and Ferruzzi M.G. Common tea formulations modulate in vitro digestive recovery of green tea catechins. Mol Nutr Food Res 2007;51(9):1152-1162. doi:10.1002/mnfr.200700086

Huang W-Y., Lin Y-R., Ho R-F., Liu H-Y., and Lin Y-S. Effects of water solutions on extracting green tea leaves. ScientificWorldJournal 2013;Article ID 368350. doi:10.1155/2013/368350

Sharma V.K., Bhattacharya A., Kumar A. and Sharma H.K. Health benefits of tea consumption. Trop J Pharm Res 2007;6(3):785-792.

Foods high in anthocyanins, their absorption and metabolism

Together with catechins and proanthocyanidins, anthocyanins and their oxidation products are the most abundant flavonoids in the human diet.
Examples of anthocyanin rich foods are:

  • certain varieties of grains, such as some types of pigmented rice (e.g. black rice) and maize (purple corn);
  • in certain varieties of root and leafy vegetables such as aubergine, red cabbage, red onions and radishes, beans;
  • but especially in red fruits.

Example of anthocyanin rich food
Anthocyanins are also present in red wine; as the wine ages, they are transformed into various complex molecules.
Anthocyanin content in vegetables and fruits is generally proportional to their color: it increases during maturation, and it reaches values up to 4 g/kg fresh weight (FW) in cranberries and black currants.
These polyphenols are found primarily in the skin, except for some red fruits, such as cherries and red berries (e.g. strawberries), in which they are present both in the skin and flesh.
Glycosides of cyanidin are the most common anthocyanins in foods.

CONTENTS

Anthocyanin rich fruits

  • Berries are the main source of anthocyanins, with values ranging between 67 and 950 mg/100 g FW.
  • Other fruits, such as red grapes, cherries and plums, have content ranging between 2 and 150 mg/100 g FW.
  • Finally, in fruits such as nectarines, peaches, and some types of apples and pears, anthocyanins are poorly present, with a content of less than 10 mg/100 g FW.

Cranberries, besides their very high content of anthocyanins, are one of the rare food that contain glycosides of the six most commonly anthocyanidins present in foods: pelargonidin, delphinidin, cyanidin, petunidin, peonidin, and malvidin. The main anthocyanins are the 3-O-arabinosides and 3-O-galactosides of peonidin and cyanidin. A total of 13 anthocyanins have been detected, mainly 3-O-monoglycosides.

Anthocyanin absorption

Until recently, it was believed that anthocyanins, together with proanthocyanidins and gallic acid ester derivatives of catechins, were the least well-absorbed polyphenols, with a time of appearance in the plasma consistent with the absorption in the stomach and small intestine. Indeed, some studies have shown that their bioavailability has been underestimated since, probably, all of their metabolites have not been yet identified.
In this regard, it should be underlined that only a small part of the food anthocyanins is absorbed in their glycated forms or as hydrolysis products in which the sugar moiety has been removed. Therefore, a large amount of these ingested polyphenols enters the colon, where they can also suffer methylation, sulphatation, glucuronidation and oxidation reactions.

Anthocyanins and colonic microbiota

Few studies have examined the metabolism of anthocyanins by the gut microbiota in the colon.
Within two hours, it seems that all the anthocyanins lose their sugar moieties, thus producing anthocyanidins.
Anthocyanidins are chemically unstable in the neutral pH of the colon. They can be metabolized by colonic microbiota or chemically degraded producing a set of new molecules that have not yet fully identified, but which include phenolic acids such as gallic acid, syringic acid, protocatechuic acid, vanillic acid and phloroglucinol (1,3,5-trihydroxybenzene). These molecules, thanks to their higher microbial and chemical stability, might be the main responsible for the antioxidant activities and the other physiological effects that have been observed in vivo and attributed to anthocyanins.

References

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

de Pascual-Teresa S., Moreno D.A. and García-Viguera C. Flavanols and anthocyanins in cardiovascular health: a review of current evidence. Int J Mol Sci 2010;11:1679-1703. doi:10.3390/ijms11041679

Escribano-Bailòn M.T., Santos-Buelga C., Rivas-Gonzalo J.C. Anthocyanins in cereals. J Chromatogr A 2004:1054;129-141. doi:10.1016/j.chroma.2004.08.152

Han X., Shen T. and Lou H. Dietary polyphenols and their biological significance. Int J Mol Sci 2007;9:950-988. doi:10.3390/i8090950

Manach C., Scalbert A., Morand C., Rémésy C., and Jime´nez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79(5):727-47 doi:10.1093/ajcn/79.5.727

Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients 2010;2:1231-1246. doi:10.3390/nu2121231

Tea polyphenols: bioactive compounds from leaves of tea plant

The leaves of the tea plant, Camellia sinensis, and tea are rich in bioactive compounds.
More than 4000 molecules have been found in the beverage.
Approximately one third of these compounds are polyphenols, the most important molecules in determining nutritional value and health benefits of the beverage.
Tea polyphenols are mostly flavonoids, such as catechins in green tea (e.g. EGCG), and thearubigins and theaflavins in black tea.

Epigallocatechin gallate, a catechin, and one of the tea polyphenolsOther bioactive compounds present in tea are:

  • alkaloids, such as caffeine, theophylline and theobromine;
  • amino acids, and among them, theanine (r-glutamylethylamide), that is also a brain neurotransmitter and one of the most important amino acids in green tea;
  • proteins;
  • carbohydrates;
  • chlorophyll;
  • volatile organic molecules, that is, compounds that easily produce vapors and contribute to the odor of the beverage;
  • fluoride, aluminum and trace elements.

These molecules provide the nutritional value of the tea, affecting human health in many ways. Their composition is highly influenced by processing of tea leaves.

Biological activities of polyphenols

Polyphenols, both in vivo and in vitro, have a broad spectrum of biological activities such as:

  • antioxidant properties;
  • reduction of various types of tumors;
  • inhibition of inflammation;
  • protective effects against hyperlipidemia and diabetes.

Therefore, they have a protective role against the development of many diseases.
Thanks to the abundance of tea polyphenols, there has been a growing interest in recent years about the possible preventive effects of beverage against several diseases, particularly cardiovascular disease, for example in the development and progression of atherosclerosis.

Mechanisms of action of tea polyphenols

Currently, there is limited information on how tea polyphenols exert their effects at cellular level.
It seems, at least in vitro, that catechins in green tea, and theaflavins and thearubigins in black tea are the bioactive compounds responsible for the physiological effects and health benefits of tea.
And among the observed mechanisms by which tea polyphenols act at the cellular level, in addition to the antioxidant effect, it has been observed, as a consequence of polyphenol binding to specific receptors on the cell membrane, changes in the activity of various protein kinases, and growth and transcriptional factors.
Moreover, it seems that these molecules, or at least EGCG, may enter the cell and directly interact with their intracellular specific targets.

References

Dwyer J.T. and Peterson J. Tea and flavonoids: where we are, where to go next. Am J Clin Nutr 2013;98:1611S-1618S doi:10.3945/ajcn.113.059584

Grassi D., Desideri G., Di Giosia P., De Feo M., Fellini E., Cheli P., Ferri L., and Ferri C. Tea, flavonoids, and cardiovascular health: endothelial protection. Am J Clin Nutr 2013;98:1660S-1666S doi:10.3945/ajcn.113.058313

Lambert J.D. Does tea prevent cancer? Evidence from laboratory and human intervention studies. Am J Clin Nutr 2013;98:1667S-1675S doi:10.3945/ajcn.113.059352

Lenore Arab L., Khan F., and Lam H. Tea consumption and cardiovascular disease risk. Am J Clin Nutr 2013;98:1651S-1659S doi:10.3945/ajcn.113.059345

Lorenz M. Cellular targets for the beneficial actions of tea polyphenols. Am J Clin Nutr 2013;98:1642S-1650S doi:10.3945/ajcn.113.058230

Sharma V.K., Bhattacharya A., Kumar A. and Sharma H.K. Health benefits of tea consumption. Trop J Pharm Res 2007;6(3):785-792.

Yuan J-M. Cancer prevention by green tea: evidence from epidemiologic studies. Am J Clin Nutr 2013;98:1676S-1681S doi:10.3945/ajcn.113.058271

Isoflavones: chemical structure, foods and health effects

Isoflavones are colorless polyphenols belonging to the class of flavonoids.
Unlike the majority of the other flavonoids, they have a restricted taxonomic distribution, being present almost exclusively in the Leguminosae or Fabaceae plant family, mainly in soy.
Since legumes, soy in primis, are a major part of the diet in many cultures, these flavonoids may have a great impact on human health.
They are also present in beans and broad beans, but in much lower concentrations than those found in soy and soy products.
Also red clover or meadow clover (Trifolium pratense), another member of Leguminosae family, is a good source.
Currently, they are not found in fruits and vegetables.

Together with phenolic acids, such as caffeic acid and gallic acid, and quercetin glycosides, they are the most well-absorbed polyphenols, followed by flavanones and catechins (but not gallocatechins).

In plants, some isoflavones have antimicrobial activity and are synthesized in response to attacks by bacteria or fungi; thus they act as phytoalexins.

CONTENTS

Chemical structure of isoflavones

While most flavonoids have B ring attached to position 2 of C ring, isoflavones have B ring attached to position 3 of C ring.

Basic skeleton structure of isoflavones
Fig. 1 – Basic Skeleton of Isoflavones

Even if they are not steroids, they have structural similarities to estrogens, particularly estradiol. This confers them pseudohormonal properties, such as the ability to bind estrogen receptors; therefore, they are classified as phytoestrogens or plant estrogens. The benefits often ascribed to soy and soy products (e.g. tofu) are believed to result from the ability of isoflavones to act as estrogen mimics .
It should be underlined that the binding to estrogen receptors seems to lose strength with time, therefore their potential efficacy should not be overestimated.
In foods, they are present in four forms:

  • aglycone;
  • 7-O-glucoside;
  • 6′-O-acetyl-7-O-glucoside;
  • 6′-O-malonyl-7-O-glucoside.

Soy isoflavones: genistein, daidzein and glycitein

Soy and soy products, such as soy milk, tofu, tempeh and miso, are the main source of isoflavones in the human diet.
The isoflavone content of soy and soy products varies greatly as a function of growing conditions, geographic zone, and processing; for example, in soy it ranges between 580 and 3800mg/kg fresh weight, while in soy milk it range between 30 and 175 mg/L. The most abundant isoflavones in soy and soy products are genistein, daidzein and glycitein, generally present in a concentration ratio of 1:1:0,2.; biochanin A and formononetin are other isoflavones present in less concentrations.

Structural formulas of isoflavones genistein, daidzein, glycitein, biochanin A, formononetin
Fig. 2 – Isoflavones

The 6′-O-malonyl derivatives have a bitter, unpleasant, and astringent taste; therefore they give a bad flavor to the food in which they are contained. However, being sensitive to temperature, they are often hydrolyzed to glycosides during processing, such as the production of soy milk.
The fermentation processes needed for the preparation of certain foods, such as tempeh and miso, determines in turn the hydrolysis of glycosides to aglycones, i.e. the sugar-free molecule.
Isoflavone glycosides present in soy and soy products can also be deglycosylated by β-glucosidases in the small intestine.
The aglycones are very resistant to heat.
Although many compounds present in the diet are converted by intestinal bacteria to less active molecules, other compounds are converted to molecules with increased biological activity. This is the case of isoflavones, but also of prenylflavonoids from hops (Humulus lupulus), and lignans, that are other phytoestrogens.

Phytoestrogens and menopause

Vasomotor symptoms, such as night sweats and hot flashes, and bone loss are very common in perimenopause, also called menopausal transition, and menopause. Hormone replacement therapy (HRT) has proved to be a highly effective treatment for the prevention of menopausal bone loss and vasomotor symptoms.
The use of alternative therapies based on phytoestrogens is increased as a result of the publication of the “Women’s Health Initiative” study, that suggests that hormone replacement therapy could lead to more risks than benefits, in particular an increased risk of developing some chronic diseases.
Soy isoflavones are among the most used phytoestrogens by menopausal women, often taken in the form of isoflavone fortified foods or isoflavone supplements.
However, many studies have highlighted the lack of efficacy of soy isoflavones, and red clover isoflavones, even in large doses, in the prevention of vasomotor symptoms (hot flushes and night sweats) and bone loss during menopause.

References

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

Han X., Shen T. and Lou H. Dietary polyphenols and their biological significance. Int J Mol Sci 2007;9:950-988. doi:10.3390/i8090950

Lethaby A., Marjoribanks J., Kronenberg F., Roberts H., Eden J., Brown J. Phytoestrogens for menopausal vasomotor symptom. Cochrane Database of Systematic Reviews 2013, Issue 12. Art. No.: CD001395. doi:10.1002/14651858.CD001395.pub4

Manach C., Scalbert A., Morand C., Rémésy C., and Jime´nez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79(5):727-47 doi:10.1093/ajcn/79.5.727

Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients 2010;2:1231-46. doi:10.3390/nu2121231

Green tea benefits for health

Tea drinking, particularly green tea, has always been associated, at least in East Asia cultures (mainly in China and Japan) with health benefits. Only recently, the scientific community has begun to study the health benefits of tea consumption, recognizing its preventive value in many diseases.

Benefits in preventing cancer

Several epidemiological and laboratory studies have shown encouraging results with respect to possible preventive role of tea, particularly green tea and its catechins, a subgroup of flavonoids (the most abundant polyphenols in human diet) against the development of some cancers like:

  • oral and digestive tract cancers;
  • lung cancer among those who have never smoked, not among smokers.

Tea polyphenols, the most active of which is epigallocatechin-3-gallate (EGCG), seem to act not only as antioxidants, but also as molecules that, directly, may influence gene expression and diverse metabolic pathways.

Green tea and cardiovascular disease

Cardiovascular disease is the main cause of deaths worldwide, particularly in low- and middle-income countries, with an estimate of about 17 million deaths in 2008 that will increase up to 23.3 million by 2030.
Daily tea consumption, especially green tea, seems to be associated with a reduced risk of developing cardiovascular disease, such as hypertension and stroke.
Among the proposed mechanisms, the improved bioactivity of the endothelium-derived vasodilator nitric oxide (NO), due to the action of tea polyphenols that enhance nitric oxide synthesis, and/or decrease superoxide-mediated nitric oxide breakdown seem to be important.

Green tea and antioxidant properties

Tea polyphenols may act, in vitro, as free radical scavengers.
Since radical damage plays a pivotal role in the development of many diseases such as atherosclerosis, rheumatoid arthritis, cancer, or in ischemia-reoxygenation injury, tea polyphenols, particularly green tea catechins, may have a preventive role.

Benefits in weight loss and weight maintenance

Green tea, but also oolong tea, that is, catechins and caffeine rich teas, has a potential thermogenic effect. This has made them a potential tool for:

  • weight loss, by increasing energy expenditure and fat oxidation;
  • weight maintenance, ensuring a high energy expenditure during the maintenance of weight loss.

Indeed, it has been shown that green tea and green tea extracts are not an aid in weight loss and weight maintenance, since:

  • they are not able to induce a significant weight loss in overweight and obese adults;
  • they are not helpful in the maintenance of weight loss.

Green tea and preventing dental decay

Animal and in vitro studies have shown that tea, and in particular its polyphenols, seems to possess:

  • antibacterial properties against pathogenic action of cariogenic bacteria, as Streptococcus mutans, particularly green tea EGCG;
  • inhibitory action on salivary and bacterial amylase (it seems that black tea thearubigins and theaflavins are more effective than green tea catechins);
  • it is able to inhibit acid production in the oral cavity./li>

All these properties make green tea and black tea, beverages with potential anticariogenic activity.

References

Arab L., Khan F., and Lam H. Tea consumption and cardiovascular disease risk. Am J Clin Nutr 2013;98:1651S-1659S doi:10.3945/ajcn.113.059345

Dwyer J.T. and Peterson J. Tea and flavonoids: where we are, where to go next. Am J Clin Nutr 2013;98:1611S-1618S doi:10.3945/ajcn.113.059584

Goenka P., Sarawgi A., Karun V., Nigam A.G., Dutta S., Marwah N. Camellia sinensis (Tea): implications and role in preventing dental decay. Phcog Rev 2013;7:152-6 doi:10.4103/0973-7847.120515

Grassi D., Desideri G., Di Giosia P., De Feo M., Fellini E., Cheli P., Ferri L., and Ferri C. Tea, flavonoids, and cardiovascular health: endothelial protection. Am J Clin Nutr 2013;98:1660S-1666S doi:10.3945/ajcn.113.058313

Hursel R. and Westerterp-Plantenga M.S. Catechin- and caffeine-rich teas for control of body weight in humans. Am J Clin Nutr 2013;98:1682S-1693S doi:10.3945/ajcn.113.058396

Hursel R., Viechtbauer W. and Westerterp-Plantenga M.S. The effects of green tea on weight loss and weight maintenance: a meta-analysis. Int J Obesity 2009;33:956-961 doi:10.1038/ijo.2009.135

Jurgens T.M., Whelan A.M., Killian L., Doucette S., Kirk S., Foy E. Green tea for weight loss and weight maintenance in overweight or obese adults. Editorial group: Cochrane Metabolic and Endocrine Disorders Group. 2012:12 Art. No.: CD008650 doi:10.1002/14651858.CD008650.pub2

Lagari V.S., Levis S. Phytoestrogens for menopausal bone loss and climacteric symptoms. J Steroid Biochem Mol Biol 2014;139:294-301 doi:10.1016/j.jsbmb.2012.12.002

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Tea: cultivation, processing and preparation

Tea is an aromatic infusion extracted from the dried leaves of Camellia sinensis, a member of the Theaceae family.
Tea is a beverage with very ancient origins, dating back to almost 4000 years ago, and it is one of the most consumed beverage worldwide, particularly in Asia, with an estimated per capita consumption of approximately 0.12 L/d.
Owing to its high consumption, even small effects on person’s health could have large effects on public health.

Cultivation of Camellia sinensis

Camellia sinensis is an evergreen plant, native to South, East, and Southeast Asia, which is currently cultivated in at least 30 countries, mainly in tropical or sub-tropical climates, even if some varieties grow in Cornwall, in Europe, and Washington State, in the USA.
In nature, if left undisturbed, Camellia sinensis can grow up to 15-20 meters (49-65 ft), while in plantations it is generally pruned to height less than a meter and a half, that is, like a small tree or bush, to facilitate cultivation and harvesting of the leaves.
It can also be cultivated in mountain, up to 1500-2000 meters (4900-6550 ft) above sea level. Many of the high-quality teas are produced from mountain crops, as the plant grows more slowly acquiring a better flavor.
Currently, the two most used varieties, of the four ones recognized, are:

  • Camellia sinensis var. sinensis, native to China;
  • Camellia sinensis var. assamica, native to India.

Processing of leaves

All the types of teas commercially available are produced from fresh leaves of the plant. During harvesting, young leaves are generally picked, as the older ones are considered to be inferior in quality.
The differences between the types of teas, e.g. green tea, oolong tea and black tea, depend on how Camellia sinensis leaves are processed after harvesting, since processing may cause a different degrees of oxidation of the substances present, in particular of catechins, a flavonoid subgroup, and the main responsibles of the benefits of green tea.
The organoleptic characteristics of the different types of tea are influenced, in addition to the processing of the leaves, even from the cultivar, the characteristics of the soil where the plant grown up, the methods of cultivation, the altitude, the climate, and the time of year in which the harvest of the leaves occurs.

How to prepare a perfect cup of tea

  • Due to the sensitiveness of dried leaves, it is good to store the packaging in cool dry place, free of fragrances that may alter its aroma.
  • Use fresh water and warm it to a temperature of 95-100°C for black tea, and about 90°C for green tea.
  • In order not to alter flavor, it is advisable to use a ceramic or porcelain teapot, avoiding those of steel. For teapot washing, avoid detergents, preferring water plus baking soda.
  • To prevent sudden changes in water temperature during the infusion, it is advisable to preheat the teapot pouring a bit of boiling water. Then, emptied the pot, add hot water (about 200-250 mL/filter)
  • How many filters/g of leaves to use? Typically, a filter (about 1.5-2 g) per person, or a teaspoon of loose tea leaves per person.
    If you prepare the beverage for some people, you add a filter/teaspoon more than the number of persons.
  • The infusion time should not exceed 10 minutes in order to avoid the development of bitter flavors; it should be 3-4 minutes for black tea, and 2-3 minutes for green tea.
    If you are using filters, you should remove them at the end of the infusion time.
    Approximately 30% of the material present in the leaves is extracted in the water.

Now, it’s time to enjoy your tea.

References

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Li S., Lo C-Y., Pan M-H., Lai C-S. and Ho C-T. Black tea: chemical analysis and stability. Food Funct 2013;4:10-18 doi:10.1039/C2FO30093A

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